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A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy
Abstract
Dendritic cells (DCs) can induce tumor- or pathogen-specific T cell responses in humans. We comprehensively compared the clinically available DC maturation stimuli for their ability to promote uniformly mature DCs that elicit higher levels of T cell responses. We compared the standard maturation stimulus, autologous monocyte-conditioned medium (MCM), with a synthetic double stranded RNA (poly I:C), soluble CD40 ligand trimer, and a defined cocktail of cytokines (TNF-alpha, IL-1 beta, IL-6) and PGE(2) to promote mature phenotype and function in human monocyte-derived DCs. The cocktail was the most efficient despite the lack of induction of IL-12p70. While these results support the use of the MCM-mimic cocktail in clinical DC immunotherapy trials, the roles of it's individual constituents remain to be completely defined.
... Still, the most common DC vaccination approach is based on MoDCs (26,27), and they are typically matured by a cytokine cocktail consisting of IL-1β/TNF-α/IL-6/PGE 2 (28). Although tumor therapy with cocktail-matured MoDCs has proven to be successful in melanoma patients, it is still unclear how such cytokine cocktail-matured MoDCs that are unable to produce IL-12 (29) are readily able to induce Th1 responses in these patients (27,29,30). Also, the use of BM-DC from IL-12-deficient mice for vaccination against Leishmania infection indicated that the development of Th1 responses relied on an undetermined source of IL-12 production by the recipient mice, not the injected DCs (31). ...
... Still, the most common DC vaccination approach is based on MoDCs (26,27), and they are typically matured by a cytokine cocktail consisting of IL-1β/TNF-α/IL-6/PGE 2 (28). Although tumor therapy with cocktail-matured MoDCs has proven to be successful in melanoma patients, it is still unclear how such cytokine cocktail-matured MoDCs that are unable to produce IL-12 (29) are readily able to induce Th1 responses in these patients (27,29,30). Also, the use of BM-DC from IL-12-deficient mice for vaccination against Leishmania infection indicated that the development of Th1 responses relied on an undetermined source of IL-12 production by the recipient mice, not the injected DCs (31). ...
... This study also sheds light on the question why the IL-1β/TNF-α/IL-6/PGE 2 matured vaccine DCs are successful in Th1 priming, despite a lack of IL-12-producing capacity (29,30). This was also reported for BM-DC immunization of Leishmania major-challenged mice, where IL-12 production by BM-DCs was not required and, rather, recipient IL-12 was mandatory. ...
Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node-resident DCs for Th1 induction. While migration of the injected DCs was essential for antigen delivery to the lymph node, the injected DCs contributed only partially to Th0 priming and were unable to instruct Th1 generation. Instead, we provide evidence that the lymph node-resident XCR1+ DCs are activated by the injected DCs to present the cognate antigen and release IL-12 for Th1 polarization. The timing of interactions in the draining lymph nodes appeared step-wise as (a) injected DCs with cognate T cells, (b) injected DCs with bystander DCs, and (c) bystander DCs with T cells. The transcriptome of the bystander DCs showed a downregulation of Treg- and Th2/Th9-inducing genes and self-antigen presentation, as well as upregulation of MHC class II and genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory DCs direct T cell priming and bystander DC activation, but not Th1 polarization, which is mediated by endogenous IL-12p70+XCR1+ resident bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.
... Currently, at least five different human DC vaccine maturation protocols have been used in clinical trials. These protocols include a four-cytokine cocktail (TNF-α, IL-6, IL-1β, and PGE2) (Lee et al., 2002;Scandella et al., 2004;Batich et al., 2017); an α-DC cytokine cocktail (TNF-α, IL-1β, IFN-α, IFN-γ, and poly I:C) (Mailliard et al., 2004;Lee et al., 2008;Park et al., 2011;Akiyama et al., 2012); LPS plus IFN-γ (Dohnal et al., 2007), TNF-α plus PGE2 (Holtl et al., 1999); and TriMix DC (electroporation of a mixture of CD40L, CD70, and a constitutively active form of Toll-like receptor 4 (caTLR4) mRNAs into immature DCs) (Van Nuffel et al., 2012;Benteyn et al., 2013). These protocols use combinations of DC maturation signals, such as pathogen-associated molecular patterns (LPS and poly I:C), T cell-dependent signals (IFN-γ and CD40L), and inflammatory cytokines (TNF-α, IL-6, and IL-1β), to upregulate the expression of costimulatory molecules, HLA, and chemokine receptors. ...
... These protocols are likely to have distinct capabilities to modulate human DC function. For example, the four-cytokine cocktail has been demonstrated to induce the upregulation of DC maturation markers but no IL-12p70 (Lee et al., 2002). The α-DC cytokine cocktail matured DCs can produce high level of IL-12p70, but show lower efficiency to express exogenous mRNA genes (Bontkes et al., 2007). ...
... Adherent cells were cultured with 15 ml AIM-V media (Invitrogen) containing 800 U/ml GM-CSF and 500 U/ml human IL-4 at 37 • C for 6 days (immature DCs) (Nair et al., 2012). On day 7, cells were collected and seeded in 12well plates at 1 × 10 6 cells/ml and then activated with different maturation stimuli (mDCs): four-cytokine cocktail (160 ng/ml IL-6, 5 ng/ml IL-1β, 5 ng/ml TNF-α, and 1 µg/ml PGE2) (Lee et al., 2002;Scandella et al., 2004;Batich et al., 2017); α-DCcytokine cocktail (10 ng/ml IL-1β, 50 ng/ml TNF-α, 3,000 IU/q IFN-α, 1,000 IU/ml IFN-γ, and 20 µg/ml poly I:C) (Mailliard et al., 2004;Lee et al., 2008;Park et al., 2011;Akiyama et al., 2012); 100 ng/ml LPS plus 1,000 IU/ml IFN-γ (Dohnal et al., 2007); and 10 ng/ml TNF-α plus 10 µg/ml PGE2 (Holtl et al., 1999). ...
Dendritic cell (DC)-based vaccination is a promising immunotherapeutic strategy for cancer. However, clinical trials have shown only limited efficacy, suggesting the need to optimize protocols for human DC vaccine preparation. In this study, we systemically compared five different human DC vaccine maturation protocols used in clinical trials: (1) a four-cytokine cocktail (TNF-α, IL-6, IL-1β, and PGE2); (2) an α-DC-cytokine cocktail (TNF-α, IL-1β, IFN-α, IFN-γ, and poly I:C); (3) lipopolysaccharide (LPS)/IFN-γ; (4) TNF-α and PGE2; and (5) TriMix (mRNAs encoding CD40L, CD70, and constitutively active Toll-like receptor 4 electroporated into immature DCs). We found that the four-cytokine cocktail induced high levels of costimulatory and HLA molecules, as well as CCR7, in DCs. Mature DCs (mDCs) matured with the four-cytokine cocktail had higher viability than those obtained with the other protocols. Based on these features, we chose the four-cytokine cocktail protocol to further improve the immunizing capability of DCs by introducing exogenous genes. We showed that introducing exogenous Bcl-2 increased DC survival. Furthermore, introducing IL-12p70 rescued the inhibition of IL-12 secretion by PGE2 without impairing the DC phenotype. Introducing both Bcl-2 and IL-12p70 mRNAs into DCs induced enhanced cytomegalovirus pp65-specific CD8⁺ T cells secreting IFN-γ and TNF-α. Taken together, our data suggest that DC matured by the four-cytokine cocktail combined with exogenous Bcl-2 and IL-12p70 gene expression represents a promising approach for clinical applications in cancer immunotherapy.
... The optimal maturation stimuli should induce high expression of MHC-I and MHC-II molecules, co-stimulatory molecules (e.g., CD40, CD80, CD86), and high secretion of Th1 inflammatory cytokines (e.g., IFN-γ, IL-12). The current standard maturation mix contains TNF-α, IL-1β, IL-6, and PGE2 [142]. Although this mix could efficiently upregulate DC surface maturation markers, it fails to induce IL-12 production [142]. ...
... The current standard maturation mix contains TNF-α, IL-1β, IL-6, and PGE2 [142]. Although this mix could efficiently upregulate DC surface maturation markers, it fails to induce IL-12 production [142]. Nonetheless, this cytokine cocktail is able to induce uniform DC maturation, as well as high levels of T cell proliferation and priming [142], and has thus been selected as a gold standard for maturation in many studies. ...
... Although this mix could efficiently upregulate DC surface maturation markers, it fails to induce IL-12 production [142]. Nonetheless, this cytokine cocktail is able to induce uniform DC maturation, as well as high levels of T cell proliferation and priming [142], and has thus been selected as a gold standard for maturation in many studies. Our group demonstrated that simulating WTL-pulsed DCs with LPS and IFN-γ led to strong IL-12p70 and IP-10 productions, as well as highly efficient MLRs by these activated DCs [143]. ...
Although different types of therapeutic vaccines against established cancerous lesions in various indications have been developed since the 1990s, their clinical benefit is still very limited. This observed lack of effectiveness in cancer eradication may be partially due to the often deficient immunocompetent status of cancer patients, which may facilitate tumor development by different mechanisms, including immune evasion. The most frequently used cellular vehicle in clinical trials are dendritic cells (DCs), thanks to their crucial role in initiating and directing immune responses. Viable vaccination options using DCs are available, with a positive toxicity profile. For these reasons, despite their limited therapeutic outcomes, DC vaccination is currently considered an additional immunotherapeutic option that still needs to be further explored. In this review, we propose potential actions aimed at improving DC vaccine efficacy by counteracting the detrimental mechanisms recognized to date and implicated in establishing a poor immunocompetent status in cancer patients.
... In the laboratory, TLR agonists such as LPS (TLR4), poly IC (TLR3), and resiquimod (TLR7) are commonly used as DC activators. In the clinic, a commonly used maturation cocktail of proinflammatory cytokines TNFα, IL-1β, and IL-6 combined with prostaglandin E 2 (PGE 2 ) was initially established as the gold standard for maturation [106]. This cocktail induced the upregulation of MHC class I and II molecules, CD40, CD80, CD86, and CCR7 but failed to effectively induce IL-12p70 [106]. ...
... In the clinic, a commonly used maturation cocktail of proinflammatory cytokines TNFα, IL-1β, and IL-6 combined with prostaglandin E 2 (PGE 2 ) was initially established as the gold standard for maturation [106]. This cocktail induced the upregulation of MHC class I and II molecules, CD40, CD80, CD86, and CCR7 but failed to effectively induce IL-12p70 [106]. However, when compared with other DC maturation stimuli (CD40L trimer, poly IC, and LPS), the cytokine cocktail induced the most uniform maturation in terms of upregulation of DC maturation markers, with the highest yield and recovery; it also stimulated the highest levels of allogeneic T cell proliferation and cytokine production, and it induced priming of Th1 responses [106]. ...
... This cocktail induced the upregulation of MHC class I and II molecules, CD40, CD80, CD86, and CCR7 but failed to effectively induce IL-12p70 [106]. However, when compared with other DC maturation stimuli (CD40L trimer, poly IC, and LPS), the cytokine cocktail induced the most uniform maturation in terms of upregulation of DC maturation markers, with the highest yield and recovery; it also stimulated the highest levels of allogeneic T cell proliferation and cytokine production, and it induced priming of Th1 responses [106]. Other studies have indicated that PGE 2 may induce differentiation of regulatory T cells and Th2 responses [107], IDO expression [108], and is responsible for the lack of IL-12p70 production [109]. ...
Immunotherapy using dendritic cell (DC)-based vaccination is an approved approach for harnessing the potential of a patient's own immune system to eliminate tumor cells in metastatic hormone-refractory cancer. Overall, although many DC vaccines have been tested in the clinic and proven to be immunogenic, and in some cases associated with clinical outcome, there remains no consensus on how to manufacture DC vaccines. In this review we will discuss what has been learned thus far about human DC biology from clinical studies, and how current approaches to apply DC vaccines in the clinic could be improved to enhance anti-tumor immunity.
... Instead, for maturation, a much wider variety of cytokine cocktails have been used by different groups [10]. In particular, a cocktail mixture containing tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and prostaglandin E 2 (PGE2), is currently considered the gold standard for DC maturation and had been largely used in the context of anti-cancer therapeutic vaccines [19,20]. This mix is able to efficiently induce expression of common DC surface maturation markers, uniform DC maturation, as well as high levels of T cell proliferation and priming [19]. ...
... In particular, a cocktail mixture containing tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and prostaglandin E 2 (PGE2), is currently considered the gold standard for DC maturation and had been largely used in the context of anti-cancer therapeutic vaccines [19,20]. This mix is able to efficiently induce expression of common DC surface maturation markers, uniform DC maturation, as well as high levels of T cell proliferation and priming [19]. However, DCs matured with this mixture failed to produce significant levels of interleukin-12 (IL-12) a crucial signal for T cell activation and Th1 differentiation [21]. ...
In the last 20 years, dendritic cells (DCs) have been largely used as a platform for therapeutic vaccination in cancer patients. However, despite its proven safety and ability to induce cancer specific immune responses, the clinical benefits of DC-based immunotherapy are currently very limited. Thus, novel approaches are still needed to boost its efficacy. Our group recently showed that squaric acid treatment of antigens is an important adjuvant that can increase vaccine-induced downstream immune responses and therapeutic outcomes. Here we further improved this dendritic cell vaccine formulation by developing a new method for differentiating and maturing DCs from their bone marrow precursors. Our data demonstrate that bone marrow-derived DCs differentiated with GM-CSF and IL-15 and matured with a maturation cocktail in two steps present a more mature and immunogenic phenotype, compared to standard DC preparations. Further suppression of the prostaglandin E2 pathway achieved even more immunogenic DC phenotypes. This vaccine was more potent at delaying tumor growth, improved animal survival and induced a more immunogenic and Th1-skewed T cell response in an ovarian cancer mouse model. These promising results support future efforts for the clinical translation of this approach.
... Upon interacting with infectious agents, immature DCs subsequently mature, a process that includes DC surface remodeling, increased antigen presentation, and the production of numerous cytokines (22). Mature DCs upregulate chemokine receptors (e.g., CCR7) that aid in their migration to lymph nodes as well as costimulatory molecules (e.g., CD80, CD86, CD40) that are required for activating antigen-specific T cells in the lymph node and inducing T cell proliferation and effector functions (23,24). In this way, DCs are key players in linking innate and adaptive immunity. ...
... Since we observed that both infected and bystander DCs are capable of activation, the bystander DCs are most likely undergoing cytokine-mediated maturation (51,52). Of note, it has been shown that cytokine-induced maturation of DCs and subsequent neoantigen pulsing promotes DC priming of naive CD4 + T cells toward a Th1 response (23). ...
Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.
... Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97,98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97,98). ...
... The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97,98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97,98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99,100). ...
Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.
... Although tumor lysates contain multiple epitopes of tumor antigens, many components released from tumor cell lysis may inhibit DC maturation, thereby affecting their ability to effectively present antigens. Most studies have improved the anti-tumor immune effect of the DC vaccine through combination with immune adjuvants [16][17][18] but few have attempted to eliminate the possible components of whole tumor lysate that may inhibit the maturation of DCs to improve the clinical efficacy of DC vaccines. ...
Objective This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell (DC) vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma. Methods Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail. The modified tumor lysate was generated by differential centrifugation. The maturity markers of DCs in each group, namely the modified tumor lysate, tumor lysate, and negative and positive control groups, were assessed using flow cytometry. Furthermore, their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE. IFN-γ secretion levels were measured with ELISA. Intracellular reactive oxygen species were measured using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The results were statistically analyzed using an unpaired Student's t-test and were considered significant at P < 0.05. Results Compared with tumor lysate-pulsed DCs, modified tumor lysate-pulsed DCs had a higher expression of maturity markers: CD1a (7.38 ± 0.53% vs. 4.47 ± 0.75%) and CD83 (19.81 ± 4.09% vs. 9.64 ± 1.50%), were better capable of stimulating lymphocyte proliferation [proliferation index (PI): 8.54 ± 0.16 vs. 7.35 ± 0.05], secreting IFN-γ, and inducing stronger in-vitro cytotoxic T lymphocyte (CTL) cytotoxicity against glioblastoma cells. In addition, we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs. Conclusion Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines, and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate. Abstract Glioblastoma is the most common primary malignant tumor of the central nervous system with an annual incidence rate of 3-5/100 000 and a poor prognosis of 14.6 months, accounting for about 50% of all gliomas [1-2]. Although there are numerous studies on glioblastoma, the anatomical location of the central nervous system limits the application of locoregional treatment. In addition, the tumor is characterized by endogenous radio-and chemoresistance, heterogeneity, and an immunosuppressive microenvironment; these factors limit the progress of glioblastoma treatment. The standard treatment is to safely maximize surgical resection with adjuvant radiotherapy and chemotherapy [3-4]. Other adjuvant treatments include tumor treating fields, immunotherapy, gene therapy, and molecular targeted therapy, among which immunotherapy has great potential in the treatment of glioblastoma. As the most effective antigen-presenting cells, dendritic cells (DCs), bridge the innate and adaptive immune system [5-7]. DCs are cultured in vitro for maturation, sensitized with tumor-associated antigens or tumor-specific antigens, and then injected back into Downloaded from
... Maturation of DCs can be achieved in many different ways, but still, there is no consensus on adequate maturation stimuli. Gold standard for maturation of DCs used to include a wellknown cocktail of proinflammatory cytokines TNF-α, IL-1β and IL-6 with PGE2 (28). This way matured DCs highly express CD40, CD80, CD86, MHC class I and II, but fail to induce IL-12 production, important for the development of Th1 antitumor response. ...
... Although tumor lysates contain multiple epitopes of tumor antigens, many components released from tumor cell lysis may inhibit DC maturation, thereby affecting their ability to effectively present antigens. Most studies have improved the anti-tumor immune effect of the DC vaccine through combination with immune adjuvants [16][17][18] but few have attempted to eliminate the possible components of whole tumor lysate that may inhibit the maturation of DCs to improve the clinical efficacy of DC vaccines. ...
Objective
This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell (DC) vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma.
Methods
Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail. The modified tumor lysate was generated by differential centrifugation. The maturity markers of DCs in each group, namely the modified tumor lysate, tumor lysate, and negative and positive control groups, were assessed using flow cytometry. Furthermore, their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE. IFN-γ secretion levels were measured with ELISA. Intracellular reactive oxygen species were measured using 2’, 7’-dichlorofluorescein diacetate (DCFDA) staining. The results were statistically analyzed using an unpaired Student's t -test and were considered significant at P < 0.05.
Results
Compared with tumor lysate-pulsed DCs, modified tumor lysate-pulsed DCs had a higher expression of maturity markers: CD1a (7.38 ± 0.53% vs . 4.47 ± 0.75%) and CD83 (19.81 ± 4.09% vs . 9.64± 1.50%), were better capable of stimulating lymphocyte proliferation [proliferation index (PI): 8.54 ± 0.16 vs . 7.35 ± 0.05], secreting IFN-γ, and inducing stronger in-vitro cytotoxic T lymphocyte (CTL) cytotoxicity against glioblastoma cells. In addition, we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs.
Conclusion
Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines, and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate.
... Although they are not the only innate immune cell with the capability to activate T cells, DCs are one of the most proficient, requiring only a select few for the activation of naïve T cells, initiating the adaptive immune response [59]. However, to accomplish this process of DC-mediated T cell activation, the cells must undergo maturation through the increased surface presentation of MHC-II and co-stimulatory molecules (CD80 and CD86) [37,[60][61][62]. As mentioned before, GXM has suppressive effects on the host immune response and prevents the phagocytosis of cryptococcal cells, and it non-specifically downregulates T cell proliferation [27]. ...
Cryptococcus neoformans is an opportunistic fungal pathogen that causes over 180,000 annual deaths in HIV/AIDS patients. Innate phagocytes in the lungs, such as dendritic cells (DCs) and macrophages, are the first cells to interact with the pathogen. Neutrophils, another innate phagocyte, are recruited to the lungs during cryptococcal infection. These innate cells are involved in early detection of C. neoformans, as well as the removal and clearance of cryptococcal infections. However, C. neoformans has developed ways to interfere with these processes, allowing for the evasion of the host’s innate immune system. Additionally, the innate immune cells have the ability to aid in cryptococcal pathogenesis. This review discusses recent literature on the interactions of innate pulmonary phagocytes with C. neoformans.
... Mature DCs also upregulate costimulatory molecules such as CD40 that play a key role in the activation of antigen-specific T-cells in the lymph nodes. 15,16 Human monocyte-derived DCs (MDDCs), generated from peripheral blood mononuclear cells (PBMCs) from healthy blood donors, have been characterized for their ability to support robust infection of DENV and are the primary model system for studying DENV infections ex-vivo. 17,18,19 There is currently no globally licensed therapeutics, and the currently licensed vaccine has variable protection against each of the four DENV serotypes. ...
Annually, roughly 2.5 billion people are at risk for dengue virus (DENV) infection, and the incidence of infection has increased 30-fold since its discovery in the 1900s. At present, there are no globally licensed antiviral treatments or vaccines that protect against all four of the DENV serotypes. The NIAID Live Attenuated Tetravalent Vaccine (LATV) dengue vaccine candidate is composed of variants of three DENV serotypes attenuated by a 30 nucleotide (Δ30) deletion in the 3' untranslated region and a fourth component that is a chimeric virus in which the prM and E genes of DENV-2 replace those of DENV-4 on the rDEN4Δ30 backbone. The vaccine candidate encodes the non-structural proteins of DENV-1, DENV-3, and DENV-4, which could be of critical importance in the presentation of DENV-specific epitopes in a manner that facilitates antigen presentation and confers higher protection. Our findings demonstrate that the attenuation mechanism (Δ30) resulted in decreased viral infectivity and replication for each vaccine virus in monocyte-derived dendritic cells but were able to generate a robust innate immune response. When tested as monovalent viruses, DEN-4Δ30 displayed the most immunogenic profile. In addition, we found that the tetravalent DENV formulation induced a significantly greater innate immune response than the trivalent formulation. We demonstrate that the presence of two components with a DENV-4Δ30 backbone is necessary for the induction of RANTES, CD40, IP-10, and Type I IFN by the tetravalent formulation. Finally, we found that the DEN-4Δ30 backbone in the DENV-2 component of the vaccine enhanced its antigenic properties, as evidenced by enhanced ability to induce IP-10 and IFNα2 in monocyte-derived dendritic cells. In sum, our study shows that the Δ30 and Δ30/Δ31 mutations attenuate the DENV vaccine strains in terms of replication and infectivity while still allowing the induction of a robust innate immune response.
... In order to improve the efficacy of DC vaccines, researchers have made various attempts to improve the clinical treatment effect of DC vaccine. For example, TLR agonists (lipopolysaccharide, polyI:C, and Resiquimod, etc.) or inflammatory cytokines (TNFα, IL-1β, and IL-6, etc.) are used to activate DCs in vitro [14] . Fusion of DCs and tumor cells into DC-tumor hybrid cells can produce stronger cellular immunity, compared with the mixture of DCs and tumor cells [15] . ...
Dendritic cells (DCs)‐based tumor vaccines have the advantages of high safety and rapid activation of T cells, and have been approved for clinical tumor treatment. However, the conventional DC vaccines have some severe problems, such as poor activation of DCs in vitro, low level of antigen presentation, reduced cell viability, and difficulty in targeting lymph nodes in vivo, resulting in poor clinical therapeutic effects. In this research, magnetic nanoparticles Fe3O4@Ca/MnCO3 were prepared and used to actively and efficiently deliver antigens to the cytoplasm of DCs, promote antigen cross‐presentation and DC activation, and finally enhance the cellular immune response of DC vaccines. The results show that the magnetic nanoparticles can actively and quickly deliver antigens to the cytoplasm of DCs by regulating the magnetic field, and achieve cross‐presentation of antigens. At the same time, the nanoparticles degradation product Mn²⁺ enhanced immune stimulation through the interferon gene stimulating protein (STING) pathway, and another degradation product Ca²⁺ ultimately promoted cellular immune response by increasing autophagy. The DC vaccine constructed with the magnetic nanoparticles can more effectively migrate to the lymph nodes, promote the proliferation of CD8⁺ T cells, prolong the time of immune memory, and produce higher antibody levels. Compared with traditional DC vaccines, cytoplasmic antigen delivery with the magnetic nanoparticles provides a new idea for the construction of novel DC vaccines.
... LPS is a major inflammatory component of gram-negative bacteria that binds to TLR4 and triggers the release of proinflammatory cytokines, the upregulation of costimulatory molecules, and the activation of antigen presentation on APCs, inducing both innate and adaptive immune responses (44). On the other hand, the cocktail containing TNFa, IL-1b, IL-6 and PGE2 has been described to induce Th1-polarized immune responses (46). These observations suggest that PODXL could exert tolerogenic or anti-inflammatory effects in immature DCs, so that its downregulation in response to inflammatory stimuli would favor an effective immune response. ...
Podocalyxin (PODXL), a cell surface sialomucin expressed in diverse types of normal and malignant cells, mediates cellular adhesion to extracellular matrix and cell-to-cell interaction. A previous study reported the expression of PODXL protein on monocytes undergoing macrophage differentiation, yet the expression of this molecule in other antigen presenting cells (APCs) and its function in the immune system still remain undetermined. In this study, we report that PODXL is expressed in human monocyte-derived immature dendritic cells at both the mRNA and protein levels. Following dendritric cells maturation using pro-inflammatory stimuli, PODXL expression level decreased substantially. Furthermore, we found that PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways. Our results revealed a polarized distribution of PODXL during the interaction of APCs with CD4⁺ T cells, partially colocalizing with F-actin. Notably, PODXL overexpression in APCs promoted their interaction with CD4⁺ T cells and CD8⁺ T cells and decreased the expression of MHC-I, MHC-II, and the costimulatory molecule CD86. In addition, PODXL reduced the translocation of CD4⁺ T-cell centrosome toward the APC-contact site. These findings suggest a regulatory role for PODXL expressed by APCs in immune responses, thus representing a potential target for therapeutic blockade in infection and cancer.
... Furthermore, a previous study assessing the immunological function of DCs in breast cancer patients pointed out that peripheral and lymph nodal DCs were unable to induce a proper T cell response due to a lowered expression of the a major histocompatibility class II molecule (MCH II) as well as the costimulatory molecule CD86 (Satthaporn et al. 2004). Due to the recurring inefficient activation of T cells by DCs in several types of cancer, one strategy has been to develop DCs vaccines (Lee et al. 2002). Briefly, this consists of taking immature DCs from the patient and putting them in contact with the TAAs ex vivo, ensuring a proper processing and presentation of the TAA by the DCs. ...
The extracellular matrix molecule tenascin-C (TNC) promotes tumor progression and metastasis by poorly understood mechanisms. I used a novel breast progression model based on a syngeneic orthotopic tumor cell grafting approach and identified TNC as an important regulator of tumor growth. I document that TNC promotes the battle between tumor regression and growth, where combined expression of tumor cell- and host-derived TNC induces tumor cell rejection. Tumor cell-derived TNC may elicit regression by induction of an antigen presenting signature (APS) expressed by the host, which correlates with better breast cancer patient survival. Tumor-cell derived TNC also triggers CXCL12 expression, thereby causing trapping of CD8+ T cells in the surrounding TNC matrix tracks. TNC binds CXCL12, and combined TNC/CXCL12 attracts and immobilizes CD8+ T cells. Inhibition of the CXCL12 receptor CXCR4 causes tumor regression that is accompanied by massive infiltration of CD8+ T cells and cell death inside the tumor cell nests. Altogether,TNC-triggered CXCL12 signaling may dampen CD8+ T cell function where physical trapping of CD8+ T cells in the TNC matrix may have implications for immune cell therapies. Our results and new tumor model, offer novel opportunities for preclinical cancer research and cancer patient therapy, by triggering the “good” and blocking the “bad” actions of TNC. In particular, overcoming the immune suppressive action of TNC, through inhibition of CXCR4, could be a useful approach.
... (iii) The efficiency of DC migration: DCs injected back into patients should migrate to the lymphoid organs to stimulate T cells to achieve effective immune responses, and some proinflammatory cytokines, such as prostaglandin E 2 (PGE 2 ), could promote the migration of DCs to some extent (70)(71)(72). However, selective migration of DCs and their residence in nonlymphoid and lymphoid organs are tightly regulated events. ...
In the past few decades, great progress has been made in the clinical application of dendritic cell (DC) vaccines loaded with personalized neoantigens. Personalized neoantigens are antigens arising from somatic mutations in cancers, with specificity to each patient. DC vaccines work based on the fundamental characteristics of DCs, which are professional antigen-presenting cells (APCs), responsible for the uptake, processing, and presentation of antigens to T cells to activate immune responses. Neoantigens can exert their antitumor effects only after they are taken up by APCs and presented to T cells. In recent years, neoantigen-based personalized tumor therapeutic vaccines have proven to be safe, immunogenic and feasible treatment strategies in patients with melanoma and glioblastoma that provide new hope in the treatment of cancer patients and a new approach to cure cancer. In addition, according to ClinicalTrials.gov, hundreds of registered DC vaccine trials are either completed or ongoing worldwide, of which 9 are in early phase I, 191 in phase I, 166 in phase II and 8 in phase III. Hundreds of clinical studies on therapeutic tumor vaccines globally have proven that DC vaccines are stable, reliable and very safe. However, in this process, many other factors still limit the effectiveness of the vaccine. This review will focus on the current research progress on personalized neoantigen-pulsed DC vaccines, their limitations and future research directions of DC vaccines loaded with neoantigens. This review aims to provide a better understanding of DCs biology and manipulation of activated DCs for DCs researchers to produce the next generation of highly efficient cancer vaccines for patients.
... The most commonly used preparation involves the reinfusion of ex-vivo derived DC pulsed with tumor-associated antigens (TAAs) or tumor cell lysates and stimulated with a defined maturation cocktail. The gold standard maturation cocktail included the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in combination with prostaglandin-E2 (PGE2) [11,12]. ...
Dendritic cells (DCs) play a critical role in the regulation of adaptive immune responses, furthermore they act as a bridge between the innate and the adaptive immune systems they have been ideal candidates for cell-based immunotherapy of cancers and infections in humans. The first reported trial using DCs in 1995, since they have been used in trials all over the world for several of indications, including cancer and human immunodeficiency virus infection. Generally, for in vitro experiments or for DCs vaccination monocyte-derived dendritic cells (moDCs) were generated from purified monocytes that isolated from peripheral blood by density gradient centrifugation. A variety of methods can be used for enrichment of monocytes for generation of clinical-grade DCs. Herein we summarized up to date understanding of systems and inputs used in procedures to differentiate DCs from blood monocytes in vitro.
... We generate a cytokine master mix before adding the cytokines to the wells containing DCs. Various DC maturation cocktails have been reported, including: (1) AIM-V medium containing GM-CSF (800 U/ml), IL-4 (500 U/ml), TNF-α (5 ng/ml), IL-1β (5 ng/ml), IL-6 (150 ng/ml), and prostaglandin E2 (PGE2; 1 μg/ml; Lee et al., 2002) and ...
T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor‐reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen‐specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single‐cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor‐reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Generation of human autologous dendritic cells from monocytes
Basic Protocol 2 : In vitro priming and expansion of human antigen‐specific T cells
Basic Protocol 3 : Cloning of antigen‐specific T cell receptors based on single‐cell VDJ sequencing data
Basic Protocol 4 : Validation of T cell receptor expression and functionality in vitro
Basic Protocol 5 : Rapid expansion of T cell receptor–transduced T cells and human T cell clones
... These mo-DCs secrete high levels of IL-12p70 and prime T cells to preferentially activate Th1 type and cytotoxic T lymphocyte (CTL) responses [38]. The gold standard maturation cocktail to obtain completely mature mo-DCs includes the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, IL-1β, and IL-6, as well as prostaglandin E2 (PGE2), in different combinations [40]. This approach is the most commonly used, even though its actual advantage relative to the use of GM-CSF/IL-4 alone has not been conclusively demonstrated. ...
The safety and feasibility of dendritic cell (DC)-based immunotherapies in cancer management have been well documented after more than twenty-five years of experimentation, and, by now, undeniably accepted. On the other hand, it is equally evident that DC-based vaccination as monotherapy did not achieve the clinical benefits that were predicted in a number of promising preclinical studies. The current availability of several immune modulatory and targeting approaches opens the way to many potential therapeutic combinations. In particular, the evidence that the immune-related effects that are elicited by immunogenic cell death (ICD)-inducing therapies are strictly associated with DC engagement and activation strongly support the combination of ICD-inducing and DC-based immunotherapies. In this review, we examine the data in recent studies employing tumor cells, killed through ICD induction, in the formulation of anticancer DC-based vaccines. In addition, we discuss the opportunity to combine pharmacologic or physical therapeutic approaches that can promote ICD in vivo with in situ DC vaccination.
... The DC vaccines involve the ability of these cells to act as an antitumoral effector in both CTLs and NK cells, in order to eradicate malignant cells (Kirkwood et al., 2012). There are several types of DC-vaccines, being the most frequently used the reinfusion of ex vivo derived DC pulsed with tumor-associated antigens (TAAs) or tumor cell lysates and stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) (Lee et al., 2002;Koski et al., 2008;Anguille et al., 2014). The DC-based immunotherapy efficiency may be enhanced using immune checkpoint inhibitors, such as anti PD-1 or anti-CTLA-4 antibodies (Mastelic-Gavillet et al., 2019). ...
Memory formation, guided by microbial ligands, has been reported for innate immune cells. Epigenetic imprinting plays an important role herein, involving histone modification after pathogen-/danger-associated molecular patterns (PAMPs/DAMPs) recognition by pattern recognition receptors (PRRs). Such “trained immunity” affects not only the nominal target pathogen, yet also non-related targets that may be encountered later in life. The concept of trained innate immunity warrants further exploration in cancer and how these insights can be implemented in immunotherapeutic approaches. In this review, we discuss our current understanding of innate immune memory and we reference new findings in this field, highlighting the observations of trained immunity in monocytic and natural killer cells. We also provide a brief overview of trained immunity in non-immune cells, such as stromal cells and fibroblasts. Finally, we present possible strategies based on trained innate immunity that may help to devise host-directed immunotherapies focusing on cancer, with possible extension to infectious diseases.
... DC/DC leugenerating-protocols contain combinations of different response modifiers, including (1) cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or Interleukin 4 (IL-4) that induce differentiation of myeloid progenitor cells; or (2) Calcium-Inophore (A23187) as a cytokine free DC/DC leu -generating method; (3) bacterial or nucleic stimulation with danger signaling effects, such as Picibanil (OK432), a lysis product from the streptococcus pyogenes or Polyinosinic:polycytidylic acid (poly I:C); and, (4) substances inducing maturation of DC/DC leu , such as Prostaglandin E 2 (PGE 2 ) or Tumor-necrosis-factor alpha (TNF-α) [17,20]. Furthermore, Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), Interferon gamma (IFN-γ), Interferon alpha (IFN-α), and Fms-related tyrosine kinase 3 ligand (FLT3-L) were used and analyzed in different protocols to generate DCs and/or DC leu from leukemic or healthy PBMCs [17,[20][21][22][23][24]. ...
Dendritic cells (DCs) and leukemia-derived DC (DCleu) are potent stimulators of various immunoreactive cells and they play a pivotal role in the (re-) activation of the immune system. As a potential treatment tool for patients with acute myeloid leukemia, we developed and analyzed two new PGE1-containing protocols (Pici-PGE1, Kit M) to generate DC/DCleu ex vivo from leukemic peripheral blood mononuclear cells (PBMCs) or directly from leukemic whole blood (WB) to simulate physiological conditions. Pici-PGE1 generated significantly higher amounts of DCs from leukemic and healthy PBMCs when compared to control and comparable amounts as the already established protocol Pici-PGE2. The proportions of sufficient DC-generation were even higher after DC/DCleu-generation with Pici-PGE1. With Kits, it was possible to generate DCs and DCleu directly from leukemic and healthy WB without induction of blast proliferation. The average amounts of generated DCs and DCleu-subgroups were comparable with all Kits. The PGE1 containing Kit M generated significantly higher amounts of mature DCs when compared to the PGE2-containing Kit K and increased the anti-leukemic-activity. In summary PGE1-containing protocols were suitable for generating DC/DCleu from PBMCs as well as from WB, which reliably (re-) activated immunoreactive cells, improved the overall ex vivo anti-leukemic activity, and influenced cytokine-release-profiles.
... In vitro maturation stimuli for DC subsets include Resiquimod, lipopolysaccharides (LPS) and polyinocinic:polycytidilic acid -each of these is a TLRs agonist. Another maturation stimulus that is very commonly employed is in the form of a cocktail of several proinflammatory cytokines inclusive of IL-6, IL-1β, prostaglandin E2 and TNF-α [38,40]. ...
A wide array of therapeutic strategies has been implemented against cancers, yet their clinical benefit is limited. The lack of clinical efficacy of the conventional treatment options might be due to the inept immune competency of the patients. Dendritic cells (DCs) have a vital role in initiating and directing immune responses and have been frequently used as delivery vehicles in clinical research. The recent clinical data suggest the potential use of DCs pulsed with nucleic acid, especially with RNA holds a great potential as an immunotherapeutic measure with compare to other cancer therapeutics. This review mainly deals with the DCs and their role in transfection with RNA in cancer immunotherapy.
... Conventional differentiation cocktails, including PGE-2, are clearly suboptimal due to impaired induction of IL-12p70 expression and their propensity to induce T regulatory cells. 41 A more recent method using TNF, IL-1β, poly I:C, IFN-α, and IFN-γ to generate αDC1 cultures has overcome some of these issues, resulting in cells that produce IL-12, migrate, activate CD8 + T cells and are safe in vivo. 42,43 However, it is not clear to what extent this method promotes enrichment for CD103 + / CD141 + DCs (or functional equivalents), which have been reported to be the antigen presenting cells capable of transporting intact antigens to lymph nodes and activating tumor-specific CD8 T-cell-mediated immune responses. ...
Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103⁺/CD141⁺ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8⁺ (but not CD4⁺) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8⁺ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.
... The most commonly used preparation involves the reinfusion of ex-vivo derived DC pulsed with tumor-associated antigens (TAAs) or tumor cell lysates and stimulated with a defined maturation cocktail. In the earlier trials, the gold standard maturation cocktail included the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in combination with prostaglandin E2 (PGE2) (8)(9)(10). However, despite the important roles of PGE2 in promoting DC migration (11) and in enhancing T cell proliferation (12), it has also been shown that PGE2 may induce differentiation of regulatory T cells (13), increase the expression of the pro-tolerogenic enzyme indoleamine 2,3-dioxygenase (IDO) (14), and may limit IL-12p70 production (15). ...
With the advent of combined immunotherapies, personalized dendritic cell (DC)-based vaccination could integrate the current standard of care for the treatment of a large variety of tumors. Due to their proficiency at antigen presentation, DC are key coordinators of the innate and adaptive immune system, and have critical roles in the induction of antitumor immunity. However, despite proven immunogenicity and favorable safety profiles, DC-based immunotherapies have not succeeded at inducing significant objective clinical responses. Emerging data suggest that the combination of DC-based vaccination with other cancer therapies may fully unleash the potential of DC-based cancer vaccines and improve patient survival. In this review, we discuss the recent efforts to develop innovative personalized DC-based vaccines and their use in combined therapies, with a particular focus on ovarian cancer and the promising results of mutanome-based personalized immunotherapies.
... Following maturation with canine IL-1β, TNF-α, IL-6, and PGE 2 [27,28], the adherent cell population adopted a characteristic dendritic cell morphology (Fig. 1a). Subsequent analysis by flow cytometry indicated that > 90% of the cells in the monocyte gate were CD11c + , CD40 + , and CD80 + (Fig. 1b, c), suggestive of a genuine dendritic cell phenotype. ...
Angiosarcoma is a deadly neoplasm of the vascular endothelium. Metastatic disease is often present at diagnosis, and 5-year survival is only 10–35%. Although there exist no immunocompetent mouse models of angiosarcoma with which to study immune-based approaches to therapy, angiosarcoma is a major killer of companion dogs, responsible for up to 2% of all canine deaths in some susceptible breeds or an estimated 120,000 per year in the US. The canine disease (HSA) often presents in the spleen as acute hemoabdomen secondary to splenic rupture. Even if life-saving splenectomy is performed, median overall survival (OS) is only 48 days, and 1-year survival is negligible. Here we report the analysis of a pilot phase I open-label trial of chemo-immunotherapy performed on consecutively presenting splenectomized canines with histologically verified HSA. Subjects received an abbreviated course of low-dose doxorubicin plus alpha interferon and an autologous dendritic cell-therapy reported to enhance durable CD8+ memory. Disease was monitored monthly by abdominal ultrasound, chest X-ray, and echocardiogram. Median OS in the per protocol population was 109 days including one of five animals that died cancer-free at 16 months after documented resolution of relapsed disease. These results indicate that therapeutic administration of chemo-immunotherapy is both feasible and safe, substantiating the rationale for additional veterinary and human clinical studies.
... The commonly used maturation cocktail for MoDC comprises the proinflammatory cytokines TNF-α, IL-1ß, and IL-6 combined with PGE2, which was established as the "gold standard" for MoDC maturation (the so-called "standard DC" or "sDC") (65). sDC upregulate major histocompatibility complex (MHC) class I and II molecules, costimulatory molecules and CCR7 but fail to induce IL-12p70 production, probably due to PGE2 (66)(67)(68). The removal of PGE2 from these cocktails generates MoDC with similar profiles but low CCR7 expression and subsequent decreased migration to the lymphoid organs (69). ...
Harnessing dendritic cells (DC) to treat HIV infection is considered a key strategy to improve anti-HIV treatment and promote the discovery of functional or sterilizing cures. Although this strategy represents a promising approach, the results of currently published trials suggest that opportunities to optimize its performance still exist. In addition to the genetic and clinical characteristics of patients, the efficacy of DC-based immunotherapy depends on the quality of the vaccine product, which is composed of precursor-derived DC and an antigen for pulsing. Here, we focus on some factors that can interfere with vaccine production and should thus be considered to improve DC-based immunotherapy for HIV infection.
... In clinic several TLRs have been used in combination based on the principle that concurrent activation of multiple TLRs can augment the effects of individual TLR stimulation (Boullart et al. 2008;Napolitani et al. 2005). As such, a cocktail of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in combination with prostaglandin E 2 was initially established as the gold standard for DC maturation (Lee et al. 2002). ...
Today, cancers pose a major public health burden. Although a myriad of cancer treatments are available, only a few have achieved clinical efficacy. This is partly attributed to cancers capability to evade host immunity by converting dendritic cells (DCs) from potent stimulators to negative modulators of immunity. Dendritic cell-based immunotherapy attempts to resolve this problem by manipulating the functional characteristics of DCs. Plant-derived polysaccharides (PDPs) can stimulate the maturation of DCs conferring on them the capacity to present internalised tumorigenic antigens to naïve T cells and subsequently priming T cells to eliminate tumours. PDPs have been used as immune modulators and later as anti-cancer agents by Traditional Chinese Medicine practitioners for centuries. They are abundant in nature and form a large group of heterogeneous though structurally related macromolecules that exhibit diverse immunological properties. They can induce antigen pulsed DCs to acquire functional characteristics in vitro which can subsequently be re-introduced into cancer patients. They can also be used as adjuvants in DC-based vaccines or independently for their intrinsic anti-tumour activities. Clinically, some in vitro generated DCs have been shown to be both safe and immunogenic although their clinical application is limited in part by unsatisfactory functional maturation as well as impaired migration to draining lymph nodes where T cells reside. We review the relative potencies of individual PDPs to induce both phenotypic and functional maturation in DCs, their relative abilities to activate anti-cancer immunity, the possible mechanisms by which they act and also the challenges surrounding their clinical application.
... In our experiments, the expression of Toll-like receptors 2, 3 and 4 was observed in hiPSDCs, and constant maturation occurred by the addition of these cytokines. In particular, in hiPSDCs matured with cytokine cocktails, the expression of CD83 and CCR7 and the secretion of IL-12 (p70) were higher than in hiPSDCs matured with other maturation factors, as previously reported in hMoDCs ( Supplementary Fig. 1a,b) [26][27][28] . Based upon our preliminary data, we used the cytokine cocktails with rhIL-6, rhTNFα, rhIL-1β and PGE2 as optimal stimuli of hiPSDCs. ...
Clinical application of dendritic cell (DC) vaccine therapy is hindered by the need for a large quantity of DCs generated from peripheral blood monocytes of the patient. We investigated whether genetically modified human induced pluripotent stem cell (iPSC)-derived dendritic cells (hiPSDCs) expressing carcinoembryonic antigen (CEA) could induce CEA-specific cytotoxic T cells in a human model and whether genetically modified mouse iPSDCs (miPSDCs) expressing CEA showed an actual antitumor effect using a CEA transgenic mouse model. We differentiated hiPSDCs from iPSCs of three healthy donors and transduced CEA cDNA into the hiPSDCs. The surface marker expression, cytokine secretion and migratory capacity of the hiPSDCs were equivalent to those of human monocyte-derived DCs (hMoDCs). Cytotoxic T cells activated by hiPSDCs-CEA exhibited CEA-specific cytotoxic activity against the target cells expressing CEA. Furthermore, in the CEA transgenic mouse model, cytotoxic T cells activated in mice immunized with miPSDCs-CEA displayed CEA-specific cytotoxic activity against MC38-CEA. In the subcutaneous tumour model, vaccination with miPSDCs-CEA achieved a significant growth inhibitory effect on MC38-CEA. No adverse events caused by the administration of miPSDCs were observed. Genetic modification of iPSDCs, inducing the expression of CEA, is a promising tool for clinical applications of vaccine therapy for treating gastrointestinal cancer patients.
... In clinical trials of melanoma (37) and glioblastoma (38), favorable outcomes were observed to be related to DC1-derived IL-12p70 production and Th1-polarized immunity. Similar to previous studies (39,40), the present study found that although DCs matured with a different time span secreted higher levels of IL-12p40, and secreted little bioactive IL-12p70. Therefore, studies to further improve the capacity of DCs to produce bioactive IL-12p70 are necessary. ...
Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and prostaglandins E2 is considered as the standard cocktail for maturing dendritic cells (DCs). However, the appropriate time span for DC maturation with the standard cocktail remains unclear. The present study aimed to compare the differences between DCs matured with the standard cocktail for 24 and 48 h, respectively, and determine whether 24‑h stimulation was sufficient for DC maturation. The findings demonstrated that, compared with DCs matured for 48 h, the levels of cluster of differentiation (CD)80, CD83, CD86 and programmed death-ligand 1 expression in DCs matured for 24 h were relatively lower. However, with the exception of CD80 whose mean fluorescence intensity (MFI) was higher in DCs matured for 48 h, the MFI values of other surface markers were comparable. Notably, the MFI of CD40 was higher in DCs matured for 24 h. In addition, the viability, T cell stimulatory capacity in allogeneic mixed lymphocyte reaction and cytokine production, including IL-12p40, IL-12p70 and IL-10, were all comparable between DCs matured for 24 and 48 h, respectively. These results indicated that 24-h stimulation may be sufficient for DC maturation when using the standard cocktail.
... In fact, the DC maturation cocktail that was used acts as pathogen-associated molecular patterns (PAMPS), nucleotides, inflammatory cytokines or cell damage and activates a complex DC maturation program consisting of the upregulation of maturation markers such as CD83, co-stimulatory markers (such as CD80, CD86, CD40), and HLA markers, among others, and of the secretion of T cell polarizing cytokines, efficiently activating antigen-specific T cells [20][21][22]. ...
We performed a retrospective genome-wide association study in HIV-infected individuals who were treated with dendritic cell-based immunotherapy in clinical trials performed by two research groups (Spain and Brazil). We aimed to identify host genetic variants influencing treatment response. The Illumina Human Core Exome 12 v 1.0 Bead Chip with over 250,000 markers was used to analyze genetic factors affecting treatment response. Additionally, we performed a meta-analysis of the results obtained from Spanish and Brazilian patients. We identified a genetic variation (rs7935564 G allele) in TRIM22 gene, which encodes TRIM22 protein acting like a HIV restriction factor, as being associated with good response to dendritic cell-based immunotherapy. We then verified the impact of TRIM22 rs7935564 SNP in susceptibility to HIV infection and disease progression by assessing the influence of biogeographic ancestry in the distribution of allelic and genotype frequencies in three populations from Italy, Brazil and Zambia. TRIM22 rs7935564 genotyping indicated association of G rs7935564 allele with long-term non-progression of HIV disease in Italian patients, thus corroborating our hypothesis that it is involved as a restriction factor in dendritic cell-based immunotherapy response. TRIM22 rs7935564 polymorphism was associated with good response to dendritic cell-based immunotherapy. We hypothesize that in selecting patients for treatment, there is a possible bias related to the natural presence of restriction factors that are genetically determined and could influence final outcome of therapy.
... mDC responses to stimuli differentially shape innate and adaptive immunity and influence HIV susceptibility [2,6,11,16]. Diverse microbial products, cytokines, endogenous ligands, and pathogens mature mDCs to differing degrees and with different qualities, giving rise to diverse DC phenotypes that variably direct T cell fate, HIV capture, and the outcome of HIV infection in DCs and the CD4 + T cells they encounter [2,11,13,[17][18][19][20][21][22][23][24][25][26][27]. Another layer of complexity in the outcome is imparted by the timing of DC maturation with respect to HIV and T cell exposure [17,28]. ...
Myeloid dendritic cells (mDCs) contribute to both HIV pathogenesis and elicitation of antiviral immunity. Understanding how mDC responses to stimuli shape HIV infection outcomes will inform HIV prevention and treatment strategies. The long double-stranded RNA (dsRNA) viral mimic, polyinosinic polycytidylic acid (polyIC, PIC) potently stimulates DCs to focus Th1 responses, triggers direct antiviral activity in vitro, and boosts anti-HIV responses in vivo. Stabilized polyICLC (PICLC) is being developed for vaccine adjuvant applications in humans, making it critical to understand how mDC sensing of PICLC influences HIV infection. Using the monocyte-derived DC (moDC) model, we sought to describe how PICLC (vs. other dsRNAs) impacts HIV infection within DCs and DC-T cell mixtures. We extended this work to in vivo macaque rectal transmission studies by administering PICLC with or before rectal SIVmac239 (SIVwt) or SIVmac239ΔNef (SIVΔNef) challenge. Like PIC, PICLC activated DCs and T cells, increased expression of α4β7 and CD169, and induced type I IFN responses in vitro. The type of dsRNA and timing of dsRNA exposure differentially impacted in vitro DC-driven HIV infection. Rectal PICLC treatment similarly induced DC and T cell activation and pro- and anti-HIV factors locally and systemically. Importantly, this did not enhance SIV transmission in vivo. Instead, SIV acquisition was marginally reduced after a single high dose challenge. Interestingly, in the PICLC-treated, SIVΔNef-infected animals, SIVΔNef viremia was higher, in line with the importance of DC and T cell activation in SIVΔNef replication. In the right combination anti-HIV strategy, PICLC has the potential to limit HIV infection and boost HIV immunity.
... We obtained these highly matured mDCs by the established method. 36 Since expression levels of CD86 and CD83 could be referred to as "intermediate," we think that these IL-32-derived CD14 ¡ HLA-DR high CD11c C cells are not completely matured, but only show "differentiation toward mature mDCs." Taken together, differentiation of IL-32-exposed monocytes to DCs or macrophages would depend on other cytokines present in the microenvironment. ...
Mycosis fungoides progresses from patch to tumor stage by expansion of malignant T-cells that fail to be controlled by protective immune mechanisms. In this study, we focused on IL-32, a cytokine highly expressed in Mycosis fungoides lesions. Depending on the other cytokines (IL-4, GM-CSF) present during in vitro culture of healthy volunteers' monocytes, IL-32 increased the maturation of CD11c+ myeloid dendritic cells and/or CD163+ macrophages, but IL-32 alone showed a clear ability to promote dendritic cell differentiation from monocytes. Dendritic cells matured by IL-32 had the phenotype of skin-resident dendritic cells (CD1c+), but more importantly also had high expression of indoleamine 2,3-dioxygenase. The presence of dendritic cells with these markers was demonstrated in Mycosis fungoides skin lesions. At a molecular level, indoleamine 2,3-dioxygenase mRNA levels in Mycosis Fungoides lesions were higher than those in healthy volunteers increased with disease progression in Mycosis fungoides lesions and there was a high correlation between indoleamine 2,3-dioxygenase and IL-32 expression. In contrast, Foxp3 mRNA levels decreased from patch to tumor stage. Increasing expression of IL-10 across Mycosis fungoides lesions was highly correlated with IL-32 and indoleamine 2,3-dioxygenase, but not with Foxp3 expression. Thus, IL-32 could contribute to progressive immune dysregulation in Mycosis fungoides by directly fostering development of immunosuppressive myeloid dendritic cells or macrophages, possibly in association with IL-10.
... Finalmente, se han reportado individuos expuestos seronegativos (HESN), que, a pesar de haber estado expuestos de manera persistente al virus por contacto sexual o parenteral, no tienen evidencia clínica de la infección (12). La existencia de individuos que resisten la infección por el VIH ha despertado el interés científico, dado que a partir de la caracterización de los mecanismos responsables del control viral se han propuesto alternativas terapéuticas, algunas de las cuales hacen parte del esquema actual de tratamiento de algunos pacientes infectados con el VIH (13,14). ...
Antiviral innate mechanisms have a potential use in developing preventive and therapeutic strategies against HIV. Specifically, antiviral soluble factors have been evaluated in multiple investigations, based on their capacity to inhibit different steps of the viral cycle, and to increase the host immune response. Among these factors, TRIM-5α, APOBEC3G, SAMHD1, ELAFIN,
SERPINA1 and SLPI are of particular interest, as they can act directly on the viral particle or the cell, or promote the production of molecules related to the viral immune response. Some of these factors have been associated with a low risk of HIV infection or slow progression to AIDS. Evaluation of mechanisms exhibited by antiviral proteins is a requirement for developing new therapeutic alternatives.
... Both IL-6 and PGE-2 were reported to inhibit the differentiation of MoDCs [111,112], but stimulate their maturation [113,114]. IL-6 inhibits the differentiation of DCs, by switching the differentiation of monocytes [115] and HSCs [116] towards macrophages. The effect depends on the upregulation of the macrophage-colony stimulating factor (M-CSF) receptor expression by monocytes and autocrine production of M-CSF [111]. ...
Mesenchymal stem/stromal cells (MSCs), having both multi-potent differentiation potential and prominent immunomodulatory properties, are seen as a very powerful tool for the therapy of diseases characterized by tissue damage and/or unregulated immune responses. Dendritic cells (DCs) are key immunoregulatory cells at the crossroads between immunity and tolerance, able to fine-tune the whole immune response via regulation of adaptive immunity. Therefore, untangling the complex interactions between DCs and MSCs is crucial for understanding various mechanisms involved in the pathogenesis of immune-related diseases and for the discovery of new therapeutic targets for advanced treatment procedures. From this perspective, we reviewed the data that have been obtained to date regarding the complex effects of MSCs on DC development and functions, delineating the abundant mechanisms involved in these interactions. Additionally, we have pointed out to additional mechanisms of MSC/DC cross-talk that have not been directly proven, but that could have a significant role, not only in DC functions and the maintenance of immune homeostasis, but also in migration, differentiation and the functions of MSCs. For now, we know that MSCs have much more influence on DCs than vice versa, so more studies should be done in order to fully understand this cross-talk.
Objective:
Canine splenic hemangiosarcomas (HSA) are malignant mesenchymal tumors with a high tendency for metastasis. Median survival times after splenectomy followed by adjuvant chemotherapy usually range between 5 and 8 months. The aim of this prospective randomized double-blinded study was to examine the efficacy of a commercially available dendritic cell therapy (PetBioCell) following splenectomy. In addition, possible side effects of this therapy were evaluated.
Material and methods:
Twenty-one dogs with histologically confirmed splenic HSA without metastasis (stages I or II) were included in the study. Ten dogs received the dendritic cell therapy, and 11 dogs received a placebo. Injections were administered according to the manufacturer's instructions monthly for the first 3 months and then every 3 months until death. Survival times and toxicoses of both groups were compared.
Results:
Follow-up data were available for all 21 patients; the observation period ranging until euthanasia or metastasis-related death. One patient that had received the dendritic cell therapy was euthanized due to prostatitis and experienced the longest survival time (668 days). One dog in the placebo-group lived for 448 days after splenectomy. The median survival times in the dendritic cell therapy and the placebo group amounted to 74 and 126 days, respectively. There was no significant difference in tumor-free interval (t(18) = 1.4, p = 0.911) and survival times (t(19) = -0.094, p = 0.463) between the 2 groups. Toxicoses reported in both groups were mild and self-limiting.
Conclusion:
Immunotherapy using autologous, immature and unprimed dendritic cells according to the PetBioCell method failed to show efficacy on tumor-free interval and survival time in the presented dog population with splenic hemangiosarcoma.
Today, treatment options for cancer patients typically include surgery, radiation therapy, immunotherapy, and chemotherapy. While these therapies have saved lives and reduced pain and suffering, cancer still takes millions of lives every year around the world. Researchers are now developing advanced therapeutic strategies such as immunotherapy, targeted therapy, and combination nanotechnology for drug delivery. In addition, the identification of new biomarkers will potentiate early-stage diagnosis. Molecular Targets and Cancer presents information about cancer diagnosis and therapy in a simple way. It covers several aspects of the topic with updated information on par with medical board levels. The book features contributions from experts and includes an overview of cancer from basic biology and pathology, classifications, surveillance, prevention, diagnosis, types of cancer, treatment and prognosis. The second part of this book discusses specialized topics in clinical oncology which include the pathophysiology of various types of cancer, cancer screening, different types of cancer surgery, cancer stem cell-targeted immunotherapy, nanotechnology for precision medicine applications in cancer and cancer surveillance. This comprehensive guide is a valuable resource for oncologists, researchers, and all medical professionals who work in cancer care and research.
The second leading cause of cancer death in women worldwide is breast cancer (BC), and despite significant advances in BC therapies, a significant proportion of patients develop metastasis and disease recurrence. Currently used treatments, like radiotherapy, chemotherapy, and hormone replacement therapy, result in poor responses and high recurrence rates. Alternative therapies are therefore needed for this type of cancer. Cancer patients may benefit from immunotherapy, a novel treatment strategy in cancer treatment. Even though immunotherapy has been successful in many cases, some patients do not respond to the treatment or those who do respond relapse or progress. The purpose of this review is to discuss several different immunotherapy approaches approved for the treatment of BC, as well as different strategies for immunotherapy for the treatment of BC.
At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient’s own cells, bringing personalized, targeted therapy for cancer one step closer.
During the ex vivo generation of anti-cancer dendritic cell (DC)-based vaccines, their maturation still represents one of the most crucial steps of the manufacturing process. A superior DC vaccine should: possess extensive expression of co-stimulatory molecules, have an exceptional type-1 polarization capacity characterized by their ability to produce IL-12p70 upon contact with responding T cells, migrate efficiently toward chemokine receptor 7 (CCR7) ligands, and have a superior capacity to activate cytotoxic T cell responses. A major advance has been achieved with the discovery of the next generation maturation protocol involving TLR-3 agonist (poly I:C), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IFN-α, and has since been known as α-type-1 maturation cocktail. We demonstrate how this combination can be greatly enhanced by the inclusion of a TLR-8 stimulation (R848), thereby contributing to potentiation between different TLR signaling pathways. For maximum efficiency, TLR-3 stimulation should precede (termed pre I:C) the stimulation with the R848/TNF-α/IL-1β/IFN-α/IFN-γ cocktail. When compared to DCs matured with α-type-1 maturation cocktail (αDCs), DCs matured with pre I:C/R848/TNF-α/IL-1β/IFN-α/IFN-γ (termed zDCs) displayed higher expression of CD80 and CD86 co-stimulatory molecules. Importantly, after CD40-ligand stimulation, which simulates DC-T cell contact, zDCs were much more proficient in IL-12p70 production. In comparison to αDCs, zDCs also displayed a significantly greater migratory capacity toward chemokine ligands (CCL)19 and CCL21, and had a significantly greater allo-stimulatory capacity. Finally, zDCs were also superior in their capacity to induce melanoma-specific CD8+ T cells, CD8+ T cell proliferation, and cytotoxic T cells, which produced approximately two times more IFN-γ and more granzyme B, than those stimulated with αDCs. In conclusion, we present a novel and superior DC maturation cocktail that could be easily implemented into next generation DC vaccine manufacturing protocols in future trials.
Immunotherapy with antigen-specific T cells is a promising, targeted therapeutic option for patients with cancer as well as for immunocompromised patients with virus infections. In this review, we characterize and compare current manufacturing protocols for the generation of T cells specific to viral and non-viral tumor associated antigens. Specifically, we discuss: (i) the different methodologies to expand virus-specific T cell (VST) and non-viral tumor-associated antigen specific T cell (TAA-T) products, (ii) the immunological principles involved when developing such manufacturing protocols and (iii) proposed standardized methodologies for the generation of polyclonal, polyfunctional antigen-specific T cells irrespective of donor source. Ex vivo-expanded cells have been safely administered to treat numerous patients with virus-associated malignancies, hematologic malignancies, and solid tumors. Hence, we have performed a comprehensive review of the clinical trial results evaluating the safety, feasibility, and efficacy of these products in the clinic. In summary, this review seeks to provide new insights regarding antigen-specific T cell technology to benefit a rapidly expanding T cell therapy field.
Allogeneic stem cell transplantation (alloSCT) is the treatment of choice for many patients with acute myeloid leukemia (AML) and myelodysplastic syndrome. The presentation of leukemic or allospecific antigens by malignant blasts is regarded as a crucial trigger for an effective allogeneic immune response. Conversely, insufficient stimulatory capacity by the leukemic blasts is thought to be a relevant escape mechanism from cellular immunotherapy (alloSCT). Our purpose was to test, whether the ability of malignant blasts to differentiate in vitro toward dendritic cells of leukemic origin (DCleu) is associated with clinical outcome. We isolated leukemic blasts from peripheral blood or bone marrow of AML and myelodysplastic syndrome patients before alloSCT (n=47) or at relapse after alloSCT (n=22). A panel of 6 different assays was used to generate DCleu in vitro. Results were correlated with clinical outcome. DCleu could be generated from all 69 samples. Significantly higher mean frequencies of DCleu were found in clinical long-term responders versus nonresponders to SCT (76.8% vs. 58.8%, P=0.006). Vice versa, the chance for response to SCT was significantly higher, if a DCleu+/dendritic cells (DC) ratio of >50% could be reached in vitro (P=0.004). Those patients were characterized by a longer time to relapse (P=0.04) and by a higher probability for leukemia-free survival (P=0.005). In vitro generation of DC and DCleu from leukemic blasts correlated with the clinical outcome. This observation may support a role of leukemic antigen presentation by "leukemia-derived DC" for the stimulation of an allogeneic immune response in AML.
Clinical approval of the immune checkpoint blockade (ICB) agents for multiple cancer types has reinvigorated the long-standing work on cancer vaccines. In the pre-ICB era, clinical efforts focused on the Ag, the adjuvants, the formulation, and the mode of delivery. These translational efforts on therapeutic vaccines range from cell-based (e.g., dendritic cells vaccine Sipuleucel-T) to DNA/RNA-based platforms with various formulations (liposome), vectors (Listeria monocytogenes), or modes of delivery (intratumoral, gene gun, etc.). Despite promising preclinical results, cancer vaccine trials without ICB have historically shown little clinical activity. With the anticipation and expansion of combinatorial immunotherapeutic trials with ICB, the cancer vaccine field has entered the personalized medicine arena with recent advances in immunogenic neoantigen-based vaccines. In this article, we review the literature to organize the different cancer vaccines in the clinical space, and we will discuss their advantages, limits, and recent progress to overcome their challenges. Furthermore, we will also discuss recent preclinical advances and clinical strategies to combine vaccines with checkpoint blockade to improve therapeutic outcome and present a translational perspective on future directions.
Auf Dendritische Zellen (DCs) basierende Vakzinen hängen von der Qualität der DC-Reifung ab, um Antigenpräsentation, Kostimulation, Lymphknotenmigration und, im Faller einer T-Helfer-1 (Th1) Polarisierung, die Freisetzung von IL-12 zu induzieren. Die Herstellung des heterodimeren IL-12p70 durch injizierte DC wurde klassisch als Schlüsselfaktor beschrieben, der für die Erzeugung einer polarisierten Th1 Immunreaktion erforderlich ist. Dennoch induzieren DCs, die IL-12 nicht ausscheiden können (z. B. nach Reifung des Cytokin-Cocktails), Th1 polarisierte Immunantwortenin Mäusen und Menschen. Da zuvor auch beschrieben wurde, dass DCs in der Lage sind, andere DCs auf Bystander-Weise zu aktivieren, haben wir hier die DC-Quelle der IL-12 Produktion für die Th1-Polarisation in einem murinen DC-Vakzinemodell untersucht. Die Migration der injizierten, aus murinem Knochenmark generierten DCs (BM-DCs) war für den Antigentransport in den Lymphknoten wesentlich. Sie trugen jedoch nur teilweise zur Antigenpräsentation bei und induzierten nur einen nicht polarisierten Th0-Zustand der T-Zellen, die IL-2 produzierten, aber kein IFN-. Stattdessen deuten die Daten daraufhin, dass endogene dermale migrierende XCR1+ DCs als Bystander-DCs zur Antigenpräsentation beitragen und IL-12 für die Th1 Polarisation bereitstellten. Die genetische Ablation von migrierenden DCs und speziell von XCR1+ migrierenden DCs hebt das Th1 Priming vollständig auf, Die Kinetik der Wechselwirkungen in den drainierenden Lymphknoten erfolgt schrittweise, indem i) injizierte DCs mit verwandten T-Zellen, ii) injizierte DCs mit Bystander XCR1+ DCs und iii) Bystander XCR1+ DCs mit T-Zellen in Kontakt treten. Das Transkriptom der Bystander-DCs zeigte eine Herunterregulierung von Treg- und Th2/Th9-induzierenden Genen und eine Hochregulierung der für die Th1- Induktion erforderlichen Gene. Zusammen zeigen diese Daten, dass injizierte reife migrierende BM-DCs das T-Zell-Priming und die Bystander-DC-Aktivierung steuern, nicht jedoch die Th1-Polarisation, die durch endogene IL-12p70+ XCR1+ Bystander-DCs vermittelt wird. Unsere Ergebnisse sind von Bedeutung für klinische Studien mit Vakzine-DCs, bei denen endogene DCs durch eine Chemotherapie funktionell beeinträchtigt werden können.
Background:
Medulloblastoma is the most common malignant brain tumor in childhood and adolescence. Although some patients present with distinct genetic alterations, such as mutated TP53 or MYC amplification, pediatric medulloblastoma is a tumor entity with minimal mutational load and low immunogenicity.
Methods:
We identified tumor-specific mutations using next-generation sequencing of medulloblastoma DNA and RNA derived from primary tumor samples from pediatric patients. Tumor-specific mutations were confirmed using deep sequencing and in silico analyses predicted high binding affinity of the neoantigen-derived peptides to the patients' human leukocyte antigen molecules. Tumor-specific peptides were synthesized and used to induce a de novo T-cell response characterized by interferon gamma and tumor necrosis factor alpha release of CD8+ cytotoxic T cells in vitro.
Results:
Despite low mutational tumor burden, at least two immunogenic tumor-specific peptides were identified in each patient. T cells showed a balanced CD4/CD8 ratio and mostly effector memory phenotype. Induction of a CD8-specific T-cell response was achieved for the neoepitopes derived from Histidine Ammonia-Lyase (HAL), Neuraminidase 2 (NEU2), Proprotein Convertase Subtilisin (PCSK9), Programmed Cell Death 10 (PDCD10), Supervillin (SVIL) and tRNA Splicing Endonuclease Subunit 54 (TSEN54) variants.
Conclusion:
Detection of patient-specific, tumor-derived neoantigens confirms that even in tumors with low mutational load a molecular design of targets for specific T-cell immunotherapy is possible. The identified neoantigens may guide future approaches of adoptive T-cell transfer, transgenic T-cell receptor transfer or tumor vaccination.
Monocyte-derived dendritic cell (moDC)-based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. In vitro culture conditions used to generate moDCs can be inconsistent, and moDCs generated in vitro are less effective than natural DCs. On the basis of our study highlighting the ability for certain kinase inhibitors to enhance tumor antigenicity, we therefore screened kinase inhibitors for their ability to improve DC immunogenicity. We identified AKT inhibitor MK2206, DNA-PK inhibitor NU7441, and MEK inhibitor trametinib as the compounds most effective at modulating moDC immunogenicity. The combination of these drugs, referred to as MKNUTRA, enhanced moDC activity over treatment with individual drugs while exhibiting minimal toxicity. An evaluation of 335 activation and T-cell-suppressive surface proteins on moDCs revealed that MKNUTRA treatment more effectively matured cells and reduced the expression of tolerogenic proteins as compared with control moDCs. MKNUTRA treatment imparted to ICT107, a glioblastoma (GBM) DC-based vaccine that has completed phase II trials, an increased ability to stimulate patient-derived autologous CD8+ T cells against the brain tumor antigens IL13Rα2(345-354) and TRP2(180-188)In vivo, treating ICT107 with MKNUTRA, prior to injection into mice with an established GBM tumor, reduced tumor growth kinetics. This response was associated with an increased frequency of tumor-reactive lymphocytes within tumors and in peripheral tissues. These studies broaden the application of targeted anticancer drugs and highlight their ability to increase moDC immunogenicity.
Introduction:
Paramunity-inducing-Factors (PINDs) consist of attenuated/inactivated viruses of various poxvirus-genera, used in veterinary medicine as non-antigen-specific, non-immunising stimulators of the innate immune system against infectious and malignant diseases. Their danger-signaling-interactions were tested for their capacity to improve leukemic antigen-presentation on DC generated from AML-patients' blasts ('DCleu') and DC-stimulation/activation of antileukemic T-cells.
Methods:
We analyzed, whether the addition of PINDs during DC cultures (15 healthy, 22 leukemic donors) and mixed lymphocyte culture (MLC, n = 15) with autologous (n = 6), allogeneic (n = 2) or T-cells after stem cell transplantation (SCT; n = 7) would alter the quality and quantity of DC, the composition of T-cell-subsets, and/or their antileukemic functionality (AF) as studied by FACS and functional Fluorolysis-cytotoxicity-assays.
Results:
Effects on 1. DC-cultures: PINDs in DC-cultures lead to increased proportions of mature DC and DCleu, but reduced proportions of viable and overall, as well as TLR4- and TLR9-expressing DC. 2. MLC: PINDs increased early (CD8+) T-cell activation (CD69+), but reduced proportions of effector-T-cells after MLC 3. AF: Presence of PINDs in DC- and MLC-cultures reduced T-cells' as well as innate cells' antileukemic functionality. 4. Cytokine-release profile: Supernatants from PIND-treated DC- and MLC-cultures resembled an inhibitory microenvironment, correlating with impaired blast lysis.
Conclusions:
Our data shows that addition of PINDs to DC-cultures and MLC result in a "blast-protective-capacity" leading to impaired AF, likely due to changes in the composition of T-/innate effector cells and the induction of an inhibitory microenvironment. PINDs might be promising in treating infectious diseases, but cannot be recommended for the treatment of AML-patients due to their inhibitory influence on antileukemic functionality.
Dendritic cells (DCs) are essential in immunity owing to their role in activating T cells, thereby promoting antitumor responses. Tumor cells, however, hijack the immune system, causing T cell exhaustion and DC dysfunction. Tumor-induced T cell exhaustion may be reversed through immune checkpoint blockade (ICB); however, this treatment fails to show clinical benefit in many patients. While ICB serves to reverse T cell exhaustion, DCs are still necessary to prime, activate, and direct the T cells to target tumor cells. In this review we provide a brief overview of DC function, describe mechanisms by which DC functions are disrupted by the tumor microenvironment, and highlight recent developments in DC cancer vaccines.
Exploitation of the patient’s own immune system to induce antitumor immune responses using dendritic cell (DC) immunotherapy has been established in early clinical trials as a safe and promising therapeutic approach for cancer. However, their limited success in larger clinical trials highlights the need to optimize DC vaccine preparations. This chapter describes the methodologies utilized for the preparation of the DC vaccine most commonly used in clinical trials. Optional variations at different stages in DC vaccine preparation, based on the nature of antigen, delivery of antigen, maturation stimuli, and mode of administration for DC vaccines, are also presented for consideration as these are often dependent on the disease setting, desired immune response, and/or resources available.
We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels ofbioactive IL-12. Other stimuli such as microbial agents, TNF-cx or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregu-lating the expression ofICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-~/pro-duction by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.
The fungus Candida albicans behaves as a commensal as well as a true pathogen of areas highly enriched in dendritic cells, such as skin and mucosal surfaces.
The ability of the fungus to reversibly switch between unicellular yeast to filamentous forms is thought to be important for
virulence. However, whether it is the yeast or the hyphal form that is responsible for pathogenicity is still a matter of
debate. Here we show the interaction, and consequences, of different forms of C. albicans with dendritic cells. Immature myeloid dendritic cells rapidly and efficiently phagocytosed both yeasts and hyphae of the
fungus. Phagocytosis occurred through different phagocytic morphologies and receptors, resulting in phagosome formation. However,
hyphae escaped the phagosome and were found lying free in the cytoplasm of the cells. In vitro, ingestion of yeasts activated
dendritic cells for interleukin (IL)-12 production and priming of T helper type 1 (Th1) cells, whereas ingestion of hyphae
inhibited IL-12 and Th1 priming, and induced IL-4 production. In vivo, generation of antifungal protective immunity was induced
upon injection of dendritic cells ex vivo pulsed with Candida yeasts but not hyphae. The immunization capacity of yeast-pulsed dendritic cells was lost in the absence of IL-12, whereas
that of hypha-pulsed dendritic cells was gained in the absence of IL-4. These results indicate that dendritic cells fulfill
the requirement of a cell uniquely capable of sensing the two forms of C. albicans in terms of type of immune responses elicited. By the discriminative production of IL-12 and IL-4 in response to the nonvirulent
and virulent forms of the fungus, dendritic cells appear to meet the challenge of Th priming and education in C. albicans saprophytism and infections.
Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell–inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell–derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon γ effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.
The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.
Chronic fatigue syndrome (CFS) is a physically debilitating illness associated with immunologic abnormalities, viral reactivation,
and impairment of cognition. In a randomized, multicenter, placebo-controlled, double-blind study of 92 patients meeting the
CFS case definition of the Centers for Disease Control and Prevention, the response of several laboratory and clinical variables
to an antiviral and immunomodulatory drug, poly(I) · poly(C12U), was determined. Measures of clinical response included Karnofsky performance score, a cognition scale derived from a self-administered
instrument assessing symptomatology (SCL-90-R), an activities of daily living scale, and exercise treadmill performance. After
24 weeks, patients receiving poly(I) · poly(C12U) had higher scores for both global performance and perceived cognition than did patients receiving placebo. In particular,
patients given poly(I) · poly(C12U) had increased Karnofsky performance scores (P < .03), exhibited a greater ability to do work during exercise treadmill testing (P = .01), displayed an enhanced capacity to perform the activities of daily living (P < .04), had a reduced cognitive deficit (P = .05), and required less use of other medications (P < .05).
Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.
The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.
Mature human dendritic cells can be generated in substantial numbers from nonproliferating progenitors in human blood using a two-step protocol. T cell-depleted mononuclear cells are first cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 (IL-4) and then exposed to monocyte conditioned medium (MCM). The dendritic cells generated using this approach are rendered terminally mature and are the most potent antigen presenting cells identified to date in humans. We sought to characterize factors in MCM that induce the terminal differentiation of dendritic cells. MCM contained substantial, although varying, quantities of several factors including tumor necrosis factor-alpha, IL-1beta, IL-6, and interferon-alpha. However, none of the four factors, individually or in various combinations, could fully substitute for the MCM to generate irreversibly differentiated dendritic cells. The yields, percentage of cells expressing the mature phase marker CD83, and mixed leukocyte reaction-stimulatory function were lower when defined cytokines were used in the place of MCM. Therefore, the full maturation of dendritic cells, because it entails changes in many known cell and molecular properties, requires a number of different cytokines that are released in tandem from appropriately stimulated monocytes. We propose that MCM-matured dendritic cells will be the most effective adjuvants for immunotherapy in vivo.
The early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MPhi) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MPhi are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon gamma priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MPhi to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.
Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.
Although in vivo priming of CD8+ cytotoxic T lymphocytes (CTLs) generally requires the participation of CD4+ T-helper lymphocytes, the nature of the 'help' provided to CTLs is unknown. One widely held view is that help for CTLs is mediated by cytokines produced by T-helper cells activated in proximity to the CTL precursor at the surface of an antigen-presenting cell (APC). An alternative theory is that, rather than being directly supplied to the CTL by the helper cell, help is delivered through activation of the APC, which can then prime the CTL directly. CD40 and its ligand, CD40L, may activate the APC to allow CTL priming. CD40L is expressed on the surface of activated CD4+ T-helper cells and is involved in their activation and in the development of their effector functions. Ligation of CD40 on the surface of APCs such as dendritic cells, macrophages and B cells greatly increases their antigen-presentation and co-stimulatory capacity. Here we report that signalling through CD40 can replace CD4+ T-helper cells in priming of helper-dependent CD8+ CTL responses. Blockade of CD40L inhibits CTL priming; this inhibition is overcome by signalling through CD40. CD40-CD40L interactions are therefore vital in the delivery of T-cell help for CTL priming.
Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.
To generate an immune response, antigen-specific T-helper and T-killer cells must find each other and, because they cannot detect each other's presence, they are brought together by an antigen-loaded dendritic cell that displays antigens to both. This three-cell interaction, however, seems nearly impossible because all three cell types are rare and migratory. Here we provide a potential solution to this conundrum. We found that the three cells need not meet simultaneously but that the helper cell can first engage and 'condition' the dendritic cell, which then becomes empowered to stimulate a killer cell. The first step (help) can be bypassed by modulation of the surface molecule CD40, or by viral infection of dendritic cells. These results may explain the long-standing paradoxical observation that responses to some viruses are helper-independent, and they evoke the possibility that dendritic cells may take on different functions in response to different conditioning signals.
Activation of immature dendritic cells (DC) in peripheral tissues induces their migration to lymph nodes and their maturation into CD83+ DC, which are able to prime naive T cells. The inflammatory cytokines IL-1beta and TNF-alpha induce mature DC, which can secrete IL-12 and promote the development of Th0/Th1-biased cells. DC maturation factors with a Th2-promoting function have not been described. Here we show that PGE2, although it does not induce final DC maturation by itself, synergizes with IL-1beta and TNF-alpha, and allows their effectiveness at 100-fold lower concentrations. While being phenotypically identical with the DC matured in the presence of high concentrations of IL-1beta and TNF-alpha alone, DC matured in the additional presence of PGE2 show impaired IL-12 production and bias naive Th cell development toward the Th2. The ability of DC to produce IL-12 is also suppressed by IL-10, which in contrast to PGE2, inhibits their maturation. The differences in the ability to produce IL-12, established during the final DC maturation, are stable after the removal of modulatory factors. Importantly, fully mature DC become unsusceptible to PGE2 and IL-10. This indicates that the levels of IL-12 production in vivo, in mature DC interacting with Th cells within the lymph nodes, are mainly predetermined at the stage of immature DC in peripheral tissues. These data imply that the character of pathogen-induced local inflammatory reaction can "instruct" local DC to initiate Th1 or Th2-biased responses.
IL-12 is a key cytokine in the development of Th1 responses. IL-12 production by antigen-presenting cells (APC) can be induced by the interaction between CD40 on the APC and CD40 ligand (CD40L) expressed on T cells after activation. Our previous study indicated that in dendritic cells (DC), the only APC that can activate naive T(h) cells efficiently, the mere CD40 engagement is insufficient to induce IL-12 production. The aim of the present study was to dissect the conditions for efficient IL-12 production by DC further. Using populations of naive and memory Th cells, recombinant CD40L, neutralizing and blocking antibodies, and by determining IFN-gamma production and CD40L expression levels, we here show that T cell-induced IL-12 production by DC results from the action of two signals, mediated by CD40L and IFN-gamma, and that the inability of naive T(h) cells to induce IL-12 production resides in their inability to produce IFN-(gamma). Other factors than CD40L and IFN-gamma can provide the required signals for IL-12 production by DC, as either factor could be replaced by lipopolysaccharide (LPS). The two-signal requirement proved unique for the production of IL-12, since either CD40 engagement or LPS was sufficient for the efficient production of tumor necrosis factor-alpha, IL-8 and the p40 subunit of IL-12, and may be considered as a safety mechanism for optimal control of potentially harmful T(h)1 responses.
Bone marrow-derived dendritic cells (BmDC) are potent APC and can promote antitumor immunity in mice when pulsed with tumor Ag. This study aimed to define the culture conditions and maturation stages of BmDC that enable them to optimally function as APC in vivo. BmDC cultured under various conditions (granulocyte-macrophage CSF (GM-CSF) or GM-CSF plus IL-4 alone or in combination with Flt3 ligand, TNF-alpha, LPS, or CD40 ligand (CD40L)) were analyzed morphologically, phenotypically, and functionally and were tested for their ability to promote prophylactic and/or therapeutic antitumor immunity. Each of the culture conditions generated typical BmDC. Whereas cells cultured in GM-CSF alone were functionally immature, cells incubated with CD40L or LPS were mature BmDC, as evident by morphology, capacity to internalize Ag, migration into regional lymph nodes, IL-12 secretion, and alloantigen or peptide Ag presentation in vitro. The remaining cultures exhibited intermediate dendritic cell maturation. The in vivo Ag-presenting capacity of BmDC was compared with respect to induction of both protective tumor immunity and immunotherapy of established tumors, using the poorly immunogenic squamous cell carcinoma, KLN205. In correspondence to their maturation stage, BmDC cultured in the presence of CD40L exhibited the most potent immunostimulatory effects. In general, although not entirely, the capacity of BmDC to induce an antitumor immune response in vivo correlated to their degree of maturation. The present data support the clinical use of mature, rather than immature, tumor Ag-pulsed dendritic cells as cancer vaccines and identifies CD40L as a potent stimulus to enhance their in vivo Ag-presenting capacity.
Activation of immature CD83- dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1 beta and TNF-alpha-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-gamma. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-gamma, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-gamma R (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureus Cowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.
For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.
Dendritic cells (DCs) are potent antigen-presenting cells that initiate protective T-cell immunity in mice. To study the immunogenicity of DCs in humans, we injected 9 healthy subjects subcutaneously with a control injection of autologous monocyte-derived, mature DCs, followed 4-6 weeks later by DCs pulsed with keyhole limpet hemocyanin (KLH), HLA-A*0201-positive restricted influenza matrix peptide (MP), and tetanus toxoid (TT). Four more subjects received these antigens without DCs. Injection of unpulsed DCs, or antigens alone, failed to immunize. Priming of CD4(+) T cells to KLH was observed in all 9 subjects injected with KLH-pulsed DCs, and boosting of TT-specific T-cell immunity was seen in 5 of 6 subjects injected with TT-pulsed DCs. Injection of antigen-pulsed DCs led to a severalfold increase in freshly isolated MP-specific, IFN-gamma-secreting CD8(+) T cells in all 6 HLA-A*0201-positive subjects, as early as 7 days after injection. When T cells were boosted in culture, there was an increase in MHC tetramer-binding cells and cytotoxic T cells after DC vaccination. These data provide the first controlled evidence of the immunogenicity of DCs in humans, and demonstrate that a single injection of mature DCs rapidly expands T-cell immunity.
Dendritic cells (DCs) are considered to be promising adjuvants for inducing immunity to cancer. We used mature, monocyte-derived DCs to elicit resistance to malignant melanoma. The DCs were pulsed with Mage-3A1 tumor peptide and a recall antigen, tetanus toxoid or tuberculin. 11 far advanced stage IV melanoma patients, who were progressive despite standard chemotherapy, received five DC vaccinations at 14-d intervals. The first three vaccinations were administered into the skin, 3 x 10(6) DCs each subcutaneously and intradermally, followed by two intravenous injections of 6 x 10(6) and 12 x 10(6) DCs, respectively. Only minor (less than or equal to grade II) side effects were observed. Immunity to the recall antigen was boosted. Significant expansions of Mage-3A1-specific CD8(+) cytotoxic T lymphocyte (CTL) precursors were induced in 8/11 patients. Curiously, these immune responses often declined after the intravenous vaccinations. Regressions of individual metastases (skin, lymph node, lung, and liver) were evident in 6/11 patients. Resolution of skin metastases in two of the patients was accompanied by erythema and CD8(+) T cell infiltration, whereas nonregressing lesions lacked CD8(+) T cells as well as Mage-3 mRNA expression. This study proves the principle that DC "vaccines" can frequently expand tumor-specific CTLs and elicit regressions even in advanced cancer and, in addition, provides evidence for an active CD8(+) CTL-tumor cell interaction in situ as well as escape by lack of tumor antigen expression.
Dendritic cells (DC) are key initiators of primary immune responses. Myeloid DC can secrete IL-12, a potent Th1-driving factor, and are often viewed as Th1-promoting APC. Here we show that neither a Th1- nor a Th2-inducing function is an intrinsic attribute of human myeloid DC, but both depend on environmental instruction. Uncommitted immature DC require exposure to IFN-gamma, at the moment of induction of their maturation or shortly thereafter, to develop the capacity to produce high levels of IL-12p70 upon subsequent contact with naive Th cells. This effect is specific for IFN-gamma and is not shared by other IL-12-inducing factors. Type 1-polarized effector DC, matured in the presence of IFN-gamma, induce Th1 responses, in contrast to type 2-polarized DC matured in the presence of PGE2 that induce Th2 responses. Type 1-polarized effector DC are resistant to further modulation, which may facilitate their potential use in immunotherapy.
The activation of dendritic cells (DC) to produce interleukin 12 (IL-12) is thought to be a key step in the initiation of cell-mediated immunity to intracellular pathogens. Here we show that ligation of the C-C chemokine receptor (CCR) 5 can provide a major signal for the induction of IL-12 synthesis by the CD8 alpha+ subset of DC and that this pathway is important in establishing interferon gamma-dependent resistance to the protozoan parasite Toxoplasma gondii. These findings support the concept that the early induction of chemokines by invading pathogens is a critical step not only for the recruitment of DC but also for the determination of their subsequent immunologic function.
It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.
Dendritic cell (DC) vaccination, albeit still in an early stage, is a promising strategy to induce immunity to cancer. We explored whether DC can expand Ag-specific CD8+ T cells even in far-advanced stage IV melanoma patients. We found that three to five biweekly vaccinations of mature, monocyte-derived DC (three vaccinations of 6 x 106 s.c. followed by two i.v. ones of 6 and 12 x 106, respectively) pulsed with Mage-3A2.1 tumor and influenza matrix A2. 1-positive control peptides as well as the recall Ag tetanus toxoid (in three of eight patients) generated in all eight patients Ag-specific effector CD8+ T cells that were detectable in blood directly ex vivo. This is the first time that active, melanoma peptide-specific, IFN-gamma-producing, effector CD8+ T cells have been reliably observed in patients vaccinated with melanoma Ags. Therefore, our DC vaccination strategy performs an adjuvant role and encourages further optimization of this new immunization approach.
Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. In this study we investigated the effects of various prostaglandins (PG) on the stimulatory capacity of DC. DC were generated from peripheral progenitor cells in the presence of IL-4 and GM-CSF and stimulated with IL-1, IL-6 and TNF-alpha on day 7. Simultaneously, PG (PGD(2), PGE(1), PGE(2), PGF(2 alpha), PGI(2)) were added at various concentrations (10(-5) to 10(-9) M) on day 7. In all experiments, PGE(2) had the most potent influence on the maturation of the DC, followed by other PG in the order PGE(1) > PGD(2) > PGF(2 alpha) > PGI(2). In addition, the expression of the surface molecules CD40, CD54, CD58, CD80, CD83, CD86 and the MHC class II molecules was upregulated after stimulation with PG. Analysis of DC supernatants after treatment with PG demonstrated significantly higher amounts of the proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha, and IL-12. Addition of PG to DC induced a markedly enhanced proliferation of both naive and activated CD4(+) and CD8(+) T cells in alloantigen-induced MLR assays. Assessment of coculture supernatants after restimulation revealed significantly higher amounts of the Th1-cytokines IL-2 and IFN-gamma and only minimal amounts of IL-4 compared to control cells. No production of IL-10 was observed. The effects of PG on the maturation of DC and enhanced T-cell proliferation could be mimicked by db-cAMP and forskolin, indicating that they were due to elevated cAMP levels. Collectively, our data show that members of the PG family promote the differentiation of DC and enhance their capacity to induce a Th1 immune response.
The functional properties of dendritic cells (DCs) are strictly dependent on their maturational state. To analyze the influence of the maturational state of DCs on priming and differentiation of T cells, immature CD83(-) and mature CD83(+) human DCs were used for stimulation of naive, allogeneic CD4(+) T cells. Repetitive stimulation with mature DCs resulted in a strong expansion of alloreactive T cells and the exclusive development of T helper type 1 (Th1) cells. In contrast, after repetitive stimulation with immature DCs the alloreactive T cells showed an irreversibly inhibited proliferation that could not be restored by restimulation with mature DCs or peripheral blood mononuclear cells, or by the addition of interleukin (IL)-2. Only stimulation of T cells with mature DCs resulted in an upregulation of CD154, CD69, and CD70, whereas T cells activated with immature DCs showed an early upregulation of the negative regulator cytotoxic T lymphocyte-associated molecule 4 (CTLA-4). These T cells lost their ability to produce interferon gamma, IL-2, or IL-4 after several stimulations with immature DCs and differentiated into nonproliferating, IL-10-producing T cells. Furthermore, in coculture experiments these T cells inhibited the antigen-driven proliferation of Th1 cells in a contact- and dose-dependent, but antigen-nonspecific manner. These data show that immature and mature DCs induce different types of T cell responses: inflammatory Th1 cells are induced by mature DCs, and IL-10-producing T cell regulatory 1-like cells by immature DCs.
Dendritic cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal antigen presentation and T-cell activation. High cell surface expression of CD83 is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83 expression on DCs. We hypothesized that interleukin 12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating antigen-specific T cells. We used combinations of signaling through CD40, using CD40 ligand trimer (CD40L), and interferon gamma to demonstrate that CD83 expression is necessary but not sufficient for optimal production of IL-12 by DCs. Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals. By intracellular cytokine detection, we determined that only a subset of cells that express high levels of CD80 and CD83 generate large amounts of IL-12. DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T cell in vitro. These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.
Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days +/- monocyte-conditioned media, yielding mature (CD83(+)/dendritic cell-lysosome-associated membrane glycoprotein(+)) and immature (CD83(-)/dendritic cell-lysosome-associated membrane glycoprotein(-)) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.
Immunostimulatory properties of dendritic cells (DCs) are linked to their maturation state. Injection of mature DCs rapidly enhances antigen-specific CD4+ and CD8+ T cell immunity in humans. Here we describe the immune response to a single injection of immature DCs pulsed with influenza matrix peptide (MP) and keyhole limpet hemocyanin (KLH) in two healthy subjects. In contrast to prior findings using mature DCs, injection of immature DCs in both subjects led to the specific inhibition of MP-specific CD8+ T cell effector function in freshly isolated T cells and the appearance of MP-specific interleukin 10-producing cells. When pre- and postimmunization T cells were boosted in culture, there were greater numbers of MP-specific major histocompatibility complex tetramer-binding cells after immunization, but these had reduced interferon production and lacked killer activity. These data demonstrate the feasibility of antigen-specific inhibition of effector T cell function in vivo in humans and urge caution with the use of immature DCs when trying to enhance tumor or microbial immunity.
Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.
Signaling lymphocyte activation molecule (SLAM), a 70-kDa costimulatory molecule that mediates CD28-independent proliferation of T cells and IFN-gamma production, has been identified on human T cells, immature thymocytes, and a subset of B cells. We have found that SLAM is expressed on mature but not immature dendritic cells (DC). However, the SLAM-associated protein, is missing in DC. SLAM surface expression is strongly up-regulated by IL-1beta. Addition of IL-1beta to the DC maturation mixture also increases the stimulatory properties of DC. These findings provide a new marker for DC maturation and help to explain two areas of DC biology. First, SLAM is a receptor for the measles virus, previously shown to infect DC. Second, SLAM could possibly contribute to the enhanced immunostimulatory functions of DC that are observed following the addition of IL-1.
By using adoptive transfer of Ag-loaded bone marrow-derived dendritic cells (BMDC), we have established an in vivo model of CTL priming. Activation of CTL in these experiments required both CD4(+) T cells and CD154, demonstrating that this model reflects CD4(+) T cell-dependent dendritic cell (DC) licensing. Because IL-12 has been suggested to play an important role in CTL activation by DC, we examined the ability of BMDC to prime CTL in the complete absence of IL-12 using p40-deficient mice. We observed that the absence of IL-12 does not affect the phenotype or allostimulatory function of BMDC after in vitro maturation. Moreover, there was no difference in the ability of Ag-loaded DC to elicit CTL cytotoxicity, whether the Ag was delivered by virus infection or peptide pulsing. Equal frequencies of Ag-specific, IFN-gamma-secreting CD8(+) T cells developed in both wild-type and IL-12-deficient backgrounds. Finally, CTL generated in the IL-12-deficient environment were capable of protecting immunized mice against tumor challenge, demonstrating that these CTL were fully functional, despite the absence of IL-12 during the maturation process in vivo. These results indicate that IL-12 is not critical for the development of IFN-gamma secreting, CD8(+) T cells and that another mechanism must be used by licensed DC to prime and activate CTL.
Dendritic cells (DCs) may arise from multiple lineages and progress through a series of intermediate stages until fully mature, at which time they are capable of optimal antigen presentation and T-cell activation. High cell surface expression of CD83 is presumed to correlate with full maturation of DCs, and a number of agents have been shown to increase CD83 expression on DCs. We hypothesized that interleukin 12 (IL-12) expression would be a more accurate marker of functionally mature DCs capable of activating antigen-specific T cells. We used combinations of signaling through CD40, using CD40 ligand trimer (CD40L), and interferon gamma to demonstrate that CD83 expression is necessary but not sufficient for optimal production of IL-12 by DCs. Phenotypically mature DCs could be induced to produce high levels of IL-12 p70 only when provided 2 simultaneous stimulatory signals. By intracellular cytokine detection, we determined that only a subset of cells that express high levels of CD80 and CD83 generate large amounts of IL-12. DCs matured with both signals are superior to DCs stimulated with the individual agents in activating antigen-specific T cell in vitro. These findings have important implications regarding the identification, characterization, and clinical application of functionally mature DCs.
The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3β and the CXC chemokine stromal cell-derived factor (SDF)-1α. Maturation of DC induced by culture in lipopolysaccharide, TNF-α or IL-1β reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3β and SDF-1α. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.
Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-γ from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-α production is induced by day 3 post infection. Not only do these data satisfactorily explain the dfferences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.
Interleukin-12 p70 (IL-12p70) heterodimer, composed of p35 and p40 subunits, is a major Th1-driving cytokine, promoting cell-mediated immunity. in contrast, IL-12p40 homodimer, secreted by APC in the absence of p35 expression, and free p40 monomer do not mediate IL-12 activity but act as IL-12 antagonists. Here it is reported that prostaglandin E-2 (PGE(2)), an inflammatory mediator with a previously known Th2-driving function, dose-dependently enhances the IL-12p40 mRNA expression and the secretion of IL-12p40 protein in human tumor necrosis factor-alpha (TNF alpha)-stimulated immature dendritic cells (DCs). This effect is selective and is not accompanied by the induction of IL-12p35 expression or by secretion of IL-12p70 heterodimer, Inability of TNF alpha /PGE(2) to induce IL-12p70 was not compensated by interferon gamma (IFN gamma), which strongly enhanced the lipopolysaccharide (LPS)-induced IL-12p70 production. In addition to the selective induction of IL-12p40 in TNF alpha -stimulated Dos, PGE(2) inhibited the production of IL-12p70 and IL-12p40 in DCs stimulated with LPS or CD40 ligand, These data suggest an additional level of the Th2-promoting activity of PGE(2), via selective induction of IL-12p40, Selective induction of IL-12p40 and suppression of bioactive IL-12p70 may have negative impact on anticancer vaccination with PGE(2)-matured DCs, (Blood.2001;97:3466-3469) (C) 2001 by The American Society of Hematology.
Dendritic cells (DCs) have been shown to enhance anti-tumor immune responses in several preclinical models. Furthermore, DC-like function can be elicited from peripheral blood monocytes cultured in vitro with interleukin-4 and granulocyte-macrophage colony-stimulating factor. For this reason, a phase 1 study was initiated at the Surgery Branch of the National Cancer Institute to test the toxicity and biological activity of the intravenous administration of peripheral blood monocyte-derived DCs. The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n = 3) was treated with 6 x 10(7) DCs and a second cohort (n = 5) with 2 x 10(8) DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n = 2)2 x 10(8) DCS were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases. A relatively large number of DCs can be safely administered intravenously. The poor clinical outcome of this study perhaps could be explained by the type of protocol used for DC maturation: the route of administration, or both. For this reason, this clinical protocol was interrupted prematurely, whereas other strategies for DC preparation and route of administration are being investigated at the authors' institution.
CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-γ intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 109 to 1012 of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.
Infection with Leishmania, an obligate intracellular parasite of mononuclear phagocytes, stimulates the production of IFN-gamma from NK cells, via a pathway which is dependent upon IL-12 and IL-2. IL-12 is also essential for the development of host protective T cell responses to this parasite. However, previous in vitro studies have indicated that macrophages fail to make IL-12 following infection with Leishmania, and that subsequent to infection, macrophages become refractory to normal IL-12-inducing stimuli. We have used an in situ approach to attempt to resolve this apparent paradox, and by immunostaining for IL-12 p40 protein, we now demonstrate for the first time, that dendritic cells (DC) are the critical source of early IL-12 production following Leishmania infection. IL-12 production by DC is transient, peaking at 1 day post infection and returning to the levels seen in uninfected mice by day 3. Although resident tissue macrophages fail to produce IL-12 after Leishmania infection, these cells are not totally refractory to cytokine inducing stimuli, as TNF-alpha production is induced by day 3 post infection. Not only do these data satisfactorily explain the differences between in vivo and in vitro data by identifying the cellular source of IL-12, but they also suggest a novel model for NK cell activation; namely that in response to pathogens which fail to trigger IL-12 production by macrophages, DC-T cell clusters provide the microenvironment for initial NK cell activation.
We investigated the possibility that T helper cells might enhance the stimulatory function of dendritic cells (DCs). We found that ligation of CD40 by CD40L triggers the production of extremely high levels of bioactive IL-12. Other stimuli such as microbial agents, TNF-alpha or LPS are much less effective or not at all. In addition, CD40L is the most potent stimulus in upregulating the expression of ICAM-1, CD80, and CD86 molecules on DCs. These effects of CD40 ligation result in an increased capacity of DCs to trigger proliferative responses and IFN-gamma production by T cells. These findings reveal a new role for CD40-CD40L interaction in regulating DC function and are relevant to design therapeutic strategies using cultured DCs.
Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34 + hematopoietic progenitors in presence of GM-CSF+TNFet for 12 d. The present study demonstrates that cord blood CD34 + HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CDla and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CDS0, CD83, CD86, CD58, high HLA class II). CDla + precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14 + progenitors mature into CDla + DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIlla described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA + naive T cells. Interestingly, the CD14 + precursors, but not the CDla + precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T ceils. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14+-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.
Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte-conditioned medium (MCM) and FCS-free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)-alpha, IL-1beta and IL-6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E2 to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF-alpha/IL-1/IL-6 or MCM alone induced CD4+ T cells that release intermediate levels of interferon (IFN)-gamma and no IL-4 or IL-10. Production of IFN-gamma was significantly induced by addition of PGE2, while no effect on production of IL-4 or IL-10 was observed. Even more striking differences were observed for CD8+ T cells. While MCM conditions only induced IFN-gamma(low), IL-4(neg) cells, TNF-alpha/IL-1/IL-6 promoted growth of IFN-gamma(intermediate), IL-4(neg) CD8+ T cells. Addition of PGE2 again only further polarized this pattern enhancing IFN-gamma production by alloreactive CD8+ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF-alpha/IL-1/IL-6 can substitute for MCM and that addition of PGE2 further enhances the yield and quality of DC generated. TNF-alpha/IL-1, IL-6 + PGE2-cultured DC seem to be optimal for generation of IFN-gamma-producing CD4/CD8+ T cells.
The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3beta and the CXC chemokine stromal cell-derived factor (SDF)-1alpha. Maturation of DC induced by culture in lipopolysaccharide, TNF-alpha or IL-1beta reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3beta and SDF-1alpha. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.
Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.
HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay.
Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase.
In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays.
Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.
The potential to harness the potency and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow-derived dendritic cells, phenotypically distinct and extremely potent antigen-presenting cells, to present tumor-associated antigens and thereby generate tumor-specific immunity. Support for this strategy comes from animal studies that have demonstrated that dendritic cells, when loaded ex vivo with tumor antigens and administered to tumor-bearing hosts, can elicit T cell-mediated tumor destruction. These observations have led to clinical trials designed to investigate the immunologic and clinical effects of antigen-loaded dendritic cells administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include antigen selection, methods for introducing the antigen into MHC class I and II processing pathways, methods for isolating and activating dendritic cells, and route of administration. Although current dendritic cell-based vaccination methods are cumbersome, promising results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immunotherapeutic strategies that take advantage of the antigen presenting properties of dendritic cells may ultimately prove both efficacious and widely applicable to human tumors.
Understanding the control exerted by cytokines on T helper cell subsets 1 and 2 (TH1-TH2) development has progressed to a fairly satisfying knowledge of intracellular signals and transcription factors. Less is understood about the molecular basis of TH1-TH2 development exerted by other parameters, such as how the antigen presenting cell can influence this process. Recent work suggests that dendritic cell subsets contribute significant polarizing influences on T helper differentiation, but how this comes about is less clear. In some cases known pathways may be used, as in the dendritic cell subset 1 exerting TH1 polarization by interleukin 12 (IL-12) production and STAT4 activation. In others, the effects are still in need of explanation.
Major insights into events that control Th1/Th2 differentiation have been acquired recently, and highlight the role of Type I IFN in Th1 generation, by inducing up-regulation of the IL-12 receptor beta(2) subunit. IFN-alpha induces responsiveness to IL-12, and here we have investigated the source and the circumstances under which IFN-alpha is produced, in the absence of viral infections. Human dendritic cells (DC) were co-cultured with autologous T cells activated by cross-linking the CD3 complex. DC were also cultured with L cells expressing human CD40 ligand (CD40L). Our results show that large amounts (>200 IU IFN-alpha from 2.5x10(4) cells) of IFN-alpha are produced by DC following interaction with stimulated T cells. Similar effects were observed when DC were cultured with CD40L-expressing transfectants, although the amount of IFN-alpha produced was reduced, suggesting that the CD40-CD40L interaction may be important. These results show that stimulated T cells can solicit the signals from DC that allow their polarization towards a Th1 phenotype. Type I DC produce Type I IFN not only following viral infection but also during an immunological interaction and this may be the basic mechanism that assists in the development of a Th1 response.
Interleukin-12 p70 (IL-12p70) heterodimer, composed of p35 and p40 subunits, is a major Th1-driving cytokine, promoting cell-mediated immunity. In contrast, IL-12p40 homodimer, secreted by APC in the absence of p35 expression, and free p40 monomer do not mediate IL-12 activity but act as IL-12 antagonists. Here it is reported that prostaglandin E(2) (PGE(2)), an inflammatory mediator with a previously known Th2-driving function, dose-dependently enhances the IL-12p40 mRNA expression and the secretion of IL-12p40 protein in human tumor necrosis factor-alpha (TNFalpha)-stimulated immature dendritic cells (DCs). This effect is selective and is not accompanied by the induction of IL-12p35 expression or by secretion of IL-12p70 heterodimer. Inability of TNFalpha/PGE(2) to induce IL-12p70 was not compensated by interferon gamma (IFNgamma), which strongly enhanced the lipopolysaccharide (LPS)-induced IL-12p70 production. In addition to the selective induction of IL-12p40 in TNFalpha-stimulated DCs, PGE(2) inhibited the production of IL-12p70 and IL-12p40 in DCs stimulated with LPS or CD40 ligand. These data suggest an additional level of the Th2-promoting activity of PGE(2), via selective induction of IL-12p40. Selective induction of IL-12p40 and suppression of bioactive IL-12p70 may have negative impact on anticancer vaccination with PGE(2)-matured DCs. (Blood. 2001;97:3466-3469)