No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Regulation of T cell proliferation by IL-7
Abstract
The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.
... Synergizes with IL-2 and IL-12 functions (84,85) Induces T-cell proliferation and inhibits apoptosis, preserves memory T-cells, and induces B-cell maturation and isotype switching (86)(87)(88) Activates both Th1 and Th2 subtypes and shows pleiotropic role (89,90) Stimulates Th1 response, IL-12 production and downregulates IL-4+ Th2 cells (86,91) IL-22 Promotes inflammatory response and is crucial in tissue repair (92,93) Protects the liver from chronic infections (94) Induces the production of antimicrobial peptide-β-defensin (95) Complementary to Th1 cytokines and requires IL-6 for production (96)(97)(98) IL-7 Induces proliferation of thymocytes, NK and mature T-cells, and production of cytotoxic T-cells (99)(100)(101)(102)(103)(104) Promotes the synthesis and secretion of IL-6, TNF-α, IL-1α, IL-1β, and MIP-113 by monocytes ...
... IL-7 is a 17 kDa glycoprotein derived from bone marrow stromal cells (285) and regulates a wide variety of functions including multiple effects on B-cells and proliferation of thymocytes (99)(100)(101), NK cells (102) and mature T-cells (103). IL-7 induces the production of cytotoxic T-cells with alloreactive, antitumor, and antiviral activities (104). ...
Leishmaniasis is a parasitic disease of humans, highly prevalent in parts of the tropics, subtropics, and southern Europe. The disease mainly occurs in three different clinical forms namely cutaneous, mucocutaneous, and visceral leishmaniasis (VL). The VL affects several internal organs and is the deadliest form of the disease. Epidemiology and clinical manifestations of VL are variable based on the vector, parasite (e.g., species, strains, and antigen diversity), host (e.g., genetic background, nutrition, diversity in antigen presentation and immunity) and the environment (e.g., temperature, humidity, and hygiene). Chemotherapy of VL is limited to a few drugs which is expensive and associated with profound toxicity, and could become ineffective due to the parasites developing resistance. Till date, there are no licensed vaccines for humans against leishmaniasis. Recently, immunotherapy has become an attractive strategy as it is cost-effective, causes limited side-effects and do not suffer from the downside of pathogens developing resistance. Among various immunotherapeutic approaches, cytokines (produced by helper T-lymphocytes) based immunotherapy has received great attention especially for drug refractive cases of human VL. Therefore, a comprehensive knowledge on the molecular interactions of immune cells or components and on cytokines interplay in the host defense or pathogenesis is important to determine appropriate immunotherapies for leishmaniasis. Here, we summarized the current understanding of a wide-spectrum of cytokines and their interaction with immune cells that determine the clinical outcome of leishmaniasis. We have also highlighted opportunities for the development of novel diagnostics and intervention therapies for VL.
... 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.
... Indeed, IL-7 was then shown to stimulate the growth of T cell progenitors as well (for a review see reference 3), thus emerging as a major regulator of early B and T cell development. More recently it has become evident that under certain conditions IL-7 can also stimulate the growth of mature peripheral T cells and T clones (4)(5)(6)(7)(8). This finding prompted us to follow up our incidental observation that murine keratinocytes seem to express IL-7 mRNA when studied by RT-PCR analysis (9). ...
... Our finding that IL-7 mKNA is expressed in diseased epidermis (probably by keratinocytes) raises the issue of a potential role of IL-7 in skin diseases. The observation that IL-7 has costimulatory activity on purified mature T cells and is a T cell growth factor for in vivo primed antigenspecific T cells (4)(5)(6)(7)(8) suggests that keratinocyte-derived IL-7 might be pathogenetically significant in a variety of inflammatory cutaneous diseases. IL-7 might, however, also be an important progression factor in cutaneous T cell lymphoma as it has been shown to be a growth factor for various lymphoma cells, including those of cutaneous T cell lymphoma (10)(11)(12)(13). ...
Interleukin 7 (IL-7) was originally identified as a growth factor for B cell progenitors, and subsequently has been shown to exert proliferative effects on T cell progenitors and mature peripheral T cells as well. Constitutive IL-7 mRNA expression so far had been demonstrated in bone marrow stromal cell lines, thymus, spleen, and among nonlymphoid tissues in liver and kidney. Here we show that both murine and human keratinocytes express IL-7 mRNA and release IL-7 protein in biologically relevant amounts. The physiological or pathological relevance of keratinocyte-derived IL-7 is presently unknown. Our finding that keratinocytes can produce IL-7 in concert with reports that IL-7 is a growth factor for in vivo primed antigen-specific T cells, as well as for T lymphoma cells suggests, however, that keratinocyte-derived IL-7 is important in the pathogenesis of inflammatory skin diseases and cutaneous T cell lymphoma.
... B cell progenitors (1,2) and subsequently shown to be a pleiotropic cytokine that has both proliferative and differentiative effects on ceils of various lineages in vitro. In particular, Ib7 supports proliferation of mature CD4 + and CD8 § cells (3)(4)(5), and induces differentiation of cytotoxic T cells, LAK cell activity (6)(7)(8), and tumoricidal activity of macrophages (9). We have investigated the possibility as to whether any of the IL-7-induced cellular functions alone or in concert could induce an antitumor reaction in vivo. ...
... T cells play the central role in the antitumor response because it is completely abrogated in mice depleted of T cells. This is in agreement with the direct proliferative effects on both activated CD4 § and CD8 + T cells described for IL-7 in vitro (4,5) and may explain the observed T cell infiltrate in the tumor. Depletion of either T cell subset revealed the absolute dependence of the IL-7-mediated antitumor effect on CD4 + cells. ...
The potential of interleukin 7 (IL-7) to induce an antitumor response in vivo was analyzed. Therefore, the IL-7 gene was expressed in the plasmacytoma cell line J558L. Although the growth of IL-7-producing cells was not retarded in vitro, the IL-7-producing cells were completely rejected upon injection into mice. Tumor rejection was observed only in syngeneic but not in nude mice. The tumor-suppressive effect could be abolished by the parallel injection of an anti-IL-7 monoclonal antibody. Immunohistochemical analysis revealed IL-7-dependent infiltration of the tumor tissue by CD4+ and CD8+ T lymphocytes, and also type 3 complement receptor-positive (CR3+) cells, predominantly macrophages. Depletion of T cell subsets in tumor-bearing mice showed the absolute dependence of the antitumor response on CD4+ cells, whereas tumor rejection was unaffected by depletion of CD8+ cells. In addition to CD4+ cells, CR3+ cells were also needed for tumor rejection. The antitumor effect of IL-7 was confirmed by expression of the IL-7 gene in a second tumor cell line of different cellular origin. Together, our results demonstrate that a high local IL-7 concentration at the tumor site obtained by tumor cell-targeted gene transfer leads to tumor rejection involving a cellular mechanism that seems to be different from the ones observed in analogous experiments with other cytokines.
... 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. ...
... 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.
... Now more commonly known as interleukin (IL)-7, its functional significance and therapeutic potential continue to be investigated. As a pleiotropic cytokine, IL-7 is involved in early B and T cell development (5)(6)(7)(8)(9)(10), growth, maintenance and differentiation of thymocytes (11)(12)(13)(14) and peripheral T-cell homeostasis (15)(16)(17). IL-7 expression has been detected in numerous types of tissue, including bone marrow (5), thymus gland (9,12), liver, kidney, spleen (8), intestine (18,19) and skin (6,19,20). ...
Methods of identifying chronic wounds that will heal in a timely, coordinated fashion and those that will not, together with novel therapeutic strategies, are vital for progression in the field of wound healing. Interleukin (IL)-7 has been associated with various biological and pathological processes. The present study explored the potential role of IL-7 in wound healing. IL-7 expression levels were examined in a clinical cohort of chronic wounds using reverse transcription-quantitative polymerase chain reaction and immunohistochemical staining analysis. The impact of recombinant human IL-7 (rhIL-7) on the growth and migrational rates of HaCaT keratinocyte cells was subsequently examined using in vitro growth and electric cell-substrate impedance sensing functional assays. The mRNA expression levels of IL-7 were increased in the healed chronic wound tissue samples, compared with non-healed chronic wound tissue samples, although the difference was not statistically significant. Similarly, immunohistochemical analysis revealed a greater staining intensity of IL-7 in the healed chronic wound tissue sections compared with the non-healed tissue sections. Treatment with rhIL-7 did not affect HaCaT cell growth rates, but was shown to enhance cell migration, an effect that could be further enhanced through the addition of inhibitors of neuronal Wiskott-Aldrich syndrome protein and protein kinase B. The data of the present study suggest that the expression levels of IL-7 may be increased in healing chronic wounds, and thus IL-7 may have a role in this process, potentially through its effects on the cellular migration of keratinocytes.
... 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.
... Recently, after the identification of a soluble molecule with pre-B cell growth factor activity, IL-7 was cloned (6)(7)(8) and shown to promote the proliferation of murine thymocytes (9)(10)(11), mature T cells (12)(13)(14), as well as the generation of lytic CTL with alloreactivity from thymocytes (15). We have recently shown that II.-7 was capable of generating antitumor CTL with similar cytolytic activity and enhanced specificity in StCr release when compared to those generated with IL-2 (Jicha, D. L., S. Schwarz, J. J. MulE, and S. A. Rosenberg, manuscript submitted for publication). ...
Interleukin 7 (IL-7) is a 25-kD cytokine that was initially described as a pre-B cell growth factor. This cytokine has also been shown to have T cell proliferative and differentiation effects. In this report, we demonstrate that antitumor cytotoxic T lymphocytes (CTL) generated by secondary in vitro sensitization of draining lymph node cells in IL-7 are effective in treating 3-day syngeneic methylcholanthrene (MCA) sarcoma pulmonary metastases in mice. In vivo titrations comparing IL-7 to IL-2 antitumor CTL show that they have equivalent potency in adoptive immunotherapy. IL-7 antitumor CTL generated against MCA sarcomas of weak immunogeneity are also tumor specific in their in vivo efficacy. This study represents the first successful use of a cytokine other than IL-2 for the generation of cells with in vivo efficacy in cellular adoptive transfer.
... 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. ...
... Thus, if signaling through IL-7R induces TCR␥ rearrangement, then both the stimulus for rearrangement and the enzymatic machinery necessary to carry out TCR rearrangement were present within the intestine of ATxBM B6 mice. Moreover, IL-7 and/or SCF synthesized by intestinal epithelium could have stimulated expansion of TCR␥␦ T cells with productive rearrangements, resulting in the substantial numbers of IEL isolated (17,31,47). ...
... Although this result could have been due to the ability of these cytokines to promote T cell proliferation, it is also consistent with the idea that these cytokines promoted activated T cell survival. This conclusion is supported by IL-4 being more active in vivo than IL-2, in spite of the fact that IL-2 is a better inducer of T cell division and by the fact that IL-7, a cytokine with limited ability to stimulate division of activated T cells (45), has effects that are similar to those of IL-4 (S.D. and A.T.V., unpublished observations). ...
Many antigen-specific T cells die after exposure to antigen in animals. These cells also die if they are isolated from animals
shortly after activation and cultured. Various cytokines were tested for their ability to interfere with this in vitro death. Surprisingly, tumor necrosis factor α and other inflammatory cytokines did not prevent the in vitro death of activated T cells, even though these cytokines do prevent activated T cell death in animals. Therefore, the inflammatory
cytokines probably act on T cells in vivo via an intermediary factor. Four cytokines, interleukin (IL)-2, IL-4, IL-7, and IL-15, did prevent activated T cell death
in vitro, with IL-4 and IL-15 more effective than IL-2 or IL-7. These cytokines share a component of their receptors, the common γ
chain, γc. Therefore, their collective ability to protect activated T cells from death may be mediated by signals involving
γc. To assess their activity in vivo, two of the cytokines, IL-2 and IL-4, were expressed in animals at local sites of superantigen responses. Both cytokines
increased the numbers of T cells found at the local sites 14 days later. Interleukin 4 was more effective than IL-2, even
though IL-2 stimulates T cell proliferation better than IL-4. This result suggested that IL-4 and related cytokines can promote
T cell survival in vivo as well as in vitro. The ability of these cytokines to prevent the death of activated T cells may be important at certain stages of immune responses
in animals.
... In contrast, IL-7Rα is lost soon after activation and its re-expression much later in the response correlates with development of memory 1. Functionally, however, differences between IL-7 and IL-15 activity are less clear. Both cytokines are found to promote survival 7, 10, 11 and proliferation 2, 7, 12 during formation and maintenance of memory. At a molecular level, both induce expression of the anti-apoptotic molecule, Bcl2 10, 13. ...
Interleukin (IL)-7 and IL-15 have non-redundant roles in promoting development of memory CD8(+) T cells. STAT5 is activated by receptors of both cytokines and has also been implicated as a requirement for generation of memory. To determine whether STAT5 activity was required for IL-7 and IL-15-mediated generation of memory, we expressed either wild type (WT) or constitutively active (CA) forms of STAT5a in normal effector cells and then observed their ability to form memory in cytokine replete or deficient hosts. Receptor-independent CA-STAT5a significantly enhanced memory formation in the absence of either cytokine but did not mediate complete rescue. Interestingly, WT-STAT5a expression enhanced memory formation in a strictly IL-7-dependent manner, suggesting that IL-7 is a more potent activator of STAT5 than IL-15 in vivo. These data suggest that the non-redundant requirement for IL-7 and IL-15 is mediated through differential activation of both STAT5-dependent and STAT5-independent pathways.
... As example, primary T cells placed in single-cell suspensions die within a few days of standard culture (Tan et al., 2001) or poorly proliferate when these cultures are augmented with IL-7 alone (Armitage et al., 1990;Rathmell et al., 2001). In fact, many in vitro studies report that IL-7 promotes proliferation when in the presence of additional signaling factors or co-stimulation (Grabstein et al., 1990;Costello et al., 1993;Tan et al., 2001). Hence, in the absence of other stimuli, in vitro studies of IL-7 have revealed only its survival function, while it is the in vivo studies that suggest a proliferative activity. ...
Interleukin-7 (IL-7) increases lymphocyte numbers, a critical feature of immune reconstitution, through mechanisms that are still poorly understood. Part of the problem is that IL-7 is produced in limited amounts by non-lymphoid cells, making in vivo studies of the cytokine's activity a challenge. To overcome this, we developed an in vitro system by which lymphocytes from secondary immune organs could be cultured to produce IL-7 responsive cells. Using this method, we showed that CD8(hi)CD44(hi) T cells accumulate in culture with IL-7 from a population of lymph node or splenic cells. These results were validated when a similar lymphocyte subset was found in mice expressing a constitutively active form of STAT5b, a key transducer of IL-7 signals. Interestingly, IL-7-expanded cells also up regulated the activation marker, CD69. The IL-7-derived CD44(hi)CD69(hi) cells were not generated from naïve cells, but expanded from an existing population, since culture in IL-7 of naïve lymphocytes from OT-1/Rag1(-/-) mice did not produce CD44(hi)CD69(hi) cells. Using the in vitro culture system to study lymphocytes from mice deficient in the apoptotic protein, BIM, we were able to attribute the expansion of CD8(hi)CD44(hi)CD69(hi) T cells to the proliferative and not survival activity of IL-7. The in vitro culture system provides an important new methodology to examine the activities of this essential as well as immunotherapeutic cytokine.
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.
Overview
IL-7 is a member of the family of cytokines with four anti-parallel α helixes that bind Type I cytokine receptors. It is produced by stromal cells and is required for development and homeostatic survival of lymphoid cells.
Genomic architecture
Interleukin 7 (IL7) human IL7: gene ID: 3574 on ch 8; murine Il7 gene ID: 16,196 on ch 3.
Protein
Precursor contains a signal sequence, mature human IL-7 peptide 152aa, predicted 17.4kd peptide, glycosylated resulting in 25kd. Crystal structure: http://www.rcsb.org/structure/3DI2.
Regulation of IL-7 production
Major producers are stromal cells in thymus, bone marrow and lymphoid organs but also reported in other tissues. Production is primarily constitutive but reported to be affected by IFNγ and other factors.
IL-7 receptors
Two chains IL-7Rα (IL-7R) and γc (IL-2RG). Human IL-7R: gene ID 3575 on ch 5; human IL2RG: gene ID 3561 on ch X; mouse IL-7R: gene ID 16,197 on ch 15; murine Il2rg gene ID 16,186 on ch X. Member of γc family of receptors for cytokines IL-2, −4, −9, −15, and −21. Primarily expressed on lymphocytes but reports of other cell types. Expression in T-cells downregulated by IL-7. Low expression on Tregs, no expression on mature B-cells. Crystal structure: http://www.rcsb.org/structure/3DI2.
IL-7 receptor signal transduction pathways
Major signals through JAK1, JAK3 to STAT5 and through non-canonical STAT3, STAT1, PI3K/AKT and MEK/ERK pathways.
Biological activity of IL-7
Required for survival of immature thymocytes, naïve T-cells, memory T-cells, pro-B-cells and innate lymphocytes. Pharmacological treatment with IL-7 induces expansion of naïve and memory T-cells and pro-B-cells.
Abnormalities of the IL-7 pathway in disease
Deficiencies in the IL-7 pathway in humans and mice result in severe combined immunodeficiency due to lymphopenia. Excessive signaling of the pathway in mice drives autoimmune diseases and in humans is associated with autoimmune syndromes including multiple sclerosis, type 1 diabetes, rheumatoid arthritis, sarcoidosis, atopic dermatitis and asthma. Mutations in the IL-7 receptor pathway drive acute lymphoblastic leukemia.
Clinical applications
IL-7 has been evaluated in patients with cancer and shown to expand lymphocytes. It accelerated lymphocyte recovery after hematopoietic stem cell transfer, and increased lymphocyte counts in AIDS patients and sepsis patients. Monoclonal antibodies blocking the IL-7 receptor are being evaluated in autoimmune diseases. Cytotoxic monoclonals are being evaluated in acute lymphoblastic leukemia. Drugs blocking the signal transduction pathway are being tested in autoimmunity and acute lymphoblastic leukemia.
Interleukin-7 (IL-7) is a potent stimulator of pre-B-lymphocyte proliferation. Pre-B cells transformed by a variety of oncogenes including those of the ABL protein tyrosine kinase family were screened for endogenous IL-7 mRNA expression by polymerase chain reaction and a sensitive bioassay for secreted IL-7. Some v-abl but none of the BCR/ABL, v-src, v-fms, v-myc, v-ras, or v-raf transformants analyzed contained elevated IL-7 transcripts. None of the cell lines secreted detectable bioactivity. We overexpressed IL-7 via a retroviral vector in an IL-7-dependent pre-B cell line to assess the potential for autocrine growth stimulation and malignant transformation. We achieved dramatic deregulation of IL-7 translational suppression by removing portions of the 5' flanking region. Levels of IL-7 expression much greater than those needed to establish factor-independent growth did not induce colony formation in agar by IL-7-expressing pre-B cell lines, and the majority of these lines were nontumorigenic in syngeneic mice. The same pre-B cell line transformed by v-abl displayed a highly malignant phenotype while containing dramatically lower IL-7 transcript levels. We conclude that endogenous IL-7 expression is not a necessary event in transformation of pre-B cells, nor is it sufficient to explain the malignant phenotype in v-abl-transformed cells. Up regulation of endogenous IL-7 expression in some transformed pre-B cells may be one of several synergistic events which can lead to malignant conversion.
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.
Studies on human B-cell development have been hampered by the lack of reproducible culture techniques to induce pre-B cells to differentiate into Ig-secreting plasma cells. Here, we describe that highly purified surface (s) mu-, cytoplasmic (c) mu+, CD10+, CD19+ human pre-B cells derived from fetal bone marrow (BM) differentiate with high frequencies into Ig-secreting plasma cells, when cocultured with activated, cloned CD4+ T cells and with interleukin-4 (IL-4). Production of IgM, total IgG, IgG4, and IgE in pre-B-cell cultures was detected, indicating that the cells also underwent Ig isotype switching. Pre-B-cell differentiation occurred in the absence of BM stromal cells, IL-7, and stem cell factor (SCF). However, IL-7 significantly enhanced the levels of Ig produced, whereas SCF was ineffective. Neutralizing anti-IL-4 monoclonal antibodies (MoAbs) completely inhibited pre-B-cell differentiation showing the specificity of the reaction. Intact CD4+ T- cell clones could be replaced by membrane preparations of these cells, indicating that the costimulatory signals provided by the activated CD4+ T cells are contact-mediated. In contrast, anti-CD40 MoAbs failed to provide the costimulatory signal required for pre-B-cell differentiation, which may be related to the very low expression of CD40 on fetal BM B cells. Activated CD4+ T cells and IL-4 also induced s mu expression and Ig synthesis in cultures initiated with pre-B cells that had been preincubated in medium for 2 days, and from which spontaneously emerging s mu+ B cells were removed by using a fluorescence-activated cell sorter. These results support the notion that the Ig synthesis observed in pre-B-cell cultures was not caused by outgrowth and differentiation of cells that spontaneously matured into s mu+ B cells. In addition, IL-4 and CD4+ T cells strongly enhanced CD40 and HLA-DR expression on the majority of cultured pre-B cells, further indicating that CD4+ T cells and IL-4 activate bona fide pre-B cells. Taken together, these data indicate that activated CD4+ T cells and IL-4 can provide all the necessary signals required for human pre-B cells to differentiate into Ig-secreting plasma cells.
The biologic effects of interleukin-7 (IL-7) and the expression of specific IL-7 membrane receptors on isolated neoplastic cells from previously untreated patients with chronic lymphocytic leukemia as well as acute leukemias were investigated in vitro. Leukemic cells were incubated for up to 6 days with various concentrations of IL-7 (0.01 to 2,000 U/mL). Neoplastic cells of the T- or B-phenotype from chronic as well as from acute leukemias proliferated in a dose-dependent manner. Cells from acute myeloid leukemias also proliferated in response to IL- 7. An optimal proliferative effect was achieved between 96 and 120 hours with 200 U/mL IL-7. Combinations of IL-7 with IL-2 and tumor necrosis factor-alpha showed an additive effect on [3H]TdR incorporation. IL-7 binding assays gave a value of approximately 33 to 180 high-affinity (kd approximately 20 pmol/L) binding sites/cell and approximately 241 to 3,280 low-affinity (kd approximately 600 pmol/L) binding sites/cell. Receptor expression correlated with the proliferation in response to IL-7. These data indicate that IL-7 can induce proliferation of relatively mature tumor cells, and that this effect is not restricted to the lymphoid lineage.
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.
Previous thymic studies detected a subcapsular A2B5 + and Thy-1+, TE4+, Vimentin+, Cytokeratin+, so-called “endocrine” RE cell or nurse cell (TNC) subpopulation within the cortical RE cell network. Secretion of multiple in situ active, autocrine growth factors and a humoral chemotactic factor by the cells of ectomesenchymal origin allows the commencement of immigration of hematopoietic stem cells. The thymic lymphopoiesis is initiated by the immigration of pluripotent (with cellular immunophenotype TdT+, Ki67+, CD3−, CD7+, CD34+, CD38+, CD44+, CD45+ or T200+), but already to T lymphocyte cell lineage committed hematopoietic stem cells during the 6–7th week of ontogenesis. CD2, a 50–55 kD, glycoprotein is the first intrathymic, early differentiation antigen expressed during the 8–9th ontogenetic weeks. This antigen also serves as a cell surface component of the alternative or antigen independent pathway of thymocyte activation. The 10th week is defined as the first expression of CD4 and CD8 antigens, which determine the basic, characteristic dichotomy of the T lymphocytes. The induction of the initial proliferative wave of immature cortical thymocytes is carried out by the LFA-3 (CD58) adherence molecules, the receptors of CD2 antigens located on reticuloepithelial cells. Because of the extremely high proliferation rate the thymic mass markedly expands in all dimensions, numerous microlobules are formed. Between the 13th to 16th week, the typical thymic cell environment is formed and the first Hassall’s bodies are developed. The outer layer of the Hassall’s bodies contains hypertrophized TE8+, TE16+ and TE19+ reticulo-epithelial cells, with an active secreting cytoplasmic structure.
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.
Aim: To study induced phosphorylation and activation of JAK3 in cutaneous T-cell lymphoma (CTCL) by interleukin-7 ( IL27) . IL27 stimulates proliferation of several benign and malignant lymphocyte populations including tumor cells from patients with CTCL. The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors A novel JAK family member JAK3 ,which is expressed in natural killer (NK) and activated T cells ,is coupled functionally and physically with the interleukin-2 ( IL-2) receptor ( IL-2R) in those cells.Methods :Cells isolated from peripheral blood of CTCL patients were treated with IL-7 in vitro for 1 ,5 ,10 ,15 and 20 min ,respectively. Immunoprecipitating the lysates was performed with anti-JAK3 and anti-p59fyn antibodies and Western blotting with anti-phosphorylated tyrosine and anti-JAK3 antibodies. Re sults : IL-7 induced tyro
sine phosphorylation of JAK-3 in CTCL cells. Anti-p59fyn antibody co-immunoprecipitated with JAK3. But it was not found that IL-12 could induce phosphorylation of JAK3. Conclusion : These results suggest that lL-7 may play a role in pathophysiology of CTCL ,and indicate that the IL-7 receptor ( IL-7R) mediates the activation of the tyrosine phosphorylation signal transduction pathway.
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.
Cytokines, chemokines and their receptors are important factors that influence the pathogenesis of HIV-1 in vivo. HIV-1 infection stimulates the production of cytokines and chemokines from a great variety of cell types, which may either induce or inhibit viral replication. Furthermore, chemokine receptors act as viral co-receptors for viral entry into cells. Co-receptor expression and co-receptor availability are important determinants of the susceptibility to infection. Cytokines and chemokines modulate coreceptor expression and thus influence HIV pathogenesis. In this review, we describe the importance of the distribution of chemokines and their receptors in the primary infection and in the later evolution of the disease, as well as the effect of relevant cytokines in viral replication and in the regulation of immune cell homeostasis. Finally, we also discuss the use of cytokines and chemokines as therapeutic agents.
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 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.
Great strides have been made in the chemotherapeutic treatment of acute lymphocytic leukemia (ALL) and moderate success has been made in treatment of acute myelogenous leukemia (AML). At this time, progress using chemotherapy has plateaued. Novel approaches to these diseases are required. Recent experiments have shown that several poorly immunogenic solid tumors can be recognized by MHC-class I restricted CD8+ cytotoxic T lymphocytes (CTL) if the tumors are engineered by genetransfer to produce one of several cytokines, including IL-2, IL-4, IL-6, IL-7, GM-CSF, TNF-α, IFN-α and -γ, or B7/BB1. The local secretion of lymphokines, critical for CTL activation, appears to bypass a deficient helper T-cell arm of the immune system. In addition, secretion of cytokines by the tumor cells stimulates the host immune system to identify and kill the untransduced parental cells upon subsequent reimplantation; and even of potential more importance, a rejection of established cancer can occur by inducing a systemic anticancer immune response by vaccination with cytokine transduced tumor cells. We studied two different murine myeloid leukemia models using WEHI3 and C1498 cell lines, transduced with a retroviral vector coding for human IL-7 (JZEN hIL/tk neo), resulting in cytokine production up to 13 ng/106 cells/24 hrs. NIH-3T3-fibroblasts, transduced with a vector coding for human IL-2 (G1Na CV hIL-2), producing up to 21 ng/106 cells/24 hrs, mixed with parental WEHI3 leukemia were used for vaccination-studies as well. Vaccination with IL-7 producing WEHI3 clones (weekly s.c. injections of 106 cells over a period of one month) resulted in a 43% survival of mice, subsequently challenged with a lethal dose of parental leukemic cells (5]104 i.v.). Vaccination with a mixture of IL-2 producing NIH-3T3-fibroblasts and parental WEHI3 cells resulted in a systemic protection of 60% of lethally challenged mice. The same experiments performed with the C1498-model showed a strong inherent immunogenicity of irradiated parental cells, as 4 out of 5 mice survived in this group. Surprisingly, all mice died in the group vaccinated with an IL-7 producing subclone. This illustrates that the process of selecting a high-cytokine producing subclone can result in clones that no longer represent the antigenic spectrum of the parental cells.
Taken together, we show that induction of a specific anti-leukemia immune response may be effective treatment for AML, especially when the tumor burden is low.
Cytotoxic T lymphocytes (CTL) were induced from the peripheral blood mononuclear cells of 6 cancer patients using tumor necrosis factor-α (TNF α). Initially, TNF α (100 and 1, 000U/ml) was added at the beginning of mixed culture with tumor lymphocytes for 3 days. Then, TNF α was removed and the cells were stimulated with an immobilized anti-CD3 monoclonal antibody (1μg/ml) and interleukin-2 (1, 000U/ml) for 3-7 days.
Subsequently, the anti-CD3 antibody was removed, and the cells were incubated with 500U/ml of interleukin-2. Cells from 2 patients showed autologous tumor-killing activity before incubation with TNF α, and this activity was unchanged by TNF α. In 2 of the 4 patients without pre-existing cytotoxic T lymphocyte (CTL) activity, there was an increase of this activity after incubation with TNF α. Both CTL activity and CTL proliferation increased in accordance with the TNF α concentration.
Flow cytometric analysis revealed an increase of CD8+Leu15- cells. However, the increase in cytotoxic activity also occurred when allogeneic cancer cells were identified as target cells, suggesting that TNF α did not increase tumor specificity although it increased autologous tumor-killing acitivity. Since a high CTL activity specific to autologous tumor cells is necessary for successful CTL therapy, this subject requires further investigation.
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.
Interleukin-7, originally described as a factor controlling the survival of B-cell progenitors, has been shown by gene knock-out technology to be a non-redundant cytokine. Of all single cytokine knock-out mice, those in which the IL-7 gene has been ablated show a profound defect in lymphocyte development. Likewise, mice in which signals emanating from the corresponding receptor, whether it be by ablation of the unique alpha or common gamma chain of the receptor, or by interference with downstream signalling elements generated by this receptor complex, also show profound defects in lymphocyte differentiation. Transgenic mice over-expressing the IL-7 gene also show profound changes in lymphocyte development which, in some instances can result in the development of lymphoid tumours. Here, we review some of these aspects of IL-7 biology with particular reference to an IL-7 over-expressing transgenic mouse line in which the IL-7 transgene is controlled by the mouse MHC class II promoter.
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.
Interleukin-7 (IL-7) is essential for both T cell and B cell development. Recent studies have suggested that IL-7 also functions as a survival-promoting factor for resting and activated T cells. In this study we examined the effects of IL-7 on survival and cytotoxicity of tumor-specific CD8+ cytotoxic T lymphocyte (CTL) clones established and maintained either with IL-2 alone or with a combination of IL-2 and IL-7. While the CTL clones cultured in IL-2 alone died around day 10, the CTL clones cultured in the presence of IL-2 and IL-7 survived for more than 4 weeks after seeding. The long-term survival of the latter was correlated with the presence of IL-7 in the medium. In addition, IL-7 alone prolonged survival of other IL-2-dependent CTL clones after the removal of IL-2. IL-7 maintained the CTLs in G1 arrest after a slight proliferation during the initial phase during which low-level but sustained DNA synthesis was observed. However, there was no direct correlation between DNA synthesis and enhancement of long-term survival by IL-7 as demonstrated by the inhibiting proliferation of the CTL clones with the protein kinase inhibitor genistein. During long-term survival in the presence of IL-7, the cytotoxic activities of the CTL clones decreased gradually to background levels although they were restored soon after the next passage. These results suggested that IL-7 had the ability to set machinery in motion against apoptosis in the IL-2-dependent CTL clones. Such an effect of IL-7 might play a role in vivo in the process leading activated T cells to the resting, that is, memory state.
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.
The low-affinity receptor for IgE, CD23, has been described in several pathological conditions. However, the factors involved in the upregulation or downregulation of this receptor are still debated. We studied the effect of interleukin 7 (IL-7) on the production of CD23 in normal PBT cells stimulated with PMA + Ca2. The results demonstrate that cytoplasmic CD23 level was significantly augmented by costimulation with PMA + Ca2plus IL-7 (1000 U/ml). Using an intracytoplamatic cytometric analysis, an accumulation of intracellular CD23 was observed at 48 hr in the presence of IL-7. This appears to have a profile different from the CD23 surface expression peaking at 72 hr of culture. We were also able to show that sCD23 was specifically increased by IL-7 and occurred with an early peak at 72 hr and a late peak at 120 hr of culture. The increased release and the biphasic production of sCD23 may reside in an accelerated degradation of the receptor due to an excessive accumulation of it. Restimulation of CD4+T cells with PMA + Ca2without IL-7 changed the profile of sCD23 production showing a second peak at 144 hr of culture. The induction of IL-7 on CD23 production appears to be independent of IL-2, IL-4, IL-9, and IL-15. Indeed, the addition of specific mAbs anti-IL-2, -IL-4, -IL-9, -IL-15, or anti-IL-2R was unable to block the effect of IL-7 on CD23. The addition of IL-7 to specific subset CD4+CD23+was able to augment the adhesiveness of T cells to parenchymal cell monolayers. The use of different cytokine (IL-2, IL-4, IL-9, IL-15) resulted in no increase of adhesiveness. In contrast, the addition of IL-7 to a different T cell subset (i.e., CD4+CD23−) was unable to rescue the lack of adhesiveness observed in these cells. The adhesion molecules LFA-1 and VLA-4 were responsible for the augmented adhesiveness of activated CD4+CD23+T cells cultured in the presence of IL-7. Blocking experiments with anti-LFA-1β, VLA-4α, anti-LFA-1β plus VLA-4α mAbs, or anti-ICAM-1 mAb added to the monolayers resulted in a complete inhibition of adhesion to parenchymal monolayers. In contrast, the addition of anti-IL-7 or anti-IL-7R mAbs was able to block the augmented adhesiveness of CD4+CD23+cells to monolayers observed in the presence of IL-7. A significant augmentation of LFA-1 and VLA-4 was observed in cells cultured in the presence of IL-7. Taken together these findings point to the likelihood that IL-7 is responsible for the observed quantitative difference in the level of adhesion molecules and may open a new role of CD23 in the immune regulation.
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.
The chapter focuses on the common γ chain for multiple cytokine receptors. The signal transducers are composed of both common and specific molecules for the cytokines, resulting in overlapping and pleiotropic functions on various target cells in similar situations to other cytokines that share the common receptor subunits. Elucidation of the modes of signal transduction from IL-2Rγ together with the accompanied subunits will lead to the demonstration of regulatory mechanisms of early T-cell development as well as cellular responses to the ligands. Since no intrinsic effectors function has been seen with cytokine receptors sharing IL-2Rγ, signal transducers are assumed to be associated with the cytoplasmic domains of the receptor subunits. Molecular identification of IL-2Rγ has led to knowledge for understanding the structures and signal-transducing functions of various cytokine receptors, and particularly has made a great contribution toward elucidation of the molecular mechanisms of human XSCID occurrence. IL-BRγ, so-called as the “γc-chain”, is utilized as a common receptor subunit among multiple cytokines such as IL- 2, IL-4, IL-7, IL-9, and IL-15. Mutations of IL-2Rγ in patients with XSCID cause dysfunction of these cytokines, resulting in impairment of early T-cell development, a typical feature of XSCID. These cytokine receptors contain specific subunit(s) for each receptor along with the γc-chain. The γc-chain participates in increasing ligands-binding affinities, except for IL-9, and in intracellular signal transduction for all these cytokines.
Activation of human peripheral blood T cells renders them capable of proliferating to IL-2, -4, and -7, and upregulates the receptors for IL-2 and -4. In this study the effect of activation on the receptor for IL-7 has been investigated. Scatchard analysis showed dual affinity binding of IL-7 to peripheral blood mononuclear cells (PBMC). Furthermore, activation of PBMC with anti-CD3 antibodies resulted in a 4-fold downregulation of both the high and low affinity IL-7 receptors. SDS - PAGE analysis of [I-125]IL-7 cross-linked resting PBMC revealed a major complex of 104/107 kDa (reduced/non-reduced) and a minor complex of 184/178 kDa (reduced/non-reduced). In contrast, cross-linking of activated PBMC revealed a third prominent complex of 93 kDa (non-reduced) not seen on unstimulated cells. This 93 kDa complex was observed on purified activated peripheral blood T cells and T cell blasts. Moreover, on a panel of IL-7 responsive T cell clones the 93 kDa complex was the only major cross-linked product observed. These results demonstrate that T cell activation causes changes in both the level of expression of the IL-7 receptor and the nature of the proteins associated with the receptor. It is postulated that these changes in receptor structure may be related to the acquisition of responsiveness to the IL-7 growth signal.
Summary Interleukin 7 (IL-7) was originally identified as a growth factor for B cell progenitors, and subsequently has been shown to exert proliferative effects on T cell progenitors and mature peripheral T cells as well. Constitutive IL-7 mKNA expression so far had been demonstrated in bone marrow stromal cell lines, thymus, spleen, and among nonlymphoid tissues in liver and kidney. Here we show that both murine and human keratinocytes express IL-7 mRNA and release IL-7 protein in biologically relevant amounts. The physiological or pathological relevance of keratinocyte- derived IL-7 is presently unknown. Our finding that keratinocytes can produce IL-7 in concert with reports that IL-7 is a growth factor for in vivo primed antigen-specific T cells, as well as for T lymphoma cells suggests, however, that keratinocyte-derived IL-7 is important in the pathogenesis of inflammatory skin diseases and cutaneous T cell lymphoma.
Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV- positive patients. There was a negative correlation between circulating IL-7 levels and CD4 T-cell count in HIV-positive patients (r 0.621; P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/l than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.
We have recently shown that activation of T cells causes structural changes in the interleukin-7 receptor (IL-7R) (Foxwell et al. Int. Immunol. 1992. 4: 277). Unactivated cells expressed a receptor characterized as a cross-linked protein of 107-kDa whereas activated cells had reduced levels of this 107-kDa complex and now express a major cross-linked product of 93 kDa. These changes in receptor expression were concomitant with the acquisition of IL-7 growth responsiveness by activated T cells. In this study, the effect of the potent immunosuppressive agents cyclosporin A and FK506 on the activation-induced responsiveness to IL-7-driven proliferation and the concomitant changes in receptor structure have been investigated. Cyclosporin A and FK506 suppressed the expression of the 93-kDa complex and the loss of the 107-kDa complex on activated cells. The presence of exogenous IL-7 inhibited the effects of the drugs on IL-7R structure, allowing expression of the 93-kDa complex. Expression of the 93-kDa complex could also be induced either by ionomycin or phorbol esters. As observed for other T cell activation parameters, only those which induced a calcium signal (ionomycin) but not protein kinase C (phorbol esters) were sensitive to the drugs. In all studies, the expression of the 93-kDa complex correlated with the ability of cells to proliferate to IL-7, and thus these results further support the hypothesis that the 93-kDa form of the IL-7R is required to transmit the cytokine's growth signal. Moreover, these data suggest that activation-induced transcriptional events are required for the expression of the 93-kDa complex and the down-regulation of the 107-kDa complex. As reported for IL-2R and IL-4R, our data also show that the expression of another T cell growth factor receptor is sensitive to the effects of cyclosporin A and FK506. These observations also have important implications for reported cyclosporin A effects on the thymus where IL-7 can act as a growth factor for thymocytes.
Previous studies have documented the effects of IL2 on the growth and effector function of tumor-infiltrating lymphocytes (TIL) in cancer patients. Since IL7 is known to induce T- and NK-cell responses in the peripheral blood, we examined the immuno-enhancing effects of IL7 on TIL derived from human renal-cell carcinoma (RCC). Whereas IL2 induced the growth of freshly isolated TIL in vitro, IL7 was ineffective alone and failed to increase the total number of cells proliferating to IL2. However, IL7 did provide a proliferative signal to TIL that were initially expanded in culture with either IL2 or IL2/IL7 for 2 weeks. IL7 also induced the proliferation of CD4+ and CD8+ TIL lines that have specificity for RCC. The proliferative response induced by IL7 was independent of IL2, since anti-IL2 antibodies did not block IL7-induced proliferation of TIL. IL7 did cooperate with anti-CD3 stimulation for the induction of proliferation; however, the magnitude of this interaction was variable and the response usually additive. In addition, IL7 synergized with anti-CD3 to induce the secretion of IFNγ from short-term-cultured TIL and from a TIL line. Although IL7 did not promote the development of a tumor-specific T-cell response from IL2-expanded TIL, IL7 enhanced lymphokine-activated killer (LAK) activity from some short-term-cultured TIL. These results illustrate that IL7 can potentiate the growth and production of IFNγ from RCC-reactive TIL and, to a lesser extent, enhance IL2-induced LAK activity of TIL.
Expressed on leukocytes, beta2 integrins (CD11/CD18) are involved in leukocyte function and trafficking. Using a CD18-deficient (CD18-/-) mouse model, the role of beta2 integrins for lymphocyte differentiation and recirculation was herein investigated. CD18-/- mice revealed a defect in distribution of naïve lymphocytes, which were reduced in numbers in axillary and inguinal lymph nodes (LN). In contrast, cervical LN (cLN) were signi¬ficantly enlarged and harbored elevated numbers of unconventional T cell receptor (TCR) alpha/beta or TCR gamma/delta double negative (DN) T cells. As demonstrated by adoptive transfer experiments, a selective homing of CD18-/- lymphocyte to pLN of wild type recipients was not the reason for this increase in cellularity. But unconventional DN T cells were found to be increased in cLNs due to a massive local expansion demonstrated by in vivo incorporation of thymidine analogue BrdU. In accordance, CD18-/- DN T cells showed an activated phenotype and recirculated through non-lymphoid organs resembling anti¬gen-experienced T and NKT cells. CD18-/- TCR alpha/beta DN T cells readily pro¬liferated upon IL-2, IL-7 and IL-15, known to regulate homeostasis or antigen-induced expansion of NKT and TCR gamma/delta T cells. In accordance with the assumption that CD18-/- TCR alpha/beta DN T cells are expanding effector cells, they also lacked suppressive function on proliferating WT T cell in vitro. Interestingly, CD18-/- TCR alpha/beta T cells revealed general characteristics of NKT cells but lacking re¬activity for CD1d/alpha-galactosylceramide showed that they were not type I NKT cells. Nevertheless, the possi¬bility that CD18-/- TCR alpha/beta DN T cells are a different subset of NKT cells cannot be fully excluded, currently. However, lack of IL-4 and IFN-gamma cytokine secretion after stimulation showed that CD18-/- TCR alpha/beta DN T cells may have different functional properties to NKT and TCR gamma/delta T cells in innate im¬munity. Collectively, DN T cells accumulated either due to antigen-dependent and/or homeostatic expansion in the periphery, likely driven by a cytokine imbalance in CD18-/- mice. The presented data indicates that CD18-/- TCR alpha/beta DN T cells, like NKT and TCR gamma/delta T cells, may be a unique subset of unconventional T cells at the interface to innate immunity accumulating compensatory to the impaired function of adaptive immunity in CD18-/- mice.
Drugs targeting memory lymphocytes may allow for a better control of rejection in transplantation, particularly in immunized patients. In this article the rationale of targeting interleukin 7 receptor alpha (IL-7Ralpha), a molecule expressed by both memory and naive T cells, is reviewed in the context of transplantation. Whereas naive T cells are partly responsible for acute rejection and are targeted by current immunosuppressive drugs that block costimulatory signals (cyclosporine A, anti-CD3 antibody, anti-CD52 antibody, anti-thymocyte globulin, etc.), memory T cells are resistant to costimulation blockade. As such, memory cells are an obstacle to experimental tolerance induction and may be involved in chronic rejection. There is thus much scientific interest in developing molecules able to target these cells. The role of the IL-7/IL-7Ralpha pathway in transplantation rejection has been suggested by the effect of an anti-IL-7 monoclonal antibody which, when associated with costimulation blockade, prolonged heart allograft survival in mice. Here the hypothesis that targeting IL-7Ralpha would preserve effector T cells that are less dependent on IL-7 for survival while sparing regulatory CD4+ CD25high IL-7Ralpha(low) T cells is discussed. An anti-IL-7Ralpha antibody could also help achieve allograft tolerance by reducing alloreactive cells.
The induction of cytokine secretion by human peripheral blood (PB) T cells was examined. Highly purified T cells stimulated with interleukin 7 (IL-7), in the absence of co-mitogen, secreted IL-2, IL-4, IL-6 and interferon gamma (IFN-gamma) upon restimulation with phorbol ester and ionomycin. In contrast, induction of T-cell cultures initiated with IL-2 or IL-4 yielded only low levels of IL-6 and virtually undetectable levels of IL-4 or IFN-gamma, while IL-2 secretion was reduced. No difference was seen in the ability of CD4+ and CD8+ subpopulations, grown in IL-7, to produce cytokines. In contrast, subdivision of T cells into memory and naive populations using the CD45RO monoclonal antibody (mAb) UCHL1, revealed that almost all of the potential to secrete IL-4 and IL-6 in response to IL-7 resided in the CD45RO+ memory population. Stimulation of cytokine-secreting cells appeared to be a direct effect of IL-7 as neutralizing antibodies directed against IL-2 and IL-4 had no effect on the levels of cytokines produced. The differences observed in the ability of IL-2, IL-4, and IL-7 to potentiate cytokine production was supported by measurement of cytokine mRNA levels by PCR. The elevated levels of cytokine secretion seen in cells cultured with IL-7 was not due simply to increased viability in these cultures compared with those containing IL-2 or IL-4, as these populations showed comparable cloning frequencies in phytohemagglutinin (PHA) + IL-2. These results demonstrate that IL-7, in the absence of co-mitogen, is a potent initial stimulus for multiple cytokine production by human T cells upon restimulation.
Current assays for chicken interleukin-2 (IL-2) utilize mitogen-activated lymphocytes. However, very high inter-assay variability and sporadic high background proliferation limit their usefulness. In view of the above, several Marek's disease virus (MDV)-transformed T-cell lines (which grow well in a serum-supplemented medium) were tested for a response to chicken IL-2 when grown in serum-free media. Five of six lines examined showed a dose-dependent proliferative response to chicken T-cell conditioned media. One line, MDCC-CU14, was chosen for further studies. In addition to the tumor cells' dose-dependent responses to semi-purified chicken IL-2, they expressed T-cell activation antigens on the cell surface. Furthermore, the level of surface expression was enhanced on cells provided IL-2. Co-incubation of the tumor cells with monoclonal antibody INN-CH-16 (specific for an antigen on the surface of activated T-cells) and IL-2 resulted in a modulation of lymphokine-induced proliferation. Together, these data suggest that signalling mechanisms in MDV T-cell tumors are intact and that these lines can be used as an assay for chicken T-cell lymphokines. Furthermore, they provide an interesting model for the study of avian and mammalian T-cell transformation. Implications for the study of Marek's disease are also discussed.
Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.
Recently, we described a murine helper T cell-derived molecule with T cell growth factor activity that is functionally and structurally distinct from IL-2, IL-4, and other known growth factors. This molecule, designated P40, was identified as a glycoprotein capable of supporting antigen-independent growth of certain helper T cell clones. Here, we report the cloning and expression of a cDNA for this new growth factor. The predicted mature protein is a cationic cysteine-rich polypeptide of 14 kD without significant homology to previously sequenced proteins.
Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell-stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes.
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.
Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative ⁵¹Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20–40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures.
When spleen cells from mice immunized with allogeneic tumor cells 2–4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm.
Effective supernatants (SUP), which potentiate mouse T-cell responses to phytohemagglutin (PHA), are obtained from cells of several species (human, rabbit, rat, mouse) and indeed from syngeneic spleen, thymus, or bone marrow cells. Unstimulated cells release some SUP activity but more is produced after stimulation. Lipopolysaccharide (LPS) produced very active SUP in all cultures tested. PHA was similarly active on human leukocytes only, whereas concanavalin A (Con A) gave highly efficient SUP only with mouse spleen cells. SUP production is not correlated with a mitotic response of the donor cells and is observed in cultures unable to respond mitotically to the stimulant. Adherent mouse spleen cell populations, consisting largely or entirely of macrophages, produce active SUP, while nonadherent cells do not. Similarly, purification of human peripheral leukocytes on nylon columns, with removal of macrophages and other adherent cells, destroys their ability to produce SUP. The importance of indirect effects in stimulating mitotic responses of T cells is emphasized by the fact that the mitotic response of mouse thymocytes to LPS and its ability to potentiate the response of these cells to PHA disappears with removal of adherent cells from the thymocyte population. Conversely the production of SUP from spleen cells stimulated by Con A requires the presence of T cells.
We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.
The roles of Ia+ accessory cells in H-2-restricted stimulation of antigen-specific T cell proliferation were explored in an in vitro model. L-glutamic acid60-L-alanine30-L-tyrosine10-(GAT) primed BALB/c nylon wool-passed T cells were depleted of Ia+ antigen-presenting cells (APC) by treatment with monoclonal anti-Ia antibody plus complement. Such cells failed to respond to soluble GAT, or to soluble GAT in the presence of phorbol myristic acetate (PMA), which is known to stimulate production of, or replace, IL-1 in vitro. Addition of gamma-irradiated syngeneic spleen cells reconstituted the response to soluble GAT, but addition of ultraviolet (UV) light-irradiated spleen cells did not, even in the presence of PMA. Preincubation of cells with GAT for 24 hr, followed by washing, then gamma irradiation, generated a cell population able to stimulate GAT-primed T cells to proliferate. The same pulsed cells exposed to UV irradiation failed to stimulate T cell responses unless PMA was added to the cultures. The relevant cells in this UV-irradiated population are Ia+. It is concluded that a finite period of time for interaction of metabolically intact APC with antigen is required before creation of an appropriate (Ia + antigen) signal recognized by the T cell. In addition to such Ia-restricted antigen presentation, however, a 2nd nonspecific signal, again requiring metabolically active APC for elaboration, is necessary for detectable T cell activation. These studies thus define 3 separable activities of APC during the process of H-2 restricted T cell activation.
A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.
Macrophages handle extracellular proteins and secrete diverse bioactive molecules and, therefore, influence the physiology
of many tissues. They also have an important immunoregulatory role. The immune response to proteins involves the activation
of the T helper subset of lymphocytes. The T helper cell is activated only when it interacts with the protein displayed on
the surface of a macrophage or other accessory cell. This interaction involves restrictive proteins encoded in the major histocompatibility
gene complex as well as growth-differentiating proteins.
Interleukin 7 (IL-7) is a 25-kDa cytokine which was purified and its corresponding cDNA was cloned based upon its ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also function as a costimulator with Con A for the proliferation of T lymphocytes by inducing the production of interleukin 2 (IL-2). We demonstrate here that IL-7 in combination with phorbol 12-myristate 13-acetate can directly drive the proliferation of purified T cells and that this response is not inhibited by cyclosporine A or by antibodies to IL-2 and IL-4. Stimulation of T cells with phorbol myristate acetate and IL-2, IL-4, or IL-7 prepared T cells to respond to any of the three lymphokines. Although T cells activated in vitro by anti-CD3 or allogeneic cells failed to proliferate when challenged with IL-7, T cells primed in vivo to the same stimuli demonstrated a significant proliferative response when restimulated in vitro with IL-7. IL-7 can, therefore, function both as a growth factor for T cells in an IL-2-independent manner and as a competence factor for the induction of lymphokine responsiveness. The ability to induce IL-7 responsiveness via stimulation of the T-cell receptor complex in vivo, but not in vitro, raises the possibility that IL-7 may play a role in T-cell growth and differentiation in vivo.
Human peripheral blood T lymphocytes were treated with recombinant interleukin 2, mitogens, and dexamethasone. The resulting accumulation of mRNA for interleukin 2 (IL 2), the interleukin 2 receptor (IL 2R), and interferon-gamma (IFN-gamma) was measured. IL 2 was found to regulate the levels of each of these mRNA. The expression of mRNA for IL 2, IL 2R, and IFN-gamma correlated very well with the levels of protein observed. In populations of peripheral blood T lymphocytes, the production of IL 2 and IFN-gamma were not necessarily coordinately expressed. The sequential expression of these mRNA was investigated in order to determine whether they might be independent of the action of IL 2. IFN-gamma and IL 2 mRNA showed biphasic accumulations. IL 2 mRNA accumulated very rapidly, within 60 min after mitogen stimulation and before any detectable IL 2R mRNA accumulation. Similarly, IFN-gamma mRNA accumulated rapidly, simultaneously with IL 2 mRNA. This early peak of IFN-gamma mRNA, therefore, is likely to be independent of IL 2 action. Both IL 2 and IFN-gamma mRNA then showed later peak times of accumulation. IL 2 mRNA levels peaked at 5 hr after mitogen stimulation, whereas IFN-gamma mRNA levels peaked at 20 hr. IL 2R mRNA continued to accumulate for the full 40 hr of these kinetic experiments. The later accumulations of IFN-gamma and IL 2R mRNA and the resulting expression of the corresponding proteins may therefore be dependent on the earlier production of IL 2 and its subsequent interaction with the IL 2R on the surface of such activated T cells.
The role of IL 1 in the activation of IL 4-producing murine T cell clones was investigated by using a calcium ionophore (ionomycin) or a phorbol ester (12-O-tetradecanoylphorbol 13-acetate; TPA) as T cell receptor-independent costimuli. The use of these pharmacologic agents to investigate IL 1-mediated T cell activation revealed two distinct mechanisms of activation. IL 1 in combination with ionomycin (iono/rIL 1) stimulated a proliferative response that was associated with the production of IL 4 as measured by lymphokine bioassay and mRNA studies. Furthermore, inhibition of this proliferative response with an anti-IL 4 monoclonal antibody or cyclosporine indicated that IL 4 functions as an autocrine growth factor. In contrast, IL 1 synergized with TPA (TPA/rIL 1) to induce proliferation in the absence of either IL 4 or IL 2 gene transcription or lymphokine secretion. The IL 4-independence of this activation mechanism was further supported by the failure of both anti-IL 4 antibodies and cyclosporine to inhibit the response. In addition, activation by TPA/rIL 1 caused no detectable alteration in cytoplasmic calcium levels. Both IL 4-dependent and IL 4-independent activation responses were associated with the expression of functional receptors for IL 2 as well as IL 4. Characterization of these activation responses suggests that the synergistic activity of IL 1 during T cell activation is multipotential. The nature of an IL 1-dependent T cell growth response, therefore, may vary depending on the balance of intracellular signals generated concurrently through the T cell receptor complex and other regulatory surface molecules.
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.
Reversed-phase high-performance liquid chromatography has been used to purify to homogeneity two different lymphokines. Human IL-2 was purified on a C8 reversed-phase column in pyridine-acetate-propanol followed by chromatography on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile. Protein sequence analysis of in situ-generated cyanogen bromide peptides obtained from this preparation established the homogeneity of this material and confirmed the amino acid sequence predicted from the published DNA sequence. Murine CSF-2 alpha was purified on a C18 reversed-phase column in trifluoroacetic acid-acetonitrile followed by chromatography on the same column in pyridine-acetate-propanol. The final preparation yielded a single band on a sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 24,500.
Interleukin 2 (IL-2) has an important role in the regulation of the expression of IL-2 receptors and the synthesis of gamma interferon (IFN-gamma) by T lymphocytes. IL-2 is required for the optimum expression of IL-2 receptors on activated T lymphocytes and for maximum synthesis of IFN-gamma in vitro. Dexamethasone, an immunosuppressant drug that inhibits IL-2 synthesis, diminished the expression of IL-2 receptors and the synthesis of IFN-gamma. Anti-Tac, a monoclonal antibody known to prevent the binding of IL-2 to its receptor without inhibiting IL-2 synthesis, down-regulated the expression of the receptor and partially inhibited synthesis of IFN-gamma. In a population of T lymphocytes prevented from synthesizing IL-2 by dexamethasone and incapable of using IL-2 as a result of blockage of IL-2 receptors by anti-Tac, the number of receptor-bearing cells and receptor density were diminished. Anti-Tac in combination with dexamethasone also exerted a synergistic effect on IFN-gamma synthesis, inhibiting it almost completely. The inhibitory effect of dexamethasone IFN-gamma synthesis may be of clinical importance, since IFN-gamma activates macrophages and thereby triggers one of the defense mechanisms against bacterial infections.
Several laboratories have recently demonstrated that the requirement for macrophages in mitogen-induced production of murine T-cell interleukin 2 (IL-2; formerly referred to as "T-cell growth factor") could be circumvented by using the macrophage-derived peptide interleukin 1 (IL-1; formerly referred to as "lymphocyte-activating factor"). Using two cloned T-cell lymphomas, we investigated the mechanism through which IL-1 exerted its effect on IL-2 production. One of the cell lines used (LBRM-33 5A4) produces large concentrations of IL-2 upon mitogen stimulation, whereas the second (LBRM-33 1A5) is incapable of producing IL-2 in response to mitogen. It was observed that addition of purified IL-1 to nonproducer 1A5 cells converted them to a state in which subsequent mitogen stimulation triggered production of IL-2. The concentration of IL-2 produced by IL-1 treated 1A5 cells was equivalent in magnitude to that generated by mitogen-stimulated 5A4 cells (500-1000 units/ml, or approximately 1000 times the concentration of IL-2 contained in conventional preparations of murine mitogen-conditioned medium). The observations that (i) brief exposure to IL-1 was sufficient for 1A5 cell conversion to IL-2 production and (ii) IL-1 could actively be absorbed from culture medium by live or fixed 1A5 cells led us to propose the existence of IL-1 receptors on responsive 1A5 cells. On the basis of these experiments, we have postulated that IL-1 mediates its effect on immune reactivity (enhancement of thymocyte mitogenesis and induction of antibody and cytotoxic T cell responses) by maturation of a subset of immature T cells to the point where they are capable of IL-2 production. Subsequent release of IL-2 after ligand activation allows for clonal expansion of activated T cells which mediate particular effector functions.
Recombinant interleukin-7. pre-B cell growth factor, has costimulatory activity on purified mature T cells
- . J Mochizuki
- P J Eisenman
- A E Conlon
- Namen
Mochizuki. J. Eisenman, P. J. Conlon, and A. E. Namen. 1989.
Recombinant interleukin-7. pre-B cell growth factor, has costimulatory activity on purified mature T cells. J. Exp. Med. 169:707.
growth of helper T cells
- J Kaye
- S S B Gillis
- E M Mizel
- T R Shevach
- C A Malek
Kaye, J., S. Gillis. S. B. Mizel. E. M. Shevach. T. R. Malek, C. A.
growth of helper T cells. J. Exp. Med. 164:580.
Downloaded from http://journals.aai.org/jimmunol/article-pdf/144/8/3015/1047445/3015.pdf by guest on 01 January 2023
Uytten-IL-4-independent mechanisms
- A Goethals
- J C Renauld
- E Van Roose
A. Goethals, J. C. Renauld, E. Van Roose, C. Uytten-IL-4-independent mechanisms. J. Immunol. 139: 1532.
guantitation and cloning of cytolytic T lymphocytes and their precursors
- H R Macdonald
- J-C J-E Cerrotini
- J L Ryser
- C Maryanski
- M B Taswell
- K T Widmer
- Brunner
MacDonald, H. R., J-C., Cerrotini. J-E. Ryser, J. L. Maryanski, C.
Taswell. M. B. Widmer. and K. T. Brunner. 1980. guantitation and
cloning of cytolytic T lymphocytes and their precursors. lmrnunol.
Rev. 51:93.