Phosphatidylinositol 3-kinase inhibitors prevent mouse cytotoxic T-cell development in vitro

To become competent killer cells, CD8(+) T cells require stimulation through signal transduction pathways associated with the T-cell receptor, costimulatory molecules such as CD28, and cytokine receptors such as the interleukin (IL)-2 receptor. We used wortmannin and LY294002, two inhibitors of phosphatidylinositol 3-kinase (PI3-K), to study the role of PI3-K in mouse cytotoxic T-lymphocyte (CTL) induction in response to mitogenic anti-CD3 antibody. Anti-CD3-induced CD8(+) T-cell proliferation and CTL development were inhibited dose dependently by both PI3-K inhibitors. IL-2 synthesis by anti-CD3-activated CD8(+) T cells was also diminished by PI3-K inhibition. PI3-K inhibition resulted in a modest decrease in anti-CD3-induced CD4(+) T-cell proliferation but failed to affect IL-2 expression by anti-CD3-activated CD4(+) T cells. PI3-K inhibition during CTL induction resulted in decreased levels of mRNAs coding for granzyme B, perforin, and Fas ligand. In addition, CTL induced in the presence of PI3-K inhibitors failed to conjugate normally with P815 target cells. Exogenous IL-2 did not reverse the effects of PI3-K inhibition on CD8(+) T-cell proliferation and CTL induction. These results support the conclusion that PI3-K activation is involved in T-cell receptor, CD28, and IL-2 receptor signaling of CD8(+) T cells. PI3-K is, therefore, an important component of multiple signal transduction pathways involved in CTL generation.     

协同刺激分子4-1BB和CD28对人外周血不同T细胞亚群的激活作用

目的 :探讨 4 1BB/ 4 1BBL协同刺激信号在CD4 和CD8 T细胞活化、增殖中的作用 ,并与CD2 8/B7信号作比较。方法 :用抗CD3单抗 (mAb)刺激人外周血单个核细胞 (PBMC)。用阻断型抗 4 1BBLmAb和抗CD80mAb ,分别阻断 4 1BB/ 4 1BBL和CD2 8/B7 1协同刺激信号。利用流式细胞术 (FCM)检测CD4 T细胞、CD8 T细胞的增殖率、CD8/CD4T细胞的比值变化和细胞分泌IFN γ的情况。结果 :用抗 4 1BBLmAb和抗CD80mAb阻断相应的协同刺激途径后 ,CD4 和CD8 T细胞的增殖和细胞分泌IFN γ的水平均明显下降。培养 8d,抗CD3mAb单独刺激组CD8/CD4T细胞的比值为 1.98± 0 .0 6 ;抗 4 1BBLmAb阻断组CD8/CD4T细胞的比值下降为 0 .96±0 .0 3;而在抗CD80mAb阻断组 ,其比值上升为 2 .6 9± 0 .16。结论 :4 1BB分子可在CD4 T细胞和CD8 T细胞的活化、增殖中提供协同刺激信号。 4 1BB分子所介导的协同刺激信号 ,在CD8 T细胞活化及增殖中发挥了更为重要的作用 ;而CD2 8分子所介导的协同刺激信号则更有利于CD4 T细胞的活化     

Both CD28 ligands CD80 (B7-1) and CD86 (B7-2) activate phosphatidylinositol 3-kinase, and wortmannin reveals heterogeneity in the regulation of T cell IL-2 secretion

In this report, the co-stimulatory signals provided by CD80(B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb.We demonstrate that while both anti-CD3 and anti-CD28 antibodiesinduced activation of phospholnositide (PI) 3-kinase, the kineticsof activation differed. Anti-CD28 produced a sustained activationof PI 3-kinase while anti-CD3 induced activation was transient.Both B7-1 and B7-2 could induce prolonged activation of PI 3-kinase.The co-stimulatory effects of B7-1 and B7-2 were dependent onCD28 cross-linking, based on complete inhibition of PI 3-kinaseactivation by CD28 antibody Fab fragments. While Jurkat T cellsco-stimulated with anti-CD3 and B7-1 or B7-2 secreted high levelsof IL-2, there were distinct effects of anti-CD28 mAb and B7-1or B7-2 on IL-2 secretion in conjunction with protein kinaseC activation. To assess functional effects of CD28 ligation,pharmacologic inhibitors of PI 3-kinase were evaluated. In Jurkatcells, efficient inhibition of PI 3-kinase activation afterB7-2 stimulation was achieved using wortmannin; however, weobserved a surprising increase in IL-2 secretion after B7 oranti-CD28 stimulation. The effect of wortmannin was concentrationdependent. Moreover, the effect was specific for receptor-mediatedactivation as wortmannin did not enhance phorbol ester pluslonomycin-induced IL-2 secretion. Another inhibitor of PI 3-kinase,LY294002, also resulted in augmentation of anti-CD28-inducedIL-2 secretion by Jurkat cells. The effects of wortmannin onIL-2 secretion were also examined in primary T cells. In markedcontrast, wortmannin resulted in a potent inhibition of anti-CD3plus B7-1 or anti-CD28-induced IL-2 secretion while phorbolester plus lonomycin-induced IL-2 secretion was wortmannin resistant.Together these observations demonstrate that signal transductionby both B7-1 and B7-2 involves PI 3-kinase, and that PI 3-kinaseor other wortmannin-sensitive targets are important for IL-2secretion. Finally, treatment of Jurkat cells with PI 3-kinaseinhibitors alone was sufficient to induce low levels of IL-2secretion. This is consistent with the notion that a wortmannin-sensitivetarget such as PI 3-kinase may down-regulate IL-2 secretionin Jurkat cells.     

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3-kinase association to CD28 and cytokine production

CD28 is a 44-kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC₅₀ of wortmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.     

Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin

T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.     

Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.     

A cell-based artificial antigen-presenting cell coated with anti-CD3 and CD28 antibodies enables rapid expansion and long-term growth of CD4 T lymphocytes

We compared the ability of two genetically modified myeloid cells, K562 and U937, to serve as artificial antigen-presenting cells (aAPC). Both aAPC were stably transfected with the low-affinity Fcgamma receptor CD32 (K32/U32 cells). K32 cells loaded with anti-CD3 and anti-CD28 Ab (K32/CD3/28) induced more rapid CD4 T-cell expansion than CD3/28-coated beads. In contrast, U32/CD3/28 induced high levels of CD4 T-cell thymidine uptake but were unable to sustain long-term T-cell expansion. K32 cells, but not U32 cells, loaded with anti-CD3 alone also stimulated CD4 T-cell growth and IL-2 secretion, indicating the expression of additional costimulatory molecules on K32 cells. We found constitutive expression of B7-H3 and a strong upregulation of mRNA encoding for IL-15, PD-L1, and PD-L2 after coculture with CD4 T cells activated by K32/CD3/28 but not U32/CD3/28. We conclude that K32 aAPCs are a robust system for clinical scale ex vivo expansion of CD4 T cells.     

Activation requirements for the induction of CD4+CD25+ T cell suppressor function

The in vivo differentiation/survival of CD4(+)CD25(+) T suppressor cells is dependent on IL-2 and CD28-mediated costimulatory signals. To determine the cytokine and costimulatory requirements for CD25(+) T cells in vitro, we established a two-stage culture system where CD25(+) T cells were activated in a primary culture. In the subsequent culture, activated CD4(+)CD25(+) cells were then mixed with responders in order to assess their suppressor function. Pre-culture of CD25(+) T cells with anti-CD3 alone resulted in poor survival and minimal induction of suppressor activity. Pre-culture in the presence of anti-CD3 and IL-2 or IL-4, but not IL-6, IL-7, IL-9, IL-10 or IL-15, resulted in proliferation of the CD25(+) cells and induction of potent suppressor function. Inhibition of the interaction of CD28 or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) with CD80/CD86 in the pre-culture of CD4(+)CD25(+) cells did not prevent the induction of suppressor function. Furthermore, the inhibition of costimulatory signals did not inhibit the ability of fresh CD25(+) T cells to inhibit CD8(+) responders under conditions where activation of the responders was independent of CD80/CD86. These studies support the view that activation of CD25(+) T cells requires IL-2/IL-4 for their survival/differentiation into effector cells, but is independent of CD28/CTLA-4-mediated costimulation.     

Positive and negative regulation of IL-12 receptor expression of naive CD4(+) T cells by CD28/CD152 co-stimulation

IL-12 is a critical cytokine for polarizing naive CD4(+) T cells toward Th1 subset. Therefore, it is important to elucidate the mechanism of IL-12R expression of naive CD4(+) T cells. In this report, we present evidence to show that expression of both IL-12Rbeta1 and beta2 mRNA is regulated by signals mediated through CD28 and CD152. Naive CD4(+) T cells stimulated with anti-CD3 alone neither expressed IL-12Rbeta2 mRNA nor bound detectable level of rIL-12, although they expressed a very low level of IL-12Rbeta1 mRNA when stimulated with a high dose of anti-CD3. Expression of IL-12Rbeta1 and beta2 mRNA was induced by the co-ligation of CD3 and CD28, and it was down-regulated by the ligation of CD152. CD28 ligation induced not only IL-12Rbeta1 and beta2 mRNA expression, but also enhanced IFN-gammaR to mediate up-regulation of IL-12R by IFN-gamma.     

Lipid phosphatases in the regulation of T cell activation: living up to their PTEN-tial

Summary: The initiating events associated with T activation in response to stimulation of the T cell antigen receptor (TCR) and costimulatory receptors, such as CD28, are intimately associated with the enzymatically catalyzed addition of phosphate not only to key tyrosine, threonine and serine residues in proteins but also to the D3 position of the myo‐inositol ring of phosphatidylinositol (PtdIns). This latter event is catalyzed by the lipid kinase phosphoinositide 3‐kinase (PI3K). The consequent production of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ serves both to recruit signaling proteins to the plasma membrane and to induce activating conformational changes in proteins that contain specialized domains for the binding of these phospholipids. The TCR signaling proteins that are subject to regulation by PI3K include Akt, phospholipase Cγ1 (PLCγ1), protein kinase C ζ (PKC‐ζ), Itk, Tec and Vav, all of which play critical roles in T cell activation. As is the case for phosphorylation of protein substrates, the phosphorylation of PtdIns is under dynamic regulation, with the D3 phosphate being subject to hydrolysis by the 3‐phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), thereby placing PTEN in direct opposition to PI3K. In this review we consider recent data concerning how PTEN may act in regulating the process of T cell activation.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

CD28 costimulation is required not only to induce IL-12 receptor but also to render janus kinases/STAT4 responsive to IL-12 stimulation in TCR-triggered T cells

The activation of resting T cells for the acquisition of various functions depends on whether CD28 costimulatory signals are provided upon T cell receptor stimulation. Here, we investigated how CD28 costimulation functions to allow TCR-triggered resting T cells to acquire IL-12 responsiveness. When T cells are stimulated with low doses of anti-CD3 mAb, CD28 costimulation was required for the optimal levels of IL-12 receptor (IL-12R) expression. However, stimulation of T cells with high doses of anti-CD3 alone induced comparable levels of IL-12R expression to those induced upon CD28 costimulation. Nevertheless, there was a substantial difference in IL-12 responsiveness between these two groups of T cells: compared to anti-CD28-costimulated T cells, T cells that were not costimulated with anti-CD28 exhibited decreased levels of Janus kinases (JAK) JAK2/TYK2 and STAT4 phosphorylation and IFN-y production following IL-12 stimulation. Importantly, STAT6 phosphorylation following IL-4 stimulation was not decreased in anti-CD28-uncostimulated T cells. These resutls indicate that CD28 costimulation not only contributes to up-regulating IL-12R expression but is also required to render JAKs/STAT4 responsive to IL-12 stimulation.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Activation-induced expression of MICA on T lymphocytes involves engagement of CD3 and CD28

MICA is an HLA-related cell stress-regulated antigen recognized by cytotoxic cells expressing the NKG2D molecule. Although resting lymphocytes do not express MICA, it can be induced on PHA-activated T cells. Here, we demonstrate by Western blot that MICA is induced on allogeneic-activated CD4(+) and CD8(+) T lymphocytes. Blocking activation with anti-HLA class I, anti-HLA-DR, or anti-CD86 mAb affected the expression of MICA slightly. When T cells were stimulated with anti-CD3 or anti-CD28 mAb plus PMA, a sustained up-regulation of MICA was observed by Western blot, RT-PCR, and flow cytometry. The expression of MICA reached a plateau at day 4 after CD3 engagement and at day 3 after anti-CD28/PMA stimulation. Conversely, the proliferative response reached a peak at day 4. Hence, CD3 or CD28 engagement induces MICA expression on T lymphocytes. This activation-induced expression might participate in NKG2D-mediated cytotoxicity toward activated T cells to maintain homeostasis during an ongoing immune response.     

Costimulatory molecules and cytokine production by T lymphocytes in common variable immunodeficiency disease

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by hypogammaglobulinaemia and recurrent infections. Although early works pointed to a primary B-lymphocyte defect as a cause of the disease, a failure in T-lymphocyte cooperation has also been suggested. T cells exert their costimulatory function through either membrane costimulatory molecules or secreted cytokines, both having an influence in the development of the humoral response. The aim of our study was to evaluate whether an abnormal expression and induction of costimulatory molecules or alterations in the production of cytokines by T cells cause deficient T/B cooperation in CVID patients. We studied the expression and upregulation of costimulatory molecules (CD28, CD40L/CD154 and CTLA-4/CD152) and production of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) in purified T lymphocytes from CVID patients stimulated with optimal doses of anti-CD3 or suboptimal doses of anti-CD3 and anti-CD28. Stimulated T cells from CVID patients expressed normal levels of CD28, CD40L/CD154 and CTLA-4/CD152 when compared with controls. Except for higher production of IL-4 after stimulation with anti-CD3, T cells of CVID patients produced similar amounts of cytokines compared with controls. An imbalance between costimulatory molecules expression (CD28, CD40L/CD154 and CTLA-4/CD152) and cytokine production by T cells does not explain a deficient cooperation between T and B cells in this group of CVID patients.     

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of na?ve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Na?ve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for na?ve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when na?ve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.     

Association of T cell antigen CD7 with type II phosphatidylinositol-4 kinase,a key component in pathways of inositol phosphate turnover

CD7 is a 40-kDa glycoprotein that is expressed on prothymocytes and persists during T cell differentiation. CD7 has been demonstrated to generate, like other costimulatory molecules, intracellular signals that modulate T cell function. However, although it binds to phosphatidylinositol 3-kinase (PI 3-kinase), the signaling events mediated by CD7 are not completely understood. In this context, phosphatidylinositol 4-kinase (PI 4-kinase) is a key enzyme involved in a variety of events, from the modeling of the actin cytoskeleton to the activation of protein kinase C. In this study, we show for the first time that PI 4-kinase of 55 kDa can associate with CD7. The enzyme activity was insensitive to wortmannin, but was inhibited by adenosine, a characteristic for type II PI 4-kinase. Together, our findings demonstrate that type II PI 4-kinases are integral components of the CD7 signaling pathway and may play a role of CD7 in co-stimulation and thymic differentiation.     

In vitro induction of regulatory T cells by anti-CD3 antibody in humans

Therapy with anti-CD3 antibody is effective in controlling models of autoimmune diseases and can reverse or prevent rejection of grafts. We studied the in vitro immunomodulatory effect of anti-CD3 treated human T cells. CD4(+) T cells were stimulated with plate-bound anti-CD3 and cultured for 12 days after which they were cultured with autologous peripheral blood mononuclear cells (PBMCs) and stimulated with soluble anti-CD3. We found that CD4(+) T cells that were stimulated with anti-CD3 (T(alphaCD3)) markedly suppressed the proliferation and cytokine production of autologous PBMCs. These regulatory T cells were not induced by incubation with isotype control (T(control)) antibody or when anti-CD3 was combined with high doses of anti-CD28 (T(alphaCD3/CD28)). T(alphaCD3) regulatory cells were anergic and produced lower levels of IFN-gamma, TNF-alpha and IL-2, and higher levels of TGF-beta than T(control) or T(alphaCD3/CD28). There were no differences in the expression of CD25 or CTLA4 on T(alphaCD3) as compared to T(control) or T(alphaCD3/CD28), and CD4(+) CD25(-) T(alphaCD3) cells were identical to CD4(+) CD25(+) T(alphaCD3) cells in their in vitro suppressive properties. Recombinant IL-2 in vitro abrogated the suppressive effect of T(alphaCD3). The suppressive effect was not related to apoptosis, was independent of HLA since T(alphaCD3) also suppressed allogeneic PBMCs, and was not related to soluble factors. Finally, no suppression was observed when non-T cells were removed from culture or when cultures were stimulated with plate-bound anti-CD3, consistent with the ability of T(alphaCD3) to downregulate CD80 on dendritic cells in co-culture experiments. Thus, we have identified human T cells with strong in vitro regulatory properties induced in vitro by anti-CD3 which appear to act in a non-HLA restricted fashion by affecting antigen presenting cells.