Interleukin 2-driven T lymphocyte proliferation is dependent upon a surface antigen distinct from the interleukin 2 receptor: requirements for inhibition of T-cell proliferation by monoclonal antibody 5C3

A monoclonal antibody (5C3) to an antigen expressed on activated guinea pig T lymphocytes that did not react with the interleukin 2 (IL-2) receptor, but inhibited IL-2-driven proliferative responses has been previously characterized. The present study provides further analysis of the inhibitory capacity of 5C3 for T-cell proliferation and of the relationship between the expression of the antigen defined by 5C3 and the capacity of cells to respond to IL-2. 5C3 inhibited proliferation of T-cell blasts to IL-2-containing fluids when added as late as 8 hr prior to termination of a 26-hr culture. 5C3 pretreatment of the IL-2-responsive blast cells was also sufficient to detect significant inhibition of proliferation. FACS analysis of these blasts indicated that maximal 5C3 binding was required for pretreatment to result in inhibition of IL-2-driven proliferation. Delayed addition of 5C3 to culture or pretreatment with 5C3 of responding cells also resulted in inhibition of proliferation of immune T lymphocytes to antigen-pulsed-presenting cells. Lastly, although modulated 5C3- blasts failed to proliferate to IL-2, induction of the 5C3-bearing molecule on these 5C3- blasts correlated with restoration of the ability of these cells to proliferate to IL-2. Collectively, these results further support the hypothesis that monoclonal antibody 5C3 interferes with a critical signal in the IL-2 growth pathway.     

Stimulation of human T cells via anti-T cell receptor monoclonal antibody BMA031: distinct cellular events involving interleukin-2 receptor and lymphocyte function antigen 1

We have analyzed activation of resting human T cells by anti-T cell receptor (TCR) monoclonal antibody (mAb) BMA031, a murine mAb of the G2b isotype. Human peripheral blood lymphocytes (PBL) respond to anti-TCR mAb by short-term proliferation in vitro and by acquisition of responsiveness to interleukin 2 (rIL-2) in the absence of detectable IL-2 production. Cell depletion and limiting dilution experiments indicate that anti-TCR mAb +/- rIL-2 stimulation covers a substantial portion of human T cells, including CD4+ and CD8+ cells. Enhancement by rIL-2 of anti-TCR mAb-induced proliferation is blocked by anti-IL-2 receptor (IL-2R, p55) mAb, while anti-TCR mAb-induced proliferation is not. In contrast, anti-TCR mAb-induced proliferation is blocked by anti-lymphocyte function antigen 1 (LFA-1, CD11a) mAb and is not demonstrable in PBL from two patients with severe congenital LFA-1 deficiency, not even in the presence of irradiated LFA-1+ PBL. We conclude that stimulation of resting human T cells by anti-TCR mAb BMA031 enables dissociation of distinct steps in T cell activation that specifically require participation of IL-2R (p55) and LFA-1 cell surface molecules in a mutually exclusive way.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytometrically detected specific binding of interleukin 2 to cells.

We have developed a new technique for detecting binding of interleukin 2 (IL-2) to cells. This technique involves incubating the cells with IL-2 and then analysing the cell surface with specific anti-IL-2 antibodies and flow cytometry. This binding was only detected on tumor cells that possessed the p55 subunit of the IL-2 receptor. The role of p55 was ascertained by inhibition of the binding with a monoclonal antibody to p55. Although p55 is necessary for cytometrically detected IL-2 binding, further studies demonstrated that p55 is not sufficient. Thus, cytometrically-detected binding is likely to involved the contribution of other IL-2 surface receptors. Interleukin-2 binding to peripheral blood T lymphocytes and to a non-transformed T-cell clone was also detected cytometrically and it was shown that this binding is regulated by the activation status of the cells. Whereas IL-2 binding to quiescent T cells could not be detected, upon activation abundant binding was seen. The functional consequences of this type of cellular binding were studied. Interleukin-2 binding to cells during a short pulse was shown to have significant long-term consequences both for T-cell proliferation and for the enhancement of major histocompatibility complex (MHC)-non-restricted cytotoxicity.     

Interleukin-2 receptor beta chain as a possible target for low doses of mafosfamide

The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Ralpha mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the alpha and beta chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rbeta mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R beta chain.     

Anti-interleukin 2 receptor antibody suppresses murine diabetic insulitis and lupus nephritis

In order to assess the importance of interleukin 2 receptor (IL-2R)-positive activated lymphocytes and macrophages in the pathogenesis of autoimmunity, we tested the prophylactic therapeutic efficacy of an anti-IL-2R (M7/20) monoclonal antibody, which recognizes the 55-kDa subunit of the heterodimeric IL-2R in two distinct models: the nonobese diabetic mouse and the NZB x NZW F1 hybrid with lupus. Treatment with anti-IL-2R monoclonal antibody suppressed autoimmune insulitis in nonobese diabetic mice and lupus nephritis in the NZB x NZW F1 hybrid. These studies indicate that highly selective targeting to activated lymphocytes and macrophages expressing the IL-2R provides a discrete method of dampening autoimmunity.     

Identification of a novel protein capable of interacting with the IL-3 receptor

IL-3 has numerous functions in hematopoiesis yet its receptor has not been fully characterized. We have developed two mAb, 4G8 and 2F2, that markedly inhibited IL-3-dependent proliferation whereas only marginally affecting IL-2 or IL-4-induced proliferation. On Western blots, both antibodies identified the same protein, which varied in size from 115 to 145 kDa in six cell lines tested. The 4G8/2F2 Ag was detected at moderate density, on a wide variety of cells including IL-3-dependent cell lines and T lymphocytes. Radioligand binding studies revealed that 4G8, but not 2F2, could inhibit the binding of 125I-IL-3 to the high affinity IL-3R. These data suggest that the mAb 4G8 and 2F2 recognize different epitopes on the same Ag, and suggest furthermore that the inhibition of IL-3-dependent proliferation mediated by 2F2, in particular, does not occur via inhibition of ligand binding. Neither antibody showed an enhanced level of fluorescent staining of Cos 7 cells transfected with the low affinity IL-3R cDNA. In addition, 4G8 did not inhibit IL-3 binding to L cells transfected with the cloned IL-3R or IL-4R despite the fact that 4G8 was expressed on these cells. These data suggest that the 4G8/2F2 Ag is a unique cell surface protein that can interact with the endogenous functional IL-3R.     

IL-4/B cell stimulatory factor 1 stimulates T cell growth by an IL-2-independent mechanism

Resting T cells are stimulated to synthesize DNA by IL-4 and phorbol myristate acetate (PMA). This response of T cells to IL-4 plus PMA is independent of the action of IL-2 as judged by 1) the lack of IL-2 in supernatants of stimulated cells, 2) the failure to detect IL-2 mRNA in stimulated cells by in situ hybridization, 3) the inability of anti-IL-2R antibody and of anti-IL-2 antibody to block responses to IL-4 plus PMA, and 4) the failure of cyclosporin A to block responses. T cells also respond to anti-CD3 antibodies and IL-4 in the presence of anti-IL-2R antibodies. IL-4 stimulation of growth of the long term T cell line HT-2 also appears to be independent of the action of IL-2. No IL-2 mRNA is found in IL-4-stimulated HT-2 cells by Northern blotting; the response of HT-2 cells to IL-4 is not blocked by anti-IL-2R antibodies; the response of HT-2 cells to IL-4 is not inhibited by cyclosporin A. Although IL-4 stimulation of T cells is independent of IL-2, IL-4 plus PMA treatment of resting T cells does cause enhanced expression of IL-2R and prepares cells to proliferate to IL-2 alone. In both these properties IL-4 resembles IL-2. These experiments lead us to conclude that IL-4 can act as an alternative to IL-2 as authentic T cell growth factor.     

Resistance in murine schistosomiasis is contingent on activated IL-2 receptor-bearing L3T4+ lymphocytes, negatively regulated by Lyt-2+ cells, and uninfluenced by the presence of IL-4

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. C57BL/6 mice were depleted in vivo of L3T4+, Lyt-1+, Lyt-2+, IL-2R+ cells, or IL-4 by administration of appropriate mAb. Resistance and various correlative parameters of the immune response were studied in normal, depleted, and congenitally athymic mice. Depletion of T lymphocytes by anti-L3T4 or anti-IL-2R mAb reduced the development and expression of resistance, IgG2a and IgE antibody formation, and delayed type hypersensitivity reactivity against schistosome Ag. Depletion with anti-IL-4 antibody led to profound suppression of IgE-eosinophil-mediated antibody-dependent cell-mediated cytotoxicity and passive cutaneous anaphylaxis responses against the parasite and no effect on IgG2a antibody, Ag-mediated blast transformation, or resistance. Depletion of Lyt-2+ cells produced augmented development and expression of resistance and an increase in the immunological parameters of anti-schistosome reactivity. These studies suggest that protective immunity to S. mansoni in mice, induced by irradiated cercariae, is dependent on L3T4+, IL-2R+ lymphocytes and negatively regulated by Lyt-2+ cells. IL-4 does not appear to be essential for the development of resistance but is essential for the IgE response to the parasite.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Attenuation of bleomycin-induced pneumopathy in mice by monoclonal antibody to interleukin-12

We previously demonstrated essential roles of the Fas-Fas ligand (FasL) pathway in bleomycin-induced pneumopathy in mice. T lymphocytes and natural killer cells express FasL on activation and use it as a cytotoxic effector molecule. Because interleukin (IL)-12 is known to play a critical role in cell-mediated immunity, we investigated whether anti-IL-12 antibody treatment suppresses the development of this model. The anti-IL-12 antibody treatment decreased the number of apoptotic cells and the degree of inflammation and fibrosis in lung tissue. The results of RT-PCR showed that IL-12p40, IL-12 receptor (R) beta2, interferon-gamma, tumor necrosis factor-alpha and FasL mRNAs were upregulated after bleomycin instillation. The upregulation of FasL, IL-12Rbeta2, and tumor necrosis factor-alpha mRNA expression in lung tissue was suppressed by anti-IL-12 antibody treatment. The results of enzyme-linked immunosorbent assay showed that the levels of IL-12p40, but not of IL-12p70, were increased in lung tissue after bleomycin instillation. Although the increase in IL-12Rbeta2 mRNA levels suggests that the T helper type 1 cell response may participate in lung injury, the increase in IL-12p40 supports T helper type 2 cell predominance in the fibrotic process of this model. The administration of anti-IL-12 antibody could be a novel therapy against lung injury and pulmonary fibrosis.     

Thymic stroma-derived T cell growth factor (TSTGF). IV. Capacity of TSTGF to promote the growth of L3T4- Lyt-2- thymocytes by synergy with phorbol myristate acetate or various IL

The present study investigates the role of thymic stroma-derived T cell growth factor (TSTGF) in promoting the growth of L3T4- Lyt2- (double-negative) thymocytes. Partially purified TSTGF samples were prepared from the culture supernatant of a newly established thymic stromal cell line, MRL104.8a. The TSTGF alone induced only marginal proliferation of double-negative thymocytes, whereas this factor exerted a potent growth-promoting effect on these cells in combination with PMA. Because such an enhanced proliferation was not inhibited by anti-IL-4 or anti-IL-2R antibody, this was not due to the stimulation of an autocrine mechanism involving the production and utilization of IL-4 or IL-2. In scrutinizing PMA-equivalent physiologic substance(s), IL-1 was revealed to be capable of replacing the role of PMA in the above co-stimulation cultures and including enhanced proliferation of double-negative thymocytes in combination with TSTGF. Although TSTGF plus IL-2 or IL-4 also exhibited an appreciable or moderate synergistic effect on the growth of double-negative thymocytes, its magnitude was weaker compared with that obtained by TSTGF plus IL-1. More important, the strikingly enhanced proliferation was induced in the combinations of TSTGF, IL-1, and IL-2 or IL-4 under conditions in which the proliferation induced by IL-1 plus IL-4 or IL-1 plus IL-2 was marginal or slight. Furthermore, such strongly enhanced proliferation was also observed in the double-negative thymocyte population which was additionally depleted of T3+ cells (namely, the L3T4- Lyt-2- T3- or dull population). These results indicate the crucial role of TSTGF in the proliferation of immature thymocytes by synergy with various cytokines.     

Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.     

IL-4 potentiates the IL-2-dependent proliferation of mouse cytotoxic T cells

In addition to the regulation of B lymphocyte growth and differentiation, the cytokine IL-4 (BSF-1) exerts effects on T lymphocytes and other bone marrow-derived lineages. We show here that recombinant mouse IL-4 synergizes with low levels of IL-2 to increase the yield of cytotoxic activity in a primary MLR, and the proliferation of both cloned IL-2-dependent CTL lines and cells obtained from a primary MLR. IL-4 did not induce the proliferation of any of several cloned CTL cell lines on its own. It also did not replace IL-2 in stimulating the growth or reactivation of quiescent, antigen-dependent CTL clones. However, IL-4 was synergistic with IL-2 after reactivation of the quiescent cells with antigen plus IL-2. Enhancement by IL-4 of the IL-2-driven proliferation of an antigen-independent line was blocked by the addition of anti-IL-4 monoclonal antibody. Although incubation of the CTL clones with IL-4 or with IL-2 plus IL-4 induced a transient increase in the expression of the mRNA encoding the 55 kDa IL-2 receptor, no change in the number or affinity of IL-2 receptors because of IL-4 was detected. This suggests that IL-4 does not potentiate the IL-2 response by altering IL-2 receptor levels. Instead, we propose that the synergistic effect of IL-4 is mediated by a different signalling mechanism from that used by IL-2.     

A functional analysis of the T-cell defect in MRL-lpr mice

Autoimmune MRL-lpr mice have a defect in antigen-specific T-cell proliferation. Our studies indicate that this defect is caused by the massive expansion in MRL-lpr mice of a unique T-cell subset which is unresponsive to antigenic signals. Pharmacologic doses of PGE1 suppress this lymphoid hyperplasia and thus prevent loss of T-cell functions by preventing numeric dilution of normal T cells by defective T cells. The inability of the unique subset of T cells to respond to antigenic signals cannot be corrected by the addition of interleukin 2 (IL-2), implying that additional cellular properties are required to initiate proliferation. While the vast majority of freshly harvested MRL-lpr T cells lack IL-2 receptors (R) as measured by anti-IL-2R monoclonal antibody staining, a large fraction of nonstimulated, cultured (48 hr) MRL-lpr T cells, but not MRL-++ T cells, express IL-2R. These experiments suggest that MRL-lpr cells are activated in vivo but an undefined suppressive influence prevents detection or expression of IL-2R until these cells are explanted and cultured.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

Purified Protein from Salmonella typhimurium Inhibits the Interleukin-2 Response of Murine Splenic T-Lymphocytes Activated with Anti-CD3 Antibody

Previously, we demonstrated that the immunosuppression induced by a purified preparation of Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) can be observed in terms of suppression of the proliferation of murine spleen cells stimulated with a mitogenic lectin. In the present study, I observed that STI inhibited the interleukin-2 (IL-2) response of purified murine splenic T lymphocytes stimulated with anti-CD3 antibody. The flow cytometric analysis of IL-2 receptor (IL-2R) expression on T cells showed that STI specifically suppressed the expression of IL-2Rβ and IL-2Rγ. Furthermore, when the IL-2-dependent T-cell line CTLL-2 was incubated with STI, the growth of CTLL-2 cells was significantly inhibited. These results suggest that the target cells for STI are T cells themselves, and that the suppression of T-cell proliferation induced by STI might involve a defect in the IL-2 receptor (IL-2R) function of T cells.     

Biochemical evidence for a third chain of the interleukin-2 receptor

Two receptor proteins that specifically bind interleukin-2 (IL-2) have been identified previously. The L (Tac or alpha) chain can bind IL-2 with a Kd value of 10 nM (low affinity). Although the H (beta) chain expressed on lymphocytes can bind IL-2 with a Kd value of 1 nM (intermediate affinity), transfected fibroblasts expressing the H chain cannot bind IL-2, suggesting the involvement of other lymphocyte-specific factors for the function of the H chain. To obtain direct evidence for the presence of a third component of the IL-2 receptor, we examined the IL-2 binding activity of detergent-solubilized cell membrane preparations. We found that lysates of transfected Cos7 cells expressing H chains can bind IL-2 when mixed with lysates from lymphocytes that cannot bind IL-2. Chemical cross-linking of 125I-IL-2-bound lysate mixture and subsequent immunoprecipitation with a noncompetitive anti-H chain antibody gave rise to two 125I-IL-2-bound proteins, a 56-kDa protein (p56) and the H chain, although neither the H chain nor p56 alone is able to bind IL-2. These results indicate that p56 is the IL-2 receptor third chain that is required for IL-2 binding to the H chain. A similar lysate mixing experiment also showed that p56 is involved in IL-2 binding to the high affinity IL-2 receptor by forming the quaternary complex of IL-2, p56, L chain, and H chain.