Small GTPases and LFA-1 reciprocally modulate adhesion and signaling

Summary:  Leukocyte-function-associated antigen-1 (LFA-1) is an integrin that is critical for T-cell adhesion and immunologic responses. As a transmembrane receptor and adhesion molecule, LFA-1 signals bidirectionally, whereby information about extracellular ligands is passed outside-in while cellular activation is transmitted inside-out to the adhesive ectodomain. Here, we review the role of small guanosine triphosphatases (GTPases) in LFA-1 signaling. Rap1, a Ras-related GTPase, appears to be central to LFA-1 function. Rap1 is regulated by receptor signaling [e.g. T-cell receptor (TCR), CD28, and cytotoxic T-lymphocyte antigen-4 (CTLA-4)] and by adapter proteins [e.g. adhesion and degranulation-promoting adapter protein (ADAP) and Src kinase-associated phosphoprotein of 55 kDa (SKAP-55)]. Inside-out signaling flows through Rap1 to regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP interacting adapter molecule (RIAM) that act in conjunction with the cytoskeleton on the cytosolic domain of LFA-1 to increase adhesion of the ectodomain. Outside-in signaling also relies on small GTPases such as Rho proteins. Vav-1, a guanine nucleotide exchange factor for Rho proteins, is activated as a consequence of LFA-1 engagement. Jun-activating binding protein-1 (JAB-1) and cytohesin-1 have been implicated as possible outside-in signaling intermediates. We have recently shown that Ras is also downstream of LFA-1 engagement: LFA-1 signaling through phospholipase D (PLD) to RasGRP1 was required for Ras activation on the plasma membrane following stimulation of TCR.     

CTLA-4: a key regulatory point in the control of autoimmune disease

Summary: Chronic autoimmune disease in humans is the result of a failure to control autoreactive immune cells in the periphery. This control is largely achieved by inhibition of newly activated and memory cells. A number of negative immune regulatory pathways have been characterized. The cell surface coreceptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) has emerged as a critical attenuator of T-cell activation and an essential component of the regulatory systems that serve to maintain peripheral tolerance. CTLA-4 expression is induced on the surface of T cells after they have received a costimulatory signal from antigen-presenting cells (APCs) via engagement of CD28 on the T-cell surface. CTLA-4 attenuates this costimulation by competing for CD28 ligands and through direct effects on APCs via the same ligands utilized by CD28. A large number of genetic association studies suggest that the CTLA-4 gene is a locus of susceptibility to autoimmune disease. However, specific functional defects in the CTLA-4 gene in patients have not been identified to date. Elucidating the role of CTLA-4 in immune tolerance has also led to a number of therapeutic applications, particularly in the treatment of malignancy and autoimmune disease.     

The outcome of T-cell costimulation through intercellular adhesion molecule-1 differs from costimulation through leucocyte function-associated antigen-1

Optimal T-cell activation requires both an antigen-specific and a costimulatory signal. The outcome of T-cell activation can be influenced by the nature of the costimulatory signal the T cell receives. We recently demonstrated the ability of stimulation through intercellular adhesion molecule-1 (ICAM-1), resident on the T-cell surface, to provide a second signal for T-cell activation, and have extended that work here to begin an examination of the functional outcome of this set of signals. Costimulation through ICAM-1 resulted in a greater percentage of cells having undergone more than three divisions when compared to costimulation through leucocyte function-associated antigen-1 (LFA-1). Costimulation through ICAM-1 also had an effect similar to costimulation through CD28 in its ability to down-regulate the cyclin dependent kinase inhibitor p27kip1. Costimulation through ICAM-1 provided greater protection from apoptosis than costimulation through LFA-1, especially in cells having divided more than three times. This was supported by the ability of costimulation through ICAM-1 to up-regulate the anti-apoptotic protein Bcl-2. Finally, costimulation through ICAM-1 or CD28 produced a greater number of T cells with a memory phenotype than costimulation through LFA-1.     

Non-responsiveness of antigen-experienced CD4 T cells reflects more stringent co-stimulatory requirements.

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

CD40 on NOD CD4 T cells contributes to their activation and pathogenicity

Our goals in this study were to investigate conditions under which T cells from NOD mice express CD40 and to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity in T1D. Using CD40-positive diabetogenic T cell clones and CD4 T cells from NOD mice, we examined expression of CD40 upon activation through the TCR and costimulation through either CD28 or CD40. Our results indicate that CD40 expression is increased upon activation with antigen/MHC and that activation of NOD CD4 T cells through TCR/CD40 rapidly induced CD40 expression. Furthermore, CD40 costimulation promoted T cell proliferation to the same extent as costimulation through TCR/CD28. Importantly, costimulation of CD4 T cells through CD40 also interfered with T cell homeostasis by altering regulation of CTLA-4 expression. Through CD40–CD154 blocking studies, we demonstrated that signaling between T cells through CD40 and its ligand contributes to activation of pathogenic T cells and that blocking CD40 on T cells abrogates their ability to transfer diabetes. Thus, costimulation through CD40 on NOD T cells contributes to their pathogenicity by providing additional pathways for activation and by inhibiting upregulation of CTLA-4 during T cell activation.     

CD40 on NOD CD4 T cells contributes to their activation and pathogenicity

Our goals in this study were to investigate conditions under which T cells from NOD mice express CD40 and to determine how CD40 on autoreactive CD4 T cells contributes to their pathogenicity in T1D. Using CD40-positive diabetogenic T cell clones and CD4 T cells from NOD mice, we examined expression of CD40 upon activation through the TCR and costimulation through either CD28 or CD40. Our results indicate that CD40 expression is increased upon activation with antigen/MHC and that activation of NOD CD4 T cells through TCR/CD40 rapidly induced CD40 expression. Furthermore, CD40 costimulation promoted T cell proliferation to the same extent as costimulation through TCR/CD28. Importantly, costimulation of CD4 T cells through CD40 also interfered with T cell homeostasis by altering regulation of CTLA-4 expression. Through CD40-CD154 blocking studies, we demonstrated that signaling between T cells through CD40 and its ligand contributes to activation of pathogenic T cells and that blocking CD40 on T cells abrogates their ability to transfer diabetes. Thus, costimulation through CD40 on NOD T cells contributes to their pathogenicity by providing additional pathways for activation and by inhibiting upregulation of CTLA-4 during T cell activation.     

ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells.

We have previously reported that costimulatory pathways including B7-CD28 and lymphocyte function-associated antigen-3 (LFA-3)-CD2 shape distinct activation profiles in human CD4+ T cells. We now show that superantigen (SAg), in combination with intracellular adhesion molecule-1 (ICAM-1) costimulation drives a proliferative response accompanied by high levels of interleukin-2 (IL-2) and moderate levels of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). This response profile differs from that observed in B7 or LFA-3 costimulated T cells because our previous results showed that B7-CD28 costimulation was accompanied by high levels of IL-2, IFN-gamma and TNF, whereas LFA-3 was a potent inducer of IFN-gamma and TNF, but had little influence on IL-2 production. The ICAM-1-induced IL-2 production could efficiently be abrogated with monoclonal antibody (mAb) against ICAM-1 or LFA-1, showing that the activation is dependent of a functional ICAM-1-LFA-1 pathway. SAg-induced IL-2, IFN-gamma and TNF were detected in both CD4+ and CD8+ T cells, whereas production of IL-10 was restricted to CD4+ T cells. A major finding in the present study was that ICAM-1 costimulation strongly inhibits IL-10 production in CD4+ T cells. Our data demonstrate that ICAM-1 costimulation is sufficient to induce large amounts of IL-2. The presence of ICAM-1 results in suppression of IL-10 production in T helper (Th) cells, which may favour the development of Th1 and not Th2 T cells.     

Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist

CD4(+)CD25(+) T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen-4 (CTLA-4)-specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4(+)CD25(+) T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA-4, produce IL-10 but not IL-2, and are able to suppress the proliferation of costimulated CD25-negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25(-) T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28-specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a 20-fold increase within 3 days in response to a single antibody dose. These data suggest that CD28-driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.     

Expression and functional significance of CTLA-4, a negative regulator of T cell activation

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.     

Modulation of T-cell costimulation as immunotherapy or immunochemotherapy in experimental visceral leishmaniasis

CD40 ligand (CD40L)-deficient C57BL/6 mice failed to control intracellular Leishmania donovani visceral infection, indicating that acquired resistance involves CD40-CD40L signaling and costimulation. Conversely, in wild-type C57BL/6 and BALB/c mice with established visceral infection, injection of agonist anti-CD40 monoclonal antibody (MAb) induced killing of approximately 60% of parasites within liver macrophages, stimulated gamma interferon (IFN-gamma) secretion, and enhanced mononuclear cell recruitment and tissue granuloma formation. Comparable parasite killing was also induced by MAb blockade (inhibition) of cytotoxic T lymphocyte antigen-4 (CTLA-4) which downregulates separate CD28-B7 T-cell costimulation. Optimal killing triggered by both anti-CD40 and anti-CTLA-4 required endogenous IFN-gamma and involved interleukin 12. CD40L(-/-) mice also failed to respond to antileishmanial chemotherapy (antimony), while in normal animals, anti-CD40 and anti-CTLA-4 synergistically enhanced antimony-associated killing. CD40L-CD40 signaling regulates outcome and response to treatment of experimental visceral leishmaniasis, and MAb targeting of T-cell costimulatory pathways (CD40L-CD40 and CD28-B7) yields macrophage activation and immunotherapeutic and immunochemotherapeutic activity.     

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.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/ B7 family

Positive and negative costimulation by members of the CD28 family is critical for the development of productive immune responses against foreign pathogens and their proper termination to prevent inflammation-induced tissue damage. In addition, costimulatory signals are critical for the establishment and maintenance of peripheral tolerance. This paradigm has been established in many animal models and has led to the development of immunotherapies targeting costimulation pathways for the treatment of cancer, autoimmune disease, and allograft rejection. During the last decade, the complexity of the biology of costimulatory pathways has greatly increased due to the realization that costimulation does not affect only effector T cells but also influences regulatory T cells and antigen-presenting cells. Thus, costimulation controls T-cell tolerance through both intrinsic and extrinsic pathways. In this review, we discuss the influence of costimulation on intrinsic and extrinsic pathways of peripheral tolerance, with emphasis on members of the CD28 family, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1), as well as the downstream cytokine interleukin-1 (IL-2).     

CD28 Function: A Balance of Costimulatory and Regulatory Signals

Both the recognition of MHC/antigen complex by the T-cell receptor and engagement of costimulatory molecules are necessary for efficient T-cell activation. CD28 has been widely recognized as the major costimulation pathway for naive T-cell activation, and the CD28/B7 pathway plays a central role in immune responses against pathogens, autoimmune diseases, and graft rejection. In this review, we will summarize evidence that CD28 is also prominent in the regulation of immune responses and the maintenance of peripheral tolerance. Indeed, CD28 engagement increases the expression of the down-modulatory molecule CTLA-4, induces the differentiation of Th2 cells that have a protective function in autoimmunity, and has an obligatory role in the homeostasis of regulatory T cells. Therefore, CD28/B7 interactions induce a balance of costimulatory and regulatory signals that have opposite outcomes on immune responses. This new perspective on CD28 function suggests that caution should be taken in the development of immunotherapies targeting costimulatory pathways.     

CD28 and inducible costimulator (ICOS) signalling can sustain CD154 expression on activated T cells

The biological outcome of receptor-mediated signalling often depends on the duration of engagement. Because CD40 signalling is controlled by the regulated expression of its ligand, CD154, the mechanisms that regulate CD154 expression probably determine the strength and duration of CD40 signalling. Here, we demonstrate that CD154 expression on the surface of mouse CD4 T cells can be separated into an early phase, occurring between 0 and 24 hr after T-cell activation, and a later extended phase, occurring after 24 hr. The early phase of CD154 expression did not require costimulation and was probably influenced by the strength of T-cell receptor (TCR) signalling alone. However, later CD154 expression was highly dependent on costimulation through either CD28 or inducible costimulator (ICOS). Although CD28 signalling interleukin (IL)-2 secretion, ICOS not, suggesting that costimulation enhance CD154 expression independently of IL-2 production. In fact, anti-CD28 treatment could still induce late-phase CD154 on anti-CD3-stimulated CD4 T cells expressing a mutated form of CD28 that not lead to the induction of IL-2. However, this CD154 induction was somewhat weaker than that of wild-type CD28-expressing cells, suggesting that direct signalling and IL-2-mediated signalling co-operatively responsible for the levels of CD154 induced by CD28. Finally, we show that the second phase of CD154 expression negatively regulated B-cell terminal differentiation and antibody secretion. These results demonstrate that TCR signalling and costimulation each regulate different phases of CD154 expression and control the biological outcome of CD40 signalling on B cells.     

B7-mediated costimulation can either provoke or prevent clinical manifestations of experimental allergic encephalomyelitis

T-cell activation requires signalling provided by ligation of the T-cell receptor for antigen (TCR) and a second antigen (Ag) nonspecific signal, known as costimulation. The B7 receptors, CD80 (B7-1) and CD86 (B7-2), on the Ag-presenting cell (APC), interact with T-cell CD28 or CTLA-4 to deliver a costimulatory signal, which is particularly important for Th1 activation. Experimental allergic encephalomyelitis (EAE) is an autoimmune disorder, induced by Th1 cells directed against myelin antigens that provides an in vivo model for studying the role of B7-mediated costimulation in the induction of a pathological immune response. Using a soluble fusion protein ligand for the B7 receptors, as well as specific monoclonal antibodies specific for either CD80 or CD86, it has been demonstrated that B7 costimulation plays a prominent role in determining clinical disease outcome in EAE. Here we review recent data indicating that a paradoxical exacerbation of disease as well as the expected amelioration of disease can occur with costimulatory receptor blockade.     

B7-h2 is a costimulatory ligand for CD28 in human

CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T?cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for?the costimulation of human T?cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27(kip1), and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

The effects of Cyclosporine A and azathioprine on human T cells activated by different costimulatory signals

Immunosuppression is an important treatment modality in transplantation and human diseases that are associated with aberrant T cell activation. There are considerable differences regarding the cellular processes targeted by the immunosuppressive drugs that are in clinical use. Drugs like azathioprine (Aza) mainly act by halting proliferation of fast dividing cells, whereas others like cyclosporine A (CsA) specifically target signaling pathways in T cells. Since the outcome of T cell responses critically depends on the quality and strength of costimulatory signals, this study has addressed the interplay between costimulation and the immunosuppressive agents CsA and Aza during the in vitro activation of human T cells. We used an experimental system that allows analyzing T cells activated in the presence of selected costimulatory ligands to study T cells stimulated via CD28, CD2, LFA-1, ICOS or 4-1BB. The mean inhibitory concentrations (IC(50)) for Aza and CsA were determined for the proliferation of T cells receiving different costimulatory signals as well as for T cells activated in the absence of costimulation. CD28 signals but not costimulation via CD2, 4-1BB, ICOS or LFA-1 greatly increased the IC(50) for CsA. By contrast, the inhibitory effects of Aza were not influenced by T cell costimulatory signals. Our results might have implications for combining standard immunosuppressive drugs with CTLA-4Ig fusion proteins, which act by blocking CD28 costimulation.