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Natural killer cell clones can efficiently process and present protein antigens
Abstract
NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.
... Whereas the immunomodulatory CD56 hi (CD16 − ) phenotype is considered the less mature peripheral blood NK cell subset, the cytotoxic CD56 dim (CD16 hi ) phenotype is considered the more mature one. Other NK cell subpopulations with unique functions and phenotypes have been described, including an adaptive NK cell subset with elevated CD57 expression and heightened memory-based activity (42,43), as well as HLA-DRexpressing NK cells with dendritic cell (DC)like Ag presentation capability (44,45). With this understanding of varied NK cell functions and phenotypes in mind, we performed functional, phenotypic, and transcriptional assessments of NK cells in autoantibody-mediated neurologic diseases. ...
... CD94, in contrast, is expressed by most NK cells in the peripheral blood and elevated on the immature CD56 hi NK cells (66). HLA-DR is expressed on immature NK cells with a DC-like Ag presentation capability (45). As expected, we observed that NK cells from NMOSD patients exhibited reduced intensity of CD16 (Fig. 3A), CD57 (Fig. 3B), and CX3CR1 (Fig. 3C) expression, given that the CD56 dim CD16 hi subset was reduced in these patients. ...
... Their phenotypic and transcriptional profile included reduced expression of markers associated with the CD56 dim CD16 hi NK cells, such as CX3CR1 and CD57, and elevated HLA-DR expression. HLA-DR 1 NK cells are enriched in infectious diseases and have Ag-presentation capability (44,45,7274). Our RNA sequencing data suggest that HLA-DR expression is transcriptional and not a result of trogocytosis. ...
Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell–mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/− NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/− population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16− immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/− subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/− NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.
... HLA-DR-expressing NK cells obtained in vitro under cytokine stimulation were shown to stimulate nonspecific activation and partial differentiation of CD4 + T cells (19). Several studies demonstrated the ability of HLA-DR + NK cells to stimulate specific T cell response to certain antigens, similarly to professional antigen-presenting cells (APCs): tetanus toxin and Der pI house dust mite allergen (20), HSV particles and peptides isolated from them (21), HCMV particles and HCMV-antibody complex (22). As HLA-DR-positive NK cells were shown to accumulate in M. tuberculosis infected lungs in vivo (17,18) and expand in response to BCG stimulation in vitro (6), they might play a role in the processing and presentation of mycobacterial antigens as well. ...
... Based on the published data describing antigen-specific activation of T cells by HLA-DR-expressing NK cells (20)(21)(22), we hypothesized that HLA-DR + NK cells may also be able to process and present certain mycobacterial antigens, as long as this subset expands in response to sonicate and is shown to accumulate in TB-infected lungs in vivo (17,18). In a series of experiments, NK cells were firstly pre-activated in vitro for 10 days in the presence of IL-2, IL-21, and IL-18 to increase the expression of HLA-DR and CD86 (15,19,29) (Supplementary Figure 7), then part of them was incubated for 24 h with M. tuberculosis sonicate (NKmtb) and then co-cultured with autologous CD4 + T cells. ...
... As we already mentioned above, HLA-DR + NK cells are shown to infiltrate TBinfected lungs (17,18); moreover, the described cells are predominantly CD56 bright , and this NK cell subset is able to circulate and enter regional lymph nodes, where it can possibly interact with naïve T cells too. The results obtained by us supplement the already published data describing specific activation of T cells by NK cells preincubated with various antigens: tetanus toxin and the allergen of the house dust mite Der pI (20), HSV particles and its glycoproteins (21), HCMV particles and HCMV-antibody complexes (22). Parts of the destroyed M. tuberculosis (sonicate) can be recognized and internalized by NK cells via TLR2 receptor, similarly to the fact described by Kim et al. for HSV particles (21), as long as NK cells are able to interact with the components of the bacterial cell wall through TLR2 (8). ...
NK cells play an important role in the control of tuberculosis infection: they are not only able to kill the infected cells, but also control the activity of macrophages and development of the adaptive immune response. Still, there is little information on the role of specific NK cell subsets in this network. In this study, we focused on the mycobacteria-driven responses of the NK cells expressing HLA-DR – a type of MHC class II. We have revealed that this subset is increased in the peripheral blood of patients with primary diagnosed tuberculosis, and expands in response to in vitro stimulation with ultrasonically destroyed Mycobacterium tuberculosis cells (sonicate). The expanded HLA-DR⁺ NK cells had less differentiated phenotype, higher proliferative activity and increased expression of NKp30 and NKp46 receptors. HLA-DR⁺CD56dim NK cells showed higher IFNγ production and degranulation level than the respective HLA-DR⁻ NK cells in response to both 24 h and 7 day stimulation with sonicate, while HLA-DR⁺CD56bright NK cells mostly demonstarted similar high responsiveness to the same stimulating conditions as their HLA-DR⁻CD56bright counterparts. After preliminary incubation with destroyed mycobacteria, cytokine-activated HLA-DR-expressing NK cells were able to mediate mycobacteria-induced and HLA-DR-dependent cytokine production in autologous CD4⁺ T cells. Thus, functionally active HLA-DR⁺ cells seem to be one of the NK cell subsets providing an important link to the adaptive immunity.
... 20,29,37 On the other hand, starting with the earliest studies in this field, several research groups showed the ability of HLA-DR + NK cells to process and present certain antigens on their surface and, as a result, stimulate activation and proliferation of the specific T cells. 28,[38][39][40] Finally, increased HLA-DR expression was recently detected in adaptive-like NK cells. 39 This review summarizes the currently available studies on the inci- ...
... Finally, there are a number of publications that demonstrate the ability of HLA-DR + NK cells to stimulate antigen-specific activation of T cells, that is, to carry out antigen presentation. 28,[38][39][40] In the earliest article on this subject, both freshly isolated and stimulated with IL-2 NK cells and, most importantly, in vitro cloned NK cell lines (clones) induced proliferation of the CD4 + T cells specific to tetanus toxoid (TT). 38 These populations of NK cells presented TT with different efficiencies, depending on the intensity of HLA-DR expression on their surface and the concentration of antigen in the medium, but less efficiently than professional APCs. ...
... 28,[38][39][40] In the earliest article on this subject, both freshly isolated and stimulated with IL-2 NK cells and, most importantly, in vitro cloned NK cell lines (clones) induced proliferation of the CD4 + T cells specific to tetanus toxoid (TT). 38 These populations of NK cells presented TT with different efficiencies, depending on the intensity of HLA-DR expression on their surface and the concentration of antigen in the medium, but less efficiently than professional APCs. At the same time, observed antigen presentation was HLA-restricted: only NK cell clones expressing a certain HLA-DR allele could stimulate the proliferation of the T-cell clones specific for this allele. ...
HLA‐DR‐expressing cells comprise an intriguing group of NK cells, which combine phenotypic characteristics of both NK cells and dendritic cells. These cells can be found in humans and mice; they are present in blood and tissues in healthy conditions and can expand in a spectrum of pathologies. HLA‐DR+ NK cells are functionally active: they produce proinflammatory cytokines, degranulate, and easily proliferate in response to stimuli. Additionally, HLA‐DR CD11c NK cells seem able to take in and then present certain antigens to CD4+ and CD8 CD11c T cells, inducing their activation and proliferation, which puts them closer to professional antigen‐presenting cells. It appears that these NK cells should be considerable players of the innate immune system, both due to their functional activity and regulation of the innate and adaptive immune responses. In this review, for the first time, we provide a detailed description and analysis of the available data characterizing phenotypic, developmental, and functional features of the HLA‐DR+ NK cells in a healthy condition and a disease. Review of the occurrence, special characteristics and antigen‐presenting capacity of the HLA‐DR‐expressing NK cells.
... Another mAb widely applied in cancer treatment is Trastuzumab, which binds the Her-2/Neu receptor often expressed by breast cancer cells (50). Move advanced immunotherapeutic approaches use passive antibody therapy to induce active immune responses. ...
... As demonstrated by us and others, PGE2 induces expression of CCR7 on DCs and functionally increases their migratory responsiveness to lymph node-homing chemokines in vitro (22,35,39 Our results also open the possibility to combine DC vaccination with COX2-inhibitory therapy, which has previously been shown in mouse models to enhance the efficacy of cancer vaccines (49,50). ...
... (49)(50)(51). These NK cells with DClike qualities have been described in humans and mice and have been reported as a new immune cell subpopulation referred to as NKDCs as well as IKDCs (IFN-producing killer dendritic cells)(52)(53)(54)(55)(56)(57). ...
... Another mAb widely applied in cancer treatment is Trastuzumab, which binds the Her-2/Neu receptor often expressed by breast cancer cells (50). Move advanced immunotherapeutic approaches use passive antibody therapy to induce active immune responses. ...
... As demonstrated by us and others, PGE2 induces expression of CCR7 on DCs and functionally increases their migratory responsiveness to lymph node-homing chemokines in vitro (22,35,39 Our results also open the possibility to combine DC vaccination with COX2-inhibitory therapy, which has previously been shown in mouse models to enhance the efficacy of cancer vaccines (49,50). ...
... (49)(50)(51). These NK cells with DClike qualities have been described in humans and mice and have been reported as a new immune cell subpopulation referred to as NKDCs as well as IKDCs (IFN-producing killer dendritic cells)(52)(53)(54)(55)(56)(57). ...
Mucin-1 (MUC1) is a heavily O-glycosylated, transmembrane protein that is expressed on the apical surface of most secretory epithelia. In malignantly transformed epithelia, MUC1 has lost its apical distribution, is underglycosylated and is secreted into the circulation. Due to the underglycosylation of MUC1, cancer-specific MUC1-Tn/STn antigens, which are highly immunogenic, become exposed. We aimed at developing a system that allows detection of antibodies directed to the native form of MUC1 and the underglycosylated MUC1-Tn epitopes. To this end, we made use of the Chinese Hamster Ovary (CHO) ldlD cell line stably transfected with MUC1. This cell line has a glycosylation defect, which can be reversed by addition of different monosaccharides to the cell culture and enables the production of cells expressing the MUC1-Tn glycoforms. After validation with glycospecific antibodies, the CHO-ldlD MUC1 system was used to detect serum MUC1 and MUC1-Tn antibodies. Using this system, we could confirm the presence of MUC1-Tn antibodies in the serum of a patient vaccinated with a truncated MUC1 peptide. This indicates that the CHO-ldlD MUC1 system represents a flow cytometry-based technique to detect antibodies binding to the underglycosylated MUC1 protein. This cellular system is complementary to the previously published methods to detect MUC1 serum antibodies, since the antibodies to the native protein are evaluated and therefore it can be effectively used for MUC1 antibody monitoring in vaccination studies as well as for functional assays.
... Another mAb widely applied in cancer treatment is Trastuzumab, which binds the Her-2/Neu receptor often expressed by breast cancer cells (50). Move advanced immunotherapeutic approaches use passive antibody therapy to induce active immune responses. ...
... As demonstrated by us and others, PGE2 induces expression of CCR7 on DCs and functionally increases their migratory responsiveness to lymph node-homing chemokines in vitro (22,35,39 Our results also open the possibility to combine DC vaccination with COX2-inhibitory therapy, which has previously been shown in mouse models to enhance the efficacy of cancer vaccines (49,50). ...
... (49)(50)(51). These NK cells with DClike qualities have been described in humans and mice and have been reported as a new immune cell subpopulation referred to as NKDCs as well as IKDCs (IFN-producing killer dendritic cells)(52)(53)(54)(55)(56)(57). ...
The limited response rate of cancer patients treated with dendritic cell (DC)-based vaccines indicates that vast improvements remain necessary. In many murine tumour models it has been demonstrated that the use of innate triggers (e.g. TLR triggers) in the maturation of DC results in higher efficacy. However, as few of these innate triggers are generated clinical grade, there remains a great necessity to fill the gap between fundamental mouse studies and a clinical trial in humans. In the present study we used a TLR2/4-agonist (FMKp which is available clinical grade) in combination with IFN-gamma (FIcocktail) in the maturation of elutriated monocyte-derived DC and compared it with the most used DC in current clinical trials (TNF-alpha/PGE-2, i.e. TP-cocktail). In addition to the assessment of CD4+ T cell polarizing capacity, we compared the quantity and intrinsic quality of induced CD8+ T cells of 2 different DC maturation protocols with all cells from the same donor. Besides differences in the cytokine profile, which could be coupled to increased Th1 and Th17 polarization, we demonstrate in this study that FMKp/IFN-gamma matured DC are twice as effective in inducing cytotoxic T cells against known tumor antigens. Both DCs induced phenotypically equivalent effector memory CD8+ T cells that did not show a significant difference in their intrinsic capacity to kill tumor cells. These findings point to the therapeutic applicability of FI-DC as superior inducers of functional antigen-specific T cells. Their increased chemokine secretion is suggestive of a mechanism by which these DC may compensate for the limited migration observed for all ex vivo cultured DC when applied in patients.
... In confirmation of our previous findings (Figs S2 and S3), NK cells were again more prevalent in the decidua compared with the PV (Fig. 4B). HLA-DR pos NK cells that are capable of independently presenting antigens to CD4T cells have been described (Roncarolo et al., 1991). In contrast to HLA-DR neg innate cells (Fig. 3), numerous individual APC clusters were enriched in the PV (Fig. 4C). ...
... We also identified HLA-DR pos cells, including mDCs, pDCs, B cells, macrophages and a population of HLA-DR pos NK cells. An antigen-presenting role for NK cells has been previously described (Roncarolo et al., 1991). The identification of fetal HLA-DR pos macrophages contrasts with the recent findings of Thomas et al. (2021). ...
Maintenance of healthy pregnancy is reliant on successful balance between the fetal and maternal immune systems. Although maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Consistent with recent reports in other fetal organs, T cells with memory phenotypes, though rare in abundance, were detected within the PV tissue and vasculature. Moreover, we determined T cells isolated from PV samples may be more proliferative than adult T cells at baseline after T cell receptor (TCR) stimulation. Collectively, we identified multiple subtypes of fetal immune cells within the PV and specifically highlight the enhanced proliferative capacity of fetal PV T cells.
... In 272 confirmation of our previous findings (Fig 2-3), NK cells were again more prevalent in the 273 decidua compared to the PV (Fig 4B). HLA-DR pos NK cells that are capable to independently 274 present antigens to CD4 T cells have been described (Roncarolo et al., 1991). In contrast to 275 was not certified by peer review) is the author/funder. ...
... We also report a diversity of HLA-DR pos cells present in the PV, where we identified 593 mDCs, pDCs, B cells, Mfs and a population of HLA-DR pos NK cells. An antigen-presenting role 594 for NK cells has been previously described(Roncarolo et al., 1991). The identification of fetal595 HLA-DR pos Mfs contrasts Thomas et al's recent findings showing no HLA-DR pos cells in the PV 596 core up to the 10 th week of gestation (Thomas et al., 2021). ...
Maintenance of healthy pregnancy is reliant on successful balance between the fetal and maternal immune systems. Although maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Further, we identified immunosuppressive signatures in innate immune cells and antigen presenting cells that potentially maintain immune homeostasis in utero. Consistent with recent reports in other fetal organs, T cells with memory phenotypes were detected within the PV tissue and vasculature. Moreover, we determined PV T cells could be activated to upregulate CD69 and proliferate after T cell receptor (TCR) stimulation and when exposed to maternal uterine antigens. Finally, we report that cytokine production by PV T cells is sensitive to TCR stimulation and varies between mid-gestation, preterm (26-35 weeks) and term deliveries (37-40 weeks). Collectively, we elucidated the complexity and functional maturity of fetal immune cells within the PV and highlighted their immunosuppressive potential.
... The functional significance of HLA-DR itself on NK cells is still under discussion. By now, it has been shown that NK cells can present certain antigens to T cells with the use of this molecule, for example tetanus toxin, 14 and can provide costimulatory signal for central memory T cell differentiation. 15 Still, HLA-DR may be a ligand for an unknown receptor itself. ...
... Some evidences to date demonstrate that NK cells possess an ability to present certain antigens through HLA-DR molecule. 9,14 On the other hand, we cannot firmly state that HLA-DR does not function as a ligand for an unknown receptor itself. Potential interactions of these cells with T cells or CD4 + monocytes should be studied further in more detail. ...
NK cells change their phenotype and functional characteristics during activation. In this work, we searched for a relationship of HLA-DR expression with differentiation stages and functional activity of NK cells ex vivo and stimulated in vitro with IL-2 challenged with gene modified feeder K562 cells expressing membrane-bound IL-21 (K562-mbIL21). This stimulation technique has been described for NK cell expansion in clinical use. We have observed that HLA-DR expression in freshly isolated circulating NK cells was mostly associated with less differentiated CD56brightCD57– cells, although in some individuals it could also be found in terminally differentiated CD57+ cells. Ex vivo HLA-DR+ NK cells possessed better capacity to produce IFN-γ in response to cytokine stimulation compared to their HLA-DR– counterparts. In vitro activation with IL-2 and K562-mbIL21 induces an increase in HLA-DR-positive NK cell proportion, again mostly among CD56brightCD57– NK cells. This happened in particular due to appearance of HLA-DR+ expression de novo in HLA-DR-negative cells. Acquired in vitro HLA-DR expression was associated with NK cell proliferation activity, more intense cytokine-induced IFN-γ production, increased degranulation toward feeder cells, and higher expression of CD86 and NKG2D. Thus, stimulation with IL-2/K562-mbIL21 causes a significant phenotype and functional shift during NK cell activation and expansion.
... However, a later study did not fi nd that NK cells could present soluble antigen but could confi rm indeed that these NK cells were able to stimulate T cells ( Brooks and Moore, 1986 ). Using MHC class II expressing NK cell clones, Roncarolo et al. found the ability of NK cells to present soluble antigens was dependant on the type of antigen used, suggesting that NK cells were not as effi cient in processing and presenting antigens as monocyte-derived cells ( Roncarolo et al., 1991 Presentation of superantigens on MHC class II expressing NK cells could also induce non-specifi c T cell expansion, which was suggested to play a role in the elimination of superantigen elaborating pathogens ( D'Orazio and Stein-Streilein, 1996 ). More recently, MHC class II expression was also observed on NK cells from infl amed tissues ( Hanna et al., 2004 ). ...
... Th1 cytokine production Induce Th1, protection against infection and tumours Agaugue et al. (2008) , Biron et al. (1999) , Combe et al. (2005) , Ferlazzo et al. (2004) , Kos and Engleman (1996) , Mailliard et al. (2003) , Martin-Fontecha et al. (2004) , Robbins et al. (2007) , Vankayalapati et al. (2004) Inhibiting effects of Th17 Ashkar et al. (2009) , Lo et al. (2008 Th2 cytokine production Induction of Th2 responses Agaugue et al. (2008) IL-10 and TGF β production Inhibit T cell responses Li et al. (2006) , Moore et al. (2001) Antigen presentation Presentation of antigen on MHC class II Scala et al. (1985) , Brooks and Moore (1986) , D'Orazio and Stein-Streilein (1996) , Roncarolo et al. (1991) , Hanna et al. (2004) Co-stimulation B7 family members, OX40L, CD244 Azuma et al. (1993) , Lee et al. (2003) , Hanna et al. (2004) , Zingoni et al. (2004) , Assarsson et al. (2004) , Saudemont et al. (2005) Cytotoxicity Elimination of T cells Cerboni et al. (2007) , Rabinovich et al. (2003) , Roy et al. (2008) Elimination of DC inhibits T cell activation Gilbertson et al. (1986) , Hayakawa et al. (2004) , Laffont et al. (2008) , Ruggeri et al. (2002) , Yu et al. (2006) Peritt et al. (1998) Help NK cell anti-tumour activity Shimizu and Fujii (2009) NKT Enhances NK cell responses by IFN γ production Smyth et al. (2002) t0020 t0020 ...
Natural killer (NK) cells are lymphocytes of the innate immune system that play a vital role in host defence. The primary function of NK cells is to kill infected cells and produce cytokines and chemokines during the early phase of pathogen infection. These NK cell functions are regulated by the expression of receptors that can lead to activating or inhibiting signals. Activating receptors on NK cells recognize a variety of molecular structures and use a variety of signalling pathways. As lymphocytes of the innate immune system, natural killer (NK) cells play an important role in the immunosurveillance against infection and cancers. How the NK cells respond to a specific infection can have knock-on effects on the development of the adaptive immune responses. However, T cells in the later stages of the immune response may also play a role in regulating NK cells. Activated NK cells may also stimulate T cells directly by expressing co-stimulatory molecules and MHC molecules. IFNγ from NK cells may directly polarize Th1 cell response by inducing T-bet. NK cell-mediated killing of DCs limits stimulation of T cells. NK cells can limit immunity by directly killing activated T cells. NK cell responses to healthy or infected cells are based upon recognition of a broad range of ligands, which tightly control both activating and inhibitory signals. As part of the adaptive immune system, T cells play a role in mounting cytotoxic or cytokine responses to both intracellular and extracellular infections.
... In addition, unstimulated human peripheral blood DC may lyse select squamous cell tumor targets [15]. Conversely, it is known that following prolonged in vitro activation, human NK cells gain the ability to present antigen to CD4 T cells [16,17]. However, whether a subset of freshly isolated, steady state human NK cells can possess both natural killing and APC function is uncertain. ...
... Human NK cells gain the ability to process and present protein antigens to CD4 T cells after prolonged in vitro activation with IL-2 [17]. This is accompanied by variable upregulation of MHC class II expression that correlates to the level of NK cell APC function [16]. Similarly, in vitro activated NK cell cultures have been shown to efficiently co-stimulate anti-CD3 or staphylococcal enterotoxin B-induced proliferation of autologous CD4 T cells in an OX40L-OX40 dependent manner [29]. ...
Whether a freshly isolated immune cell can be equipped with both natural killing and antigen-presenting cell (APC) function has recently become controversial in mice. We sought to probe the existence of a candidate human cell with these properties by searching for cells in healthy subjects that co-express APC surface molecules and NK cell receptors. We have found that CD3(-)CD14(-)CD19(-) mononuclear cells of human blood, spleen, liver, and lymph nodes contain two distinct populations of cells that co-express HLA-DR (DR) and CD56. Circulating CD56(+) cells expressing high levels of DR were phenotypically and functionally similar to conventional CD56(-)dendritic cells (DC). Furthermore, we demonstrate here that a separate cohort of CD56(+) cells that express low levels of DR are NK cells that possess dual function as potent killers endowed with weak APC function.
... However, expression of HLA-DR on NK cells has also been documented, where it has sometimes been used as an activation marker because of its increased expression following IL-2 stimulation [9][10][11][12][13][14][15][16][17]. HLA-DRexpressing NK cells have been shown to present tetanus toxin and house dust mite-derived peptides to T-cell clones in an HLA-DRrestricted manner [18]. Antigen bound by activating NK receptors can be internalised to class II MHC-loading compartments and processed for subsequent HLA-DR-mediated presentation leading to T-cell proliferation [19]. ...
... An important goal is to address the functional importance of HLA-DR-expressing NK cells. It has been shown previously that NK cells expressing HLA-DR can present antigen to trigger T-cell proliferation and IL-2 production [18][19]. The accumulation of HLA-DR-expressing NK cells at sites of inflammation [10,19] and correlations between their numbers and the magnitude of immunopathology (for example in IgA nephropathy [20]) suggests that they may contribute to inflammation and immunopathology, either by antigen presentation or an alternative mechanism. ...
Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-γ response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-γ response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.
... The critical importance of GATA3 was shown with anti-sense oligonucleotides inhibiting T-cell development from murine fetal liver cells in FTOC 34 . To be able to study the role of GATA3 in lymphoid development in vivo Ting and co-workers 82 performed a Rag2 -/complementation assay 83 . Homozygous GATA3 deficient embryonic stem cells were injected in a Rag2 knock-out blastocyst. ...
... Earlier cells with characteristics of both NK cells and DCs were found in humans. NK-cell clones with the ability to process and present antigens, that have cytotoxic activity and the capacity to stimulate proliferation of T-cell clones were already described back in 1991 83 . Unfortunately the ability to produce type I IFNs was not tested. ...
Proefschrift Universiteit van Amsterdam. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.
... Obwohl NK-Zellen Antigene über MHC-Klasse-I/-II präsentieren können und außerdem kostimulatorische Moleküle exprimieren (Roncarolo et al., 1991), ist die mögliche Funktion der NK-Zellen als APCs bisher unklar. Homodimer, vermutlich Korezeptor (Vitale et al., 2001b) CD226 (DNAM-1) assoziiert mit LFA-1, kann Zytotoxizität auslösen (Bottino et al., 2003;Shibuya et al., 1996;Shibuya et al., 1998;Shibuya et al., 1999) CD69 ...
... YT-Zellen exprimieren wie andere NK-Zellen kostimulatorische Moleküle und MHC-Moleküle (Klasse-I und -II) und könnten potentiell als antigenpräsentierende Zellen wirken (Roncarolo et al., 1991). Die Kokultivierung allogener PBL mit bestrahlten mocktransfizierten oder cIgTCR + YT-Zellen führte zu keiner Zunahme der Zytotoxizität der "stimulierten" PBL gegenüber den mock-und cIgTCR-transfizierten YT-Zellen. ...
Die spezifische adoptive Immuntherapie ist ein hoffnungsvoller Ansatz zur Behandlung von Tumoren. Die aufwendige individuelle Bereitstellung primärer Effektorlymphozyten könnte durch den Einsatz etablierter tumorantigenspezifischer Effektorzellinien vermieden werden. In dieser Arbeit wurde untersucht, ob sich ein Tumortargeting der humanen Natürlichen Killer-(NK)-Zellinie YT durch den Gentransfer chimärer Immunglobulin-T-Zellrezeptoren (cIgTCRs) erreichen läßt. Die cIgTCR-Konstrukte wurden aus single-chain-Fv-Fragmenten (scFv), dem IgG1-Fc-Teil und der CD3-Zeta-Signalkette erzeugt. Die scFv-Fragmente wurden aus den humanisierten Antikörpern BW431/26 und HuM195, die spezifisch für das karzinoembryonale Antigen (CEA) bzw. CD33 sind, konstruiert und zeigten als scFv-hFc-Fusionsproteine eine spezifische Bindung an Tumorzellen. Die YT-Zellen wurden mit den cIgTCR-Genkonstrukten über Elektroporation transfiziert und über immunologische Verfahren angereichert. In-vitro-Studien ergaben eine spezifische Lyse von CEA+ Kolonkarzinomzellinien durch die scBW431/26-hFcZeta+ YT-Zellen. Die Zytotoxizität korrelierte mit der Expression des cIgTCR-Antigens auf den Tumorzellen und wurde durch zirkulierendes CEA nicht gehemmt. Die scHuM195-hFcZeta+ YT-Zellen zeigten eine spezifische Lyse der CD33+ myeloischen Leukämiezellinie KG1. Die Bestrahlung wurde zur Wachstumsbegrenzung der YT-Zellen eingesetzt. Die spezifische Zytotoxizität der scBW431/26-hFcZeta+ YT-Zellen gegenüber CEA+ Tumorzellen war einen Tag nach Bestrahlung unverändert. Die Koinjektion von CEA+ Tumorzellen mit bestrahlten scBW431/26-hFcZeta+ YT-Zellen führte zu einer signifikanten Hemmung des Tumorwachstums in NOD/SCID-Mäusen. Die cIgTCR+ YT-Zellen zeigten in vitro eine geringe Sensibilität gegenüber allogenen Blutlymphozyten. Die Ergebnisse zeigen, daß die Zytotoxizität der NK-Zellinie YT tumorantigenspezifisch durch cIgTCR-Gentransfer erweitert wird und ein Potential zur Behandlung minimaler Tumorerkrankungen besteht.
... In contrast to freshly isolated, resting human NK cells, activated human NK cells can express human leukocyte antigen DR (HLA-DR), OX40 ligand, CD80, and CD86 and have been shown previously to have antigen-presenting capabilities similar to certain DC subsets. [22][23][24] Activated NK cells can also express CD4 on their surface, 25,26 a molecule typically found on a subset of CD3 ϩ T cells and monocytes/macrophages; however, CD4 has also been observed on many activated cell types including CD8 ϩ T cells, 27 B cells, 28 monocytes, 29 eosinophils, 30 and neutrophils. 31 Given the small percentage of circulating CD4 ϩ NK cells, previous studies have used in vitro stimulation protocols to expand the number of NK cells expressing CD4 25,26 ; however, these activated CD4 ϩ NK cells may not be representative of CD4 ϩ NK cells in vivo. ...
... In contrast, monocytes and dendritic cells are traditionally thought to take up foreign antigens, process them, and present the antigens to T cells. However, several reports have documented antigen presentation by activated human NK cells [22][23][24] and possibly cytolytic capabilities by DCs. [37][38][39][40][41] Furthermore, a recent report has found immunologic memory mediated by virusspecific mouse NK cells. ...
The lack of natural killer (NK) cell-specific markers, as well as the overlap among several common surface antigens and functional properties, has obscured the delineation between NK cells and dendritic cells. Here, novel subsets of peripheral blood CD3/14/19(neg) NK cells and monocyte/dendritic cell (DC)-like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56+ monocyte/DC-like cells, which lack CD7. In contrast to CD7+CD56+ NK cells, CD7(neg)CD56+ cells lack expression of NK cell-associated markers, but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7, we observed approximately 60% of CD4+CD56+ cells were CD7(neg) cells, indicating the actual frequency of activated CD4+ NK cells is much lower in the blood than previously recognized. Functionally, only CD7+ NK cells secrete gamma interferon (IFNgamma) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore, using CD7 to separate CD56+ NK cells and CD56+ myeloid cells, we demonstrate that unlike resting CD7+CD56+ NK cells, the CD7(neg)CD56+ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7(neg)CD56+ monocyte/DC-like cells, thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo.
... Although the existence of the IKDC population would make evolutionary sense, it has been controversial whether IKDCs really represent a separate DC lineage or nothing else but activated NK cells, endowed with antigen presenting capacity [18]. The latter view is supported by the fact that human NK cells have long been known to have APC-like activity [19,20]. The developmental pathways between NK cells and IKDCs also overlap as both rely on the IL- 2Rb, IL-5Rb and common c chain (and thus a functional IL-15R complex) and IL-15 for development, whereas cDCs and pDCs develop in the absence of a functional IL-15R [15,16]. ...
... NK cells have extensive trafficking capabilities and are found within non-lymphoid and lymphoid tissues, sometimes in close proximity to naı¨venaı¨ve T cells, as also shown in this study [34,37]. In vitro studies revealed that activated human NK cells and NK cell clones can process soluble antigen, up-regulate MHCII and co-stimulatory molecules, and thus stimulate naı¨venaı¨ve CD4 and CD8 T cell responses [19,20]. The functional relevance of this has been questioned in vivo, as mouse NK cells were classically not found to have direct APC function [17,18]. ...
Natural killer cells are innate effector cells known for their potential to produce interferon-gamma and kill tumour and virus-infected cells. Recently, B220(+)CD11c(int)NK1.1(+) NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220(-) NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.
... Direct interaction of APC with NK cells allows for precise, local delivery of cytokines and delivery of co-stimulatory signals which further promotes NK cell activation [43]. Additional mechanisms may operate for cells where the distinctions between APC and NK cell functions are blurred, such as for mouse activated DC subpopulations that co-express NK cell markers and demonstrate perforin-dependent killing [44] or for human activated NK cells which bear APC markers (MHCII and co-stimulatory molecules) and demonstrate capacity to present antigen to CD4 + T cells in vitro [45][46][47]. Their contribution to pathogen clearance in vivo is unclear and their functional impacts may be both pathogen-and tissue-dependent. ...
Host control of mouse cytomegalovirus (MCMV) infection of MHCII ⁻ salivary gland acinar cells is mediated by CD4 ⁺ T cells, but how they protect is unclear. Here, we show CD4 ⁺ T cells control MCMV indirectly in the salivary gland, via IFNγ engagement with uninfected, but antigen ⁺ MHCII ⁺ APC and recruitment of NK cells to infected cell foci. This immune mechanism renders direct contact of CD4 ⁺ T cells with infected cells unnecessary and may represent a host strategy to overcome viral immune evasion.
... Previous studies addressing HLA class II function on NK cells have tested soluble peptides (33,34) and Staphylococcal Enterotoxin B crosslinking (32) for triggering HLA-class II-dependent CD4+ T cell activation, hence bypassing the requirement for whole antigen uptake, processing and presentation by the NK cell. Expanded NK cell clones were shown to process and present HLA class II-dependent peptides derived from soluble proteins though failed to present whole Mycobacterium leprae (50). The herein presented results showed that NK cells can, indeed, perform these processes upon direct or antibody-aided interaction with viral preparations. ...
Natural killer (NK) cells play a dual role in the defense against viral pathogens by directly lysing infected cells as well as by regulating anti-viral T cell immunity. Infection by human cytomegalovirus (HCMV) promotes a persistent expansion of NKG2C+ adaptive NK cells which have been shown to display enhanced antibody-dependent responses against infected targets and associated to viral control in transplanted patients. Based on gene expression data showing increased transcription of CIITA and several genes related to the MHC class II pathway in adaptive NK cells, we explored their putative capacity for antigen presentation to CD4+ T cells. Phenotypic analysis confirmed a preferential steady-state expression of HLA-DR by circulating NKG2C+ adaptive NK cells in healthy individuals. Expression of HLA-DR in NKG2C+ adaptive NK cells was variable and unrelated to the expression of activation (i.e., CD69 and CD25) or differentiation (i.e., FcRγ chain, CD57) markers, remaining stable over time at the individual level. Incubation of purified NK cells with HCMV complexed with serum specific antibodies induced an up-regulation of surface HLA-DR concomitant to CD16 loss whereas no changes in CD80/CD86 co-stimulatory ligands were detected. In addition, surface CX3CR1 decreased upon antigen-loading while HLA-DR+ NK cells maintained a CCR7-, CXCR3low homing profile. Remarkably, HCMV-loaded purified NK cells activated autologous CD4+ T cells in an HLA-DR dependent manner. The fraction of T lymphocytes activated by antigen-loaded NK cells was smaller than that stimulated by monocyte-derived dendritic cells, corresponding to CD28-negative effector-memory CD4+ T cells with cytotoxic potential. Antigen presentation by NK cells activated a polyfunctional CD4+ T cell response characterized by degranulation (CD107a) and the secretion of Th1 cytokines (IFNγ and TNFα). Overall, our data discloses the capacity of NKG2C+ adaptive NK cells to process and present HCMV antigens to memory CD4+ cytotoxic T cells, directly regulating their response to the viral infection.
... A noter qu'in vitro, les IKDC spléniques sont incapables de présenter des antigènes viraux aux LT spécifiques du virus(Caminschi et al., 2007). Suite à ces quelques études, il s'est avéré que les IKDC représentent plutôt une population immature de cellules NK (J.Hanna et al., 2004) (Roncarolo et al., 1991). ...
L’infection par le virus de la grippe, ou le Myxovirus influenzae de type A (IAV), constitue l'une des causes les plus importantes de maladies des voies respiratoires dans le monde. Elle conduit également à des épidémies récurrentes avec des taux élevés de morbidité et de mortalité. Des surinfections bactériennes, principalement causées par Streptococcus pneumoniae (pneumonie), sont souvent associées à la grippe et contribuent de manière significative à l’excès de mortalité. La perturbation de l'intégrité des tissus pulmonaires et la diminution de l'immunité antibactérienne au cours de l'infection par IAV sont à l’origine de la colonisation et à la dissémination des bactéries.L'infection grippale entraîne une altération profonde du compartiment de cellules myéloïdes pulmonaires caractérisée par une altération numérique ou fonctionnelle des cellules sentinelles - les macrophages alvéolaires et les cellules dendritiques conventionnelles (cDC) - et par un recrutement de cellules myéloïdes inflammatoires -les neutrophiles, les monocytes inflammatoires ou encore les cellules dendritiques inflammatoires.Les cellules myéloïdes sont originaires de la moelle osseuse (MO). Lors d’infections, la myélopoïèse peut être profondément affectée afin de maintenir la production et la mobilisation de cellules myéloïdes inflammatoires au niveau du site d’infection. A l’heure actuelle, les conséquences de l’infection grippale sur la myélopoïèse restent encore mal connues.Dans notre projet, nous rapportons que l'infection grippale conduit à une diminution transitoire du nombre de cDC (cDC1 et cDC2) dans les poumons qui coïncide avec une chute dans la MO, du nombre de progéniteurs/précurseurs impliqués dans la génération des cDC (CDP, pre-cDC et plus particulièrement les pre-cDC1). Cette diminution de la "DCpoïèse" est associée à une accélération de la génération des monocytes, i.e. monopoïèse. La différenciation altérée des cDC est indépendante des cytokines pro-inflammatoires et n'est pas due à un dysfonctionnement intrinsèque des précurseurs de cDC. De façon intéressante, nous rapportons que ces altérations au niveau de la MO sont associées à une diminution de la production de Flt3-L ou Fms-like tyrosine kinase 3 ligand, un facteur crucial pour la différenciation des DC. La supplémentation en Flt3-L au cours de la grippe rétablit la différenciation des progéniteurs de cDC dans la MO et restaure le compartiment des cDC pulmonaires. De façon intéressante, cette restauration s’accompagne d’une protection partielle contre l’infection pneumococcique secondaire caractérisée par une réduction de la charge bactérienne, une amélioration de la pathologie pulmonaire et une survie prolongée.
... Human NK cells have been suggested to present antigen to and stimulate T cell responses against HSV and several other reports indicate that some human NK cell subsets may express MHC II and co-stimulatory molecules that stimulate T cell proliferation (12,13,15,(36)(37)(38)(39)(40). In this context, our data are the first to directly show that NK cells indeed can stimulate proliferation of alphaherpesvirus-experienced T cells upon killing of alphaherpesvirus-infected target cells. ...
Natural killer (NK) cells belong to the innate immune system and play a central role in the defense against viral infections and cancer development, but also contribute to shaping adaptive immune responses. NK cells are particularly important in the first line defense against herpesviruses, including alphaherpesviruses. In addition to their ability to kill target cells and produce interferon-γ, porcine and human NK cell subsets have been reported to display features associated with professional antigen presenting cells (APC), although it is currently unclear whether NK cells may internalize debris of virus-infected cells and whether this APC-like activity of NK cells may stimulate proliferation of antiviral T cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we show that vaccination of pigs with a live attenuated PRV vaccine strain triggers expression of MHC class II on porcine NK cells, that porcine NK cells can internalize debris from PRV-infected target cells, and that NK cells can stimulate proliferation of CD8⁺ and CD4⁺CD8⁺ PRV-experienced T cells. These results highlight the potential of targeting these NK cell features in future vaccination strategies.
... Our observations add to the growing understanding of how NK cells regulate T cell responses. NK cells can provide co-stimulation and even antigen presentation that activates T cells (49,50). Upon activation, T cells upregulate NKG2D ligands and are then reduced in number through cytotoxic action of NK cells (26)(27)(28). ...
In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4⁺ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4⁺ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.
... Subsequent studies may help to elucidate a functional role of RANKL and B7-H3 molecules in these highly proliferating NK cells. In addition, it will be interesting to verify the occurrence of a preferential expansion of HLA-DR expressing NK cells with other ex vivo expansion protocols, and whether this confers antigen presenting characteristics to NK cells, as it has been previously shown (54). Confirmation of antigen presenting properties of expanded NK cells may be further considered to extend the therapeutic potential of NK cells. ...
Clinical studies with adoptive immunotherapy using allogeneic natural killer (NK) cells showed feasibility, but also limitation regarding the transfused absolute cell numbers. First promising results with peripheral blood mononuclear cells (PBMCs) as feeder cells to improve the final cell number need further optimization and investigation of the unknown controlling mechanism in the cross-talk to NK cells. We investigated the influence of irradiated autologous PBMCs to boost NK cell proliferation in the presence of OKT3 and IL-2. Our findings demonstrate a requirement for receptor–ligand interactions between feeders and NK cells to produce soluble factors that can sustain NK cell proliferation. Thus, both physical contact between feeder and NK cells, and soluble factors produced in consequence, are required to fully enhance NK cell ex vivo proliferation. This occurred with an indispensable role of the cross-talk between T cells, monocytes, and NK cells, while B cells had no further influence in supporting NK cell proliferation under these co-culture conditions. Moreover, gene expression analysis of highly proliferating and non-proliferating NK cells revealed important phenotypic changes on 5-day cultured NK cells. Actively proliferating NK cells have reduced Siglec-7 and -9 expression compared with non-proliferating and resting NK cells (day 0), independently of the presence of feeder cells. Interestingly, proliferating NK cells cultured with feeder cells contained increased frequencies of cells expressing RANKL, B7-H3, and HLA class II molecules, particularly HLA-DR, compared with resting NK cells or expanded with IL-2 only. A subset of HLA-DR expressing NK cells, co-expressing RANKL, and B7-H3 corresponded to the most proliferative population under the established co-culture conditions. Our results highlight the importance of the crosstalk between T cells, monocytes, and NK cells in autologous feeder cell-based ex vivo NK cell expansion protocols, and reveal the appearance of a highly proliferative subpopulation of NK cells (HLA-DR⁺RANKL⁺B7-H3⁺) with promising characteristics to extend the therapeutic potential of NK cells.
... NK cells from the SV-PBS group exhibited a significant increase in expression of MHC class-II related genes at day 28, the only significant increase in MHC class-II related genes in any cell type in the unadjuvanted vaccine group at any time point (Fig 7). While not considered classical APC, NK cells have been previously reported to upregulate MHC class-II and present antigen to T cells [85][86][87]. Subjects receiving unadjuvanted vaccine did not achieve seroprotection; however, H5-specific antibody titers were increased relative to baseline, suggesting that low-level immune induction occurred. In support of this theory, Gillard et al. have previously shown that a 2-dose prime regimen with 3.75mcg of split-virus H5N1 vaccine (A/ Vietnam/1194/2004) without adjuvants led to cross-reactive seroprotection following a second 2-dose boost regimen with a heterologous 3.75mcg split-virus unadjuvanted H5N1 vaccine (A/Indonesia/5/2005) administered 6 months later [88]. ...
Background
Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood.
Objective and Methods
We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18–49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination.
Results
Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination.
Conclusions
Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed.
Trial Registration
ClinicalTrials.gov NCT01573312
... The regulation of MHC-II expression following exposition to inflammatory stimuli depends on the CIITA (36). However, although it was previously acknowledged that NK cell clones could process and present some soluble Ags such as tetanus toxoid (37), the expression of HLA-DR on NK cells was mostly considered as an activation marker (38)(39)(40)(41)(42). As previously suggested by Evans et al. (42), the identification of HLA-DR + NK cells after culture with IL-2 might result from the expansion of rare HLA-DR + cells initially present in peripheral blood NK cells. ...
NK cells are critical for innate immunity-mediated protection. The main roles of NK cells rely on their cytotoxic functions or depend on the tuning of Th1 adaptive immunity by IFN-γ. However, the precise influence of inflammatory cytokines on NK cell and CD4 T lymphocyte interactions was never investigated. In this study, we provide evidence that IL-21, a cytokine produced during chronic inflammation or infectious diseases, promotes the differentiation of a specific subset of NK cells coexpressing CD86 and HLA-DR and lacking NKp44. More importantly, IL-21-propagated HLA-DR(+) NK cells produce macrophage migration inhibitory factor and provide costimulatory signaling during naive CD4(+) T cell priming inducing the differentiation of uncommitted central memory T cells. Central memory T cells expanded in the presence of HLA-DR(+) NK cells are CXCR3(+)CCR6(-)CCR4(-)CXCR5(-) and produce IL-2, as well as low levels of TNF-α. Costimulation of CD4(+) T cells by HLA-DR(+) NK cells prevents the acquisition of effector memory phenotype induced by IL-2. Moreover, we identified this population of NK HLA-DR(+) macrophage migration inhibitory factor(+) cells in inflammatory human appendix. Collectively, these results demonstrate a novel function for IL-21 in tuning NK and CD4(+) T cell interactions promoting a specific expansion of central memory lymphocytes.
... 124 In general, interaction between NK cells and DCs play a role in the activation and maturation of DC. 125 Furthermore, activated NK cells express MHC class II and can act as APCs. 126,127 Activated NK cells indeed trigger TCR induced function of CD4 T cells dependent on the expression of OX40 ligand and B7 by NK cells. 128 ...
Natural killer (NK) cells can detect and kill tumor cells and infusion of NK cells to cancer
patients may be a promising option to treat cancer. In this context, ex vivo expansion is used to
produce large quantities of activated NK cells, because sufficient numbers of these effector cells
are essential for successful NK cell based adoptive cancer immunotherapy. The development of
efficient NK cell expansion protocols and the transfer of these protocols to clinically applicable
methods represent a major challenge. To overcome this issue, the aim of my project was to
develop a clinically applicable method that yields large numbers of highly functional NK cells.
First, a fully automated technical process was developed to activate and expand NK cells with
(interleukin) IL-2 and irradiated clinical-grade feeder cells (EBV-LCL). In comparison to the
manual procedure, the automated process yielded similar NK cells in terms of cell numbers,
surface marker profile, gene expression and in vitro effector functions. Upon expansion, NK
cells up-regulated functional surface molecules, such as TRAIL, FasL, NKG2D and DNAM-1,
they increased the production of interferon (IFN)-g and tumor necrosis factor (TNF)-a and they
became more cytotoxic against tumor cell lines. Next, because in the used protocol NK cell
expansion was restricted to a period of 2-4 weeks, a more efficient protocol for long-term
expansion was developed. Manual NK cell expansion with EBV-LCL and IL-2 induced a 22–
fold mean NK cell expansion after one week that was significantly increased to 53–fold by
addition of IL-21. Furthermore, repeated stimulation with irradiated EBV-LCL and IL-2 and
addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with
1011–fold NK cell expansion after six weeks, which is an unprecedented high expansion rate not
achieved by any other method so far. Most importantly, adoptive transfer of NK cells expanded
with this optimized protocol led to significant inhibition of tumor growth in a melanoma
xenograft mouse model, proofing the therapeutic efficacy of the ex vivo generated NK cells.
This anti-tumor efficacy was superior over that from conventionally IL-2 activated NK cells,
demonstrating that the improved NK cell expansion method enhanced not only the quantity but
also the therapeutic quality of NK cells.
In conclusion, the outcome of this project is a fully automated process for ex vivo production of
NK cells and an optimized protocol for NK cell expansion with unparalleled efficacy. The
expanded NK cells possess potent anti-tumor features and showed therapeutic efficacy in a
preclinical melanoma xenograft model. Thereby, the project serves clinical needs and makes it
possible to generate high cell doses of functional NK cells for the use in cancer immunotherapy.
... Thus, our existing in vitro cellular proliferation assays appear too insensitive. Previously, human NK cells have been shown to process and present tetanus toxoid and Der p 1 to T cells, but protein antigen from Mycobacterium leprae was poorly processed and presented (Roncarolo et al., 1991). It is also noteworthy that many of the antigens associated with type-2 immune responses are either shed by multicellular helminth parasites (Kamata et al., 1995) or are proteins with protease activity, such as Der p 1 or papain (Gough et al., 1999; (F) ILC2 number following coculture with OTIITg CD4 + T cells, as indicated. ...
Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.
... Interestingly, unique cell types conjugating properties of both NK cells and DC have previously been described (5,6). In humans, activated NK cells have been reported to express MHC class II and present antigens to T cells (7)(8)(9)(10). Conversely, in humans and in rodents, some DC subsets exhibit cytolytic activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). ...
Interferon-producing killer dendritic cells (IKDC) were first described for their outstanding anti-tumoral properties. The “IKDC” terminology implied the description of a novel DC subset and initiated a debate on their cellular lineage origin. This debate shifted the focus away from their notable anti-tumoral potential. IKDC were recently redefined as precursors to mature NK (mNK) cells and consequently renamed pre-mNK cells. Importantly, a putative human equivalent of pre-mNK cells was recently associated with improved disease outcome in cancer patients. It is thus timely to revisit the functional attributes as well as the therapeutic potential of pre-mNK cells in line with their newly defined NK-cell precursor function.
... Activation of naïve T cells is dependant on IFN- produced by NK cells [Martin-Fontecha, 2004]. Activated NK cells have also been reported to stimulate autologous CD4 T cells through direct cell to cell interactions involving the expression of OX40 ligands and CD86 [Roncarolo, 1991; Zingoni, 2004]. ...
Natural killer (NK) cells are a key component of the innate immune system that are capable of rapid recognition and elimination of target cells without prior sensitization. Compelling evidence that NK cells limit viral replication in vivo has come from studies involving cytomegalovirus (CMV) infection. Human CMV (HCMV) is a pathogen responsible for causing significant morbidity and mortality in immunocompromised individuals. Due to the strict species specificity of the CMVs, many aspects of the host response to infection have been determined using animal models. This chapter discusses some of the mechanisms used by NK cells to identify CMV- infected cells and describes the escape mechanisms employed by the CMVs to evade detection. We also consider some of the recent data suggesting that in addition to their role in directly inhibiting viral replication, NK cells have the capacity to shape the adaptive immune response elicited by CMV infection.
... The induction of specific antifungal T helper cell responses through DCs may also be influenced by NK cells, since NK cells provide antigenic cellular debris, which is internalized by maturing DCs and presented to T cells in lymph nodes (55). Interestingly, NK cells also have the capability to directly induce CD4 ϩ T cell responses by antigen presentation in a class II HLArestricted manner, as was demonstrated for NK cells presenting tetanus toxoid to tetanus-specific T cell clones (56). ...
Based on improved experimental methodologies and on a better understanding of the immune system, there is increasing insight into the antifungal activity of natural killer (NK) cells. Murine and human NK cells are able to damage fungi of different genera and species in vitro, they exert both direct and indirect antifungal activity through cytotoxic molecules such as perforin and through cytokines and interferons, respectively. On the other hand, recent data suggest that fungi exhibit immunosuppressive effects on NK cells. Whereas clear in vivo data are lacking in humans, the importance of NK cells in the host response against fungi has been demonstrated in animal models. Further knowledge of the interaction of NK cells with fungi might help to better understand the pathogenesis of invasive fungal infections and to improve treatment strategies.
... [47][48][49] HLA-expressing NK cells have been shown to present antigen to trigger T-cell proliferation and interleukin-2 production. 50,51 In addition, HLA-DR-expressing NK cells have significantly stronger cytolytic activity in comparison to other NK cells. 49 Evans et al. 49 demonstrate that the magnitude of an individual's NK-cell interferon-c response triggered by the model pathogen, Mycobacterium bovis bacillus Calmette-Guérin, was associated with the initial proportion of HLA-DR-expressing NK cells in peripheral blood mononuclear cells. ...
The chemokine receptor CCR5 is pivotal in determining an individual's susceptibility to HIV-1 infection and rate of disease progression. To establish whether population-based differences exist in cell surface expression of CCR5 we evaluated the extent of CCR5 expression across all peripheral blood cell types in individuals from two populations, South African Africans (SAA) and South African Caucasians (SAC). Significant differences in CCR5 expression, both in number of CCR5 molecules per cell (density) and the percentage of CCR5-expressing cells, were observed between the two study groups, within all cell subsets. Most notably, the percentage of all CCR5(+) cell subsets was significantly lower in SAC compared with SAA individuals (P < 0·01) among natural killer (NK) -cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) whereas CCR5 density was significantly higher in SAC compared with SAA individuals in CCR5(+) CD8(+) T-cell subsets and CCR5(+) NK-cell subsets (CD56(+) , CD16(+) CD56(+) and CD56(dim) ) (all P < 0·05). These relationships were maintained after exclusion of CCR5Δ32 heterozygous individuals (n = 7) from the SAC dataset. The SAA individuals exhibited significantly higher cell activation levels, as measured by HLA-DR expression, than SAC individuals in CD4(+) T-cell subsets (P = 0·002) and CD56(+) NK-cell subsets (P < 0·001). This study serves to demonstrate that ethnically divergent populations show marked differences in both cell activation and CCR5 expression, which are likely to impact on both susceptibility to HIV-1 infection and the rate of HIV-1 disease progression.
Prenatal arsenic exposure is a major public health concern, associated with altered birth outcomes and increased respiratory disease risk. However, characterization of the long-term effects of mid-pregnancy (second trimester) arsenic exposure on multiple organ systems is scant. This study aimed to characterize the long-term impact of mid-pregnancy inorganic arsenic exposure on the lung, heart, and immune system, including infectious disease response using the C57BL/6 mouse model. Mice were exposed from gestational day 9 till birth to either 0 or 1000µg/L sodium (meta)arsenite in drinking water. Male and female offspring assessed at adulthood (10-12 weeks of age) did not show significant effects on recovery outcomes after ischemia reperfusion injury but did exhibit increased airway hyperresponsiveness compared to controls. Flow cytometric analysis revealed significantly greater total numbers of cells in arsenic-exposed lungs, lower MHCII expression in natural killer cells, and increased percentages of dendritic cell populations. Activated interstitial (IMs) and alveolar macrophages (AMs) isolated from arsenic-exposed male mice produced significantly less IFN-γ than controls. Conversely, activated AMs from arsenic-exposed females produced significantly more IFN-γ than controls. Although systemic cytokine levels were higher upon Mycobacterium tuberculosis (Mtb) infection in prenatally arsenic-exposed offspring there was no difference in lung Mtb burden compared to controls. This study highlights significant long-term impacts of prenatal arsenic exposure on lung and immune cell function. These effects may contribute to the elevated risk of respiratory diseases associated with prenatal arsenic exposure in epidemiology studies and point to the need for more research into mechanisms driving these maintained responses.
Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating central nervous system condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that [i] cytotoxicity is not incumbent on MOG autoantibody quantity alone, [ii] engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not, [iii] the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not, and [iv] all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing MOGAD patients. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.
NK cells are members of the innate immunity and play a central role in the defense against viral infections and cancer development, but also contribute to triggering and shaping adaptive immune responses. Human NK cells may express MHC II and costimulatory molecules, including CD86, CD80, and OX40 ligand, which allows them to stimulate the CD4⁺ T-cell response. In contrast, murine NK cells do not express MHC II or costimulatory molecules. Upon activation, mouse NK cells can acquire these molecules from dendritic cells (DCs) via intercellular membrane transfer, which leads to suppression of DC-induced CD4⁺ T-cell responses rather than stimulation of T-cell responses. Previous studies showed that porcine NK cells can express MHC II molecules, but it was unknown if porcine NK cells also express costimulatory molecules and whether NK cells may affect T-cell proliferation. We found that primary porcine NK cells express functional MHC II molecules and costimulatory CD80/86, particularly upon activation with IL-2/IL-12/IL-18, and that they are able to stimulate T-cell proliferation. In addition, we show that porcine NK cells are able to internalize antigens derived from killed target cells in an actin polymerization-dependent process. All together, these results indicate that porcine NK cells possess properties associated with APCs, which allows them to stimulate T-cell proliferation.
Background:
House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking.
Objective:
To comprehensively analyse the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function.
Methods:
Proteomic analysis (liquid chromatography-tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity, and putative function analyses were performed in silico according to Gene Ontology annotations.
Results:
Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T cell response, underlining the heterogeneity of T cell responses to HDM allergens.
Conclusions and clinical relevance:
Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM.
Defining NK cells has been challenging in many veterinary species. Although several groups have described putative NK cell populations, there is still no consensus on a definition of NK cells in the dog. In the present study, canine NK cells are characterized as CD3-GranzymeB+ cells, further divided into a NCR1+ and a NCR1- subset. All dogs examined displayed both subsets in blood, although of quite variable magnitude. Following vaccination an increase was observed in the CD3- NCR1- cell population in blood, but not in the CD3- NCR1+ population. Non-B non-T cell cultures stimulated with IL-2 and IL-15 were dominated by CD3-GranzymeB+ cells after approximately 2 weeks and a large proportion of the CD3-GranzymeB+ cells expressed NCR1. IL-12 stimulation lead to a further upregulation resulting in an almost uniform expression of NCR1. The cultured cells expressed MHC class II, showed a variable expression of CD8 and were negative for CD4 and CD21. The cultures were able to kill known NK cell targets, and NCR1 was shown to be a major activating receptor. A large proportion of the NCR1+ cells, but none of the NCR1- cells, produced IFNγ in response to IL-12 stimulation. These results show that NCR1 defines two subsets of canine NK cells, likely to represent different activation stages, and that NCR1 acts as an activating receptor on canine NK cells.
The two main proteolytic machineries of eukaryotic cells, lysosomes and proteasomes, receive substrates by different routes. Polyubiquitination targets proteins for proteasomal degradation, whereas autophagy delivers intracellular material for lysosomal hydrolysis. The importance of autophagy for cell survival has long been appreciated, but more recently, its essential role in both innate and adaptive immunity has been characterized. Autophagy is now recognized to restrict viral infections and replication of intracellular bacteria and parasites. Additionally, this pathway delivers cytoplasmic antigens for MHC class II presentation to the adaptive immune system, which then in turn is able to regulate autophagy. At the same time, autophagy plays a role in the survival and the cell death of T cells. Thus, the immune system utilizes autophagic degradation of cytoplasmic material, to both restrict intracellular pathogens and regulate adaptive immunity.
Natural killer (NK) cells are a key component of the innate immune system that are capable of rapid recognition and elimination of target cells without prior sensitization. Compelling evidence that NK cells limit viral replication in vivo has come from studies involving cytomegalovirus (CMV) infection. Human CMV (HCMV) is a pathogen responsible for causing significant morbidity and mortality in immunocompromised individuals. This chapter discusses some of the mechanisms used by NK cells to identify CMV-infected cells and describes the escape mechanisms employed by the CMVs to evade detection. Human cytomegalovirus (HCMV) is a common human pathogen typically encountered during childhood. Primary HCMV infection is rapidly controlled by the immune system but not eliminated, resulting in the establishment of a latent infection that persists for the life of the host. Reactivation of HCMV in healthy individuals is usually asymptomatic. However, in immune-compromised patients, HCMV reactivation is a significant clinical problem causing diseases such as interstitial pneumonitis, encephalitis and retinitis. Since CMVs are strictly species specific, animal models have been used to investigate various aspects of viral pathogenesis. In particular, the study of murine CMV (MCMV) has provided valuable insights into how the immune system responds to CMV infection and has helped to define the immune evasion mechanisms used by CMV to ensure that viral replication proceeds successfully. NK cells play a critical role in controlling immune responses, including the resolution of such responses.
Innate lymphoid cells (ILCs) are a new class of immune cells that include natural killer (NK) cells and appear to be the innate counterparts to CD4+ helper T cells and CD8+ cytotoxic T cells based on developmental and functional similarities. Like T cells, both NK cells and other ILCs also show connections to the major histocompatibility complex (MHC). In human and mouse, NK cells recognize and respond to classical and nonclassical MHC I molecules as well as structural homologues, whereas mouse ILCs have recently been shown to express MHC II. We describe the history of MHC I recognition by NK cells and discuss emerging roles for MHC II expression by ILC subsets, making comparisons between both mouse and human when possible.
Dendritic cells (DCs) play a central role in the immune response by presenting antigens to naïve T cells and therefore, triggering the adaptive arm of the immune response. Interferon-producing killer dendritic cell (IKDC) is a multi-tasking cell that shares phenotypic and functional features of both natural killer (NK) cells and DC. Following activation by toll-like receptor (TLR) ligands or tumor cells, IKDC develops cytotoxic properties and subsequently matures into a DC-type of cell able to present antigen to naïve T cells. This "bi-typic" function is associated with innate and adaptive immunologic features, which mark these unique APCs as an attractive direct link between natural and acquired immunity. DCs are promising vectors for the design of effective anti- tumor immunotherapies. They are potent adjuvants because of their immunostimulatory effects on T cells and their coordinated cellular cooperation with all the cellular elements of the immune response. However, tumor generate an immunosuppressive environment with over-expression of the signal transducer and activator of transcription (Stat)-3, leading to production of cytokines such as IL-10 or TGFβ, recruitment of myeloid-derived suppressor cells (MDSC) and tumor -associated macrophages (TAM), or expression of B7-H1, among other mechanisms. This generates inadequately stimulated DCs, which suppress effector responses. Therefore, the new trend in cancer immunotherapy is the combination of DC vaccine or T cell therapy with a chemotherapy causing immunogenic tumor death, with DC activation, enhanced antigen cross-presentation or reduction of the immunosuppressive process.
NK cells are effector lymphocytes of the innate immune system that control tumors and microbial infection by limiting their spread and subsequent tissue damage. Recently, more and more evidence has been obtained that NK cells also display a potent regulatory function by secreting various cytokines or cell-to-cell contact and thus regulate innate and adaptive immune responses and maintain immune homeostasis. In this review, we summarize the progress in studying the positive and negative regulatory effects of NK cells in immune responses as well as the NK subsets identified in humans and mice.
B7/BB1 is a membrane differentiation antigen expressed on activated B cells, macrophages, and dendritic cells that binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T cell activation initiated via the CD3/T cell receptor complex. We now report that B7 is also expressed on activated human peripheral blood T cells, CD4 T cell clones, CD8 T cell clones, and natural killer cell clones. B7 appears relatively late after T cell activation, can be detected on both CD4 and CD8 T cell subsets, and is present on antigen-specific, major histocompatibility complex-restricted CD4 and CD8 T cell clones. Expression of B7 on activated T cells was confirmed by immunoprecipitation from 125I-labeled activated T cells and by detection of B7 transcripts. A B7+ CD4+ T cell clone was able to stimulate a primary allogeneic mixed lymphocyte response using small, resting peripheral blood T cells as responders. The alloantigen-induced proliferative response and cytokine production was partially inhibited by anti-B7 monoclonal antibody. Since activated T cells can coexpress both CD28 and its counter-receptor, B7, this suggests that activated T cells may be capable of autocrine costimulation via the CD28 activation pathway.
MOLECULES encoded by the human GDI locus on chromosome 1 (ref. 33) are recognized by selected CD4−8− T-cell clones expressing either αβ or γδ T-cell antigen receptors1,2. The known structural resemblance of GDI molecules to antigen-presenting molecules encoded by major histocompatibility complex (MHG) genes on human chromosome 6 (refs 3, 4, 34, 35), suggested that GDI may represent a family of antigen-presenting molecules separate from those encoded in the MHC1,5,6. Here we report that the proliferative and cytotoxic responses of human CD4−8− αβTCR+ T cells specific for Mycobacterium tuberculosis can be restricted by GDlb, one of the four identified protein products of the GDI locus. The responses of these T cells to M. tuberculosis seemed not to involve MHG encoded molecules, but were absolutely dependent on the expression of GDlb by the antigen-presenting cell and involved an antigen processing requirement similar to that seen in MHC class Il-restricted antigen presentation. These results provide, to our knowledge, the first direct evidence for the proposed antigen-presenting function of GDI molecules and suggest that the GDI family plays a role in cell-mediated immunity to microbial pathogens.
Background. Human natural killer (NK) cells mediate spontaneous cytotoxicity against tumor cells and represent the main precursors of lymphokine-activated killer (LAK) cell activity. A comparison of some aspects of NK and LAK cell activity was undertaken in 85 preoperative patients with breast cancer and 75 healthy donors.
Methods. NK cell activity (tested in 18-hour cultures of effector peripheral blood mononuclear cells [PBMC] with K562 or MOLT-4 tumor target cells) was significantly diminished in these patients as it was the fully mature LAK cell activity (i.e., interleukin-2 (IL-2)-in-duced cytotoxicity in PBMC) against NK resistant target cells. Using immunoenzymatic methods we showed that the reduced NK cell activity was due to abnormally high levels of prostaglandin E2 (PGE2) produced by monocytes in culture.
Results. PGE2 was found to suppress the production of IL-2 in these cultures. Removal of monocytes from PBMC restored to almost normal levels the deficient NK and LAK cell activity in patients with breast cancer and was also associated with a normalization in the levels of PGE2 and IL-2. Indomethacin and gamma-interferon (IFN-γ) increased the NK and LAK cell activity in these patients up to the levels of healthy donors. When highly purified CD56+ cells (obtained by an immunomagnetic isolation technique) were used as effector cells, no differences in LAK cell activity could be noticed between healthy donors and patients with cancer. FACS and northern blot analyses demonstrated a PGE2-mediated down-regulation of IL-2 receptor (IL-2R) expression on CD56+ cells that correlated with reduced LAK cell activity. This inhibitory effect of PGE2 was noticeable in long-term LAK cultures and was abrogated in the presence of IFN-γ or indomethacin.
Conclusion. This study may have important implications in the potentiation of NK and LAK cell activity for immunotherapeutic protocols in patients with breast cancer.
Natural killer (NK) cells contribute to not only innate but also to adaptive immunity by interacting with dendritic cells (DCs) and T cells. All activated human NK cells express HLA-DR and can initiate MHCII-dependent CD4(+) T-cell proliferation; however, the expression of MHCII by mouse NK cells and its functional significance are controversial. In this study, we show that NK-DC interactions result in the emergence of MHCII-positive NK cells. Upon in vitro or in vivo activation, mouse conventional NK cells did not induce MHCII transcripts, but rapidly acquired MHCII protein from DCs. MHCII H2-Ab1-deficient NK cells turned I-A(b)-positive when adoptively transferred into wild-type mice or when cultured with WT splenic DCs. NK acquisition of MHCII was mediated by intercellular membrane transfer called "trogocytosis," but not upon DAP10/12- and MHCI-binding NK cell receptor signaling. MHCII-dressed NK cells concurrently acquired costimulatory molecules such as CD80 and CD86 from DCs; however, their expression did not reach functional levels. Therefore, MHCII-dressed NK cells inhibited DC-induced CD4(+) T-cell responses rather than activated CD4(+) T cells by competitive antigen presentation. In a mouse model for delayed-type hypersensitivity, adoptive transfer of MHCII-dressed NK cells attenuated footpad swelling. These results suggest that MHCII-dressed NK cells generated through NK-DC interactions regulate T cell-mediated immune responses.
Les maladies allergiques sont en constante augmentation tant en prévalence qu'en gravité. Les mécanismes physiopathologiques connus impliquent l'induction d'une réponse Th2 par les cellules dendritiques, conduisant à une production d'IgE et une inflammation. L'immunité innée a récemment été mise en avant dans le contrôle de l'immunité adaptative, spécifique de l'antigène. Cependant, le rôle des cellules Natural Killer (NK), cellules de l'immunité innée connues essentiellement pour leurs fonctions anti-tumorales et anti-microbiennes, est encore inconnu dans la pathologie allergique. Néanmoins, quelques études ont montré une modification de certaines sous-populations de cellules NK chez les patients asthmatiques allergiques. Le but général du travail de thèse était d'étudier le rôle des cellules NK dans l'asthme allergique. Dans un premier temps, les variations quantitatives et qualitatives des cellules NK, ainsi que l'effet de leur déplétion ont été évalués dans l'asthme expérimental. Dans un deuxième temps, l'effet de CCL18, chimiokine impliquée dans l'asthme allergique, a été étudié sur les cellules NK de sujets non allergiques et de sujets allergiques. Tout d'abord, le premier modèle murin d'asthme expérimental utilisé était celui très largement décrit chez les souris BALB/cByJ, consistant en deux sensibilisations systémiques d'ovalbumine (OVA) et d'alum suivies de trois provocations allergéniques d'OVA par aérosols. Chez les souris sensibilisées et provoquées à l'OVA, une augmentation du nombre de précurseurs de cellules NK, mais également du nombre de cellules NK, et plus particulièrement des cellules NK immatures, a été observé dans les ganglions médiastinaux (ganglions drainant les poumons). Celle-ci était accompagnée d'une augmentation du pourcentage de cellules NK ayant incorporé du BrdU. Outre ces variations quantitatives, nous avons également montré que les cellules NK étaient activées. Dans les poumons, le nombre de cellules NK n'était pas significativement modifié chez les souris sensibilisées et challengées à l'OVA comparativement aux souris contrôle. Néanmoins, une diminution non significative du nombre de cellules NK, et plus particulièrement des cellules NK les plus matures chez les souris sensibilisées et provoquées à l'OVA a été observée. De plus, comme dans les ganglions médiastinaux, les cellules NK pulmonaires étaient activées. Nous avons ensuite évalué l'effet de la déplétion des cellules NK par administration de l'anticorps anti-ASGM1 avant les provocations allergéniques sur l'inflammation pulmonaire allergique. Chez les souris sensibilisées et provoquées à l'OVA, la déplétion en cellules NK diminuait significativement l'éosinophilie dans les lavages bronchoalvéolaires, sans affecter pour autant l'hyperréactivité bronchique et les taux sériques d'immunoglobulines spécifiques de l'OVA (IgE, IgG2a et IgG1) (Article soumis). La déplétion des cellules NK par l'anti-ASGM1 n'étant pas totale (73%), nous avons choisi de reproduire ces expériences chez les souris NKDTR qui expriment le récepteur de la toxine diphtérique dans le promoteur du gène NKp46, permettant la déplétion spécifique et plus efficace (>90%) des cellules NK (Collaboration Pr E Vivier et Dr T Walzer). Ces souris étant de fond génétique C57BL/6, nous avons mis au point un autre modèle de sensibilisation allergique pulmonaire. Les souris ont reçu deux sensibilisations systémiques d'ovalbumine (OVA) et d'alum suivies de deux séries de trois provocations allergéniques d'OVA par voie intranasale. Dans les ganglions médiastinaux, contrairement au modèle précédent, aucune modification du nombre de cellules NK et de la distribution des sous-populations n'a été observée chez les souris sensibilisées et provoquées à l'OVA. D'un point de vue phénotypique, chez les souris sensibilisées et provoquées à l'OVA, les cellules NK étaient activées et l'expression membranaire du CD107a augmentée témoignant ainsi d'une dégranulation. Dans les poumons, le pourcentage de cellules NK les plus matures était diminué chez les souris sensibilisées et provoquées à l'OVA. Les cellules NK pulmonaires étaient également activées et l'expression membranaire de CD107a augmentée. Le pourcentage de cellules NK exprimant l'IFN-g était diminué. Concernant CCL18, cette dernière est une chimiokine produite préférentiellement au niveau du poumon. Dans le laboratoire, il a été montré que la production de CCL18 était augmentée chez les patients asthmatiques, et qu'elle recrutait les lymphocytes Th2 et les basophiles, suggérant le rôle de cette chimiokine dans l'asthme allergique. Notre objectif était d'évaluer la réponse des cellules NK isolées du sang périphérique de sujets allergiques vis-à-vis de CCL18 et de la comparer à celle de sujets non allergiques. Le récepteur de CCL18 étant encore inconnu, nous avons analysé son expression par les cellules NK par la fixation de CCL18 biotinylé. L'étude en cytométrie en flux a révélé que des cellules NK de donneurs allergiques et non allergiques exprimaient un récepteur pour CCL18. De plus, nous avons montré que les cellules NK de sujets allergiques et non allergiques migraient en réponse à CCL18, et ce dans une voie dépendante des protéines G. L'attraction des cellules NK par CCL18 était dépendante du donneur, mais indépendante du statut allergique. Aucune attraction préférentielle d'une sous-population de cellules NK (CD56hi ou CD56lo) par CCL18 n'a été mise en évidence. CCL18 était également capable de stimuler la cytotoxicité des cellules NK de sujets allergiques et de donneurs non allergiques, vis-à-vis des cellules cibles Jurkat. Les réponses étaient variables en fonction du donneur, mais une fois encore étaient indépendantes du statut allergique. Enfin, CCL18 n'induisait pas de production de cytokines (IFN-g et TNF-a) par les cellules NK de sujets allergiques et non allergiques. Au vu de l'ensemble de ces résultats, nous pouvons conclure que les cellules NK de sujets allergiques sont attirées et activées par CCL18 de façon similaire aux cellules NK de sujets non allergiques. En résumé, ces travaux ont permis de montrer que, dans un modèle murin d'asthme allergique, les cellules NK s'accumulent dans les ganglions médiastinaux et semblent réguler l'éosinophilie pulmonaire. Nous avons également montré que les cellules NK de sujets allergiques ne présentaient pas de dysfonctionnement dans la réponse vis-à-vis de CCL18 comparativement aux sujets non-allergiques.
This work is centered on the study of the NKG2 C-type lectin-like receptors on NK and CD4+T cells. We provide evidence supporting that CD4+T cells specific for Human Cytomegalovirus (HCMV) may express different NK cell receptors, and demonstrate that the C-type lectin-like receptor NKG2D is expressed on cytotoxic CD4+T cells with an effector/memory phenotype, enhancing their TCR-dependent proliferation and cytokine production. A second part of the work is centered on the study of the CD94/NKG2 receptors on NK cells. We show that NKG2A can be induced on NKG2C+ NK cells upon activation with rIL-12 or when cocultured with HCMV-infected dendritic cells, and that NKG2A expression inhibits the response of NKG2C+NK clones against HLA-E-expressing targets, providing a potential regulatory feedback mechanism to control cell activation. Altogether, our results support that expression of NKG2 C-type lectin like receptors may be shaped during the course of viral infections, providing mechanisms to finely regulate both NK and CD4+T cell functions.Aquesta tesi es centra en lestudi dels receptors lectina de tipus C NKG2 en cèllules Natural Killer i T CD4+. Demostrem que les cèllules T CD4+ específiques pel Cytomegalovirus Humà poden expressar diferents receptors NK, i que el receptor lectina tipus C NKG2D sexpressa en cèllules citotòxiques i de memòria, potenciant la proliferació i secreció de citocines depenent del TCR. La segona part daquesta tesi es centra en lestudi de lexpressió dels receptors CD94/NKG2 en cèllules NK. Mostrem com lexpressió de CD94/NKG2A sindueix en cèllules CD94/NKG2C+ estimulades amb IL-12 o cultivades amb cèllules dendrítiques infectades pel Cytomegalovirus Humà, i que lexpressió de CD94/NKG2A inhibeix la resposta de clons NK CD94/NKG2C+ envers dianes HLA-E+, constituint un possible mecanisme de feedback negatiu per controlar lactivació cellular. En resum, els nostres resultats demostren que lexpressió dels receptors lectina tipus C NKG2 pot ser modificada durant les infeccions víriques consitutint un possible mecanisme per regular la resposta tant de cèllules NK com T CD4+.
IFN-producing killer dendritic cells (IKDC) represent a recently discovered cell type in the immune system that possesses a number of functions contributing to innate and adaptive immunity, including production of type 1 and 2 IFNs, interleukin (IL)-12, natural killing, and ultimately antigen presentation to naïve T cells. Here, we compared in vitro and in vivo responses of mouse IKDC, conventional dendritic cells (DC), and natural killer (NK) cells to murine cytomegalovirus infection and found distinct functions among these cell subsets. Upon recognition of infected fibroblasts, IKDC, as well as NK, produced high level of IFN-gamma, but unlike NK, IKDC simultaneously produced IL-12p40 and up-regulated MHC class II (MHC-II) and costimulatory molecules. Using MHC-II molecule expression as a phenotypic marker to distinguish activated IKDC from activated NK, we further showed that highly purified MHC-II(+) IKDC but not NK cross-present MHC class I-restricted antigens derived from MCMV-infected targets to CD8(+) T cells in vitro and in vivo. Our findings emphasize the unique nature of IKDC as a killer antigen-presenting cell directly linking innate and adaptive immunity.
The degradation of cellular proteins in fibroblasts, both those of rapid and those of slow turnover rates, was inhibited by low concentrations of chloroquine or neutral red in the medium. Cells inhibited by chloroquine can be inhibited further by fluoride. Chloroquine was taken up by the fibroblasts and the concentration in the cells reached several hundred times that in the medium. Isopycnic fractionation studies showed that within the cells the chloroquine was concentrated in the lysosomes, and that these chloroquine-containing lysosomes had a lower equilibrium density than the lysosomes of untreated cells. Chloroquine, at concentrations attained inside the lysosomes, inhibited cathepsin B1 but not cathepsin D. It is concluded that chloroquine impairs the breakdown of cellular proteins after these have entered the lysosome system, probably through inhibition of cathepsin B1.
In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.
Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.
The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.
In the present study we have identified and characterized three subpopulations of peripheral blood NK cells based on the surface expression of CD56 and CD16. We have designated these subsets CD16neg, CD16dim, and CD16bright according to the relative surface density of CD16. The CD16bright subset comprised about 10% to 15% of PBL, whereas the CD16dim and CD16neg subsets comprise less than 1% of the total lymphocytes. A detailed characterization of these subsets revealed both similarities and differences. The three subsets shared a great deal of phenotypic similarity, expressing CD2, CD7, CD11b, CD38, CD45R, CD18, and the p75 IL-2R on the majority of the cells in each subset. There were, however, several prominent phenotypic differences, particularly in the expression of CD57, CD11c, CD44, CD25, Leu-8, L263, and L265. The CD16neg cells were morphologically large agranular lymphocytes and demonstrated low levels of non-MHC restricted cytolysis of NK-sensitive tumor lines. The CD16dim and CD16bright subsets were large granular lymphocytes and revealed potent cytotoxicity against NK-sensitive targets. All subsets demonstrated IL-2-dependent activation and proliferation; however, the CD16dim and CD16neg subsets were preferentially responsive to very low concentrations of rIL-2. Although rIL-4 effectively inhibited the IL-2-induced cytolytic activation of all three NK cell subsets, only the CD16bright cells showed rIL-4 inhibition of IL-2 dependent proliferation. Cytokine transcription was also differentially regulated in the NK cell subsets after rIL-2 activation. Although TNF-alpha was equally transcribed in each subsets, IFN-gamma and serine protease-HF were preferentially transcribed in the CD16bright NK cells. Based on these results, we propose that these NK cell subsets represent portions of the NK cell differentiation pathway present in the peripheral blood.
The requirements for activated, Ia-positive human T cells to present antigen were examined. Although activated T cells could present allo-Ia antigens, activated T cells could not present native, soluble protein antigens. We have now shown that activated T cells can present denatured protein antigens to stimulate proliferation of antigen-specific T-cell lines. Since denatured antigen may represent a processed form of antigen, the data suggest that activated T cells can present antigen but may not be able to process antigen as efficiently as other presenting cells. We have also shown that antigen-specific T-cell lines, which are also Ia positive, are able to present antigen to themselves, if the antigen is in a denatured form. Autopresentation requires a critical minimal cell number to stimulate proliferation, even with denatured antigen. The ability of activated T cells to present antigen may reflect an important amplification or feedback mechanism of immune regulation.
Recent experiments have implicated intracellular events in the formation of the MHC class II-peptide complexes recognized by CD4-positive T cells. These data raise the possibility that the intracellular association of class II with the non-polymorphic glycoprotein, invariant chain (Ii), may regulate the interaction between processed antigen and MHC class II molecules. To address this possibility, we have generated a series of transfected fibroblast cell lines that express class II with and without Ii. Although the presence of Ii does not seem to affect the ability of the cells to process and present intact antigen, Ii-negative cells express an altered form of class II at the cell surface. This modified conformation of class II in Ii-negative cells is detectable by an increase in the ability to present antigenic peptides to T cells and a decrease in the binding of several class II-specific monoclonal antibodies.
Article de synthese sur: - l'effet de la fixation specifique de l'antigene aux immunoglobulines de membrane, aux recepteurs Fc, et autres molecules de la surface cellulaire sur la presentation de l'antigene; - le mecanisme de la fixation et de la degradation de l'antigene
Prompted by our recent finding that lymphokine-activated killer (LAK) cells mediate both veto and natural suppression, we tested the ability of adoptively transferred LAK cells to block two in vivo alloreactions which complicate bone marrow transplantation: resistance to transplanted allogeneic bone marrow cells, and lethal graft-vs-host disease. Adoptive transfer of either donor type B6D2 or recipient-type B6 lymphokine-activated bone marrow cells, cells found to have strong LAK activity, abrogated or inhibited the resistance of irradiated B6 mice to both B6D2 marrow and third party-unrelated C3H marrow as measured by CFU in spleen on day 7. The ability of lymphokine-activated bone marrow cells to abrogate allogeneic resistance was eliminated by C lysis depletion of cells expressing asialo-GM1, NK1.1, and, to a variable degree, Thy-1, but not by depletion of cells expressing Lyt-2, indicating that the responsible cells had a LAK cell phenotype. Similar findings were obtained by using splenic LAK cells generated by 3 to 7 days of culture with rIL-2. Demonstration that allogeneic resistance could be blocked by a cloned LAK cell line provided direct evidence that LAK cells inhibit allogeneic resistance. In addition to inhibiting allogeneic resistance, adoptively transferred recipient-type LAK cells prevented lethal graft-vs-host disease, and permitted long term engraftment of allogeneic marrow. Irradiation prevented LAK cell inhibition of both allogeneic resistance and lethal graft-vs-host disease. These findings suggest that adoptive immunotherapy with LAK cells may prove useful in preventing graft rejection and graft-versus-host disease in human bone marrow transplant recipients.
Studies of cytotoxicity by human lymphocytes revealed not only that both allogeneic and syngeneic tumor cells were lysed in a non-MHC-restricted fashion, but also that lymphocytes from normal donors were often cytotoxic. Lymphocytes from any healthy donor, as well as peripheral blood and spleen lymphocytes from several experimental animals, in the absence of known or deliberate sensitization, were found to be spontaneously cytotoxic in vitro for some normal fresh cells, most cultured cell lines, immature hematopoietic cells, and tumor cells. This type of nonadaptive, non-MHC-restricted cellmediated cytotoxicity was defined as “natural” cytotoxicity, and the effector cells mediating natural cytotoxicity were functionally defined as natural killer (NK) cells. The existence of NK cells has prompted a reinterpretation of both the studies of specific cytotoxicity against spontaneous human tumors and the theory of immune surveillance, at least in its most restrictive interpretation. Unlike cytotoxic T cells, NK cells cannot be demonstrated to have clonally distributed specificity, restriction for MHC products at the target cell surface, or immunological memory. NK cells cannot yet be formally assigned to a single lineage based on the definitive identification of a stem cell, a distinct anatomical location of maturation, or unique genotypic rearrangements.
To trigger class II-restricted T cells, antigen presenting cells have to capture antigens, process them and display their fragments in association with class II molecules. In most species, activated T cells express class II molecules; however, no evidence has been found that these cells can present soluble antigens. This failure may be due to the inefficient capture, processing or display of antigens in a stimulatory form by T-cells. The capture of a soluble antigen, which is achieved by nonspecific mechanisms in macrophages and dendritic cells, can be up to 10(3) times more efficient in the presence of surface receptors, such as surface immunoglobulin on B cells that specifically bind antigen with high affinity. We asked whether T cells would be able to present soluble antigens that bind to their own surface molecules. Here we show that such antigens can be effectively processed and presented by both CD4+- and CD8+-bearing human T cells. This indicates that T cells are fully capable of processing and displaying antigens and are mainly limited in antigen presentation by their inefficiency at antigen capture.
This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.
The processing and presentation of whole irradiated Mycobacterium tuberculosis (Mtbγ) and its purified protein derivative (PPD) by the peripheral blood monocytes from healthy Bacillus Calmette Guérin (BCG)-vaccinated individuals was investigated. To study processing and presentation as events distinct from T cell recognition and proliferation, monocytes were pulsed with antigens for varying time intervals and fixed. The kinetics of presentation indicate that up to 2 h was required for effective presentation of PPD and 2–4 h for Mtbγ, and that the ability to activate T cells declined as the time interval for which pulsing occurred was increased, so that responses were abolished by 8–10 h. Prefixed monocytes could not present Mtbγ and PPD to T cells indicating that processing was an essential requisite. Lysosomotropic agents chloroquine, monensin, and leupeptin inhibited the presentation of these antigens suggesting the role of lysosomes/endosomes in processing. Furthermore, monocytes incubated with optimal concentration of antigens for different lengths of time released determinants which were still antigenic but circumvented the need for any further processing.
Addition of nonprimed syngeneic monocytes, both untreated or paraformaldehyde fixed to cells which had been pulsed and fixed, restored the responses even at the later time periods when responses were not detected. This second interaction of the monocyte with T cells was not major histocompatibility complex restricted in that the addition of monocytes from another donor was equally effective.
The ability of large granular lymphocytes (LGL) to function as antigen-presenting cells (APC) in the proliferative response to the soluble bacterial antigen streptolysin O (SLO) was investigated. Despite the fact that a subset of LGL isolated by sorting peripheral blood lymphocytes with the B73.1 monoclonal antibody on a fluorescence-activated cell sorter (FACS-IV) expressed MHC Class II molecules of the DP, DQ and DR subregion loci, presentation of SLO by LGL was not demonstrated. Thus, T-cell populations containing LGL but carefully depleted of monocytes, isolated either by sorting using the FACS-IV or by SRBC-rosetting, were unresponsive to antigenic stimulation with SLO. Application of exogenous interleukin-1 to FACS-IV-isolated LGL-containing T-cell populations did not elicit presentation of SLO by the LGL. In vitro activation with phytohaemagglutinin and interleukin-2, which induced Class II expression in T-cell populations, resulted in an increased expression of Class II molecules of the DP, DQ and DR specificities on LGL. Although such activated T-cell and LGL populations were incapable of presenting SLO to freshly isolated antigen-non-responsive T cells, both activated populations were able to act as stimulators in an allogeneic mixed lymphocyte reaction. The ability of highly Class II-positive activated LGL to present membrane-bound antigens suggests that their inability to present a soluble antigen may be related to the absence of effective antigen sequestration and/or processing mechanisms.
The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.
We studied the antigen-presenting capacity of mouse L fibroblasts transfected with genes encoding Ia polypeptides of the major histocompatibility complex (MHC). These cells function as efficient antigen-presenting cells (APC) in stimulating peptide antigen-specific MHC-restricted proliferation of long-term T-cell lines, thus establishing the capacity of Ia-expressing L-cell transfectants to present antigens to apparently normal T cells. However, in contrast to splenic APC, L-cell transfectants fail to present native hen egg-white lysozyme to the same T cells. Since this result is similar to that obtained with physiologic APC pretreated to prevent antigen degradation, it suggests that L-cell transfectants, without such pretreatments, may be compromised in their ability to process native lysozyme. However, since such transfectant cells have been shown to present other complex polypeptides such as keyhole limpet hemocyanin, a random copolymer of glutamic acid, alanine, and tyrosine, and influenza virus neuraminidase, this observation suggests that protein antigens differ in the stringency of processing requirements.
I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.
The functional significance of multiple cells--among lymphoid and nonlymphoid cells--capable of having Ia molecules on their membranes must be critically addressed. Ia is absolutely required before a cell can interact with helper T cells, but it is not clear whether the presence of this protein is all that is needed for antigen presentation. Indeed, at present, except for the macrophage, few cells have been studied for antigen presentation using a wide range of protein antigens, either soluble or particulate. On the basis of the studies discussed in the first section, it appears that the recruitment of most helper-T cell clones takes place by APC that can internalize and process the protein antigens, be they soluble or part of the structure of microorganisms. The fact that helper T cells are programmed to recognize antigen in the context of Ia, and therefore on an APC such as the macrophage, forces recognition of antigens that are altered or processed. Indeed, proteins in their native state may not remain membrane-bound for long periods; the T cells, therefore, have the opportunity to recognize the altered fragments. To this issue is added the requirement for the T-cell receptor to interact with Ia molecules. The available information, therefore, leads one to conclude that APC deficient in their capacity to internalize and process proteins will not be able to present them. The finding that small peptides from a previous catabolism of proteins can be presented without further handling implies that APC with limited processing capacity could be involved in presentation of such small peptides. The different Ia-positive APC of the lymphoid organs may interact to different extents with protein antigens and collaborate with each other to bring about an effective stimulation of the clones of helper T cells. The macrophage, being the most ubiquitous cell and the one capable of interacting with many proteins, is our candidate as the major APC involved in the recruitment and enlargement of clones T cells. The observations that macrophages can release proteins partially altered implies that there may be cooperativity among the various APC. Data for this have been obtained. Most likely B cells will be found to have a limited capacity to present all antigens because of their inherent difficulties in internalizing large particulate materials. In such instances, B cells may interact with the solubilized proteins released by the macrophages. The same may apply to the Langerhans/dendritic cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Supernatants of hybridomas from fused spleen cells of mice immunized with either a T4+ or a T8+ CTL clone, were screened for their ability to inhibit the cytotoxic activity of the T4+ and the T8+ CTL clone. Eight monoclonal antibodies (MAbs) with blocking activity were obtained and a preliminary characterization of these antibodies was carried out. The MAbs SPV-L1 and SPV-L5 were found to react with thymocytes and all peripheral blood leukocytes. SPV-L1 and SPV-L5 precipitated a molecular complex consisting of two noncovalently bound chains of molecular weights of 95 and 160 kD indicating that they recognize the human equivalents of the recently described leukocyte function associated (LFA-1) antigens. The SPV-L1 and SPV-L5 antibodies inhibited the reactivity of six CTL clones tested in this study. In addition, SPV-L1 and L5 blocked the lectin dependent cellular cytotoxicity mediated by these CTL clones. Natural killer cell activity mediated by fresh peripheral blood lymphocytes and activated killer cell activity generated in MLC both measured against K562 was also inhibited by SPV-L1 and SPV-L5. Two other antibodies, SPV-L3 and SPV-L4 blocked strongly the cytotoxic activity of two T4+ CTL clones, whereas the reactivity of the three T8+ CTL clones and one T4+ CTL clone was not or only moderately inhibited. These antibodies recognized an Ia antigen as judged from tissue distribution and immunoprecipitation studies. However, these latter studies also suggest that SPV-L3 and SPV-L4 recognize HLA-DC rather than HLA-DR antigens. Finally, three antibodies (SPV-T3a, SPV-T3b, SPV-T3c) were obtained directed against the T3 molecular complex and one MAb (SPV-T8) was found to react with T8. The anti-T3 reagents were shown to be mitogenic for peripheral blood lymphocytes, with the exception of SPV-T3a. The results presented here indicate that the antigens recognized by SPV-L1 and SPV-L5 are involved in various cytotoxic reactions. In contrast, SPV-L3, SPV-T3 and SPV-T8 only seem to play a role in antigen specific cytotoxic reactions.
The first step in the induction of immune responses, whether humoral or cell mediated, requires the interaction between antigen-presenting cells and T lymphocytes restricted at the major histocompatibility complex (MHC). These cells invariably express MHC class II molecules (HLA-D region in man and Ia in mouse) which are recognized by T cells of the helper/inducer subset in association with antigen fragments. Interestingly, in certain pathological conditions, for example in autoimmune diseases such as thyroiditis and diabetic insulitis, class II molecules may be expressed on epithelial cells that normally do not express them. We speculated that these cells may be able to present their surface autoantigens to T cells, and that this process may be crucial to the induction and maintenance of autoimmunity. A critical test of this hypothesis would be to determine whether epithelial cells bearing MHC class II molecules (class II+ cells) can present antigen to T cells. We report here that class II+ thyroid follicular epithelial cells (thyrocytes) can indeed present viral peptide antigens to cloned human T cells.
Antibody 3G8 reacts with the receptor for the Fc fragment of aggregated IgG present on the majority of neutrophilic granulocytes and on a small proportion of lymphocytes. In this report, we compare the pattern of reactivity of antibody 3G8 on peripheral blood lymphocytes (PBL) and on polymorphonuclear leukocytes (PMN) with that of antibody B73.1, which reacts with the Fc receptor of natural killer (NK) cells or with a molecule functionally associated with it. We show that 3G8 reacts with the same PBL subset detected by antibody B73.1 and is responsible for virtually all NK cytotoxic activity. The lymphocyte subset recognized by the two antibodies has the morphology of large granular lymphocytes and includes neither B nor T cells. Our results indicate that NK cells and PMN express the same Fc receptor for immune complexes, and that B73.1 and 3G8 recognize on the same receptor two distinct epitopes that are preferentially expressed on NK cells and on PMN, respectively.
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