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Regulation of human T cell proliferation by IL-7
Abstract
The regulation of human T cell proliferation by rIL-7 was investigated. Purified peripheral blood T cells were stimulated to proliferate in a short-term assay by IL-7 in the presence of CD3 mAb or lectin. This stimulation was accompanied by a significant increase in the expression of IL-2R on both CD4+ and CD8+ T cells over that seen with mitogen alone. The proliferation of these cells in the presence of exogenous IL-7 involved both IL-2-dependent and - independent mechanisms as shown by the ability of neutralizing IL-2 antibody to partially inhibit the response. Anti-IL-4 and anti-IL-6 antibodies had no effect on IL-7-induced T cell growth. These results suggest that the costimulatory effect of IL-7 on human T cells is primarily direct, not involving other intermediate T cell growth factors. IL-7 was also found to be mitogenic in a long-term assay in the absence of any costimulus, with the onset of proliferation occurring later than that seen in the presence of mitogen. These results demonstrate that IL-7 provides a potent T cell stimulus either alone or in the presence of co-mitogen and, although this stimulus is accompanied by an increase in the level of IL-2R expression, it is not dependent on the action of IL-2 for its effect.
... Accordingly with their low CD127 expression Treg seem to be the only T cell population whose peripheral homeostasis is IL-7 independent (Liu et al., 2010) , 1989) or PMA (Chazen et al., 1989) stimulated murine T lymphocytes increased cell proliferation as reveled by a day 3 Thymidine incorporation assay. Similar results were obtained with anti-CD3 stimulation of murine (Costello et al., 1993) and human T cells (Armitage et al., 1990). Remarkably, IL-7 incubation during activation led to an increase in IL-2Rα expression at T cells surface compatible with a synergic effect of IL-7 on IL-2 signaling (Morrisey et al., 1989;Armitage et al., 1990;Grabstein et al., 1990;Costello et al., 1993). ...
... Similar results were obtained with anti-CD3 stimulation of murine (Costello et al., 1993) and human T cells (Armitage et al., 1990). Remarkably, IL-7 incubation during activation led to an increase in IL-2Rα expression at T cells surface compatible with a synergic effect of IL-7 on IL-2 signaling (Morrisey et al., 1989;Armitage et al., 1990;Grabstein et al., 1990;Costello et al., 1993). However addition of anti-IL-2 blocking antibodies suppress IL-7 effect on T cell proliferation completely after PMA stimulation (Chazen et al., 1989), partially after αCD3αCD28 stimulation (Costello et al., 1993) but has no effect during ConA, OKT3 or PHA stimulation (Morrisey et al., 1989;Armitage et al., 1990;Grabstein et al., 1990). ...
... Similar results were obtained with anti-CD3 stimulation of murine (Costello et al., 1993) and human T cells (Armitage et al., 1990). Remarkably, IL-7 incubation during activation led to an increase in IL-2Rα expression at T cells surface compatible with a synergic effect of IL-7 on IL-2 signaling (Morrisey et al., 1989;Armitage et al., 1990;Grabstein et al., 1990;Costello et al., 1993). However addition of anti-IL-2 blocking antibodies suppress IL-7 effect on T cell proliferation completely after PMA stimulation (Chazen et al., 1989), partially after αCD3αCD28 stimulation (Costello et al., 1993) but has no effect during ConA, OKT3 or PHA stimulation (Morrisey et al., 1989;Armitage et al., 1990;Grabstein et al., 1990). ...
Regulatory T cells (Treg) represent a crucial CD4 T cells subset involved in maintenance of immune-tolerance. Since their first description important efforts have been undertaken to better understand their biology, their development and their mechanisms of action. However, little is known about factors controlling Treg peripheral homeostasis. The aim of this thesis work was to better define mechanisms involved in governing Treg homeostasis and to investigate the eventual contribution of perturbation of Treg homeostasis in human disease. In the first part of this thesis work we tried to define in the murine system the role played by IL-7 in governing Treg homeostasis. We showed that Treg surface expression of CD127, the IL-7 receptor alpha chain, is finely regulated as it depends on their activation as well as on their tissue localization. More importantly, we demonstrated that Treg do express functional levels of CD127, identifying these cells as potential target of IL-7. Using both genetically modified murine models of altered IL-7 signaling and adoptive transfer models, we obtained definitive evidence for a direct role of IL-7 in governing Treg cell numbers. Finally, we demonstrated that IL-7 signaling in Treg optimizes their capacity to react to IL-2 an important cytokine regulating Treg homeostasis. In the second part of this work we investigated Treg homeostasis in the context of HIV infection. Employing for the first time in HIV infection a novel consensus Treg identification strategy and applying it to different groups of HIV infected patients, including primary infected patients and HIV controllers, we showed that HIV infection is characterized by an early and long lasting alteration of Treg homeostasis. In particular we demonstrated that effector rather than naive Treg are affected by HIV infection. Moreover, we showed that effector Treg numbers inversely correlated with HIV specific CD8 T cells responses, providing ex vivo evidence of Treg involvement in HIV immunity.
... IL7 is a stromal cell-derived cytokine that has a number of effects on lymphocytes . IL7 stimulates the growth ofpre-B cells, thymocytes, and mature T cells, and enhances the generation of CTL and lymphokine-activated killer cells (18)(19)(20)(21)(22)(23)(24) . Receptors for IL-7 have also been demonstrated on myeloid cells (25), however, until now, no activity for IL7 on monorytes/macrophages or neutrophils has been reported. ...
... Monocytes Are Less Sensitive to 11,7 than Are T Lymphocytes. We have previously shown that IL7 costimulates the proliferation ofhuman T cells and enhances CTL generation at concentrations as low as 0.1 ng/ml (21,23). To directly compare the concentrations of IL-7 required to stimulate T cells versus monocytes, we used the same preparation of IL-7 and compared its ability to either induce IL-6 secretion by monocytes or enhance CTL generation in human MLC ( Table 2). ...
... Interleukin 7 has a number of biological effects on lymphocytes and lymphocyte precursors, including stimulating the growth of pre-B cells, thymocytes, and mature T cells (18)(19)(20)(21)(22)(23)(24) . In the experiments reported herein, we demonstrate that purified rIL7 also has potent effects on human peripheral blood monocytes. ...
Peripheral blood monocytes can be induced by stimuli such as bacterial lipopolysaccharide (LPS) to secrete an array of cytokines. We have studied the effects of interleukin 7 (IL-7) on human peripheral blood mononuclear cells (PBMC) and found that IL-7 is a relatively potent inducer of IL-6 secretion IL-6 protein levels were determined either by the B9 hybridoma growth factor assay or by enzyme-linked immunosorbent assay, and mRNA for IL-6 was analyzed by Northern hybridization. Detailed examination revealed that, among PBMC, monocytes, rather than lymphocytes, were secreting IL-6 in response to IL-7. In contrast to the low concentrations of IL-7 required to stimulate T cell growth and differentiation (as low as 0.1 ng/ml), relatively high concentrations of IL-7 were necessary to induce IL-6 secretion by monocytes (at least 10 ng/ml). An optimal concentration of IL-7 (100 ng/ml) induced monocytes to secrete 10-fold more IL-6 than an optimal concentration of IL-1 beta (10 ng/ml), and almost as much as LPS. However, significantly more IL-7 than IL-1 beta was required to induce detectable levels of IL-6. The kinetics of IL-6 secretion by monocytes were identical in response to IL-7, IL-1 beta, or LPS, with IL-6 protein detectable in culture supernatants as early as 2 h after the initiation of culture. IL-4 was found to markedly inhibit the ability of IL-7 or LPS to induce IL-6 mRNA and IL-6 secretion. In addition to promoting IL-6 production, IL-7 induced the secretion of immunoreactive IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) by monocytes. IL-7 also induced monocyte/macrophage tumoricidal activity against a human melanoma cell target, an activity that may be related to the secretion of IL-1 alpha, IL-1 beta, and TNF-alpha. Finally, we used a whole blood culture system as a bridge to in vivo analysis to demonstrate that IL-7 induces cytokine secretion in the absence of culture medium, fetal calf serum, and adherence to plastic. Our data suggest that IL-7, in addition to regulating lymphocyte growth and differentiation, has potent effects on cells of the monocytic lineage. Thus, IL-7 may be an important mediator in inflammation and in the macrophage immune response to tumors.
... At present, there are several interleukin 7dependent cell lines in the world, which may be used for testing of rhIL7 biological activity: 1xN/2b (stromal cell line), 2Е8 (mouse Blymphocytes), D1 (knockout mice thymocytes), DW34 (preВcells), PB1 (preВcells), Pno (Тcells). Table 1 contains brief comparative characterization of cell lines which may be used for testing of biological activity of rhIL7 [3,8,9]. ...
... We have suggested testing the specificity of our rhIL7 in test sample versus rhIL7 (Peprotech, USA, Cat.N. 20007) in referen ce sample on human PBMCs . In accordance with requirements of national and international recommendations, RSD may not exceed 2% [1,8]. Fig. 1 shows the results of specificity testing of the method -proliferation of human peripheral blood cells at increasing rhIL7 concentration. ...
Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation characteristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.
... The injection of recombinant IL-7 (rIL-7) circumvents this problem and boosts anti-tumor T cell responses [13,14]. Since IL-7 promotes T cell survival [15,16], activation [17,18], proliferation [19] and memory T cell (T M ) formation [20] its direct action on T cells is supposed to be the major cause for its potent anti-tumor effects [21]. For the effective treatment of viral infections and cancer by ATT high numbers of adoptively transferred CD8 + cells are required in vivo [7]. ...
... Importantly, rIL-7 treatment did not affect primary EG7 growth in either host (data not shown). Several studies provided evidence that rIL-7 promotes activation, survival, function of CD8 + T cells [15][16][17][18][19]30] and memory T cell (T M ) formation [20]. So far, however, these effects were considered to result from direct effects of rIL-7 on CD8 + T cells. ...
The adoptive transfer of antigen-specific CD8+ T cells is a promising approach for the treatment of chronic viral and malignant diseases. In order to improve adoptive T cell therapy (ATT) of cancer, recent strategies aim at the antibody-based blockade of immunosuppressive signaling pathways in CD8+ T cells. Alternatively, adjuvant effects of immunostimulatory cytokines might be exploited to improve therapeutic CD8+ T cell responses. For example, Interleukin-7 (IL-7) is a potent growth, activation and survival factor for CD8+ T cells that can be used to improve virus- and tumor-specific CD8+ T cell responses. Although direct IL-7 effects on CD8+ T cells were studied extensively in numerous models, the contribution of IL-7 receptor-competent (IL-7R+) host cells remained unclear. In the current study we provide evidence that CD8+ T cell-mediated tumor rejection in response to recombinant IL-7 (rIL-7) therapy is strictly dependent on IL-7R+ host cells. On the contrary, CD8+ T cell expansion is independent of host IL-7R expression. If, however, rIL-7 therapy and peptide vaccination are combined, host IL-7R signaling is crucial for CD8+ T cell expansion. Unexpectedly, maximum CD8+ T cell expansion relies mainly on IL-7R signaling in non-hematopoietic host cells, similar to the massive accumulation of dendritic cells and granulocytes. In summary, we provide evidence that IL-7R+ host cells are major targets of rIL-7 that modulate therapeutic CD8+ T cell responses and the outcome of rIL-7-assisted ATT. This knowledge may have important implications for the design and optimization of clinical ATT protocols.
... This stromal cell-derived cytokine, termed interleukin-7 (IL-7), has been purified and molecularly cloned, and the recombinant murine and human proteins are now available [3,4]. Since IL-7 also stimulates murine pre-B cells from bone marrow [5], murine thymocytes, and, as comitogen, mature T cells [6] and induces proliferation of human T cells [7], we investigated whether IL-7 could stimulate DNA synthesis in B-lineage ALL blasts in suspension culture and also the capacity of IL-7 to induce blast cell maturation in vitro. ...
... With an arbitrary cut-off of a SI > 5,4 of 10 cALL samples (5,7,8,9) and 1 of 4 B-ALL samples (14) were stimulated by IL-7. IL-3 stimulated DNA synthesis in 5 of 9 cALL and 3 of 4 B-ALL samples. ...
Acute lymphoblastic leukemia (ALL) is a clonal disorder characterized by derangements of self-renewal and differentiation of lymphoid precursor cells in the bone marrow. Corresponding to the inconsistent stimulatory effects of the recombinant hematopoietic growth factors studied to date on B-lineage ALL blasts in vitro [1], a reproducible culture assay that supports proliferation and maturation of ALL blasts has not yet been reported.
... однако увеличение числа продуцирующих ВиЧ-1 клеток в тканях, подвергнутых воздействию иЛ-7, происходит не только из-за подавления апоптоза, но и благодаря тому, что иЛ-7 стимулировал пролиферацию CD4 + Т-клеток, как инфицированных, так и не инфицированных, о чем свидетельствуют наши данные по экспрессии ki-67. Процесс активизации пролиферации клеток под действием иЛ-7 был описан для культур изолированных клеток [17,41,[43][44][45] и в настоящее время служит основой для клинических испытаний, целью которых является увеличение числа Т-клеток у ВиЧ-1инфицированных лиц [16,17]. ...
The majority of HIV-1 infections in women transmit through vaginal intercourse, in which virus-containing semen is deposited on the cervico-vaginal mucosa. Semen is more than a mere carrier of HIV-1, since it contains many biological factors, in particular cytokines, that may affect HIV-1 transmission. The concentration of interleukin (IL)-7, one of the most prominent cytokines in semen of healthy individuals, is further increased in semen of HIV-1-infected men. Here, we investigated the potential role of IL-7 in HIV-1 vaginal transmission in an ex vivo system of human cervico-vaginal tissue. We simulated an in vivo situation by depositing HIV-1 on cervico-vaginal tissue in combination with IL-7 at concentrations comparable with those measured in semen of HIV-1-infected individuals. We found that IL-7 significantly enhanced virus replication in ex vivo infected cervico-vaginal tissue. Similarly, we observed an enhancement of HIV-1 replication in lymphoid tissue explants. Analysis of T cells isolated from infected tissues showed that IL-7 reduced CD4+ T cell depletion preventing apoptosis, as shown by the decrease in the number of cells expressing the apoptotic marker APO2.7 and the increase in the expression of the anti-apoptotic protein B-cell lymphoma (Bcl)-2. Also, IL-7 increased the fraction of cycling CD4+ T cells, as evidenced by staining for the nuclear factor Ki-67. High levels of seminal IL-7 in vivo may be relevant to the survival of the founder pool of HIV-1-infected cells in the cervico-vaginal mucosa at the initial stage of infection, promoting local expansion and dissemination of HIV infection.
... Several in vitro studies have shown that IL-7 contributes to MAIT cell effector function (51)(52)(53). On the other hand, IL-7 is well known to enhance proliferation and survival of human T cells (54) and a modest IL-7 proliferative potential has been shown among CD161 ++ , IL-18Rα + , CD45RA − , CD27 + CD8 T cells (55). Furthermore, administration of rhIL-7 promotes CD8 T cell expansion (56,57). ...
Mucosal-associated invariant T (MAIT) cells have been implicated in various forms of autoimmunity, including type 1 diabetes (T1D). Here, we tested the hypothesis that CD8 and double negative (DN) MAIT cell frequencies were altered among diagnosed T1D subjects compared to controls. To do this, we analyzed cryopreserved peripheral blood mononuclear cells (PBMCs) from age-matched T1D and control children using flow cytometry. We observed that CD8 and DN MAIT cell frequencies were similarly abundant between the two groups. We tested for associations between MAIT cell frequency and T1D-associated parameters, which could reveal a pathogenic role for MAIT cells in the absence of changes in frequency. We found no significant associations between CD8 and DN MAIT cell frequency and levels of islet cell autoantibodies (ICA), glutamate decarboxylase 65 (GAD65) autoantibodies, zinc transporter 8 (ZNT8) autoantibodies, and insulinoma antigen 2 (IA-2) autoantibodies. Furthermore, CD8 and DN MAIT cell frequencies were not significantly associated with time since diagnosis, c-peptide levels, HbA1c, and BMI. As we have examined this cohort for multiple soluble factors previously, we tested for associations between relevant factors and MAIT cell frequency. These could help to explain the broad range of MAIT frequencies we observed and/or indicate disease-associated processes. Although we found nothing disease-specific, we observed that levels of IL-7, IL-18, 25 (OH) vitamin D, and the ratio of vitamin D binding protein to 25 (OH) vitamin D were all associated with MAIT cell frequency. Finally, previous cytomegalovirus infection was associated with reduced CD8 and DN MAIT cells. From this evaluation, we found no connections between CD8 and DN MAIT cells and children with T1D. However, we did observe several intrinsic and extrinsic factors that could influence peripheral MAIT cell abundance among all children. These factors may be worth consideration in future experimental design.
... IL-7 was first identified as a B cell precursor growth factor (Namen et al, 1988a and b) and subsequently both mature T cells and thymocytes have been found to respond to IL-7 (Murray et al, 1989;Armitage et al, 1990;Fabbi et al, 1992). By contrast mature murine B cells and B cell lines have been found to express little or no IL-7 binding species (Park et al, 1990). ...
X-linked severe combined immunodeficiency (XSCID) is characterised by a failure of both humoral and cellular immunity resulting in recurrent and opportunistic infections. Affected individuals have no T cells and normal or elevated numbers of B cells, indicating a defect in T cell development. At the start of this study the gene involved in XSCID had been mapped to the region Xq 13.1-21.1. In an attempt to reduce the extent of this region, the positions of polymorphic microsatellite loci, mapping to Xq 13.2-21.1, were refined. In 1993 the gene responsible for XSCID was cloned and identified as the IL-2 receptor γ chain which was also found to be a subunit of the IL-4, IL-7, IL-9 and IL- 15 receptors and denoted γc. This new information made it possible to screen a number of XSCID patients for mutations in the γc gene using single strand conformation polymorphism (SSCP) analysis. Mutations were identified in 14 individuals and this information has also aided carrier status assignment in a number of families. Expression of mutant γc at both the mRNA and surface protein levels was investigated in a number of EBV transformed B cell lines and also primary lymphocytes from patients. Surface expression as detected by a monoclonal antibody appeared to be disrupted in the majority of XSCID cases studied. Since the γc chain is a subunit of the IL-7 receptor and IL-7 is known to have a role in T cell development, an assay for IL-7 receptor function in XSCID patients was developed. This assay utilised a whole blood culture system and IL-6 production by monocytes in response to IL-7 was measured. The preliminary results obtained may help to elucidate the role of the γc chain in T cell development. The work presented here reflects the advancement of this field from gene cloning to the development of an understanding of the role of the XSCID gene product in both the normal and disease states.
... Interleukin (IL)-7, a member of the gamma cytokine receptor family, plays important non-redundant roles in the homeostasis, proliferation, differentiation, and survival of T cells in human and in various animal models (Alves et al. 2007;Armitage et al. 1990;Carini and Essex 1994;Ibelgaufts 2007;Jicha et al. 1991;Mazzucchelli and Durum 2007;Roifman et al. 2000;Smyth et al. 1991;Soares et al. 1998;Welch et al. 1989). IL-7 signals through binding to its receptor complex which is composed of two chains: a unique alpha chain (CD127) to which IL-7 specifically binds, and a common gamma chain (CD132) which is shared with the receptors of IL-2, IL-4, IL-9, IL-15 and IL-21 (Goodwin et al. 1990;Jiang et al. 2005). ...
Interleukin-7 is essential for the development and maintenance of T cells, and the expression of the IL-7 receptor is tightly regulated at every stage of the T cell’s lifespan. In mature CD8 T cells, IL-7 plays important roles in cell survival, peripheral homeostasis, and cytolytic function. The IL-7 receptor alpha-chain (CD127) is expressed at high levels on naïve and memory cells, but it is rapidly downregulated upon IL-7 stimulation. In this study, we illustrate the dynamicity of the CD127 promoter and show that it possesses positive as well as negative regulatory sites involved in upregulating and downregulating CD127 expression, respectively. We cloned the CD127 gene promoter and identified key cis-regulatory elements required for CD127 expression in mature resting primary CD8 T cells. The core promoter necessary for efficient basal transcription is contained within the first 262 bp upstream of the TATA box. Additional positive regulatory elements are located between −1200 and −2406 bp, conferring a further 2- to 4-fold enhancement in gene expression. While transcription of the CD127 gene is increased directly through a glucocorticoid response element located between −2255 and −2269 bp upstream of the TATA box, we identified a suppressive region that lies upstream of 1760 bp from the TATA box, which is likely involved in the IL-7-mediated suppression of CD127 transcription. Finally, we illustrated IL-7 does not bias alternative splicing of CD127 transcripts in primary human CD8 T cells.
... Finally, IL-7 treatment of T cells allow their proliferation and survival without inducing their switch between naïve and memory subsets (Webb et al. 1999), (Soares et al. 1998) and only inducing a slight upregulation of CD25, CD98, CD71, CD11a and CD40-L much lower than obtain upon a TCR stimulation (Armitage et al. 1990). ...
Lentiviral vectors and their ability to transfer gene into hematopoietic stem cells are currently evaluated for the cure of several single-gene diseases (eg : B-thalassemia, Adrenoleucodystrophy, SCID). Likewise, gene transfer into B and T lymphocytes is of major interest in gene therapy and immunotherapy. We engineered new lentiviral vectors pseudotyped by some chimeric (BaEV/TR) and mutant (BaEVRLess) glycoproteins from the baboon endogenous retrovirus. We demonstrated that these new vectors can transduce more efficiently resting and mild stimulated hematopoietic stem cells than obtained with lentivectors pseudotyped by the glycoprotein G from the vesicular stomatitis virus (VSV-G). It is the same with the recently developed lentiviral vectors pseudotyped by the H and F glycoprotein from measles virus (H/F-LVs).We also compared the ability of the H/F-LVs with the BaEV/TR and BaEVRLess lentiviral vector pseudotype to transfer genes into B and T lymphocytes and into the whole T lineage. From now on, we are able to propose adapted vectors for gene transfer at each stage of differentiation from CD34+ cells to thymocytes and mature T cells. This could allow us to propose some new clinical protocols in gene therapy with a co-transplantation of genetically modified stem cells and their differentiated T progenitors in order to reduce the aplasia stage induced by current transplantation protocols.
... IL-7 also plays an important role in the activation and expansion of cytotoxic CD8 T cells. IL-7 both stimulates proliferation of CD8 T cells in a time-and dose-dependent manner [13][14][15][16] and upregulates the expression of perforin 17 and CD25, the IL-2 receptor alpha-chain. 18 Given the central role IL-7 plays in T-cell biology, it is not surprising that impaired IL-7/IL-7R signaling has been implicated in a number of disease states including cancer, 19,20 rheumatoid arthritis, 21 multiple sclerosis [22][23][24][25] and chronic viral infections such as hepatitis C 26 and human immunodeficiency virus (HIV). ...
Interleukin (IL)-7, a key immuno-regulatory cytokine, plays an essential role in peripheral T cell homeostasis and function. Signaling via the IL-7 receptor is tightly regulated and we and others have shown IL-7 provides negative feedback on its own signaling by down regulating expression of the IL-7 receptor alpha-chain (CD127) through both suppression of CD127 gene transcription and by internalization of existing CD127 proteins from the cell membrane. We show here for the first time in primary human CD8 T cells that upon stimulation with IL-7, CD127 is internalized through clathrin-coated pits, a process dependent on both lipid raft formation and the activity of dynamin. As visualized by confocal microscopy, CD127 shows increased co-localization with clathrin within 5 min of IL-7 stimulation and within 15-30 min is seen in multiple intracellular punctae co-localizing with the early endosomal marker EEA1. By two hours after addition of IL-7, CD127 staining associates with the late endosomal marker RAB7 and with the proteasomal 20S subunit. By inducing receptor internalization and translocation from early endosomes to the proteasome, IL-7 directly influences its receptor density on the cell surface and thus regulates the intensity of its own signaling cascades. Given the important role IL-7 plays in T cell development, homeostasis and function, deciphering how expression of its receptor is controlled on the cell surface is essential to understanding how T cell activity can be regulated in different microenvironments and in response to different pathogens.Immunology and Cell Biology accepted article preview online, 14 August 2015. doi:10.1038/icb.2015.80.
... Given the known ability of IL2 to promote T cell growth and differentiation, an obvious and important consideration in the present studies concerns the role of IL2 as the actual mediator of 11,7 effects . This possibility is particularly relevant in view of the recent demonstration that IL7 induces the p55 chain of the IL2R on murine and human T cells (11,12) . To address this issue, an antiserum against human IL-2 was used in culture in conjunction with IL-7. ...
The effects of purified recombinant interleukin 7 (IL-7) on the generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte culture (MLC) and on the induction of lymphokine-activated killer (LAK) cells in autologous cultures of human peripheral blood mononuclear cells were investigated. IL-7 was found to induce the generation of both CTL and LAK cells in bulk cultures. The appearance of peak CTL activity in MLC established with exogenous IL-7 was delayed in comparison with replicate cultures containing exogenous IL-2, but both cytokines stimulated quantitatively similar levels of antigen-specific lytic activity. An IL-2-neutralizing antiserum inhibited substantially, but not completely, the effect of IL-7 on CTL generation, implying the existence of both an indirect component of IL-7 activity via IL-2 utilization, as well as an IL-2-independent component. Cell surface phenotypic analysis of IL-2- or IL-7-generated CTL effector cells revealed that CD8+ cells were responsible for the vast majority of lytic activity. Limiting dilution analysis (LDA) revealed that essentially identical frequencies of CTL precursors (CTL-P) were capable of clonal expansion and/or differentiation in the presence of exogenous IL-2, IL-4, or IL-7, supporting the concept that all three of these cytokines are capable of exerting a major influence on T cell growth and differentiation. Approximately half of the CTL-P that responded in IL-7-supplemented LDA cultures did so in an IL-2-independent manner. IL-7 stimulated the development of LAK cells in autologous bulk cultures, but only weakly in comparison with IL-2. In contrast to its effects on CTL generation, the induction of LAK cells by IL-7 was virtually independent of IL-2. LAK cells induced by IL-7, like those induced by IL-2, were phenotypically heterogeneous and included CD8+, CD56+, and gamma/delta+ cells. Limiting dilution analysis indicated that IL-2 stimulated fivefold more LAK-P than IL-7 and 220-fold more than IL-4. Collectively, these data suggest that IL-7 has potent regulatory effects on human cytolytic cell populations and, either alone or in combination with other cytokines, could be important for the in vitro expansion of cells for adoptive immunotherapy.
... The role of IL-7 in the development of this B cell lineage is completely unknown. IL-7 also has been shown to stimulate the proliferation of murine (18,19) and human mature T lymphocytes (20), and thymocytes (21). IL-7 is produced by thymic stromal cells (6) as well as bone marrow-derived stromal cells, and so it might be expected that T cell precursors would require IL-7 at some stage of development. ...
The effects of interleukin 7 (IL-7) on the growth and differentiation of murine B cell progenitors has been well characterized using in vitro culture methods. We have investigated the role of IL-7 in vivo using a monoclonal antibody that neutralizes IL-7. We find that treatment of mice with this antibody completely inhibits the development of B cell progenitors from the pro-B cell stage forward. We also provide evidence that all peripheral B cells, including those of the B-1 and conventional lineages, are derived from IL-7-dependent precursors. The results are consistent with the rapid turnover of B cell progenitors in the marrow, but a slow turnover of mature B cells in the periphery. In addition to effects on B cell development, anti-IL-7 treatment substantially reduced thymus cellularity, affecting all major thymic subpopulations.
... Interleukin (IL)-7 is pivotal to T-cell survival and homeostasis and plays an important role in maintaining constant numbers of naïve and memory CD4 and CD8 T-lymphocytes in the peripheral circulation [9]. IL-7 promotes T-cell proliferation by stimulating entry into the cell cycle [10,11,12,13] and enhances Tcell survival by up regulating the anti-apoptotic factors Bcl-2 and Bcl-xL [14] while inhibiting the pro-apoptotic factors Bad and Bax [15]. IL-7 signals through the IL-7 receptor, a heterodimeric complex comprised of a unique a-chain (CD127) and the common c-chain (CD132) that is shared with the receptors for IL-2, -4, -9, -15, and -21. ...
HIV infection elicits defects in CD4 T-cell homeostasis in both a quantitative and qualitative manner. Interleukin-7 (IL-7) is essential to T-cell homeostasis and several groups have shown reduced levels of the IL-7 receptor alpha-chain (CD127) on both CD4 and CD8 T-cells in viremic HIV+ patients. We have shown previously that soluble HIV Tat protein specifically down regulates cell surface expression of CD127 on human CD8 T-cells in a paracrine fashion. The effects of Tat on CD127 expression in CD4 T-cells has yet to be described. To explore this effect, CD4 T-cells were isolated from healthy individuals and expression levels of CD127 were examined on cells incubated in media alone or treated with Tat protein. We show here that, similar to CD8 T-cells, the HIV-1 Tat protein specifically down regulates CD127 on primary human CD4 T-cells and directs the receptor to the proteasome for degradation. Down regulation of CD127 in response to Tat was seen on both memory and naive CD4 T-cell subsets and was blocked using either heparin or anti-Tat antibodies. Tat did not induce apoptosis in cultured primary CD4 T-cells over 72 hours as determined by Annexin V and PI staining. Pre-incubation of CD4 T-cells with HIV-1 Tat protein did however reduce the ability of IL-7 to up regulate Bcl-2 expression. Similar to exogenous Tat, endogenously expressed HIV Tat protein also suppressed CD127 expression on primary CD4 T-cells. In view of the important role IL-7 plays in lymphocyte proliferation, homeostasis and survival, down regulation of CD127 by Tat likely plays a central role in immune dysregulation and CD4 T-cell decline. Understanding this effect could lead to new approaches to mitigate the CD4 T-cell loss evident in HIV infection.
... In 1988, De Villartay and colleagues identied δRec and ψJα as TCRD deleting elements anking the TCRD locus, which preferentially recombine resulting in deletion of the TCRD locus ( Figure 1). 58 The signal joint TRECs (sjTRECs) produced in that rearrangement process are common to the majority of TCRαβ + T cells and speci c to phenotypically naive T cells 55,59 and might therefore be used as measure of thymic output and frequency of RTEs. ...
... In addition to playing a critical role in peripheral immune homeostasis [9,14,15,16,17,18] and the development and maintenance of T-cell memory, [19,20] IL-7 also plays an important role in the activation of CD8 T-cells in response to foreign antigen. IL-7 independently stimulates CD8 T-cell proliferation, [21,22,23,24] and potentiates cytolytic activity [25,26,27,28,29,30,31] by enhancing production of IFN-c following TCR stimulation [32,33] and by inducing accumulation of intracellular perforin. [34,35] Given the important role IL-7 plays in CD8 T-cell responses, decreased IL-7 signaling would be expected to result in impaired cell mediated immunity and inefficient control of viral pathogens including HIV. ...
Expression of the IL-7 receptor α-chain (CD127) is decreased on CD8 T-cells in HIV infected patients and partially recovers in those receiving antiretroviral therapy with sustained viral suppression. We have shown that soluble HIV Tat protein down regulates CD127 expression on CD8 T-cells isolated from healthy HIV-negative individuals. Tat is taken up by CD8 T-cells via endocytosis, exits the endosome and then translocates to the inner leaflet of the cell membrane where it binds to the cytoplasmic tail of CD127 inducing receptor internalization and degradation by the proteasome. This down regulation of CD127 by Tat results in impaired CD8 T-cell function. Interestingly, suppression of CD127 by Tat is reversible and requires the continual presence of Tat in the culture media. We thus questioned whether the low IL-7 receptor expression evident on CD8 T-cells in HIV+ patients was similarly reversible and if suppression of the receptor could be maintained ex vivo by Tat protein alone. We show here that when CD8 T-cells isolated from HIV+ patients are incubated alone in fresh medium, low CD127 expression on the cell surface recovers to normal levels. This recovery of CD127, however, is completely inhibited by the addition of HIV Tat protein to the culture media. This study then provides evidence that soluble factor(s) are responsible for low CD127 expression on circulating CD8 T-cells in HIV+ individuals and further implicates Tat in suppressing this receptor essential to CD8 T-cell proliferation and function.
... This corresponds with the finding by other authors that LAK activity from thymic and BM cells, as opposed to secondary lymphoid tissues, can not be generated by incubation with IL-7 only, or is at least significantly weaker than PBL-derived IL-7-induced LAK activity (58,60,62). Initially described as an essential cytokine for B-and T-lymphopoiesis, IL-7 is now known to modulate immune effector function of NK and LAK cells, CTL, and monocytes (62)(63)(64)(65)(66)(67). Moreover, recently, IL-7 has been reported to be critical for the development of immature NK cell colonies from BM cultures whereas others have shown these cultures to yield phenotypical but non-functional NK cells (68,69). ...
Background. We have previously shown that allogeneic bone marrow CBRl) chimeras preconditioned with total lymphoid irradiation and low-dose total body irradiation (TLI/TBI) develop a stronger graft-versus leukemia (GVL) effect than chimeras preconditioned with high-dose total body irradiation only (TBI). Here, we report on the possible role of cytokines in the mechanism underlying this GVL effect. Methods. Splenic mRNA levels of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and of inducible nitric oxide synthetase were determined by reverse transcription-poly merase chain reaction in TLI/TBI- or TBI-conditioned C3H/AKR BM chimeras challenged with AKR-type BW5147.3 leukemia cells. Ex vivo TGF-beta protein production by splenocytes was determined using ELISA. The possibility that cytokines influence the GVL effect by modulating the activity of IL-2-activated lymphocytes (LAK cells) was investigated by in vitro assays on donor-type BM cells. Results. Of all cytokine mRNA levels studied, those of TGF-beta and IL-7 were different between groups; both were significantly more elevated in TBI- than in TLI/ TBI-conditioned or normal mice. Differences were apparent after conditioning and were not influenced by additionally injected BM or leukemia cells. Cultured splenocytes of TBI-conditioned animals produced significantly more TGF-beta protein than those of TLI/TBI-conditioned ones or normal controls. r-TGF-beta but not r-IL-7 suppressed in vitro LAK activity of donor-type BM cells against BW5147.3 cells in a dose-dependent way. Conclusions. High-dose TBI-induced, host-derived splenic TGF-beta may inhibit generation of LAR cells from subsequently transplanted donor BN cells, suppressing their capacity to generate cytotoxicity upon injection of leukemia cells. The cytokine profile, induced by irradiation in host hematopoietic organs, can significantly modify posttransplant immunological processes such as the GVL,effect and graft-versus-host disease (GVHD).
Interleukin-7 (IL-7) is a versatile cytokine that plays a crucial role in regulating the immune system’s homeostasis. It is involved in the development, proliferation, and differentiation of B and T cells, as well as being essential for the differentiation and survival of naïve T cells and the production and maintenance of memory T cells. Given its potent biological functions, IL-7 is considered to have the potential to be widely used in the field of anti-tumour immunotherapy. Notably, IL-7 can improve the tumour microenvironment by promoting the development of Th17 cells, which can in turn promote the recruitment of effector T cells and NK cells. In addition, IL-7 can also down-regulate the expression of tumour growth factor-β and inhibit immunosuppression to promote anti-tumour efficacy, suggesting potential clinical applications for anti-tumour immunotherapy. This review aims to discuss the origin of IL-7 and its receptor IL-7R, its anti-tumour mechanism, and the recent advances in the application of IL-7 in tumour therapy.
p>CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. Previous data shows that distribution of antibody raised against CD40 in mice with syngeneic lymphoma resulted in a rapid cytotoxic T-cell response independent of T-helper cells. This response eradicated the established lymphoma and provided protection against secondary tumor re-challenge without further antibody treatment. Although precise mechanism of in vivo action of anti CD40 antibodies is not clear, indirect evidence suggests that dendritic cells (DCs) play a major role in establishment of the response.
The intracellular signalling events that lead to CD40 ligation-induction activation of IL6 gene transcription in a murine DC line, FSDC, were determined. IL6 RT-PCR and promoter assays established the responsiveness of FSDCs to anti CD40 antibody ligation. Further promoter assays showed that the transcription factors NFκB and AP1 are downstream transcriptional mediators of CD40 induced IL6 gene expression. Anti CD40 treatment of FSDCs stimulated increased expression of specific NFκB (p50:p65) and AP1 (c-Jun, JunB, JunD and c-Fos) DNA:protein complexes. Over-expression of an IκBα dominant negative repressor or a dominant negative JunD resulted in a strong inhibition of CD40 inducible IL6 promoter activity supporting a role of both transcription factors.
Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDCs followed by stimulation with an anti human CD40 antibody. These experiments revealed that anti-CD40 stimulation of NFκB and IL6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2. Induction of IL6 mRNA by anti CD40 treatment was found to be a transient event and was followed by a diminution of IL6 transcripts to levels below those found in unstimulated cells. This loss of IL6 expression was associated with reduced p50:p65 NFκB binding and elevated binding of CBF1 to a site overlapping the NFκB site. Over-expression of CBF1 resulted in a profound inhibition of basal and anti CD40 induced IL6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of IL6 response.</p
To better understand the regulation of interleukin-7 receptor (IL-7R) expression, we have pursued a detailed analysis of the structure of the murine and human IL-7R genes. The genes consist of eight exons, the sizes of which are conserved in mouse and human cells, spread out over 24 kbp (murine) and 19 kbp (human). A differential splicing event results in an mRNA encoding a secreted form of the human IL-7R gene. Primer extension and S1 nuclease analysis show a single transcriptional start site for the murine IL-7R gene. The 5'-flanking region of the murine IL-7R gene contains TATA- and CAAT-like sequences. The promoter region also contains a functional interferon regulatory element, to which the interferon-induced nuclear factors IRF-1 and IRF-2 are capable of binding and which is able to confer interferon-inducible expression on a heterologous gene. There are also potential binding sites for the transcription factors AP-1 and AP-2 as well as multiple glucocorticoid response elements. A fusion gene containing 2.5 kb of murine IL-7R 5' regulatory sequence linked to the bacterial chloramphenicol acetyltransferase gene directed expression of chloramphenicol acetyltransferase activity in murine pre-B-cell line 70Z/3 but not in the mouse fibroblast cell line NIH 3T3. Comparison of the murine and human IL-7R exon/intron boundaries with those of other hematopoietin receptor superfamily members whose exon/intron boundaries are also known reveals a conserved evolutionary structure.
Interleukin 7 (IL7) is a hematopoietic growth factor required for development and maintenance of lymphocytes including T cells, B cells, and NK cells. Recently, chicken IL7 (chIL7) has been cloned and studied in viral and parasite infection models. However, no monoclonal antibodies (mAb) that specifically detect chicken IL7 have been developed so far. In this study, recombinant chIL7 was expressed for immunization and mAb against chIL7 were developed and characterized to assess their immunological properties. Five mAb exhibiting specific binding to chIL7 were generated and determined their applicability in western blot, ELISA, and neutralization assay. A sandwich ELISA mAb pair that enables the measurement of chIL7 protein levels in biological samples from Eimeria-infected chickens was identified and several mAb neutralized chicken primary thymocytes proliferation mediated by chIL7. The mAb developed in this study will be valuable reagents for fundamental and applied immunological studies in poultry.
This chapter focuses on interleukin-7 (IL-7), which stimulates the growth of immature and mature T cells. IL-7 also promotes the expansion and effector function of cytolytic T cells and their precursors. Additionally, it enhances lymphokine-activated killer (LAK) cell activity in peripheral blood and can stimulate the antitumor abilities of monocytes and macrophages. The human IL-7 gene consists of six exons and five introns distributed over at least 33 kbp. The length of intron 2 is unknown but consists of at least 15 kbp. The human IL-7 cDNA contains an open reading frame spanning 531 nucleotides, encoding a protein of 177 amino acids (aa) with a calculated molecular mass of 17,400 Da. This includes a 25 aa signal sequence that is absent from the mature IL-7 protein. The 5' untranslated region of the murine cDNA contains 548 nucleotides, while the analogous region of the human cDNA spans a region of 384 nucleotides. The 3' untranslated region of the human cDNA contains 658 nucleotides and is larger than the corresponding region of the murine cDNA (579 nucleotides). IL-7 was originally detected in supernatants produced from a bone marrow stromal cell line, and it is now well established that many stromal cell lines derived from bone marrow secrete IL-7. The levels of IL-7 produced, however, are uniformly low in unmodified cell lines. Most IL-7-producing cell lines are refractive to inductive stimuli and only modest increase in IL-7 production is achieved by stimulation with lipopolysaccharide. Transcripts encoding IL-7 have been detected in murine spleen, thymus, kidney, and bone marrow and in human spleen and thymus.
The present study was aimed at identifying surface-membrane molecules involved in the regulation of human B-cell ontogeny. For this purpose, murine monoclonal antibodies (MoAbs) were generated against Pre-Alp, a pre-B acute lymphoblastic leukemia (ALL) cell line, and MoAb R34.34 was selected for further characterization. R34.34 recognized a molecule expressed on normal B-cell precursors (BCP) but not on mature B cells. The antibody also reacted with T lymphocytes, a subpopulation of monocytes from peripheral blood, and a subset of CD34+ cells. Immunoprecipitation analysis indicated that R34.34 recognizes an 80-kD molecular weight antigen. Antibody R34.34 was further found to be directed against an epitope interfering with binding of interleukin-7 (IL-7) to Pre-Alp cells. Expression cloning from a Pre-Alp cDNA library showed that R34.34 antigen is CDw127, the 75- to 80-kD IL-7 receptor. Proliferation of the B-lineage ALL cell lines Reh and Mieliki was inhibited by IL-7, and this effect was specifically reverted by MoAb R34.34. In addition, antibody R34.34 specifically inhibited IL-7- dependent proliferation of normal BCP, Pre-Alp cells, and peripheral T cells. These results imply that both inhibitory and proliferative effects of IL-7 can be mediated through the same receptor on various lineages. R34.34 antibody should be important for the analysis of signal transduction through CDw127.
In unstimulated conditions osteoclast renewal occurs as a result of the stromal cell production of the key osteoclastogenic factors, receptor activator of NFkB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Inflammation is known to cause increased osteoclastogenesis; however, the mechanisms responsible for this phenomenon are poorly understood. We now show that interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFα), cytokines typically produced in inflammatory conditions, increase the stromal cell production of IL-7. This factor, in turn, up-regulates production of osteoclastogenic cytokines by T cells leading to stimulation of osteoclast (OC) formation. Although T cells were found to produce soluble forms of both RANKL and M-CSF, saturating concentrations of osteoprotegerin failed to inhibit approximately 40% of the OC formation, suggesting that IL-7 acts via both RANKL-dependent and RANKL-independent pathways. Despite the identification of T-cell–secreted M-CSF, this cytokine was not essential for either RANKL-dependent or -independent OC formation, suggesting that T cells secrete other cytokines capable of substituting for M-CSF action. On the basis of our data, we propose a novel mechanism for inflammatory bone loss in which induction of IL-7 from stromal cells by IL-1 and TNFα leads to the production of soluble osteoclastogenic cytokines by T cells. Thus, the mechanism by which IL-7 causes bone resorption involves the activation of T cells and the T-cell–dependent augmentation of osteoclastogenesis.
The effects of granulocyte-macrophage colony-stimulating factor (GM- CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T- cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM- CSF responsive cell type, thymic AC and T cells were examined for GM- CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single- cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.
In the present report, we studied the role of the stromal-derived cytokine interleukin-7 (IL-7) in the IL-2–gene regulation in activated T lymphocytes. Production of IL-2 requires the formation of transcription factors involved in the IL-2 –gene regulation. T-cell receptor (TCR)/CD3 engagement results in the activation of nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1), and nuclear factor κB (NFκB), whereas the CD28 responsive complex (CD28RC) is activated in response to the CD28 signal. Costimulation of phytohemagglutinin/anti-CD28 activated T lymphocytes with IL-7 induces a fivefold enhanced IL-2–mRNA accumulation and a 2.5-fold enhanced protein secretion. The IL-2–gene transcription rate is increased 3.4-fold, indicating that the effect of IL-7 is in part mediated at the transcriptional level. The molecular mechanisms underlying the IL-7 effect involve the upregulation of the DNA binding activity of NFAT (60%) and AP-1 (120%), without affecting the activities of NFκB and CD28RC, which was confirmed by transfection assays. We also show that the IL-7–induced enhancement of the AP-1–DNA binding activity is not cyclosporin A-sensitive. Since AP-1 is part of the NFAT complex, we conclude that the IL-7–signaling pathway is involved in the activation of the fos and jun proteins of which AP-1 consists.
In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.
Human recombinant interleukin-7 (IL-7) was labeled with biotin and used to examine IL-7 receptor (IL-7R) expression and regulation on human primary hematopoietic cells, the monocytoid line THP1, and a range of B- and pre-B-celi lines by flow cytometry. A strong intensity of staining was observed using relatively high (greater than 1 x 10(-7) mol/L) concentrations of biotinylated IL-7 on the majority of cell types examined. This reactivity, which could be effectively competed with excess unlabeled IL-7, did not correlate with either mRNA levels for the cloned receptor or with estimates of IL-7R expression determined by [125I]IL-7 binding. Staining of cells with a titration of biotinylated IL-7 showed, at concentrations greater than 1 x 10(-7) mol/L binding with a Ka in the range of 1 x 10(6) mol/L-1, to 1 x 10(7) mol/L-1, an affinity 100 to 1,000 times lower than that reported for the cloned IL- 7 receptor. Further data suggesting the existence of a distinct low- affinity IL-7R were provided by two antibodies specific for the cloned IL-7R. Staining with these monoclonal antibodies (MoAbs) correlated with both IL-7R mRNA levels and receptor expression determined by [125I]IL-7 binding, but was not compatible with the distribution of reactivity seen with biotinylated IL-7. Using tritiated biotin to label IL-7, it was estimated that the total number of IL-7 binding sites on the cell lines examined ranged from 1 x 10(4) to at least 5 x 10(5)/cell. Cross-linking studies showed that [125I]IL-7 associated with two major proteins of approximately 62 Kd and 70 Kd on the surface of RPMI 1788 and THP1 cells, in contrast to the 75- to 80 Kd molecule characteristic of the previously cloned receptor, expressed on the surface of Daudi cells. Proliferation of THP1 cells, expressing only the low-affinity form of IL-7R and lacking detectable IL-7R mRNA, could be inhibited by the addition of IL-7 in a concentration-dependent fashion, indicating that, at least on this cell line, binding of IL-7 with a Ka of 1 x 10(6) mol/L-1 to 1 x 10(7) mol/L-1 can transduce a biological signal. Taken together, the data contained in this report demonstrate the existence of a low-affinity IL-7R, expressed in high numbers on hematopoietic cells of different lineages, which is the product of a gene distinct from that encoding the cloned IL-7R.
The current study was undertaken to assess the vaccine efficacy of Eimeria tenella EF-1α/chicken IL-7 (chIL-7) DNA vaccine when administered with Montanide Gel 01 adjuvant against live Eimeria acervulina challenge in commercial broiler chickens. The criteria used for the evaluation of vaccine efficacy were weight gain, duodenal lesion scores, oocyst counts, humoral antibody response, and duodenal proinflammatory cytokine gene expression. Chickens vaccinated with EF-1α (100 µg)/chIL-7 (20 µg) in Gel 01 PR adjuvant showed body weight gain similar to the uninfected control and higher oocyst shedding, a lower gut lesion score, and higher proinflammatory cytokine gene expression than did the infected controls. Moreover, chickens vaccinated with chIL-7 (20 µg) in Gel 01 PR adjuvant shed fewer oocysts with reduced gut lesion scores and produced higher levels of anti-EF-1α serum antibody than did the infected control. Chickens vaccinated with EF-1α (50 µg)/chIL-7 (20 µg) in Gel 01 PR adjuvant showed higher weight gains than did the infected control and shed significantly fewer oocysts than the infected control. Furthermore, chickens vaccinated with EF-1α (100 µg) in Gel 01 PR adjuvant demonstrated the lowest anti-EF-1α serum antibody levels. This study demonstrated the beneficial effects of using EF-1α and/or host cytokine chIL-7 DNA vaccine together with Gel 01 PR adjuvant to improve T-cell-mediated effector function in broiler chickens challenged with live E. acervulina.
The elaboration of an immune response to infection or tissue damage is orchestrated by a myriad of different cells and cell types, the activities of which must be under precise temporal and spatial control. On a cellular basis, this requires an efficient means of self-regulating activation programs and of communicating activities between cells. The soluble mediators of this cellular information network are the cytokines.
Melanoma is the leading cause of death among all skin cancers and its incidence continues to rise rapidly worldwide in the past decades. The available treatment options for melanoma remain limited despite extensive clinical research. Melanoma is an immunogenic tumor and great advances in immunology in recent decades allow for the development of immunotherapeutic agents against melanoma. In recent years, immunotherapy utilizing cytokines has been particularly successful in certain cancers and holds promise for patients with advanced melanoma. In this review, an overview of the current status and emerging perspectives on cytokine immunotherapy for melanoma are discussed in details. Such a study will be helpful to unveil the mysterious mask of cytokine-based immunotherapy for melanoma.
Multiple Myeloma (MM) is a B cell malignancy characterized by the bone marrow (BM) accumulation of monoclonal plasmacells. Different clinical and experimental evidences favour the view that in human MM, as in murine plasmacytoma, the disease may be disseminated by clonogenic plasmacell precursors circulating in the peripheral blood (PB). The disease is widespread to the axial skeleton by the time of diagnosis, while very few plasmacells are seen in the PB and their number becomes evident only in the terminal phases of the disease. PB lymphocytes expressing the relevant idiotype have been described and PB mononuclear cells (PBMC) may show a monoclonal immunoglobulin (Ig) gene rearrangement. Also, DNA- aneuploid cells have been detected in PB samples and several methods have been described to grow plasmacell colonies from PB. Finally, MM PBMC cultured in vitro in presence of IL3 and IL6 give rise to a variable proportion of monoclonal B cells. In this work we have asked two questions: 1) how can we investigate the presence of malignant plasmacell precursors in the PB of patients with MM ?; 2) why are these precursors selectively homing to the BM ? In order to address these questions we have used a threefold approach: a) PBMC from MM patients have been cultured in presence of different combinations of cytokines; b) PBMC from MM patients have been cultured in presence of autologous serum; c) long term culture of BM stromal cells have been established from MM patients and characterized.
Binding of a T cell to an antigen presented by an antigen presenting cell (APC) leads to mutual activation of both cell types and to the release of an array of cytokines. During the last years the increase in knowledge of the involvement of individual cytokines in the interactive process between T cells and monocytes/macrophages has been overwhelming. The fast progress is mainly due to the development of cloning methods, sequencing techniques, and production of cytokines at a large scale. This has given the opportunity to exactly define the biological properties of different factors on the various cell populations.
The development and differentiation of lymphocytes are regulated by the local microenvironment in the thymus, bone marrow and intestinal epithelium. Mice engineered to lack the transcription factor Ikaros develop normal myeloid and erythroid cells, but lack lymphoid cells1, implying the existence of a ‘lymphoid stem cell’ restricted to T, B, or NK cell lineage specific differentiation. Experimental evidence suggests that cytokines act both to regulate differentiation of lymphoid progenitors and to activate and expand mature lymphocyte populations. A unidirectional model for lymphoid differentiation suggests that lineage commitment is driven by exposure of stem cells to lineage-specific cytokines. The availability of recombinant cytokines that regulate lymphoid differentiation and function has led to their clinical application in enhancing anti-tumor immunity and immune reconstitution in the setting of bone marrow transplantation. A discussion of the effect of cytokines on T cell and Natural Killer cell differentiation and function will be the subject of this review. B cell differentiation has been reviewed elsewhere2.
It has long been recognized that the growth and differentiation of resting mature T lymphocytes into functional helper or cytolytic cells is dependent upon not only an antigenic stimulus but also soluble factors (Plate, 1976; Ryser et al., 1978). Consequently, recent attention has been focused upon the identification and further characterization of these soluble regulators. Following the description of interleukin-2 (IL-2) as a T-cell growth factor that can influence the generation of cytolytic T lymphocytes (CTL) (Gillis et al., 1978), it became apparent that several other cytokines exist which have similar activity. However, many of the early studies on T cell-active factors were clouded by the use of impure cytokine preparations and the possibility of indirect effects occurring in bulk lymphocyte cultures due to cytokine cascades. Thus, it was possible that a reputed T-cell growth factor or CTL induction factor was in fact affecting the secondary production of IL-2 or other T-cell growth factors, rather than itself directly stimulating T cells.
The study of lymphokines began in the mid 1960s, after the simultaneous discovery by David (1966) and Bloom and Bennett (1966) that in vitro activation of lymphocytes leads to the production of factors that inhibit the migration of macrophages [468]. Subsequently, DuMonde et al. (1969) coined the term lymphokines to refer to factors that modulate the growth or mobility of a variety of leukocytes [167]. Hundreds of communication molecules have now been described, which are produced by both lymphocytic and nonlymphocytic cells. Nonlymphocytic cells involved in this process include normal macrophages, fibroblasts, mast cells, and eosinophils. It was therefore proposed by Cohen and co-workers in 1977 that this entire class of mediators be termed cytokines, in recognition of the contribution from nonlymphocytic components. It is now recognized that cytokines may function in a wide range of activities, including regulation of immunological and inflammatory processes, which not only regulate normal cell growth and differentiation but also participate in repair mechanisms.
Recent studies have demonstrated that interactions between mucosal lymphocytes and intestinal epithelial cells are crucial for maintaining mucosal immunity. The mucosal lymphocytes may serve a critical role in the mucosal immune system by providing immune surveillance of epithelial cells. Although recent studies have shown that cytokines released from mucosal mononuclear cells may affect intestinal epithelial cell differentiation, the signals originating from epithelial cells that may regulate mucosal lymphocytes are yet to be defined. It has been reported that mucosal lymphocytes proliferate minimally to mitogens or stimulation through the CD3 pathway. This in vitro hyporesponsiveness of mucosal lymphocytes is supposed to be due to the absence of essential growth factor. Interleukin-7 (IL-7) is a stromal cell-derived pleiotropic cytokine with lymphoid precursor cell growth-promoting activity. The recent studies have demonstrated IL-7 mRNA expression and IL-7 protein production in the intestinal epithelial cells. These results, in concert with the findings that IL-7 receptors are expressed by mucosal lymphocytes, suggest that IL-7 regulates the proliferation of the intestinal mucosal lymphocytes. This chapter reviews the intestinal epithelial cell-derived IL-7 as a regulatory factor for the intestinal mucosal lymphocytes.
Cytokines are regulatory proteins, produced and secreted by various cells, which control immune response, hematopoiesis, inflammation, wound repair and tissue morphogesis. Cytokines may be secreted or membrane bound. Secreted cytokines may act locally as autocrine or paracrine factors or over some distance as would a hormone. Membrane bound cytokines act by cell-cell contact, communicating information from one cell to another, often bidirectionally. There are cell surface receptors for each cytokine that bind the cytokine spe-cifically. Receptor subunits may be shared between different cytokines. Binding the cytokine brings about signaling and a series of cell activating events. For many cytokine receptors (not all), this involves increased phosphorylation of certain tryrosine residues on key cellular proteins.
The epithelial cells could be an important antigen-presenting cell for controlling mucosal immune responses. Since a large number of CD3+ T cells reside in the intestinal epithelium, it is logical to investigate molecular and cellular aspects of cell to cell interactions between intraepithelial T lymphocytes and epithelial cells for mucosal immune responses. CD3+ T cells situated in the mucosal epithelium are commonly termed intraepithelial lymphocytes (IELs). Although IELs possess several unique characteristics when compared with CD3+ T cells in other organized systemic lymphoid tissues, a most profound feature of IEL is the occurrence of high numbers of γδ T cells. Thus, it has been shown that 20–80% of IELs expressed γδ heterodimer chains of T-cell receptor (TCR) dependent on the age, strain, and microenvironment. Intraepithelial γδ T cells modulate growth and differentiation of epithelial cells. Thus, these γδ T cells have been shown to produce keratinocyte growth factor which supports growth of cultured epithelial cells. Further, the disruption of TCRδ gene resulted in the reduction of epithelial cell turnover and the inhibition of MHC class II expression. These findings clearly demonstrate the importance of cell to cell interactions between mucosal T lymphocytes and epithelial cells for the intestinal immune system. This chapter summarizes the recent observations which describe molecular aspects of mucosal γδ T cell and epithelial cell interactions via particular cytokines and their receptors. Further, it provides new evidence that mucosal γδ T cells can influence the CD4+, αβ T-cell dependent IgA B-cell response.
The vast majority of Foxp3 regulatory T cells (Treg) exhibits constitutive expression of CD25 (IL-2Rα), which allows the constitution of the high affinity IL-2Rαβγ receptor, ensuring efficient IL-2 binding by Treg. Maintenance of CD25 expression at Treg surface depends on both cell intrinsic factors and environmental stimuli such as IL-2 itself. Whether other factors can participate to maintenance of CD25 expression in vivo is at present unknown. In the present work we demonstrated that IL-7, a gamma-chain cytokine exerting a crucial role in T cell development and homeostasis, is able and necessary to sustain the expression of high levels of CD25 at Treg surface. We demonstrated that, during in vitro cultures performed in the absence of IL-2, IL-7 is able to sustain CD25 expression at Treg surface through a transcriptional mechanism. By studying mice in which IL-7 signaling is either genetically impaired or increased and by employing adoptive transfer murine models, we demonstrated that IL-7 is necessary for sustained expression of CD25 at Treg surface in vivo. To ascertain the biological impact of IL-7 mediated modulation of CD25 expression, we demonstrated that IL-7 modulation of CD25 expression at Treg surface affected their ability to efficiently bind IL-2 and transduce IL-2 signaling. Finally, we demonstrated that IL-7 dependent modulation of CD25 associated with potentiated IL-2 induced expansion of Treg in vivo. Collectively, our results identify IL-7 as a necessary factor contributing to sustained CD25 expression at Treg surface in vivo thereby affecting their ability to efficiently react to IL-2.
Virus-specific cytotoxic T lymphocytes (CTLs), which kill virus-infected cells, are thought to be a major host defense against viral infections. The addition of interleukin 7 (IL-7) at the onset of mitogen-stimulated cultures resulted in a marked (up to threefold) augmentation of env-specific cytotoxicity in human immunodeficiency virus type 1 (HIV-1)-infected individuals (p < 0.001). Addition of IL-7 on day 3 or 5 produced a significant but lesser augmentation of CTL response as compared to day 0. The IL-7-induced proliferative response and augmentation of cytotoxic activity was time and dose dependent, with an optimal IL-7 concentration of 1000 U/ml. Cell surface phenotypic analysis of CTL effector cells indicates that IL-7 primarily affects the proliferation of CD8(+) T cells. Anti-IL-2 monoclonal antibody (MAb) substantially inhibited the proliferative effect of IL-2, but did not affect the proliferative effect of IL-7. Endogenous IL-2-induced generation of cytotoxic T cells was blocked by MAbs to IL-2 or IL-2R. The addition of IL-7 restored the process of conversion of precursor CTLs (pCTLs) to mature CTLs (mCTLs) and significantly enhanced specific cytolytic activity. It appears that IL-7 is a potent regulatory cytokine capable of acting independently of IL-2 in mitogen-specific activation of pCTLs to mCTLs. These data suggest that IL-7 should be considered as a potential therapeutic approach in AIDS and other infectious diseases in which CTL response declines.
Lymphokines play an important role in immune responses to viruses by modulating functions of T lymphocytes. In the present study, we examined the effects of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-7 (IL-7), and interferon gamma (IFNγ) on proliferation, cytotoxic activity and lymphokine production of a dengue virus-specific CD8+ human cytotoxic T lymphocyte (CTL) clone. IL-2 and IL-7 induced proliferation of the CD8+ CTL clone in the presence or absence of specific antigen, while IFNγ suppressed proliferation of the clone. IL-7 and IFNγ augmented dengue virus-specific cytotoxic activity without inducing non-specific cytotoxic activity, and IL-2 induced non-specific cytotoxic activity. IL-2 induced IFNγ production by the CD8+ CTL clone. IL-4 and IL-6 did not modulate the functions of the CD8+ CTL clone in these experimental conditions.
These results suggest that functions of dengue virus-specific CD8+ CTL are modulated by IL-2, IL-7 and IFNγ, and that IL-7 is a lymphokine useful to induce growth and to maintain specific cytotoxic activity of CD8+ CTL clones in vitro.
Interleukin-7 has demonstrated potent enhancing effects on the growth and differentiation of several immature cell types, including thymocytes, and on survival of resting and antigen activated T cells. In this study, we evaluated the effects of IL-7 on post-thymic antigen-specific T cells from human blood. IL-7 was found to enhance proliferation responses and IFN-γ secretion of myelin or recall Ag-specific Th1 cells through the selective up-regulation of the IL-2Rα and γ but not β chains in both an Ag-dependent and Ag-independent manner, but did not affect monocytes, B cells, or NK cells. These functions of IL-7 enhanced the detection of Th1 but not Th2 cell frequency by >2.5 fold, and promoted selection of Ag-specific Th1 cells by the limiting dilution method. Moreover, IL-7 pretreatment conferred increased resistance of CD4+ T cells to CD8+ cell lysis. These studies demonstrate that IL-7 promotes the growth and survival of circulating Ag-specific human Th1 cells through a mechanism that probably involves the γc common receptor for IL-2 family members that includes IL-7.
We have developed a new technique that allows us to quantify antigen-specific T cells, and to determine their functional phenotype and origin from naive versus memory populations. Using this methodology, we have characterized a total of 286 T-cell lines specific for myelin basic protein (MBP) and influenza hemagglutinin from 16 multiple sclerosis (MS) patients and nine healthy donors. Our data support the notion that MBP-specific T cells undergo in vivo activation in MS patients and indicate a presence of immune dysregulation that renders MS patients prone to develop autoimmunity. Our methodology offers a way to study antigen-specific T-cell characteristics as a surrogate marker in immunotherapy trials.
The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effec-tor subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-+ , but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-+ and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.
The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4⁺ T lymphocytes from HIV-1–infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4⁺ T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.
The effect of Traditional Chinese Medicine (TCM), Ekki-Youketsu-Fusei-Zai (EYFZ), on human peripheral blood lymphocytes (PBL) was investigated in vitro. MTT assay showed that EYFZ could directly stimulate PBL to proliferate without mitogen. In addition, RT-PCR and ELISA revealed that though EYFZ induced the expression of interferon gamma (INF-) mRNA and the production of INF- in culture supernatant, it did not induce the experssion or the production of interleukin-2 (IL-2) or interleukin-4 (IL-4). Moreover, we also examined which kind of immunocyte was involved in the INF- production by EYFZ in culture supernatant by inhibition experiment with monoclonal antibodies. It was shown that the EYFZ induced the production of INF- by T cell subsets of CD4 and CD8 cells. Thus, these results suggest that EYFZ has some significant stimulating
Although initially identified as a cytokine that promotes pre-B cell growth, it is now clear that IL-7 mediates effects on a wide range of cell types. Data generated during the past several years have demonstrated the exciting immunomodulatory potentials of IL-7 in T cell mediated immune responses. However, the potential use of IL-7 to promote B and T cell lymphopoiesis in clinical settings has yet to be meaningfully evaluated. Similarly, the role for IL-7 in augmenting immune responses in clinical settings to antigens expressed on tumor cells, to parasitic infections, and to viral antigens also awaits investigation. It is hoped that the results of studies in these areas will soon be undertaken to determine whether the promise shown by this cytokine in pre-clinical studies will be borne out. Certainly, the addition of a cytokine with the substantial beneficial immunomodulatory activities attributed to IL-7 in both in vitro and in vivo studies in murine systems, and in vitro studies using human lymphoid cells, would be a welcome addition in the immunotherapeutic treatment of cancer and HIV.
A cDNA encoding biologically active human interleukin 7 was isolated by hybridization with the homologous murine clone. Nucleotide sequence analysis indicated that this cDNA was capable of encoding a protein of 177 amino acids with a signal sequence of 25 amino acids and a calculated mass of 17.4 kDa for the mature protein. Recombinant human interleukin 7 stimulated the proliferation of murine pre-B cells and was active on cells harvested from human bone marrow that are enriched for B-lineage progenitor cells. Analysis of RNA by blot hybridization demonstrated the presence of two size classes of interleukin 7 mRNA in human splenic and thymic tissue.
Resting T cells proliferate in response to B cell stimulatory factor 1 (BSF-1; interleukin 4) plus phorbol myristate acetate (PMA). This response is obtained with highly purified T cells and is density independent, suggesting that accessory cells are not required. Both L3T4+ and Lyt-2+ T cells respond to BSF-1 plus PMA. Although BSF-1 alone does not cause T cell proliferation, it maintains the viability of small, dense T cells, indicating that it acts on resting T cells. Furthermore, BSF-1 is required early in the proliferative response of resting T cells to BSF-1 plus PMA, further supporting the concept that it acts on G0 or early G1 cells. However, BSF-1 is also needed late in the first round of division of T cells stimulated with BSF-1 plus PMA. Removing BSF-1 at 24 h of stimulation prevents entry into S phase. These results indicate that BSF-1 is involved in both the induction of competence and in the progression phases of T cell division.
We have used a biological assay system we developed to biochemically purify a previously uncharacterized murine lymphopoietic growth factor designated lymphopoietin 1 (LP-1). This factor is capable of stimulating the proliferation and extended maintenance of precursor cells of the B lineage. A stromal cell line producing LP-1 was established after transfection of primary stromal cultures with a plasmid encoding the transforming genes of SV40. This factor was purified to a single 25-kD species from the culture supernatant of an adherent stromal cell line. This material acts on immature lymphocytes, it binds to specific receptors on cells, and is distinct from previously described hematopoietic factors. LP-1 has been purified some 10(7)-fold with an overall recovery of 35%. The purified protein exhibits a specific activity of approximately 4 X 10(6) U/micrograms of protein and is active at a half-maximal concentration of 10(-13) M.
The effects of B cell stimulatory factor 1 (BSF-1) on the generation of human CTL and lymphokine-activated killer (LAK) cells in vitro were investigated. Both L-2 and BSF-1 were potent helper factors for the generation of antigen-specific CTL in MLC; detection of optimal BSF-1-induced CTL activity in this system occurred when BSF-1 was added to cultures after an initial period of activation during which exogenous BSF-1 was not present. In contrast to IL-2, BSF-1 failed to induce an LAK cell population, as detected with Daudi tumor targets, in cultures that had not been allosensitized. Furthermore, when both lymphokines were added together at culture initiation, BSF-1 inhibited the IL-2-driven generation of cytolytic cells. The differential ability of BSF-1 to promote the generation of CTL but not LAK could have important implications for lymphokine-mediated immunotherapy.
Recent studies suggested that the clonally unique Ti epitopes defined by non-cross-reactive monoclonal antibodies might represent the variable regions of the antigen receptor. Here we determine whether such anti-Ti antibodies could trigger clonal T cell activation. Anticlonotypic monoclonal antibodies to the 49/43-kdalton heterodimer of a given clone or antibodies to the 20/25-kdalton membrane associated monomorphic T3 molecule selectively induce proliferation and IL-2 secretion when linked to a solid support. In contrast, anti-T4 and anti-T8 antibodies under the same conditions have no effect. In conclusion, these results imply that anticlonotypic antibody functions in a fashion analogous to antigen and further support the notion that the T3-Ti molecular complex represents the antigen receptor on human T lymphocytes.
Several soluble factors have recently been associated with the proliferation and differentiation of thymus-derived lymphocytes. One of these factors present in medium conditioned by T cell mitogen-stimulated lymphocytes has the ability to promote the long-term culture of normal and antigen-specific cytotoxic T cells. We report a method to test for this proliferative stimulus in the form of a sensitive microassay based upon the tritiated-thymidine incorporation of continuous murine tumor-specific cytotoxic T cell lines (CTLL). The microassay requires microliter quantitites of sample fluid and is amenable to quantitative analysis. This highly reproducible, quantitative assay for T cell growth factor (TCGF) has allowed investigation as to the kinetics of TCGF generation and has revealed that T lymphocytes are required for its production. Further investigation has supported the notion that this nonspecies-specific factor is actively removed from tissue culture medium by the proliferation of either T cell mitogen-activated lymphocytes or CTLL.
Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.
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The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.
The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.
The capacity of human recombinant tumor necrosis factor-alpha (rTNF alpha) to modulate human T cell proliferation was examined. To examine the effect of rTNF alpha on the responding T cell directly, T cell activation was studied in the absence of viable accessory cells (AC). Highly purified AC-depleted peripheral blood T4 or T8 cells were stimulated with immobilized monoclonal antibodies to the cluster of differentiation (CD)3 molecular complex, an AC-independent stimulus. rTNF alpha augmented anti-CD3-stimulated T4 and T8 cell proliferation. The capacity of rTNF alpha to enhance T cell proliferation varied inversely with the density of immobilized anti-CD3 and the number of responding cells in each culture. The capacity of rTNF alpha to enhance antigen-induced T4 cell proliferation was also examined. Antigen-bearing paraformaldehyde-fixed antigen-presenting cells induced modest T4 cell proliferation when cultured in flat-bottomed wells; this response was enhanced by rTNF alpha. The results demonstrate that rTNF alpha has direct effects on T cells, facilitating their capacity to proliferate in response to mitogens and antigens. These data indicate that rTNF alpha may play an immunoregulatory role, enhancing the proliferation of T lymphocytes.
The events involved in the commitment and development of lymphoid lineage cells are poorly understood. We have used a recently described long-term culture system to establish a bioassay that can detect a novel growth factor capable of stimulating the proliferation of lymphoid progenitors. Using direct expression in mammalian cells we have isolated a complementary DNA clone encoding this novel haematopoietic growth factor, designated interleukin-7.
Purified peripheral murine T cells, in the presence of concanavalin A, can be activated to produce interleukin 2 (IL-2) through stimulation either with a previously described murine lymphokine designated T cell-activating factor (TAF) or with a cloned human lymphokine that has been called beta 2 interferon, B-cell-stimulatory factor 2, hybridoma growth factor, inducible 26-kDa protein, or hematopoietic colony-stimulating factor 309 by different investigators. We and others propose the designation interleukin 6 (IL-6) for the latter molecule. Our experiments demonstrate that either murine TAF or human IL-6 can restore the ability of purified T cells to proliferate in response to Con A or antibodies against the T-cell antigen receptor. Most if not all of the proliferation can be blocked by antibodies against the alpha chain of the IL-2 receptor. Furthermore, highly purified CD8- T cells can be activated by IL-6 in the presence of Con A to secrete IL-2. We propose that IL-6 and murine TAF are important "second signals" in primary antigen-receptor-dependent T-cell activation. Whether or not murine TAF is a homologue of human IL-6 remains to be determined.
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