Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

LFA-1在胸腺细胞活化信号传导中的功能研究

在ConA和固相抗CD_3单抗刺激系统中,应用抗LFA-1/ICAM-1单抗,研究其在胸腺细胞活化中的功能作用,结果证明,培养初期加入可溶性抗LFA-1可完全阻断ConA活化胸腺细胞增殖,对固相抗CD3单抗诱导的胸腺细胞活化也表现出相同的抑制效应,但对ConA刺激24h后的胸腺细胞应答以及IL-1 IL-2诱导的胸腺细胞增殖无影响。在可溶性抗LFA-1单抗的存在下,ConA诱导胸腺细胞合成IL-2和IL-6的能力显著下降,IL-2R的表达降低。此外,当用固相抗LFA-1和固相抗CD3或用二抗交联LFA-1和CD3刺激胸腺细胞时,抗LFA-1则具有明显地促增殖应答效应,单纯固相抗LFA-1刺激或交联LFA-1均无诱导活化作用,研究结果表明,LFA-1是未成熟胸腺细胞活化的重要辅助分子之一,它可参与TCR/CD3途径介导的早期活化信号的传导,并为胸腺细胞表达IL-2R 和产生IL-2可能提供复合刺激信号。     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Colitis-inducing potency of CD4+ T cells in immunodeficient, adoptive hosts depends on their state of activation, IL-12 responsiveness, and CD45RB surface phenotype

We studied the induction, severity and rate of progression of inflammatory bowel disease (IBD) induced in SCID mice by the adoptive transfer of low numbers of the following purified BALB/c CD4+ T cell subsets: 1) unfractionated, peripheral, small (resting), or large (activated) CD4+ T cells; 2) fractionated, peripheral, small, or large, CD45RBhigh or CD45RBlow CD4+ T cells; and 3) peripheral IL-12-unresponsive CD4+ T cells from STAT-4-deficient mice. The adoptive transfer into SCID host of comparable numbers of CD4+ T cells was used to assess the colitis-inducing potency of these subsets. Small CD45RBhigh CD4+ T lymphocytes and activated CD4+ T blasts induced early (6-12 wk posttransfer) and severe disease, while small resting and unfractionated CD4+ T cells or CD45RBlow T lymphocytes induced a late-onset disease 12-16 wk posttransfer. SCID mice transplanted with STAT-4-/- CD4+ T cells showed a late-onset IBD manifest > 20 wk posttransfer. In SCID mice with IBD transplanted with IL-12-responsive CD4+ T cells, the colonic lamina propria CD4+ T cells showed a mucosa-seeking memory/effector CD45RBlow Th1 phenotype abundantly producing IFN-gamma and TNF-alpha. In SCID mice transplanted with IL-12-unresponsive STAT-4-/- CD4+ T cells, the colonic lamina propria, mesenteric lymph node, and splenic CD4+ T cells produced very little IFN-gamma but abundant levels of TNF-alpha. The histopathologic appearance of colitis in all transplanted SCID mice was similar. These data indicate that CD45RBhigh and CD45RBlow, IL-12-responsive and IL-12-unresponsive CD4+ T lymphocytes and lymphoblasts have IBD-inducing potential though of varying potency.     

Signals delivered via MHC class II molecules synergize with signals delivered via TCR/CD3 to cause proliferation and cytokine gene expression in T cells.

We examined the role of MHC class II molecules in transducing signals to activated human T cells. Cross-linking of MHC class II molecules synergized with submitogenic amounts of anti-CD3 mAb in causing proliferation and secretion of the cytokines IL-2, IL-3, IFN-gamma, and TNF-alpha by MHC class II-alloreactive T cell lines. Signaling via MHC class II molecules in T cells resulted in activation of tyrosine kinases, in generation of inositol phosphates, and in Ca2+ mobilization that was abrogated by the tyrosine kinase inhibitor herbimycin A. Thus, like signaling via TCR/CD3, signaling via MHC class II molecules involved tyrosine kinase-dependent activation of phospholipase C, resulting in phosphoinositol turnover and Ca2+ flux. However the signaling pathways coupled to MHC class II molecules and to TCR/CD3 differed, because engagement of the transmembrane phosphatase CD45 inhibited Ca2+ fluxes triggered via TCR/CD3 but not Ca2+ fluxes triggered via MHC class II molecules.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.     

Inhibition by anti-HLA class II monoclonal antibodies of monoclonal antibody OKT3-induced T cell proliferation. Studies at the mRNA level

mAb to monomorphic determinants of HLA class II Ag have been shown to inhibit monocyte-dependent OKT3-induced T cell proliferation, indicating that MHC class II molecules play a regulatory role also in Ag nonrestricted, CD3-induced T cell proliferation. This effect involves several steps in the process of T cell activation and proliferation, including IL-1 beta, IL-6, and IL-2 secretion and IL-2R alpha expression. In the present study, we analyzed the effect of an anti-HLA class II mAb (Q5/6) on the mRNA expression of genes related to monocyte and T cell activation. mRNA levels for early (early c-myc, c-fos) and late (late c-myc, N-ras, c-myb) genes involved in T cell activation were determined as well as mRNA levels for IL-1 beta, IL-6, IFN-gamma, IL-2, and IL-2R alpha. The kinetics of mRNA induction for ICAM-1 was also investigated. The results show that in T lymphocytes the expression of c-fos and early c-myc mRNA was unaffected by mAb Q5/6, whereas the c-myb and N-ras mRNA levels were strongly diminished as well as those of IL-2, IL-2R alpha, and IFN-gamma mRNA. An early increase of ICAM-1 mRNA was partially inhibited. In monocytes, a marked reduction of IL-1 beta and IL-6 mRNA was found. It is concluded that the HLA class II determinant involved in the inhibition mechanism can be engaged in the control of IL-1 beta and IL-6 mRNA levels and constitute an accessory signal up-regulating IL-2 and IL-2R alpha gene activation, through a pathway not affecting c-myc and c-fos expression.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

IL-21 stimulates human myeloma cell growth through an autocrine IGF-1 loop

IL-21 is a member of the type I cytokine family related most closely to IL-2 and IL-15. IL-21 is a pleiotropic cytokine, produced by T, NKT, and dendritic cells, which modulates lymphoid and myeloid cell functions. Besides its activities on normal lymphoid cells, it has been shown that IL-21 is a growth factor for myeloma cells. In the present study, we demonstrate that IL-21 generated myeloma colonies from 9 of 24 human myeloma cell lines (HMCL) in a collagen-based assay. Of major interest, the capacity of IL-21 to stimulate clonogenicity was restricted to CD45(-) HMCL. We found that IL-21 induced tyrosine phosphorylation of STAT-3, STAT-1, and Erk1/2. Interestingly, an Akt activation was observed lately after 30 min to 1 h of IL-21 stimulation, indicating that this Akt phosphorylation could be due to an IGF-1 autocrine loop. This hypothesis was sustained both by the fact that IL-21 treatment induced an IGF-1 mRNA synthesis and that an antagonistic anti-IGF-1 receptor mAb (AVE1642) strongly inhibits the IL-21-induced clonogenicity. Thus, we demonstrated by quantitative PCR that IL-21 induced clonogenicity through an autocrine IGF-1 secretion in HMCL and primary myeloma cells. Because we have previously demonstrated that CD45 phosphatase inhibits the IGF-1 signaling, this inhibitory effect of CD45 explains why the IL-21-induced clonogenicity was restricted to CD45(-) HMCL. These results support that therapy against IGF-1R, which are presently under investigation in multiple myeloma, could be beneficial, not only to suppress IGF-1-mediated myeloma cell growth, but also IL-21-mediated myeloma cell growth.     

IL-10 and TGF-beta redundantly protect against severe liver injury and mortality during acute schistosomiasis

The cytokines IL-10 and TGF-beta regulate immunity and inflammation. IL-10 is known to suppress the extent of hepatic damage caused by parasite ova during natural infection with Schistosoma mansoni, but the role of TGF-beta is less clear. Cytokine blockade studies in mice revealed that anti-IL-10R mAb treatment during acute infection modestly increased cytokine production and liver damage, whereas selective anti-TGF-beta mAb treatment had marginal effects. In contrast, mice administered both mAbs developed severe hepatic inflammation, with enlarged, necrotic liver granulomas, cachexia, and >80% mortality by 8 wk postinfection, despite increased numbers of CD4(+)CD25(+)Foxp3(+) T regulatory cells. Blocking both IL-10 and TGF-beta at the onset of egg production also significantly increased IL-4, IL-6, TNF, IFN-gamma, and IL-17 production and markedly increased hepatic, peritoneal, and splenic neutrophilia. In contrast, coadministration of anti-IL-10R and TGF-beta mAbs had little effect upon parasite ova-induced intestinal pathology or development of alternatively activated macrophages, which are required to suppress intestinal pathology. This suggests that inflammation is controlled during acute S. mansoni infection by two distinct, organ-specific mechanisms: TGF-beta and IL-10 redundantly suppress hepatic inflammation while intestinal inflammation is regulated by alternatively activated macrophages.     

A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production

Cytokine production upon T cell activation results from the integration of multiple signaling pathways from TCR/CD3 and from costimulatory molecules such as CD28. Among these pathways, the possible role of p38 mitogen activated protein kinase (MAPK) is the least understood. Here, we used a highly specific p38 MAPK inhibitor, the SB203580 compound, to examine the role of this enzyme in the induction of various cytokines in human T cells stimulated with anti-CD3 and anti-CD28 mAb together or in combination with PMA. Cytokine induction was monitored by ELISA and at the mRNA level. While SB203580 had little effect on IL-2 production and proliferation, it significantly reduced the production of several other cytokines. The secretion of IL-4, IL-5, IL-13, and TNF-alpha was inhibited by 20-50% with modes of T cell activation involving the CD28 pathway, whereas their mRNA expression was little affected. In contrast, IFN-gamma induction via CD28/PMA or CD3/CD28, but not CD3/PMA, was markedly diminished both at the protein and at the mRNA levels. Most interestingly, SB203580 also suppressed IL-10 secretion and mRNA induction via CD28-dependent activation by 75-85% (IC50 approximately 0.2 microM). Subset analysis suggested that this inhibition did not reflect a differential effect on T cell subsets. Therefore, p38 MAPK activity appears to contribute to cytokine production, mostly via CD28-dependent signaling. Moreover, IL-10 seems to rely more on this activity than other cytokines for its induction in T cells.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

IL-10 inhibits human T cell proliferation and IL-2 production.

Human IL-10 has been reported previously to inhibit the secretion of IFN-gamma in PBMC. In this study, we have found that human IL-10 inhibits T cell proliferation to either mitogen or anti-CD3 mAb in the presence of accessory cells. Inhibited T cell growth by IL-10 was associated with reduced production of IFN-gamma and IL-2. Studies of T cell subset inhibition by human IL-10 showed that CD4+, CD8+, CD45RA high, and CD45RA low cells are all growth inhibited to a similar degree. Dose response experiments demonstrated that IL-10 inhibits secretion of IFN-gamma more readily than T cell proliferation to mitogen. In addition, IL-2 and IL-4 added exogenously to IL-10 suppressed T cell cultures reversed completely the inhibition of T cell proliferation, but had little or no effect on inhibition of IFN-gamma production. Thus, in addition to its previously reported biologic properties, IL-10 inhibits human T cell proliferation and IL-2 production in response to mitogen. Inhibition of IFN-gamma production by IL-10 appears to be independent of the cytokine effect of IL-2 production.     

1,25-Dihydroxyvitamin D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8- thymocytes

CD45R is a high molecular weight (p205/220) form of a series of transmembrane glycoproteins, collectively known as CD45 and present in some form on all lymphoid cells. We have proposed that CD45R+ thymocytes, a minority (15 to 30%) of total thymocytes, represent the generative thymic lineage whereas CD45 p180+ thymocytes are destined for intrathymic death. To test this hypothesis, we prepared human thymus fractions enriched for the expression of CD45R by exhaustive depletion of CD45 p180+ cells, as well as progenitor CD3-4-8- "multinegative" thymocytes which are predominantly CD45R+. Northern analysis of RNA extracted from CD45 p180- and multinegative thymus fractions demonstrated that these populations are enriched for cells able to synthesize mRNA encoding IL-2 and IL-2R after mitogenic stimulation, as compared to unfractionated thymus, consistent with the properties expected for generative thymocytes. Postulating that the CD45R glycoprotein might represent an important signal delivery molecule, we analyzed the ability of mAb specific for CD45 epitopes to synergize with suboptimal amounts of PHA and PMA in the stimulation of IL-2 mRNA production by multinegative thymocytes. We found that CD45R-specific mAb synergizes strongly with PHA/PMA to stimulate IL-2 and IL-2R mRNA expression. In contrast, mAb to CD45 common determinants were unable to synergize. Multinegative thymocytes depleted of all CD45 p180+ cells were compared to total multinegative cells and found to synthesize fourfold greater levels of IL-2 mRNA after stimulation with anti-CD45R mAb. This CD45 p180- multinegative subset is enriched for cells expressing a high density of CD45R, and for CD45- thymus cells, suggesting a possible enrichment for nonlymphoid cells which may play a role in the stimulation process. Our results suggest that the extended amino acid insert of CD45R plays a fundamental role in transmembrane signalling, and that CD45R may be a primary signal transducer for developing thymic progenitor cells.