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.     

Transforming growth factor-β1 induces antigen-specific unresponsiveness in naive t cells

Transforming growth factor-β1 (TGF-β1) is a cytokine with complex immunomodulatory effects including the ability to inhibit the onset or seventy of autoimmune disease. This study was designed to test the possibility that one mechanism by which TGF-β1 exerts its immunosuppressive effects is by inducing antigen (Ag)-specific unresponsiveness in CD4⁺ cells. TGF-β1 was shown here to inhibit the Ag-specific proliferation of naive CD4⁺ cells from T cell receptor (TCR) transgenic mice. More importantly, the naive CD4⁺ cells exposed to TGF-β1 and Ag, but not to TGF-β1 alone, in primary cultures were unable to proliferate or secrete IL-2 in response to a subsequent Ag challenge following removal of TGF-β1 from the cultures. Anti-CD28 mAb partially blocked the Ag-specific inactivation induced by TGF-β1 in naive CD4⁺ cells. The inhibitory effects of TGF-β1 on CD4⁺ cells are not mediated by alterations in APC costimulation since TGF-β1 did not inhibit the Ag-induced expression of MHC class II molecules, CD80 or CD86 on splenic APC. Taken together, the results suggest that the immunosuppressive activities of TGF-β1 encompass direct induction of Ag-specific unresponsiveness in naive CD4⁺ cells.     

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus

TCRαβ⁺ CD8α⁺CD8β^–intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely β2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86^neg/low and MHCI⁺. However, selective depletion of β2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.     

Distinct effects of CD86-mediated costimulation on resting versus activated human CD4+ T cells

CD80 and CD86 are important in the initiation of T cell immunity. Although their costimulatory function has long been appreciated, it remains unclear whether the biological significance of the two B7 isoforms resides in their different patterns and kinetics of expression or whether differences exist in their function. We have addressed this issue using HLA‐DR1 transfectants co‐expressing CD80, CD86, or both molecules as stimulators for naïve, memory, and activated human CD4⁺ T cells. Both CD80 and CD86 efficiently costimulated alloresponses by unseparated peripheral blood CD4⁺ T cells; however, CD86 was substantially inferior in costimulating alloresponses by separated memory T cells, and completely incompetent in costimulating three human T cell clones. Furthermore, CD80/CD86 double transfectants stimulated lower responses by the clones than cells expressing CD80 alone. That CD86 was actively inhibitory rather than merely neutral was evidenced by the increase in response to the double CD80/CD86 APC when anti‐CD86 antibody was added. Furthermore, addition of anti‐CTLA‐4 Fab to cultures of HLA‐DR1 transfectants co‐expressing CD86, fully restored the proliferative response. These results indicate that CD80 and CD86 mediate distinct signals in previously activated T cells, and demonstrate that CTLA‐4 ligation may dominate the outcome of CD86‐mediated costimulation of activated CD4⁺ T cells.     

Regulation of IFN-γ production in granulocyte-macrophage colony-stimulating factor-deficient mice

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF−/−) mice produce far lower serum levels of IFN-γ in response to LPS than GM-CSF+/+ mice. CD4⁺ and CD8⁺ T cells from LPS-injected GM-CSF−/− mice showed a deficiency in IFN-γ production and proliferative activity in response to IL-2 and IL-12, whereas IFN-γ production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF−/− mice do not have an intrinsic defect in IFN-γ production, because IL-12 injection induces the same high levels of IFN-γ in GM-CSF−/− and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF−/− T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-γ levels and T cell responses in LPS-injected GM-CSF−/− mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.     

Differential function of CD80- and CD86-transfected human melanoma cells in the presence of IL-12 and IFN-gamma

Introduction of co-stimulatory molecules like CD80 and CD86 represents a means to augment the immunogenicity of tumor cells and to induce immune responses directed at tumor antigens. Here we compared CD80- and CD86-transfected human melanoma cells to induce primary immune responses by their capacity to promote proliferation of human allogeneic resting T lymphocytes. CD80- and CD86-transfected SkMel63 melanoma cells induced T cell activation to a comparable degree, which was found to be independent of the cell surface density of these co- stimulatory molecules. Co-expression of CD80 and CD86 did not result in a synergistic increase in T cell proliferation. Both CD80 and CD86 transfectants induced the proliferation of isolated CD4+ or CD8+ T cells. Exogenous IL-2, IL-4 and tumor necrosis factor-alpha respectively enhanced primary T cell proliferation independent of CD80 or CD86 expression. Interestingly, differential activities of CD80 and CD86 were observed following stimulation of resting T cells in the presence of IL-12. Whereas IL-12 increased T cell proliferation in the presence of CD86-transfected melanoma cells, it exhibited an inhibitory function in the presence of CD80-expressing SkMel63 cells. Experimental evidence indicates that this inhibitory effect was mediated by IFN- gamma since (I) IFN-gamma secretion of stimulated T cells was augmented by IL-12, (II) exogenous IFN-gamma also inhibited T cell proliferation induced by CD80- but not CD86-transfected SkMel63 cells and (III) the inhibitory effect of IL-12 was blocked by an anti-IFN-gamma mAb.      

Evidence for quantitative and qualitative differences in functional activation of MIs-reactive T cell clones and hybridomas by antigen or TcR/CD3 antibodies

In this study, we demonstrated that some Vp6⁺, CD4⁺, Mls-l^a-specific T cell clones had cytolytic activity when stimulated with anti-T cell receptor(TcR)/CD3 monoclonal antibodies (mAb), but not with targets expressing Mls-1^a, although they produced lymphokines (interleukin 2 and interferon-y) in response to both types of stimuli. To examine the possibility that lack of cytolysis resulted from expression of the Mls-l^a antigen on merely a fraction of splenic B blasts, we (a) used the B cell lymphoma LBB.3.4.16 and (b) measured esterase secretion which is generally concurrent with cytotoxic T lymphocyte (CTL) activity. The B cell lymphoma maximally stimulated the T cell clone for interferon-y production when responding and stimulating cells were incubated at a 1:1 ratio, but it was never killed by the Mls-1^a-specific T cell clone unless TcR/CD3-specific mAb were added. Furthermore, a fivefold excess of the Mls-1^a B cell lymphoma did not induce any secretion of esterase, which was observed only in the presence of the TcR/CD3-specific mAb. Comparison of the reactivity of two Mls-1^a-specific T cell hybridomas expressing the same TcR at similar surface density, revealed both quantitative and qualitative differences between CD3-specific mAb and Mls stimulation of the hybridomas. A small quantitative difference in the sensitivity of hybridoma FJ22.5 to stimulation with V_β6 or CD3-specific mAb resulted in a marked decrease in efficiency of stimulation by Mls-1^a for interleukin 2 production and to inability to detect growth inhibition by Mls-expressing cells. A qualitative difference was observed when analyses of inositol phosphate production were performed under optimal conditions of stimulation of the highly responsive T cell hybridoma (FJ8.1): only stimulation with CD3-specific mAb, but not Mls-expressing cells, could induce detectable inositol phosphate production. Lack of cytolysis of Mls-1^a class II-expressing B cells may have evolutionary significance in view of the recent mapping of Mls to mouse mammary tumor virus genes.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1 / Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extra corporeally with antigen, induced the development of cells secreting IL-2, IFN-γ and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-γ. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to co-stimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/ B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-γ-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Increased expression of CD80, CD86 and CD70 on T cells from HIV-infected individuals upon activation in vitro: regulation by CD4+ T cells

T cells express CD28 and CD27 which transduce co-stimulatory signals after interaction with their ligands on antigen-presenting cells (APC). These ligands, CD80, CD86 and CD70, are also expressed to some extent on activated T cells. Here, we show that in human immunodeficiency virus (HIV)-infected individuals, CD28 and CD27 expression is decreased on CD8⁺ T cells. On the other hand, T cell stimulation in vitro induced high CD80, CD86 and CD70 expression on T cells from HIV-infected individuals. It appeared that an inverted CD4:CD8 T cell ratio could explain this enhanced expression of co-stimulatory ligands. Indeed, high expression levels of CD80, CD86 and CD70 were found on activated CD8⁺ T cells from HIV⁻ individuals cultured in the absence of CD4⁺ T cells. Addition of CD4⁺ T cells prevented this up-regulation. However, in HIV-infected individuals, addition of excess autologous or healthy control CD4⁺ T cells did not completely counteract up-regulation of co-stimulatory ligand expression on CD8⁺ T cells. Thus, to some extent, CD8⁺ T cells in HIV-infected individuals appeared to be refractory to CD4⁺ T cell-mediated regulation of ligand expression in vitro. Activated T cells from HIV-infected individuals and activated CD8⁺ T cells from healthy controls were able to act as accessory cells in CD3-induced T cell proliferation, which was dependent on cell-cell contact. Thus, we showed that T cells from HIV-infected individuals express enhanced levels of co-stimulatory ligands upon activation, which provides them with accessory cell properties. Enhanced stimulatory potential of these nonprofessional APC may contribute to persistently high levels of immune activation in HIV infection related to disease progression.     

Antigen-specific B cells are required for the secondary response of T cells but not for their priming

We studied the potential role of B cells in T cell responses using severe-combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B-17 mice (TG mice). These mice developed both CD4⁺ and CD8⁺ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed-type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)-2, IL-6 and interferon (IFN)-γ and the expression of IL-4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B-17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen-primed B cells, but not by naive B cells, lipopolysaccharide-activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen-primed B cells could induce T cell responses without professional antigen-presenting cells (APC). T and B cells were purified from OVA-immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN-γ in the absence of non-B APC. When anti-CD80 or anti-CD86 was added in the assay, proliferation and IFN-γ production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.     

Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an env^AKR-myc^MH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2^d.b+, I-A^d.b+, CD54⁺, CD11b⁺, CD11c⁺, 2.4G2⁺, F4/80⁺, CD44⁺, 2F8⁺, ER-MP 12^?, Sca-1⁺, Sca-2⁺, NLDC-145^?, B7.2⁺, B7.1^?, J11d^?, B220^?, Thy-1^?, and CD3^?. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-γ (IFN-γ); in contrast, stem cell factor, IL-1α and tumor necrosis factor-α had no effect. Preculture with IFN-γ was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin-producing β-cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: K^d, A^g7, E°, D^b) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM-CSF (DC^NOD). NOD DC, in contrast to CBA DC, have very low levels of intracellular I-A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal β2-microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN-γ does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DC^NOD does not map to the MHC, because the DC from the MHC congenic NOD.H2^h4 mouse (H2: K^k, A^k, E^k, D^k) shares the cell surface phenotype of the DC^NOD. DC from these NOD.H2^h4 also fail to present HEL or the appropriate HEL-peptide to an antigen-specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF-α, IL-6, low levels of IL-12(p70) and NO in response to LPS plus IFN-γ. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM-CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.     

Normal B cells fail to secrete interleukin-12

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-γ (IFN-γ)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4⁺ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-γ, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-γ. Co-culture with CD4⁺ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4⁺ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4⁺ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.     

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.     

Development of Dendritic Cells from GM-CSF-/- Mice in vitro : GM-CSF Enhances Production and Survival of Cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF^-/- mice. Multiple cultures from GM-CSF^-/- and wild type mice were established and compared for cell production. GM-CSF^-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF^-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF^-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF^-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

Cyclosporin A increases IFN-γ production by T cells when co-stimulated through CD28

Despite its calcineurin-inhibiting properties, cyclosporin A (CsA) can not inhibit IL-2 production when T cells are co-stimulated by CD80/CD86 on the antigen-presenting cells. We studied the in vitro effect of CsA on IFN-γ production. Anti-CD3 monoclonal antibody (mAb) was used as the primary stimulus for activation of purified human T cells. A stimulating anti-CD28 mAb, or CD80 or CD86 on stably transfected P815 cells, provided the co-stimulatory signal. IL-2 production was hardly affected by CsA under these stimulating conditions, while IFN-γ (at the protein and mRNA level) was markedly stimulated by CsA. The use of anti-CD3 or phorbol 12-myristate 13-acetate with ionomycin as the primary stimulus, together with co-stimulation through either CD28 or CD2 using transfectants with the appropriate ligands, allowed us to demonstrate that the resistance of IFN-γ production to inhibition by CsA required both CD3 and CD28 triggering. Inhibition of IL-10 production, and to a lesser degree of IL-4 production, by CD4⁺ cells was responsible for the enhancement of IFN-γ production in the presence of CsA. In conclusion, IFN-γ production by CD28-co-stimulated CD4⁺ T cells is resistant to inhibition by CsA and can even be facilitated by CsA as a result of removing a negative regulatory signal which is mainly IL-10 mediated. This finding might have implications for immunosuppressive strategies based upon the use of CsA.