Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.     

Molecular mechanisms involved in T cell activation. I. Evidence for independent signal-transducing pathways in lymphokine production vs proliferation in cloned cytotoxic T lymphocytes

It is well-established that activated T cells proliferate in response to interleukin 2 (IL 2) and produce various soluble lymphokines such as macrophage-activating factor (MAF) in response to antigen. Prior to investigating the molecular events involved in signaling the initiation of these responses in cloned murine cytotoxic T lymphocytes (CTL), we determined whether these responses could occur independently, and we established for each response the time during which signal transducing mechanisms may function. It was found that this cloned CTL population was in a resting state (G1 phase of cell cycle) 7 days after stimulation with antigen plus IL 2. At this time, the incubation of these resting CTL with IL 2 for 4 to 6 hr resulted in a maximal proliferative response that was not accompanied by the production of MAF. Conversely, the incubation of resting CTL with antigen or lectin (in the absence of IL 2) for at least 8 hr resulted in the maximal production of MAF at 24 hr without inducing a proliferative response. In addition, antigen or lectin, but not IL 2, triggered an immediate (less than 1 min) and sustained (at least 8 hr) mobilization of intracellular calcium. The kinetics of this calcium response paralleled the minimum time (8 hr) that was required for resting CTL to interact with either antigen or lectin in order to produce maximal titers of MAF. These results indicate that proliferation and lymphokine (MAF) production in cloned murine CTL are independent events. In these resting CTL, the signal mechanisms that mediate the production of lymphokines are most likely restricted to the initial 8 hr of stimulation by antigen or lectin and involve the rapid and prolonged mobilization of cytoplasmic calcium. Proliferative signals, however, are probably complete within 4 to 6 hr after stimulation by IL 2 and do not involve readily demonstrable fluxes of cytoplasmic calcium, as determined by the fluorescent calcium probe Quin 2.     

Inhibitory effect of cyclosporin A on the OKT3-induced peripheral blood lymphocyte proliferation

In this paper we studied the effect of cyclosporin A (CyA) on interleukin 1 (IL-1) and interleukin 2 (IL-2) production and on IL-2 receptor expression by human peripheral blood lymphocytes induced to proliferate following OKT3 monoclonal antibody stimulation. CyA inhibited T-cell proliferation in a dose-dependent manner and its effect was inversely correlated with the entity of the mitogenic signal. The drug reduced not only IL-2 synthesis but also IL-1 production. CyA was also found to be able to inhibit the expression of IL-2 receptors on T cells. By supplementing with IL-1 and/or IL-2 the cultures carried out in the presence of CyA, it became evident that the inhibition of IL-2 production mainly depended on the CyA-induced reduction of IL-1 synthesis. Thus the IL-2 production by "resting" T cells had to be considered as an IL-1-dependent event. In addition it was found that the presence of IL-1 constituted a crucial requirement for the induction and the positive modulation of IL-2 receptor expression. Although IL-2 could play a role in facilitating the expression of IL-2 receptors, its effectiveness to do so depended on the presence of IL-1. In conclusion, CyA is to be considered not only as a potent immunodepressive drug but also as a valuable tool for the study of T-cell activation and proliferation.     

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.     

Molecular mechanisms involved in T cell activation. II. The phosphatidylinositol signal-transducing mechanism mediates antigen-induced lymphokine production but not interleukin 2-induced proliferation in cloned cytotoxic T lymphocytes

The phospholipid metabolism of cloned murine cytotoxic T lymphocytes (CTL) was examined under conditions in which the induction of proliferation by interleukin 2 (IL 2) and the stimulated production of lymphokine (macrophage-activating factor (MAF] by concanavalin A (Con A) and specific antigen occurred independently of each other. Activation of the CTL by either of the latter two stimuli resulted in changes in the metabolism of phosphatidylinositol (PI) that were early (less than 2.5 min), specific, and prolonged (6 to 8 hr). These changes were primarily characterized by an increase in phosphatidic acid (PA) and PI, with a decrease in phosphatidylinositol-4,5-bisphosphate. The duration of these phospholipid responses, particularly PA and PI, approximated the minimum time of CTL-stimulus interaction required to produce maximal titers of MAF. No changes were observed in other major classes of phospholipids during 8 hr of continuous stimulation. Stimulation with an irrelevant antigen had no effect on CTL phospholipid metabolism. In contrast to specific antigen or Con A, the T cell growth factor IL 2 failed to elicit specific and early biosynthetic responses from PA and PI. Instead, there were nonspecific biosynthetic responses from all major phospholipid classes (including phosphatidylcholine and phosphatidylethanolamine, as well as PA and PI) which occurred between 1 and 6 hr after IL 2 stimulation. Both 1,2-diacylglycerol (DAG) and inositol phosphates (IP), the hydrolytic products of PI turnover, were produced in response to MAF-inducing stimuli, but neither were detected in response to the proliferative stimulus IL 2. Together, these results indicate that the hydrolysis of PI and the concomitant production of the putative second messengers DAG and IP are involved in signaling the production of lymphokines (MAF) by CTL. On the other hand, the failure of IL 2 to elicit a full-spectrum PI response suggests that signals mediating CTL proliferation may utilize an alternate and still undefined pathway.     

Natural killer (NK) cell stimulatory factor or IL-12 has differential effects on the proliferation of TCR-alpha beta+, TCR-gamma delta+ T lymphocytes, and NK cells.

We have analyzed the effects of NK cell stimulatory factor/IL-12, on proliferation of PBL and their subsets. IL-12 synergizes with lectins and phorbol diesters to induce proliferation of CD4+ and CD8+ peripheral blood T lymphocytes. In the case of phorbol-diester-induced proliferation, the effect of IL-12 is in part mediated by induced IL-2 production, as suggested by the observation that IL-12 enhances IL-2 production in these cultures and that anti-IL-2 antibodies inhibit proliferation. IL-12 synergizes also with anti-CD3 antibodies and with allogeneic stimulation in MLC in inducing T cell proliferation. IL-12 alone is mitogenic for preactivated T and NK lymphoblasts. This mitogenic effect is observed with similar doses of IL-12 on NK lymphoblasts as well as on CD4+ and CD8+ TCR-alpha beta+ and on TCR-gamma delta+ lymphoblasts. On TCR-alpha beta+ T lymphocytes the effect of IL-12 is always additive to that of IL-2 over a wide dose range. The same effect is observed on highly activated, actively proliferating NK cells. However, on NK and TCR-gamma delta+ lymphoblasts reverting to a resting state after stimulation and on a TCR-gamma delta+ acute leukemia-derived T cell line, IL-12 inhibits significantly the proliferation induced by moderate to high doses (10 to 100 U/ml) of IL-2. This inhibitory effect is, at least in part, indirect, and depends on IL-12-induced production of TNF. Neutralizing anti-TNF antibodies, but not anti-IFN-gamma and anti-transforming growth factor antibodies, restore by more than 70% the inhibition of proliferation induced by IL-12 in these cultures. However, TNF alone cannot mimic the inhibitory effect of IL-12 on the IL-2-induced proliferation of NK and TCR-gamma delta+ lymphoblasts, suggesting the involvement of additional mechanisms. The relevance of these findings for the biology of lymphocyte subsets mediating MHC nonrestricted cytotoxicity is discussed.     

Helper-independent CD8+ cytotoxic T lymphocytes express IL-1 receptors and require IL-1 for secretion of IL-2

The purpose of this study was to examine the role of IL-1 on the activation of CD8+/CD4- class I-restricted helper cell-independent cytolytic T cell (HITc) clones known to produce IL-2 and proliferate in vitro after Ag stimulation with a Friend retrovirus-induced leukemia (FBL). The functional role of IL-1 in Ag-specific proliferation and IL-2 secretion was assessed by stimulating the T cell clones with FBL either in the presence or absence of macrophages (M phi), rIL-1, or rIL-2. Resting cloned HITc cells, purified from residual accessory cells, failed to proliferate in response to FBL alone, but proliferated in response to FBL plus M phi, rIL-1 or rIL-2. Stimulation with FBL alone in the absence of M phi or IL-1 was sufficient for induction of IL-2R expression, and rendered cells responsive to IL-2, but M phi or IL-1 were also required to induce production of IL-2. The activity of IL-1 was further examined by measuring the binding of [125I]rIL-1 alpha, which demonstrated that resting cloned HITc cells expressed IL-1R that increased in number after activation with Ag. This expression of IL-1R and requirement for IL-1 by CD8+ HITc was surprising because previous studies examining T cell populations after mitogen stimulation have not detected IL-1R on the CD8+ population. Therefore, the role of IL-1 in the activation of CD8+ CTL that do not secrete IL-2 after activation was assessed. By contrast to HITc, CD8+ CTL required exogenous IL-2 to proliferate in vitro and did not express IL-1R. These data demonstrate that the subset of CD8+ T cells responsible for IL-2 production express IL-1R and that triggering this receptor with IL-1 after Ag stimulation results in the production of IL-2 and subsequent proliferation.     

Cytokine-Independent Proliferation of IL-2-Nonproducing CTL Clones in Association with High TCR-Signal Transduction Responses

Alloreactive CTL clone D2-23 proliferated in response to antigenic cells without IL-2 production. Among subclones of D2-23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fc receptors. Cyclosporin A, which totally blocks IL-2 production of Th1 cells, barely or only partially inhibited PMA- or aCD3-induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL-2 plus antigenic cells, or even IL-2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR-linked mechanism for CTL proliferation that is independent of costimulatory cell- or cytokine-mediated signaling, but is originally prepared by prior stimulation with IL-2.     

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.     

Regulation of human cytolytic lymphocyte responses by interleukin-12.

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.     

Role of uncultured human melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes.

The role of uncultured melanoma cells in the proliferation of autologous tumor-specific cytotoxic T lymphocytes (CTLs) was investigated. Uncultured autologous tumor cells by themselves induced modest, but significant, proliferation in 10 of 13 (77%) CTL clones and in only two of nine non-CTL clones. Uncultured allogenic melanoma cells mostly failed to induce CTL proliferation. Autologous tumor-induced CTL proliferation declined with increasing age of the culture. It did not correlate with IL-2 receptor-alpha expression or was not inhibited by addition of anti-IL-2 antibody to the culture. It was inhibited by pretreatment of tumor cells with anti-MHC class II, but not -MHC class I mAb. IL-2 alone was sufficient for the potent proliferation of five of nine CTL clones. In all these five CTL clones, autologous tumor cells suppressed IL-2-induced proliferation. The remaining four CTL clones, however, required both uncultured autologous melanoma cells and IL-2 for the proliferation. IL-4 or IL-6, in particular IL-6, facilitated IL-2-induced CTL proliferation, but not their cytotoxicity. In summary, uncultured melanoma cells by themselves induced modest levels of CTL proliferation in the context of MHC class II antigens, whereas they suppressed IL-2-induced CTL proliferation in more than half of the clones.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Potentiation of lymphokine-activated killer cell differentiation and lymphocyte proliferation by stimulation of protein kinase C or inhibition of adenylate cyclase

Interleukin 2 (IL-2) stimulated the differentiation of human peripheral blood leukocytes into lymphokine-activated killer cells, as well as DNA synthesis of human T lymphocytes. Both effects of IL-2 could be inhibited by prostaglandin E2, a potent stimulator of adenylate cyclase; however, the inhibitory effect of prostaglandin E2 could be overcome by increased concentrations of IL-2. The opposite effects of IL-2 and prostaglandin E2 were paralleled by their respective abilities to inhibit and stimulate cAMP production in intact cells. Other agents, which inhibit adenylate cyclase directly (somatostatin, beta-endorphin, UK 14.3041) or indirectly by activation of protein kinase C (phenylephrine), could stimulate both differentiation and proliferation. None of these agents alone or in combination were as effective as maximal concentrations of IL-2. However, all agents potentiated differentiation and proliferation induced by submaximal and maximal concentrations of IL-2. Additionally, combinations of agents which stimulated protein kinase C with those that inhibited adenylate cyclase were additive in the potentiation of IL-2-induced differentiation. Neither inhibition nor potentiation of IL-2-induced lymphokine-activated killer cell differentiation was accompanied by changes in Tac expression or gamma-interferon production. The data indicate that the stimulation of lymphokine-activated killer cell differentiation and lymphocyte proliferation in human cells share a common initial biochemical signal. Although the inhibition of adenylate cyclase is not sufficient to maximally stimulate either process and cannot bypass the requirement for IL-2, modulation of this enzyme complex, positively or negatively, can regulate the ultimate physiologic response to IL-2.     

Interleukin-6 counteracts effects of cyclosporin A on extracellular matrix metabolism by human dermal fibroblasts

Although the actions of cyclosporin (CyA) on keratinocyte are well established, little is known about its effects on dermal fibroblasts. Interleukin-6 (IL-6) is one of the inflammatory cytokines playing a pivotal role in certain skin diseases such as psoriasis. The aim of this study has been to determine whether CyA modifies the metabolism of extracellular matrix (ECM) by human fibroblasts in vitro. CyA altered the morphology of fibroblasts in the collagen matrix. Fibroblast proliferation was suppressed by CyA at 100 and 10 ng/ml. The production of type I collagen and tissue inhibitor of metalloproteinase 1 was also suppressed by CyA at 1000 ng/ml, and co-stimulation with IL-6 enhanced decreased production at 1000 and 100 ng/ml CyA. The production of matrix metalloproteinase 1 (MMP-1) was also suppressed by CyA in a dose-dependent manner. In contrast, the decreased production of MMP-1 was restored at 0.1-100 ng/ml CyA in the presence of IL-6. Regardless of the presence or absence of IL-6, the production of MMP-2 decreased at 1000 and 100 ng/ml, whereas the production of MMP-9 was unchanged. The production of transforming growth factor-beta decreased at 100 ng/ml CyA. This study thus indicates that CyA influences ECM metabolism and the proliferation of human dermal fibroblasts, and that the effects of CyA are modulated by IL-6. CyA might also, in part, improve psoriatic skin by regulating the remodeling of ECM and by its action on immunocompetent cells.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Clonal anergy blocks the response to IL-4, as well as the production of IL-2, in dual-producing T helper cell clones.

In this report we extend the in vitro clonal anergy model to examine the regulation of proliferation in T cells that secrete both IL-2 and IL-4. Newly cloned Ag-specific murine T cells are shown to depend on both IL-2 and IL-4 synthesis for maximal proliferation. Whereas IL-2 responsiveness is constitutive in these cells, IL-4 responsiveness develops only after Ag and APC stimulation. Remarkably, proliferation of these cells to Ag is sensitive to inhibition by clonal anergy, even though IL-4 synthesis remains inducible. Anergy in these cells is associated with an inability to respond to IL-4, in addition to the development of an IL-2 production defect. The results suggest that anergy induction may be capable of preventing the clonal expansion of autoreactive T cells producing both IL-2 and IL-4 in vivo.     

Virus-induced interferon alpha/beta (IFN-alpha/beta) production by T cells and by Th1 and Th2 helper T cell clones: a study of the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta on functions of different T cell populations

Spleen cells, resting T cells, activated T cells, and T cell clones characterized as type 1 (Th1) and type 2 (Th2) were investigated for their ability to produce interferon (IFN) following in vitro culture with Newcastle disease virus (NDV). All of the above cell populations, including both Th1 and Th2 T cell clones, produced high levels of IFN following in vitro culture with NDV. This IFN was characterized as a mixture of IFN-alpha and IFN-beta with IFN-alpha being the predominate species of IFN contained in the mixture. IL-2 greatly enhanced the production of IFN-alpha/beta by all cell populations in response to NDV. These different T cell populations responded very differently to the immunoregulatory actions of IFN-gamma versus IFN-alpha/beta. IFN-alpha/beta was shown to be a potent inhibitor of Con A or IL-2-induced proliferation of different T cell populations. This inhibition was not associated with a reduction in lymphokine production since spleen cells or Th1 T cell clones cultured with Con A and IFN-alpha/beta had no decrease in IL-2 or IFN-gamma production when compared to Con A-stimulated control cultures. IFN-gamma had little to no inhibitory activity on Con A-induced proliferation of spleen cells. In fact, Con A-induced proliferation was usually enhanced by IFN-gamma when nylon wool-enriched T cells were assessed. Different results were observed when IFN-gamma and IFN-alpha/beta were investigated for their ability to inhibit IL-2-induced proliferation of different T helper cell clones. IFN-gamma and IFN-alpha/beta were both capable of inhibiting IL-2-induced proliferation of T cell clones characterized as type 2 (Th2). In contrast, IFN-gamma had no effect on IL-2-induced proliferation of Th1 clones. IFN-alpha/beta, however, inhibited IL-2-induced proliferative responses of both Th1 and Th2 T cell clones. These results document the facts that (1) IFN-gamma and IFN-alpha/beta differ in their immunoregulatory actions, (2) different T cell subpopulations vary in their susceptibility to IFN-gamma regulation, and (3) virus induction of IFN-alpha/beta appears to be a ubiquitous function associated with different T cell populations.     

IL-12 augments antigen-dependent proliferation of activated T lymphocytes.

Ag-dependent T cell activation requires multiple transmembrane signals including activation of Ag-specific T cell receptor in combination with signals delivered through cytokine receptors. IL-12 is a heterodimeric cytokine involved in the regulation of NK and T lymphocyte responses. In examining the role of IL-12 in T cell activation, we found a direct relationship between Ag stimulation and IL-12-induced proliferation. Unlike IL-2, which induced proliferation of CTL either in the presence or absence of a CD3/TCR co-signal, IL-12 mediated proliferation of CTL only when the cells were recently co-stimulated with alloantigen or solid-phase anti-CD3 antibody. After culture in the absence of alloantigen or anti-CD3 for 7 to 14 days, these CTL lost the ability to proliferate to IL-12 alone. Under these conditions, however, IL-12 synergized with low-dose IL-2 to induce CTL proliferation. Restimulation with alloantigen or solid-phase anti-CD3 restored the ability of the CTL to proliferate to IL-12 alone. Not all Ag signals resulted in IL-2 independent proliferation to IL-12. For example, CTL with specificity for influenza matrix peptide proliferated best when co-cultured with peptide Ag presented on self MHC and a combination of IL-2 and IL-12. This evidence suggests that IL-12 may be useful in expanding an Ag-specific T cell population, as the culture of CTL with IL-12 and low-dose IL-2 leads to proliferation only in response to an Ag co-signal.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Human resting B lymphocytes can serve as accessory cells for anti-CD2-induced T cell activation.

Although resting B cells are poor accessory cells for signals transmitted through the TCR/CD3 complex, we report that these B cells can support T cell proliferation when T cell activating signals are delivered through CD2. This was first suggested when leucine methyl ester treatment of PBMC abolished proliferation induced by anti-CD3, but not by the accessory cell-dependent anti-CD2 mAb combination, GT2 and OKT11. Then we demonstrated that unstimulated, resting B cells could support the proliferation of both CD4+ and CD8+ T cells. Aggregated IgG inhibited proliferation, suggesting that anti-CD2 mAb bound to T cells were cross-linked by attachment to B cell FcR. Two lines of evidence suggested that lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interaction was crucial for anti-CD2-induced proliferation. First, proliferation was blocked by mAb against these adhesion molecules. Second, intercellular adhesion molecule-1 expression rapidly increased on resting B cells after the addition of anti-CD2, but not anti-CD3. This was of interest because fixed monocytes, but not fixed B cells, were able to support the proliferative response. In contrast to lymphocyte function-associated Ag-1/intercellular adhesion molecule-1, CD28/B7 interaction was not required for anti-CD2-induced proliferation, although ligation of these molecules provided important costimulatory signals for stimulation by anti-CD3. Finally, neutralizing antibodies against IL-1 alpha, IL-1 beta, and IL-6 showed only modest inhibitory effects on T cell proliferation. The addition of IL-1 and/or IL-6 to T cells failed to substitute for accessory cells and were only partially effective with fixed B cells. Further evidence of a linkage between CD2 and CD45 isoforms was obtained. Anti-CD45RA, but not anti-CD45RO, potentiated anti-CD2-induced T cell proliferation. These studies have revealed a novel role for resting B cells as accessory cells and have documented costimulatory signals that are important for this effect. Because Ag-presentation by resting B cells to T cells generally leads to T cell nonresponsiveness, it is possible that this tolerogenic signal may be converted to an activation signal if there is concurrent perturbation of CD2 on T cells.