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.     

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.     

Role of CD28/B7 costimulation in the dexamethasone-induced suppression of IFN-gamma.

In vitro exposure of peripheral blood mononuclear cells (PBMC) to glucocorticoids (GC), at concentrations observed during psychologic stress, induces a shift in the human type 1/type 2 cytokine balance toward a type 2 cytokine response. The mechanisms involved in these cytokine alterations are unknown but likely include modulation of regulatory cytokines or the interaction between the antigen-presenting cell (APC) and T lymphocyte or both. The CD28/B7 costimulation pathway has been reported to modulate the type 1/type 2 cytokine balance and may contribute to the GC-associated cytokine alterations. Therefore, we sought to determine the effect of dexamethasone (Dex) on the expression and function of the human CD28/B7 costimulatory pathway and whether these alterations contribute to the Dex-induced type 1/type 2 cytokine alterations. Dex inhibited the expression of both CD80 and CD86 on THP-1 cells, a human acute monocytic leukemia cell line, as determined by flow cytometry. Dex also inhibited the expression of CD28 and CTLA-4 on phytohemagglutinin (PHA)-stimulated CD3+ T lymphocytes, which was attenuated by the addition of interleukin-12 (IL-12). Lastly, activation of CD28 with anti-CD28 antibody attenuated the Dex-induced decrease in interferon-gamma (IFN-gamma) production by anti-CD3 antibody-stimulated PBMC. These data suggest that Dex induces a modulation of the CD28/B7 costimulatory pathway that contributes to the shift in the type 1/type 2 cytokine balance toward a predominant type 2 cytokine response.     

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     

Importance of CD80/CD86-CD28 interactions in the recognition of target cells by CD8+CD122+ regulatory T cells

CD8+CD122+ regulatory T cells are a newly identified, naturally occurring type of regulatory T cell that produce interleukin-10 (IL-10) and effectively suppress interferon-gamma (IFN-gamma) production from both CD8+ and CD4+ target cells. Molecular mechanisms responsible for the recognition of target cells by CD8+CD122+ regulatory T cells were investigated in this study by using an in vitro culture system that reconstitutes the regulatory action of these cells. CD8+CD122( regulatory T cells did not produce IL-10 and did not suppress the IFN-gamma production of allogeneic target T cells when they were stimulated by immobilized anti-CD3 antibody alone, but they clearly produced IL-10 and suppressed the IFN-gamma production of target cells when stimulated by anti-CD3 plus anti-CD28-coated beads. IFN-gamma production by major histocompatibility complex-class I-deficient T cells was also suppressed by CD8+CD122+ regulatory T cells stimulated with anti-CD3 plus anti-CD28 antibody but was not suppressed by cells stimulated by anti-CD3 alone. Experiments examining the blockade of cell surface molecules expressed on either the regulatory cells or the target cells by adding specific neutralizing antibodies in the culture indicated that CD80, CD86, and CD28 molecules were involved in the regulatory action, but cytotoxic T lymphocyte antigen-4, inducible costimulatory molecule (ICOS) and programmed death-1 (PD-1) molecules were not. Finally, CD8+CD122+ cells isolated from CD28-knockout (CD28-/-) mice showed no regulatory activity. These results indicate that CD8+CD122(+) regulatory T cells recognize target T cells via the interaction of CD80/CD86-CD28 molecules to become active regulatory cells that produce suppressive factors such as IL-10.     

Modulation of Murine Lyme Borreliosis by Interruption of the B7/CD28 T-Cell Costimulatory Pathway

Recent studies have implicated cytokines associated with Th2 cells in the genetic resistance to murine Lyme borreliosis. Because the B7/CD28 costimulatory pathway has been shown to influence the differentiation of Th-cell subsets, we investigated the contribution of the B7 molecules CD80 and CD86 to the Th2 cytokine profile and development of arthritis in BALB/c mice infected with Borrelia burgdorferi. Effective blockade of CD86/CD28 interaction was demonstrated by elimination of interleukin 4 (IL-4) and upregulation of gamma interferon (IFN-γ) responses by B. burgdorferi-specific T cells and by reduction of B. burgdorferi-specific immunoglobulin G. Despite the shift toward a Th1 cytokine pattern, which others have associated with disease susceptibility, the severity of arthritis was unchanged. Moreover, combined CD80/CD86 blockade by using anti-CD80 and anti-CD86 monoclonal antibodies or CTLA-4Ig enhanced IFN-γ production over that seen with CD86 blockade alone, yet augmentation of this Th1-associated cytokine did not enhance disease. These results demonstrate that IL-4 production by T cells in B. burgdorferi-infected BALB/c mice is dependent upon CD86/CD28 interaction and that this cytokine does not contribute significantly to host resistance to the development of arthritis. In addition, combined CD80/CD86 blockade resulted in preferential expansion of IFN-γ-producing T cells in B. burgdorferi infection, suggesting that costimulatory pathways other than B7/CD28 may contribute to T-cell activation during continuous antigen stimulation. These studies may provide insight into the role of the B7/CD28 pathway in other infectious and autoimmune diseases in which deviation of Th cell immune responses occurs and antigen is persistently present.Lyme disease is a multisystem illness due to infection with the tick-transmitted spirochete Borrelia burgdorferi. Experimental infection of laboratory mice with B. burgdorferi results in acute arthritis and carditis that reproducibly peak at 2 to 4 weeks of infection and then resolve within 3 months despite spirochete persistence (4). Studies using SCID mice, which lack functional T and B cells, have demonstrated that disease is due to the innate immunity of the host and can occur in the absence of specific immune responses (6, 28). The persistent and progressive nature of disease manifestations in SCID mice underscores the importance of T and B cells in initiating disease regression (5, 6, 28). Recent studies support the additional role of specific immunity in modulating disease severity via direct effects on spirochete burden through B. burgdorferi-specific antibodies (5) and indirectly through Th cell-associated cytokines that influence the activation of innate immune cells (14, 23). In particular, the dominance of Th1-type responses, which support macrophage activation, in patients with chronic Lyme arthritis has implicated this T-cell phenotype in the development and perpetuation of severe inflammatory disease (32, 37). Th1-type responses have also been observed during B. burgdorferi infection of C3H mice, a disease-susceptible strain, whereas Th2 responses, which promote B-cell functions, can be detected in BALB/c mice, a comparatively disease-resistant strain (14, 23). Despite the greater inflammatory response in C3H mice, their pathogen burden as assessed by quantitative PCR of spirochete DNA remains higher than that of disease-resistant mouse strains (36), suggesting that the recruitment of innate immune cells is appropriate yet ineffective at controlling infection (29).In addition to signals provided by T-cell antigen receptor engagement, the interaction of costimulatory molecules present on antigen-presenting cells (APCs) with their ligands on T cells is believed to be necessary for the initial priming of naive T cells. In particular, the B7/CD28 costimulatory pathway has been implicated in the differentiation of naive Th0 cells into Th1 and Th2 subsets (33). The mechanisms by which these molecules assist in the priming of the T-cell immune response are complex and poorly understood. Two members of the B7 family have been characterized, CD80 and CD86 (also known as B7-1 and B7-2, respectively), and differ not only in their binding properties to CD28 on T cells but also in the timing of their appearance on conventional APCs during the initiation of an immune response (11). CD86 appears earlier on the surface of mitogen-activated APCs and has a lower affinity for CD28 than does CD80. Once activated, T cells express CTLA-4, a second receptor to which both CD80 and CD86 bind with greater affinity than they bind CD28 (21). Interaction of CD80/CD86 with CTLA-4 can downregulate the T-cell immune response (35). Blockade of CD86 during the initiation of a T-cell response results in an immune response oriented toward a Th1 phenotype, whereas a similar blockade of CD80 does not consistently favor a Th2 phenotype (20). Experiments using mutant mice deficient in CD80 and/or CD86 reveal the important role of these molecules in sustaining a Th-cell phenotype and, in the case of CD86 expression, in the development of a Th2 response (20). Costimulation through the B7/CD28 pathway contributes to the expansion of autoimmune disease processes seen in experimental autoimmune encephalitis (17, 27), a predominantly Th1-associated disease, and autoimmune diabetes (19). Studies using a soluble recombinant form of CTLA-4 designated CTLA-4Ig have supported many of the observations made with anti-B7 antibodies (13, 19, 26).We have recently reported that the Th2 response of B. burgdorferi-infected BALB/c mice is preceded by a Th1 response and that the presence of interleukin 4 (IL-4) is associated with accelerated resolution of arthritis (12). A hind-foot inoculation route was used in that study so that T-cell responses could be examined in lymph nodes adjacent to joints afflicted with arthritis. We demonstrated that this route of inoculation induces moderately severe arthritis in BALB/c mice at day 14 of infection that undergoes more rapid regression than the arthritis seen in similarly infected C3H mice, in which IL-4 responses are not detectable. Previous studies have shown that treatment of mice with anti-IL-4 monoclonal antibody (MAb) exacerbates arthritis in BALB/c mice assessed at intervals corresponding to the plateau and resolution phases of disease, providing evidence that IL-4 modulates the severity of established arthritis (14, 23). The influence of Th2 cell effector functions on the development of arthritis remains unknown. In the current study, we have examined the effects of interruption of Th2 cell differentiation by B7/CD28 blockade with anti-CD80 and/or anti-CD86 MAb or CTLA-4Ig on the cytokine profiles and development of arthritis in BALB/c mice infected with B. burgdorferi.     

Critical role of activation-inducible lymphocyte immunomediatory molecule/inducible costimulator in the effector function of human T cells: a comparative in vitro study of effects of its blockade and CD28 blockade in human beings and monkeys

Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator, ICOS) is the third homolog of the "professional" costimulatory molecule, CD28. To date, the characteristics and role of AILIM/ICOS, especially in effector function of T cells, have been determined through numerous studies in vitro and in vivo using mice. Considering potential differences among species, whether the AILIM/ICOS blockade acts as an efficacious immunomodulator for human diseases remains to be elucidated. In the present study, ability of AILIM/ICOS blockade to modulate immune responses of human and monkey cells was investigated using a fully human antibody (JTA-009), comparing the effect of CD28 blockade. JTA-009 blocked the response of human and monkey T cells co-stimulated with anti-CD3 and AILIM/ICOS ligand, B7h. AILIM/ICOS and CD28 blockade both inhibited human mixed lymphocyte reaction in different fashions, as well as cytokine production in T helper (Th) 1-/Th2-type recall responses. In monkeys however, CD28 blockade by CTLA4-Ig effectively prevented mixed lymphocyte reaction to a greater extent than AILIM/ICOS blockade. These results suggest that AILIM/ICOS blockade is valuable for suppressing both primary allogenic response and recall responses of T cell in human beings, and that there are differences between human and monkey use preferences for costimulatory molecules.     

Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-beta and IL-10.

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-gamma and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-gamma by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-beta and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-beta and IL-10 significantly suppressed IFN-gamma production by MNCs. IFN-beta in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-beta and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.     

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.     

CD28-B7-mediated T cell costimulation in chronic cardiac allograft rejection: differential role of B7-1 in initiation versus progression of graft arteriosclerosis

Provision of adequate T cell costimulation is critical for the development of acute and chronic allograft rejection. We have previously reported that early blockade of CD28-B7 T cell costimulation prevents the development of graft arteriosclerosis, in the LEW into F344 rat cardiac transplant model. In this study, we used the same model to examine the requirement for CD28-B7-mediated T cell costimulation in the progression of established chronic rejection and examined the individual roles of B7-1 (CD80) and B7-2 (CD86) costimulatory molecules. Late blockade of CD28-B7 T cell costimulation by the fusion protein CTLA4Ig, which binds both CD80 and CD86, attenuated the development of transplant arteriosclerosis, mononuclear cell infiltration, and parenchymal fibrosis in this model. Selective blockade of CD80 using the mutant fusion protein Y100F was as effective as CTLA4Ig in this regard. In contrast to CTLA4Ig, blockade of CD80 alone by Y100F was ineffective at preventing early graft loss and prolonging graft survival when given early after transplantation. This study is the first to demonstrate that late blockade of CD28-B7 T cell costimulation interrupts chronic cardiac allograft rejection, and it indicates the importance of continued T cell activation in this process. This study further defines functional differences between CD80 and CD86 costimulatory molecules in vivo.     

Treatment with anti-CD86 costimulatory molecule prevents the autoimmune lesions in murine Sjögren's syndrome (SS) through up-regulated Th2 response

Intraperitoneal administration with anti-CD86 (B7.2) MoAb into the murine model for primary SS in NFS/sld mutant mice resulted in dramatically inhibitory effects on the development of autoimmune lesions, while no significant effects were observed when the mice were administered with anti-CD80 (B7.1) MoAb. We found that spleen cells in the murine SS model treated with anti-CD86 MoAb showed a significant impairment of autoantigen-specific T cell proliferation. T cell activation markers (CD44high, CD45RBlow, Mel-14low) were significantly down-regulated in the spleen cells gated on CD4 in anti-CD86-treated mice. We detected a higher level of cytokine production of IL-4 from splenic T cells in anti-CD86-treated mice, but not of IL-2, and interferon-gamma (IFN-gamma), compared with those in the anti-CD80- and PBS-treated SS model. Moreover, serum autoantibody production against alpha-fodrin autoantigen was almost entirely suppressed in anti-CD86-treated mice. These data provide strong evidence that in autoimmune exocrinopathy resembling SS in NFS/sld mutant mice, the CD86 costimulatory molecule plays a crucial role in the initiation and subsequent progression of Th1-mediated autoimmunity in the salivary and lacrimal glands.     

The costimulatory signal upregulation is associated with Th1 bias at the maternal-fetal interface in human miscarriage

PROBLEM To evaluate whether the association of the costimulatory signal regulation with T helper 1/T helper 2 (Th1/Th2) bias at maternal-fetal interface in human pregnancy loss. METHOD OF STUDY The expression of CD80 and CD86 in decidual tissues and CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4) in the decidual T cells was compared between normal early pregnancy and miscarriage by qPCR and Western blot. The cytokine production in decidual T cells was performed by flow cytometry. The correlation of costimulatory molecule expression with Th1/Th2 cytokines was analyzed. RESULTS The CD80 mRNA and protein expression showed no significant difference between normal pregnancy and miscarriage. An increase in the expression of CD28 and CD86 was accompanied by a decrease in the expression of CTLA-4 in miscarriage in comparison with the early pregnancy. The higher expression of interleukin (IL)-2 and interferon-γ (IFN-γ), and lower expression of IL-4 and IL-10 in the decidual T cells were present in miscarriage. A correlation analysis showed a significant positive correlation of CD86 and CD28 expression with the Th1 cytokine production (IL-2 and IFN-γ), a significant negative correlation of CTLA-4 expression with the Th1 cytokine production. CONCLUSION The upregualtion of costimulatory signals on T cells might form an abnormal immune microenvironment, a shift to Th1 responses, at maternal-fetal interface, which leads to human miscarriage.     

CD28 interactions with either CD80 or CD86 are sufficient to induce allergic airway inflammation in mice.

Previous studies have shown that the pan CD28/cytotoxic T lymphocyte antigen (CTL)A-4 antagonist CTLA4 immunoglobulin (Ig) inhibits eosinophilic airway inflammation in Schistosoma mansoni-sensitized and airway-challenged mice. In the present study, the importance of CD28 as well as the individual roles of CD80 and CD86 were examined in this system using wild-type and CD28 knockout (KO) mice. Unlike wild-type controls, CD28KO mice did not produce systemic IgE or eosinophilic airway inflammation after antigen challenge. However, a lymphocytic infiltrate and continued production of interferon-gamma was observed in these animals. Thus, CD28 is not essential for the initial recruitment of lymphocytes into antigen-challenged airways but critically regulates the allergic T-helper 2 phenotype. We next determined by polymerase chain reaction and flow cytometry that CD80 and CD86 molecules are constitutively expressed in the naive murine lung and on eosinophils in the allergic lung, suggesting a potential important role for both ligands in the development of asthma. Combined anti-CD80/anti-CD86 treatment throughout the antigen challenge period fully blocked the development of allergic airways, whereas a partial reduction was observed in mice treated with either anti-CD80 or anti-CD86 antibody alone. However, only anti-CD86 blocked systemic IgE production. Therefore, signaling through either CD80 or CD86 is sufficient to generate a partial local allergic response, whereas CD86 costimulation is essential to induce systemic allergic (IgE) reactions. Finally, combined anti-B7 monoclonal antibody treatment after sensitization reduced airway eosinophilia and interleukin (IL)-4/IL-5 cytokine secretion consistent with an ongoing role for CD28/B7 interactions in the effector phase of the disease. These results emphasize the importance of differential B7 expression on different cells and in different organs on subsequent CD28/B7-mediated immune events, including the potential for CD28/B7 blockade in the treatment of atopic airway disease in people.     

Modulation of cell cycle progression by CTLA4-CD80/CD86 interactions on CD4+ T cells depends on strength of the CD3 signal: critical role for IL-2

Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a well-studied T cell costimulatory receptor that is known to inhibit T cell activation. In this study, the relationship between strength of the first signal and costimulatory interactions on primary mouse CD4(+) T cells was investigated. CTLA4-CD80/CD86 interactions differentially modulate T cell cycling based on the mode of CD3 signal: Activation with plate-bound (pb) anti-CD3 generates a strong signal compared with a weak signal with soluble (sol) anti-CD3, resulting in approximately sevenfold higher amounts of interleukin (IL)-2 and an increase in cell cycling. Activation of T cells with sol anti-CD3 (weak signal) together with CTLA4-CD80/CD86 blockade lowers IL-2 production and cell cycling, demonstrating an enhancing role for these interactions. Conversely, blockade of CTLA4-CD80/CD86 interactions on T cells activated with pb anti-CD3 (strong signal) increases proliferation, which is consistent with CTLA4 as a negative regulator. Also, coculture of T cells with Chinese hamster ovary cells expressing CD80 or CD86 demonstrates that the strength of the primary signal plays an important role. It is important that modulation of IL-2 amounts leads to distinct alterations in the functional effects of CTLA4-CD80/CD86 interactions. On increasing IL-2 amounts, activation of T cells stimulated with sol anti-CD3 (weak signal) and CTLA4-CD80/CD86 blockade is greater compared with control. Concurrently, neutralization of IL-2 greatly reduces activation of T cells stimulated with pb anti-CD3 (strong signal) and CTLA4-CD80/CD86 blockade compared with control. These results underscore the importance of strength of first signal, CTLA4-CD80/CD86 interactions, and IL-2 amounts in modulating primary CD4(+) T cell responses.     

Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses

The present study analyzes the role of CD28-B7-mediated costimulation during in vitro human peripheral blood memory T cell activation by influenza A virus. Inhibition studies using the B7-binding fusion protein CTLA4Ig and antibodies against CD80 and CD86 demonstrate that CTLA4Ig and anti-CD86 inhibited influenza-specific T cell proliferation, interleukin (IL)-2 and interferon (IFN)- production, and generation of influenza-specific CD8+ CTL. The production of IL-10 and IL-18, which are known to modulate T cell immune responses, were not affected by blocking the CD28-B7 costimulatory pathway. Inhibition of diverse influenza-specific T cell functions could be reversed by the addition of exogenous IL-2 or IL-12 but not by the addition of IFN- or IL-18. Although IL-2 is known to overcome CD28-B7 costimulatory requirements, this is the first report showing that exogenous IL-12 is able to bypass CD28-B7 costimulatory blockade induced by CTLA4Ig in vitro. The induction of IFN- production with the recently described IFN- inducing cytokine IL-18 was not detected. In conclusion, these results demonstrate that CD86 represents a major costimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens. These observations may have important implications for the development of immunotherapeutic strategies in diverse immunodeficiency diseases as well as in tumor immunotherapy.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

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.     

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.     

Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.

The induction of T helper cell subsets during the course of non-lethal or lethal blood-stage Plasmodium chabaudi AS infection was investigated using inbred strains of mice which differ in the level of resistance to this intraerythrocytic parasite. Resistant C57Bl/6 mice experience a non-lethal course of infection characterized by moderate levels of both parasitaemia and anaemia and resolution of primary acute infection by 4 weeks, while susceptible A/J mice experience lethal infection with fulminant parasitaemia and severe anaemia. T helper subset function was assessed during infection by determining the kinetics of spleen cell production in vitro of the Th1-derived cytokine, interferon-gamma (IFN-gamma), and of the Th2-derived cytokine, IL-5, using sandwich ELISAs. Spleen cells from resistant C57Bl/6 mice were found to produce high levels of IFN-gamma within 1 week of infection in response to both the mitogen concanavalin A (Con A) and malaria antigen. Furthermore, CD4+ T cells were found to be the source of IFN-gamma while both CD4+ and CD8+ T cells were found to produce IL-5. Decreased IFN-gamma production after day 10 was concomitant with significant production of IL-5 between 2 and 3 weeks post infection. In contrast, spleen cells from susceptible A/J mice produced high levels of IL-5 within the first week of infection. In addition, these animals were found to have high serum levels of IL-5. These results, thus, confirm previous observations that resolution of primary blood-stage P. chabaudi infection occurs by sequential activation of Th1 CD4+ T cells followed by activation of the Th2 subset, and in addition, suggest that induction of a strong Th2 response early in infection may lead to a severe and lethal course of malaria.