Interplay between IL-2 and IL-4 in human thymocyte differentiation: antagonism or agonism

The effect of recombinant interleukin 2 (IL-2) and IL-4, as well as a combination of both lymphokines on human post-natal thymocytes at different maturation stages, was analyzed by culturing highly purified pro-T cells, pre-T cells, double-negative and double-positive thymocyte subsets in the presence of IL-2 and/or IL-4. Both IL-2 and IL-4 responsiveness are developmentally regulated in human thymocytes, since IL-2 and IL-4 responses decline with increasing thymocyte differentiation, double-positive T cells displaying far less proliferation than immature thymocytes. IL-2 and IL-4 may influence pro-T cell growth in both an antagonistic and additive fashion. At low doses, IL-4 inhibits IL-2-supported growth of pro-T cells, whereas, at higher concentrations, this inhibitory effect is masked by the ability of IL-4 to stimulate pro-T cell proliferation. In contrast to peripheral lymphocytes, IL-4 does not down-regulate the expression of the IL-2 receptor light chain on thymocytes. In pro-T cell cultures, IL-2 and IL-4 favour the differentiation of distinct cell populations, namely lymphocytes displaying preferentially a TCR alpha/beta+ and CD4+CD8- phenotype versus predominantly TCR gamma/delta+ and CD4-CD8+ cells, respectively. The effect of IL-2 dominates over that of IL-4, since the composition of cultures set up in the presence of IL-2 plus IL-4 resembles that of cells cultured with IL-2 alone. In synthesis, IL-2 and IL-4 exhibit reciprocal inter-relations in human thymocyte cultures, thus supporting the notion that these lymphokines are implicated in the complex regulation of a local cytokine network.     

Expression and biological activity of interleukin-1 receptors in mouse gamma/delta thymocytes during ontogeny.

We have recently shown that the response of mouse thymocytes to interleukin (IL)-1 + IL-2 was maximal at birth and that the responding cells displayed a CD4-CD8- T cell receptor (TcR) gamma/delta + phenotype. Unexpectedly, despite their high proportion of gamma/delta + cells, fetal thymocyte populations responded only weakly to IL-1 + IL-2. In this report, we demonstrate that the discrepancy between the day 17.5 fetal and newborn sensitivities to the combined action of IL-1 and IL-2 is a consequence of the different patterns of high-affinity IL-1 receptor (IL-1R) expression displayed by these two cell subsets. Actually, high- and low-affinity IL-1R are found in TcR gamma/delta + newborn cells and, in contrast, only low-affinity IL-1R are detectable in day 17.5 fetal cells. Our binding and functional studies strongly support the hypothesis that high-affinity IL-1R on the one hand, and low-affinity ones on the other hand, are involved in the response to IL-1 + IL-2 of newborn and day 17.5 fetal thymocytes, respectively. In addition, the high-affinity IL-1R appear to be far more efficient than the low-affinity receptors in promoting IL-2 responsiveness of thymocytes.     

Role of IL-2 in rat fetal thymocyte development

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high- affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR- P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.      

Differential effect of tumour necrosis factor on human thymocyte subpopulations.

We have studied the effect of tumour necrosis factor (TNF) on purified human thymocyte subpopulations. For this purpose human thymocytes were purified by negative selection with three rounds of several antibodies plus complement. TNF was able to co-stimulate in a dose-response manner the proliferation of single positive (SP) CD3+ CD4+ or CD3+ CD8+ thymocytes in the presence of optimal doses of interleukin-2 (IL-2), phytohaemagglutinin (PHA), anti-CD3 antibodies or phorbol esters. However, CD1+ CD3low CD4+ CD8+ cortical thymocytes did not proliferate significantly in response to any stimulus alone or in combination. The TNF proliferative effect on SP thymocytes was blocked by an anti-IL-2R alpha antibody. In addition, TNF enhanced the expression of the IL-2R alpha but not IL-2R beta on the cell surface of CD1- CD3+ SP thymocytes over the levels induced by the other primary stimuli, inducing as a consequence, an increase in the number of high affinity IL-2R. Furthermore, TNF was able to increase IL-2R alpha mRNA levels on SP thymocytes. On the other hand, TNF was mitogenic in the absence of any other stimulus for CD1- CD3- CD4- CD8- prethymocytes, as was IL-2, and this proliferation was not blocked by anti-IL-2R alpha antibodies. Furthermore, the proliferation of this subset in response to IL-2 and TNF was additive. TNF was able to increase directly the cell surface expression of both chains, IL-2R beta and IL-2R alpha, and the IL-2R alpha messenger RNA (mRNA) levels of CD1- CD3- CD4- CD8- prethymocytes. In summary, our results suggest that TNF may have an important role as a co-stimulatory signal in some human thymocyte subpopulations by inducing the expression of IL-2R.     

IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets

The specific signals inducing the growth and maturation of thymocytes remain undefined. We show here that recombinant IL-7 induces growth of fetal and adult mouse CD4-8- thymocytes. IL-7 also induces a lower but significant response in CD4+8- and CD4-8+ thymocytes. Day 14 fetal thymic lobes cultured in IL-7 for 6 days show a 2-fold increase in cell number when compared to control cultures. The thymocyte subsets that proliferate in response to IL-7 can be maintained in culture for extended periods of time. CD4-8- thymocytes maintained in IL-7 did not change their phenotype with respect to CD4 and CD8 expression. In addition, we show that the combination of IL-7 plus IL-6 provides a potent growth stimulus for CD4+8- and CD4-8+ thymocytes. A cloned thymic epithelial cell, that can be induced to express MHC class II molecules, transcribes both IL-7 and IL-6 mRNA. A cloned thymic macrophage cell line produces IL-6 but no detectable IL-7 mRNA. The pattern of biological activities present in the supernatants of these cell lines is also presented. These observations suggest that the thymic epithelial and macrophage cell types may be an in vivo source of signals which mediate thymocyte development.     

Effect of PGE2 on thymocyte proliferation induced by Con A or IL-4 + PMA

Prostaglandin E₂ (PGE₂) is known to inhibit peripheral T-lymphocyte and thymocyte proliferation activated by antigens, mitogens or anti-CD3 antibodies. In this study, we have investigated, the effect of PGE₂ on thymocyte proliferation induced by the combination of IL-4 plus PMA. PGE₂ inhibits the proliferation of thymocytes activated by ConA, whatever the culture period; in contrast PGE₂ shifts the kinetics of thymocyte proliferation after stimulation by IL-4 plus PMA, but does not sustain the proliferation beyond day 3. This effect depends upon cell density, IL-4 concentration and on the time that PGE₂ is added to the culture. By use of the cAMP inducer, forskolin, or a cAMP analog, db-cAMP, we observed the same results. PGE₂ increases the proliferation of CD8⁺ corticoresistant thymocytes (CRT) activated by IL-4 plus PMA, but inhibits that of CD4⁺CRT. These results suggest that PGE₂ can regulate thymocyte proliferation differently according to the activation pathway and the thymic subpopulations.     

CD3-mediated apoptosis of human medullary thymocytes and activated peripheral T cells: Respective roles of interleukin-1, interleukin-2, interferon-γ and accessory cells

Clonal deletion represents an important mechanism for the establishment of tolerance, by the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death, termed apoptosis, induced by mobilization of the T cell receptor (TCR) on both thymocytes and mature T cells. The mechanism which drives T cells towards cell death or cell proliferation after TCR mobilization remains unclear. We show here that the mobilization of the CD3/TCR complex of both CD4⁺ and CD8⁺ single-positive medullary human thymocytes and human mature activated T cells, in the absence of accessory cells, leads to an activation-induced cell death process by apoptosis. In both cases, apoptosis was associated with interferon (IFN)-γ gene expression and secretion in the absence of interleukin (IL)-2 gene expression; and the addition of anti-IFN-γ antibody prevented cell death. Apoptosis could also be prevented by cyclosporin A (CsA) treatment and could be re-induced by the addition of IFN-γ to CsA-treated cells. Addition of IL-2 had two different effects, it prevented apoptosis and also allowed proliferation in response to CD3 monoclonal antibody. Addition of IL-1, which induces IL-2 gene expression and secretion or addition of accessory cells, had the same preventive effect. These results suggest that the uncoupling of IFN-γ and IL-2 gene expression following CD3/TCR mobilization initiates apoptosis of human T cells at several different stages during development and activation. We propose that co-signals provided by accessory cells allow a coupling of IL-2 gene and IFN-γ gene expression, and that an essential role for IL-2 secretion in T cell activation involves the inhibition of a death program induced by IFN-γ secretion.     

Endogenously secreted IL-4 is required for mouse thymocytes to become cytotoxic. Human, but not mouse, IL-2 induces a functionally immature thymic subset to secrete IL-4 and become CTL.

Experiments described here demonstrate that the differentiation of mouse thymocytes into cytotoxic T lymphocytes (CTL) requires interleukin-4 (IL-4). To reach this conclusion, we took advantage of our discovery that human and mouse IL-2 have very different effects on the development of CTL from a functionally immature subset of thymocytes. The lobster agglutinin 1 (LAg1)-negative subpopulation of thymocytes proliferated when cultured with concanavalin A (Con A)+ human or mouse IL-2, but these cells became CTL only when cultured with Con A+ human IL-2. Furthermore, Con A+ human IL-2, but not mouse IL-2, stimulated IL-4 production by cells within this population. Con A-induced cytotoxicity by mature LAg1-positive thymocytes and normal thymocytes was also accompanied by secretion of IL-4. The anti-IL-4 mAb 11B11 inhibited induction of cytotoxicity by all thymocyte populations tested. Taken together these experiments indicate that stimuli which induce cytotoxicity by mouse thymocytes also induce the secretion of IL-4, which is necessary for the differentiation of thymocyte CTL precursors into CTL.     

Effects of exogenous cytokines on the ethanol-mediated suppression of murine thymocyte proliferation

Although attempts have been made to assess the effect of ethanol on murine thymocyte proliferation, the mechanism which accounts for the immunosuppressive effect of ethanol on the thymocyte proliferation has not been elucidated. Thus, a mouse model was used to determine (1) whether there is a similarity in the effect of ethanol exposure in vitro and in vivo on the proliferative response of thymocytes to phytohemagglutinin (PHA), (2) whether ethanol exposure affects the responsiveness of thymocytes to exogenous interleukin (IL)-I and IL-2, and (3) whether ethanol affects IL-1 production by peritoneal macrophages. We found that the proliferative response of thymocytes from mice fed on an ethanol-containing diet was significantly inhibited (P<0.05) compared to that in mice red on maltose or standard diets. We also observed that low concentrations of ethanol (12.5 mM) appeared to enhance the mitogenic response of thymocytes to PHA, but the response was not significantly greater than that of controls (P>0.05). Ethanol at higher concentrations (25–100 mM) significantly suppressed the mitogenic response of thymocytes to PHA (P<0.05) in a dose-dependent manner. Our data also revealed that (1) ethanol did not significantly suppress IL-I secretion by adherent macrophages stimulated by LPS, and (2) the addition of exogenous IL-I was insufficient to restore full responsiveness in thymocytes from ethanol-fed mice. Taken together, these results suggest that the suppressive effect of ethanol on thymocyte proliferation is not mediated by insufficient IL-1. Finally, we present novel evidence that addition of exogenous IL-2 completely restores the impaired proliferative response of thymocytes from ethanol-fed mice to control levels. In summary, our results demonstrate that ethanol inhibits thymocyte proliferation in response to PHA, and that the inhibition is not due to insufficient IL-1. We also report that addition of exogenous IL-2 is sufficient to restore full proliferative capacity to thymocytes from ethanol-fed mice.     

Cyclosporin A inhibition of macrophage colony-stimulating factor (M-CSF) production by activated human T lymphocytes

M-CSF is a pleiotropic cytokine involved in the survival, proliferation, and differentiation of cells of the monocyte/macrophage lineage. M-CSF is produced by numerous cells including CD3-activated T cells. M-CSF serum levels are increased during acute graft rejection. We tested the in vitro production of M-CSF, GM-CSF, IL-2, and IL-4 by T-cell clones costimulated by CD3 and accessory activation pathways and the effects of cyclosporin A and methylprednisolone. The nine clones studied and CD4+ cells purified from peripheral blood mononuclear cells (PBMC) spontaneously produced low levels of M-CSF, which PMA and CD3 mAb strongly enhanced. In contrast to IL-2, CD28 mAb did not further enhance this production. CsA inhibited M-CSF production by clones and purified CD4 T cells. Addition of IL-2, anti IL-2, or anti CD25 mAb to the cultures demonstrated that CsA down-regulated M-CSF synthesis by activated T cells through its inhibition of IL-2 synthesis. These results could help to better understand the complex mechanisms of acute graft rejection and immunosuppression.     

Expression and function of CD2 during murine thymocyte ontogeny

CD2, originally recognized as the sheep erythrocyte receptor of human T cells, has been implicated in early T cell development in the thymus. In this report, we examined the expression and functional role of CD2 during murine thymocyte ontogeny by using monoclonal antibodies to murine CD2. Surface expression of CD2 was first detected in Thy-1+ fetal thymocytes at day 14 of gestation and it progressively increased during CD4-CD8- phenotype. Surface IL 2 receptor (CD25) expression was readily detected in surface CD2- cells at day 13 of gestation and the majority of CD2+ cells appeared to be generated from CD25+ cells thereafter. In adult CD4-CD8- thymocytes, the expression of CD2 and CD25 was mutually exclusive. These results indicate that surface CD2 expression is not a prerequisite for CD25 induction during murine thymocyte ontogeny. This was further confirmed by fetal thymus organ culture in which anti-murine CD2 mAb was included. The antibody treatment led to a suppressed CD2 expression on thymocytes; however, there was no effect on the appearance of CD25. Moreover, no influence on the development of mature CD3+ thymocytes was observed after fetal thymus organ culture in the presence of anti-murine CD2 mAb, and a substantial number of CD3+CD2- cells was demonstrated in fetal and adult CD4-CD8- thymocytes. These findings argue against the functional relevance of CD2 expression during early T cell development as proposed in humans.     

1,25-Dihydroxyvitamin D3 (calcitriol) suppresses IL-2 induced murine thymocyte proliferation

1,25-dihydroxyvitamin D3 (Calcitriol) inhibits mitogen stimulated T cell proliferation by blocking the production of Interleukin-2 (IL-2). The present study was initiated to determine whether the action of calcitriol was limited only to inhibition of IL-2 production, or if it influenced other events as well. To avoid the use of lectins, thymocytes from CD-I Swiss mice were chosen, which proliferate in response to Interleukin-I (IL-I) or IL-2 without the addition of lectins. Calcitriol inhibited (80-90%) IL-I induced CD-I mouse thymocyte proliferation, whereas 25-OH-D3 was ineffective. Further addition of IL-I failed to overcome this suppression. Surprisingly, calcitriol also inhibited IL-2 induced thymocyte proliferation (60-80%). Further addition of IL-2, in the presence of calcitriol, was ineffective in enhancing thymocyte proliferation. Similar results were obtained with C57BL/10 mouse thymocytes. Additional studies excluded the possibilities that calcitriol mediated inhibition was due to calcitriol IL-2 binding or damage to thymocytes by calcitriol. Thus, calcitriol not only blocks IL-2 production, but these results strongly suggest, that it also interferes with IL-2-thymocyte interaction, which in turn results in inhibition of thymocyte proliferation.     

Growth of fetal thymocytes in organ culture: effect of recombinant lymphokines on thymocyte maturation

Interleukin 2 (IL2) has a dose-dependent inhibitory effect on the growth and phenotypic maturation of thymocyte populations grown in fetal thymus organ culture. Addition of IL2 (100 U/ml) to 14-day fetal thymus organ cultures induces the appearance of a population of lymphokine-activated killer (LAK) cells which lyse allogeneic, syngeneic, and syngeneic tumor cell targets. The addition of the monoclonal antibody, PC-61, blocks the IL2-dependent growth and activation of LAK cells but does not influence the maturation of CD4+ CD8+ fetal thymocytes. These data imply that IL2 is not a major regulator of normal fetal thymocyte maturation. The effects of a range of recombinant lymphokines (IL1 alpha, IL1 beta, IL3, IL4, GM-CSF, G-CSF, M-CSF) on the proliferation and phenotypic maturation of fetal 14-day thymocytes in organ culture has been analysed. Two significant changes were seen. First, IL1 alpha and IL1 beta inhibited growth and the expression of the CD4 and CD8 antigens in organ culture, and second, GM-CSF increased the expression of Mac-1+ cells. IL4, which has known T cell growth-promoting activity, IL3, G-CSF, and M-CSF did not alter either normal growth or surface antigen expression in fetal thymocytes. While some of these lymphokines may function as accessory molecules in fetal thymocyte development, our experiments suggest that they do not have a significant influence on thymocyte maturation when used alone.     

IL-4 and IL-2 promote human T-cell proliferation through symmetrical but independent pathways.

The role of IL-4 and IL-2 on normal human T-cell activation and proliferation was studied. Both IL-2 and IL-4 were unable to induce proliferation of resting T cells. Therefore, we investigated their effect and the regulation of the T-cell proliferative response in competent T cells. T cells were rendered competent following incubation with PDB/ionomycin for 30 min or suboptimal concentrations of PHA for 60 min. Cells were then washed and recultured with PDB, IL-2, or IL-4 in the second or progression phase of the culture. Cells cultured in medium alone in this phase did not proliferate. IL-2 and IL-4 independently promoted competent T cells to proliferate to a similar degree as the response to PDB and the combination of IL-2 and IL-4 was not additive. The induction of competence and subsequent responsiveness to IL-2 and IL-4 could be maintained for about 24 hr after which time they become gradually less responsive to the interleukin in the progression phase. Addition of anti-IL-2R mAb or anti-IL-2 mAb resulted in selective inhibition of IL-2-mediated proliferation only. Similarly, addition of anti-IL-4 mAb resulted only in inhibition of IL-4-mediated proliferation. Addition of IL-2 during the progression phase led to an enhancement of IL-2R (TAC) expression while IL-4 did not affect IL-2R expression. The production of IL-2 and IL-4 by competent T cells could not be enhanced by the noncorresponding lymphokine. These results on the protein level were confirmed at the mRNA level as well and demonstrated that only PDB and IL-2 could induce IL-2 mRNA and PDB and IL-4 enhanced IL-4 mRNA. The immunosuppressive drug, cyclosporin A, failed to inhibit progression triggered by PDB, IL-2 or IL-4 in competent T cells. These findings suggest that IL-2 and IL-4 trigger T-cell proliferation through symmetrical, but independent pathways.     

CD4单克隆抗体改变新生小鼠胸腺细胞亚群的分布和功能

新生期小鼠（３～７天）腹腔注入ＣＤ４ＭｃＡｂ，可引起胸腺细胞表型和功能变化。胸腺细胞表型分析显示：胸腺细胞总数、ＤＰ和ＣＤ４ＳＰ细胞明显减少；ＣＤ８ＳＰ细胞数增加；ＤＮ细胞数目变化不大。上述变化与ＣＤ４ＭｅＡｂ和胸腺细胞表面的ＣＤ４分子的结合有关。功能试验表明：实验组小鼠胸腺细胞对有丝分裂素ＣｏｎＡ、ＰＷＭ的反应性降低，混合淋巴细胞反应（ＭＬＲ）也明显低于对照组。但是胸腺细胞对异型细胞的杀伤作用则高于对照组。功能试验的结果与表型的变化是吻合的，有剂量依赖性。ＣＤ４ＭｃＡｂ引起的胸腺细胞表型和功能的变化是可以恢复的，一般于注射ＣＤ４ＭｃＡｂ后１０天，胸腺细胞亚群分布恢复或接近正常，而功能则恢复较慢。     

Intoxication of high affinity IL-2 receptor positive thymocytes blocks early stages of T cell maturation.

Early murine fetal thymocytes express functional, high affinity IL-2 receptors as determined by: (i) the presence of IL-2R beta chain (p75) mRNA; (ii) IL-2 (10 U/ml) induced cell proliferation/cellular maturation in lobe submersion cultures (LSC). Under the influence of IL-2, early thymocytes differentiate in vitro into more mature, early single positive CD4-CD8+ followed in vivo by double positive CD4+CD8+ and single positive CD4+CD8-T and CD4-CD8+ thymocytes. Specific intoxication of high affinity IL-2R positive thymocytes by recombinant interleukin-2-diphtheria toxin-related fusion protein (DAB486-IL-2) results in transient, dose dependent blockade of in vivo and in vitro thymocyte maturation. DAB486-IL-2 induced effects upon in vivo maturation are reversible within 2 weeks after cessation of drug administration. Taken together, these results demonstrate the expression of functional, high affinity IL-2 receptors on early thymocytes. Elimination of high affinity IL-2 receptor positive thymocytes with DAB486-IL-2 results in transient blockade of T cell maturation. Since DAB486-IL-2 is now in clinical trial, it is reassuring to note that it does not permanently disrupt thymic maturation.     

Plasticity in the positive selection of T cells: affinity of the selecting antigen and IL-7 affect T cell responsiveness

The current study examines how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of their thymocyte precursors. We used a thymus reaggregate culture system in which CD4(+)CD8(+) thymocytes from AND TCR transgenic mice were induced to undergo positive selection by pigeon cytochrome c (PCC) peptide or its analogs presented by I-E(k) class II MHC on a thymic epithelial cell line. When low-affinity peptide analogs drove positive selection, up to 100 microM was needed to produce >50% CD4(+) T cells, and these cells were highly responsive to PCC. In contrast, <0.2 microM high-affinity peptides was required to achieve similar selection efficiency, but the resultant cells failed to respond to PCC. However, these cells were not dead based on dye exclusion and capacity to respond to phorbal ester and to agonist if IL-2 was also present, supporting the view that non-responsiveness of cells selected on high-affinity peptides is a form of central T cell tolerance distinct from deletion. Cells selected on intermediate-affinity peptides showed variable responsiveness which was suppressed 5- to 10-fold by addition during reaggregate culture of antibody to the IL-7R. Similarly, supplementary IL-7 in the reaggregate culture produced CD4(+) T cells that were promiscuously responsive. Overall, this study demonstrates that the responsiveness of T cells is not rigidly controlled and that the presence of IL-7 during T cell development has the potential to negate central T cell tolerance and produce autoreactive T cells.     

Activation of peripheral CD8+ T lymphocytes via CD28 plus CD2: Evidence for IL-2 gene transcription mediated by CD28 activation

It is well established that peripheral CD8+ and CD4+ T cells display different requirements for in vitro activation by mitogenic mAb. Most CD4+ T cells can be activated by anti-CD3 or mitogenic combinations of anti-CD2. In contrast, CD8+ T cells display minimal responses to CD3 activation, and no proliferation is observed via CD2 activation. Purified peripheral blood CD8+ T cells, stringently depleted of APC, have been studied for their capacity to respond to mAb directed against CD3, CD2 and CD28, used alone or in combination. It is demonstrated that proliferation can be induced by co-stimulation of CD2 and CD28. This does not require autologous APC. CD8+ T cells can also be activated by the combination of anti-CD3 plus anti-CD28 in the presence of APC, but only minimal cell proliferation is obtained in the absence of APC. The response via CD2 plus CD28 is IL-2-dependent, as demonstrated by the ability of mAb against the IL-2 receptor to block proliferation, and is almost completely inhibited by cyclosporine A (CsA). These results suggest that the signal generated by stimulation of CD28 in combination with CD2 differs from that seen with CD28 activation combined with either PMA or CD3. Induction of IL-2 gene activation in CD8+, CD28+ peripheral T cells may therefore require additional "second signals", which are not necessary for activation of CD4+ cells. One such signal might be the interaction between CD28 and its natural ligand.     

TCR- and IL-1-mediated co-stimulation reveals an IL-4-independent way of Th2 cell proliferation

Previously, it has been shown that T_h1 cells, when triggeredsolely via their TCR, are blocked from proliferation in responseto IL-2. Herein, we describe a similar characteristic for T_h2cells in that immobilized mAb directed to the TCR blocked proliferationof T_h2 cells in response to IL-4. This ‘prollferatlveblock’ was observed in all four T_h2 cell clones tested,but not In a subline of one of the clones which has been culturedin vitro for several years. Addition of IL-1 neutralized theproliferatlve block in all four T_h2 cell clones. Surprisingly,blocking experiments with SIL-4R and anti-IL-4 mAb revealedthat in three out of four T_h2 cell clones this effect of IL-1was IL-4-independent and could also not be blocked by cyclosporinA (CsA). in contrast, the proliferation of one T_h2 cell clonein response to the TCR- and IL-1-mediated signals was indeedinhibited by sIL-4R, antML-4 mAb and CsA. Thus, our data illustratethat in addition to the well-known IL-4-dependent proliferation,there also exists an iL-4-lndependent, IL-1-mediated way ofT_h2 cell proliferation.     

Accessory Signalling by B7-1 for T Cell Activation Induced by Anti-CD2: Evidence for IL-2-Independent CTL Generation and CsA-Rcsistant Cytokine Production

Resting T cells can be activated by selected pairs of anti-CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti-CD28, or by the combination of IL-1β and IL-6. The present study was undertaken to investigate accessory signalling by B7-1, the natural ligandof CD28, in this pathway of T cell activation. 3T6 mouse fibrobiasts were transfected with human B7-1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti-CD2 MoAb, T cell proliferation, production of cytokines (IL-2, IL-4, IL-10, GM-CSF, IFN-α and TNF-α), and generation of cytotoxic T lymphocytes were all supported by B7-l(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti-IL-2 + anti-IL-2R MoAb revealed both IL-2-dependent and IL-2-independent CTL generation after B7-1 -mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL-2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7-1 - CD28 interaction. Anti-CD2 MoAb with B7-1 costimulation could directly induce proliferation, IL-2 production and generation of CTL activity in highly purified CD8⁺ T cells without the heip of CD4⁺ T cells. We conclude that CD28 ligation with the natural ligand B7-1 provides a strong accessory signal for CD4 and CD8 cell activation through CD2.