Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

Comparison of mitogenic responses of young and old rhesus monkey T cells to lectins and interleukins 2 and 4

Purified T cells from rhesus monkeys, like human T cells, do not show a significant mitogenic response to lectins or PMA, but when combined with PMA or accessory cells, PHA and Con A induce a vigorous mitogenic response. This response is strongly impaired in purified T cells from old rhesus monkeys compared to young T cells, from 56 to 72%, and parallels results obtained with T cell preparations containing accessory cells. Likewise, purified T cells do not respond to interleukin 2 (IL-2) or IL-4, but in the presence of PMA, a significant mitogenic response occurs in the young but not the old T cells. This response is augmented by accessory cells, but is still very deficient in the old T cells. These results show that the IL-2 independent activation of T cells triggered by IL-4, like the conventional IL-2 activation, is age impaired. The deficient response to IL-2 implies an age-related deficiency in IL-2 receptor as well in aged rhesus T cells, and may account for the less effective response of the old cells to calcium ionophore (+PMA) activation. The use of purified T cells in these studies obviate the influence of accessory cells, and thus simplify interpretation.     

Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.     

Accessory cell-derived helper signals in human T-cell activation with phytohemagglutinin: induction of interleukin 2-responsiveness by interleukin 6, and production of interleukin 2 by interleukin 1 [corrected

Interleukins (IL-) 1 and 6 have been shown to represent accessory signals for T-cell activation. In the present study, we further examined the effects of both cytokines on accessory cell-depleted human T cells stimulated with phytohemagglutinin (PHA). The addition of IL-6 to the cultures resulted in T-cell proliferation; however, IL-1 was unable to support PHA-induced T-cell growth. The addition of IL-1 consistently induced a low level of IL-2 production and strongly enhanced T-cell proliferation in the presence of IL-6. Thus, the effect of IL-1 on T-cell growth becomes apparent only in the presence of IL-6. Blocking the IL-2-receptor (IL-2R) with the monoclonal antibodies anti-Tac and MikBêta 1 (directed to the alpha and bêta chains of the IL-2R, respectively) had no effect on PHA/IL-6-supported proliferation, but completely eliminated the growth-enhancing effect of IL-1. On the other hand, a neutralizing anti-IL-4-antiserum did not affect PHA/IL-6- or PHA/IL-6/IL-1-induced proliferation. Further experiments showed that IL-6 enhances T-cell responsiveness to IL-2, as evidenced by enhanced IL-2-induced proliferation. However, we could not find an effect of IL-6 on the expression of IL-2R as measured by staining with anti-Tac and with MikBêta 1 or by binding of (125I)-IL-2 to T cells. It can be concluded from these studies that IL-1 and IL-6 have different helper effects on PHA-induced T-cell activation. In the presence of PHA, IL-6 induces limited IL-2/IL-4-independent growth, and more importantly it renders T cells responsive to IL-2. IL-1 provides a signal leading to IL-2 production. The combination of IL-1 and IL-6 represents a synergistic helper signal, leading to an IL-2-dependent pathway of proliferation.     

Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells.     

Distinct regulation of lymphokine production is found in fresh versus in vitro primed murine helper T cells

The kinetics of lymphokine RNA induction and secretion of biologically active lymphokine from CD4-enriched splenic T cell populations was investigated. Cells stimulated immediately after isolation from murine spleen ("fresh" T cells) and cells restimulated after 4 days of in vitro culture ("primed" T cells) were compared. Northern blot analysis and bioassays were used to analyze and quantitate production of eight lymphokines and the IL-2R. Fresh T cells produced high levels of IL-2 and low to moderate levels of IL-3, granulocyte/macrophage-CSF, and IFN-gamma. In vitro primed T cells produced IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte/macrophage-CSF, IFN-gamma, and high levels of IL-2R RNA. Comparison of RNA levels and bioassays of supernatants from these populations indicated that primed T cells produced at least 10-fold more of six of the lymphokines than fresh T cells. Only IL-2 was produced in near equal amounts by fresh and primed T cells. There were also marked differences in the kinetics of lymphokine production by fresh and primed CD4+ T cells. After restimulation with Con A and PMA, primed cells produced a short burst of lymphokine RNA that peaked between 7.5 and 13 h and declined after 18 h. Fresh T cells lagged in the initial production of lymphokine RNA, with levels peaking 18 to 44 h after mitogenic stimulation. Depletion of CD4+ cells indicated that cells of helper phenotype were responsible for the majority of lymphokine production from the primed cells. Thus different subpopulations of Th cells defined by their respective ability to respond either directly (fresh T cells) or only after culture and restimulation (primed T cells) show different patterns of lymphokine gene regulation. Other studies suggest that the activity of "fresh" Th cells is due to a population with a "memory" phenotype, while the cells which require culture have a "precursor" phenotype. These distinct patterns of lymphokine gene regulation in the two populations of Th cells may account in part for differences seen in the kinetics and magnitude of the naive and memory immune responses which are regulated by Th cells.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

One-signal requirement for interferon-gamma production by human large granular lymphocytes

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.     

Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody

The effect of stimulating normal type 1 murine T cell clones with anti-CD3 antibody was examined in vitro. In the absence of accessory cells, anti-CD3 antibody immobilized on plastic plates stimulated inositol phosphate production, suboptimal proliferation, IL-2 and IL-3 production, and maximal IFN-gamma production. Addition of accessory cells augmented lymphokine production and proliferation when the effects of "high-dose suppression" were relieved by removing the T cells from the antibody-coated plates. Exposure of type 1 T cell clones to immobilized anti-CD3 antibody alone rapidly induced long-lasting proliferative unresponsiveness (anergy) to Ag stimulation that could be prevented by accessory cells. This anergic state was characterized by a lymphokine production defect, not a failure of the T cells to respond to exogenous IL-2 or to express surface Ti/CD3 complexes. In addition, anergy could not be induced in the presence of cyclosporine A. These results suggest that under certain conditions anti-CD3 antibodies may have potent immunosuppressive effects independent of Ti/CD3 modulation. Furthermore, our results support a two-signal model of type 1 T cell activation in which Ti/CD3 occupancy alone (signal 1) induces anergy, whereas Ti/CD3 occupancy in conjunction with a costimulatory signal (signal 2) induces a proliferative response.     

Histamine inhibits interferon-gamma production via suppression of interleukin 2 synthesis

Histamine, a modulator of various immune functions, inhibits the production of interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) by polyclonally activated human blood mononuclear cells. The histamine-induced inhibition of IFN-gamma synthesis can be completely eliminated by the addition of recombinant IL-2. The IFN-gamma synthesis by T8+ lymphocytes is highly dependent on IL-2 supplied either by the IL-2 producing T4+ lymphocytes or through exogenous addition of recombinant IL-2. It is concluded that histamine acts primarily on the interleukin 2 synthesis by the T4+ lymphocytes and as a consequence of this inhibition, interferon-gamma production is reduced.     

Histamine modulates the production of interferon-gamma and interleukin-2 by mitogen-activated human mononuclear blood cells

Histamine inhibited the production of interferon-gamma and interleukin 2 (IL-2) induced in human peripheral blood mononuclear cells by Staphylococcal Enterotoxin A (SEA) but had no effect on the expression of IL-2 receptors. The effects on lymphokine production were dose dependent with maximal inhibition occurring at histamine concentrations of 10(-4) to 10(-6) M. The H2-agonist 4-methylhistamine but not the H1-agonist 2-methylhistamine modulated lymphokine production in a similar manner as histamine. Histamine at concentrations of 10(-3) to 10(-8) M had no inhibitory effect directly on the activity of admixed IL-2 containing medium. The inhibitory effects of histamine could be reversed by the H2-antagonist cimetidine but not by the H1-antagonist diphenhydramine. This indicates that the inhibitory effects of histamine on lymphokine production are mediated through H2-receptors on mononuclear cells.     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Bovine papilloma virus encoded E2 protein activates lymphokine genes through DNA elements, distinct from the consensus motif, in the long control region of its own genome.

Activation of T cells by antigen, lectin or a combination of phorbol ester (PMA) and calcium ionophore (A23187) leads to the induction of a set of lymphokine genes. Transfection of a human T cell leukemia cell line, Jurkat, or an African green monkey kidney cell line, CV1, with a cDNA encoding E2 protein, a trans-activator of bovine papilloma virus type 1, results in activation of interleukin 2 (IL-2), interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) genes in a transient transfection assay. 5' deletion and mutation analyses showed that the sequence between positions -60 and a TATA-like sequence is required for basic promotor function and that the sequence between positions -95 and -73 containing conserved lymphokine element 2 (CLE2) and a GC box (CLE2/GC box) mediates the positive response to E2 protein. The latter has been previously shown to respond to PMA/A23187 stimulation or to p40tax, a trans-activator encoded by human T cell leukemia virus type 1 (HTLV-I). The sequence located between -108 and -99 (CLE1) is inhibitory to E2 protein or PMA/A23187 stimulation. The combination of E2 protein and PMA/A23187 appears to eliminate an inhibitory effect of the upstream region. However, E2 protein, like p40tax, mediates a positive response through CLE1 alone linked to the basic promoter sequence. The level of activation of the long control region (LCR) by E2 protein is unaffected by the number of CLE2/GC box sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.     

Differential effect of interferon-gamma and interleukin-2 on the induction of IgA and IgM anti-dextran responses

Supernatants from T-cell lines and T-cell hybridomas can substitute for T cells in the induction of the anti-alpha(1,3) Dextran B1355 plaque-forming cell response in culture. The present study sought to define the lymphokines required for the induction of IgA and IgM anti-alpha (1,3) dextran responses. Recombinant Interferon-gamma (IFN-gamma) supported the induction of low levels of IgA anti-alpha(1,3) dextran plaque-forming cells in splenic B-cell cultures. IgA responses were substantially increased when cultures containing IFN-gamma were supplemented with an interleukin 2 (IL-2)-containing supernatant from the murine T-cell hybridoma BW.Mls, purified murine IL-2, or recombinant human IL-2. In striking contrast, IgM anti-alpha(1,3) dextran plaque-forming cells were not produced in cultures containing IFN-gamma alone or in combination with purified or recombinant IL-2. However, substantial IgM responses could be produced in cultures containing IFN-gamma and BW.Mls supernatant. This data indicates that there may be different lymphokine requirements for the induction of IgA and IgM anti-alpha(1,3) dextran B cells, or alternatively, such B cells may be in different stages of differentiation and therefore, not respond to the same lymphokines.     

Stimulation of AIDS lymphocytes with calcium ionophore (A23187) and phorbol ester (PMA): studies of cytoplasmic free Ca, IL-2 receptor expression, IL-2 production, and proliferation

We have studied whether the decreased lymphocyte proliferative responses of AIDS lymphocytes to stimulation by mitogens and antigens may be overcome when challenged with a combination of calcium ionophore A23187 and phorbol ester PMA. Comparison of the proliferative response of lymphocytes from nine patients with AIDS with the response of lymphocytes from nine control subjects showed that the response of AIDS lymphocytes was severely decreased when stimulated with PHA and no further response could be achieved by stimulation with A23187/PMA. On the other hand, no significant difference between the PHA-induced rise of cytoplasmic free calcium concentration ([Ca2+]1) in normal and AIDS lymphocytes was observed. The percentage of cells expressing IL-2 receptors (CD25) was also normal both after addition of PHA and after addition of A23187/PMA and the expression was normal on both CD4 and CD8 cells. The production of IL-2 in normal lymphocytes stimulated with A23187/PMA was 33 times higher than that after stimulation with PHA. In AIDS lymphocytes the production of IL-2 induced by all activators was severely decreased compared to control subjects, although the production of IL-2 after stimulation with A23187/PMA was higher than that in control lymphocytes after stimulation with PHA. The present study shows that a direct activation of protein kinase C combined with mobilization of cytoplasmic calcium does not overcome the lymphocyte proliferative deficiency of AIDS lymphocytes.     

FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes.

This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes.     

Characterization of accessory cell costimulation of Th1 cytokine synthesis.

We studied the capacity of macrophage and B cell lines to provide a costimulatory signal that enhances synthesis of IFN-gamma and IL-2 by mouse Th1 clones stimulated with suboptimal doses of immobilized anti-CD3 antibody. The J774 macrophage line and the CH27 B lymphoma line had the greatest costimulatory activity and routinely increased IL-2 production by 10-fold to 100-fold. Other macrophage and B cell lines had less activity and T cell lines were unable to costimulate. The J774 and CH27 lines did not costimulate IL-4 production by a Th2 clone and had only a small effect on IL-2 production by T cell hybridomas. The process of costimulation was fixation-sensitive, contact-dependent and did not involve stable cytokines present in the T cell/accessory cell conditioned media. Neutralizing antibodies for IL-1, IL-6, and TNF failed to inhibit costimulation. Antibodies to the LFA-1/ICAM-1 pair of adhesion molecules also failed to inhibit. Costimulation of IL-2 production by accessory cells was found to have a unidirectional species restriction: mouse accessory cells costimulated mouse and human IL-2-producing T cells, but human U937 cells induced with PMA were effective only for human T cells. The results indicate that accessory cells can significantly regulate Th1 effector function at the level of cytokine production.