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     

SOCS-1 protects from virally-induced CD8 T cell mediated type 1 diabetes

CD8(+) cytotoxic T lymphocytes (CTL) can rapidly kill beta-cells and therefore contribute to the development of type 1 diabetes (T1D). CTL-mediated beta-cell killing can occur via perforin-mediated lysis, Fas-Fas-L interaction, and the secretion of TNF-alpha or IFN-gamma. The secretion of IFN-gamma can contribute to beta-cell death directly by eliciting nitric oxide production, and indirectly by upregulating MHC class I and 'unmasking' beta-cells for recognition by CTL. Earlier studies in the RIP-LCMV mouse model of diabetes showed that disruption of beta-cell IFN-gamma signaling alone abolished the direct detrimental effects of IFN-gamma, but not MHC class I upregulation. Suppressor of cytokine signaling-1 (SOCS-1) represses several crucial cytokine signaling pathways simultaneously, among them IFN-gamma and IL-1-beta. We therefore evaluated the protective capacity of islet cell SOCS-1 expression in the CD8(+) mediated RIP-LCMV diabetes model. Clinical disease was prevented in over 90% of the mice. Not only absence of MHC-I and Fas upregulation, but also resistance to cytokine-induced killing of beta-cells and a complete lack of CXCL-10 (IP10) production in islets led to a lack of islet infiltration and impaired activation of autoaggressive CD4(+) and CD8(+) T-cells in these mice. Thus, SOCS expression renders beta-cells resistant to CTL attack in a mouse model of T1D.     

HIV-1 induces IL-10 production in human monocytes via a CD4-independent pathway

In HIV-infected patients, increased levels of IL-10, mainly produced by virally infected monocytes, were reported to be associated with impaired cell-mediated immune responses. In this study, we investigated how HIV-1 induces IL-10 production in human monocytes. We found that CD14(+) monocytes infected by either HIV-1(213) (X4) or HIV-1(BaL) (R5) produced IL-10, IL-6, tumor necrosis factor-alpha (TNF-alpha), and to a lesser extent, IFN-gamma. However, the capacity of HIV-1 to induce these cytokines was not dependent on virus replication since UV-inactivated HIV-1 induced similar levels of these cytokines. In addition, soluble HIV-1 gp160 could induce CD14(+) monocytes to produce IL-10 but at lower levels. Cross-linking CD4 molecules (XLCD4) with anti-CD4 mAbs and goat anti-mouse IgG (GAM) resulted in high levels of IL-6, TNF-alpha and IFN-gamma but no IL-10 production by CD14(+) monocytes. Interestingly, neither anti-CD4 mAbs nor recombinant soluble CD4 (sCD4) receptor could block IL-10 secretion induced by HIV-1(213), HIV-1(BaL) or HIV-1 gp160 in CD14(+) monocytes, whereas anti-CD4 mAb or sCD4 almost completely blocked the secretion of the other cytokines. Furthermore, HIV-1(213) could induce IL-10 mRNA expression in CD14(+) monocytes while XLCD4 by anti-CD4 mAb and GAM failed to do so. As with IL-10 protein levels, HIV-1(213)-induced IL-10 mRNA expression in CD14(+) monocytes could not be inhibited by anti-CD4 mAb or sCD4. Taken together, HIV-1 binding to CD14(+) monocytes can induce CD4-independent IL-10 production at both mRNA and protein levels. This finding suggests that HIV induces the immunosuppressive IL-10 production in monocytes and is not dependent on CD4 molecules and that interference with HIV entry through CD4 molecules may have no impact on counteracting the effects of IL-10 during HIV infection.     

4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.     

Acquisition of regulatory function by human CD8(+) T cells treated with anti-CD3 antibody requires TNF

Anti-CD3 mAb can modulate graft rejection and attenuate autoimmune diseases but their mechanism(s) of action remain unclear. CD8(+) T cells with regulatory function are induced in vitro by Teplizumab, a humanized anti-CD3 antibody and inhibit responses of autologous and allogeneic T cells. They inhibit CD4(+) T-cell proliferation by mechanisms involving TNF and CCL4, and by blocking target cell entry into G2/M phase of cell cycle but neither kill them, nor compete for IL-2. CD8(+) Treg can be isolated from peripheral blood following treatment of patients with Type 1 diabetes with Teplizumab, but not from untreated patients. The induction of CD8(+) Treg by anti-CD3 mAb requires TNF and signaling through the NF-κB cascade. The CD8(+) Treg express CD25, glucocorticoid-induced TNF receptor family, CTLA-4, Foxp3, and TNFR2, and the combined expression of TNFR2 and CD25 identifies a potent subpopulation of CD8(+) Treg. These studies have identified a novel mechanism of immune regulation by anti-CD3 mAb and markers that may be used to track inducible CD8(+) Treg in settings such as chronic inflammation or immune therapy.     

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.     

beta-cell antigen-specific CD56(+) NKT cells from type 1 diabetic patients: autoaggressive effector T cells damage human CD56(+) beta cells by HLA-restricted and non-HLA-restricted pathways

Studies of type 1 diabetes indicate that autoaggressive T cells specific to beta-cell antigens, reaching certain threshold levels, may play critical roles in the development of the disease. Flow cytometric analyses found that autoreactive T-cell lines from patients induced by beta-cell antigens consisted of four major subsets (CD4(+)CD56(-), CD4(+)CD56(+), CD8(+)CD56(-), and CD8(+)CD56(+)) and that CD56(+) NKT cells might be derived from CD56(-) T cells. Moreover, the proportion of CD56(+) NKT cells in the T-cell lines was influenced by time course of repeated antigen stimulation. beta-cell antigen-specific CD56(+) NKT (CD4(+) or CD8(+)) cells were more aggressive (HLA-restricted and -unrestricted) effector cells lysing target cells such as K562, Jurkat, P815 plus anti-CD3 antibody, and autologous B cells sensitized by beta-cell peptides, when compared with their CD56(-) counterparts. beta-cell antigen- specific CD4(+)CD56(+) NKT cells showed non-HLA-restricted cytotoxicity to human beta cells, insulinoma cell line CM, and to islet cell lines TRM-6 and HP62 expressing CD56 but not to four CD56(-) pancreatic cell lines of non- islet origin. The CD4(+)CD56(+) NKT cells showed stronger cytotoxicity to CM, TRM-6 and HP62 cells than did CD4(+)CD56(-) T cells. Moreover, isotope-unlabelled CD56(+) cells and anti-CD56 antibodies were able to inhibit cytotoxicity of CD4(+)CD56(+) NKT to CD56(+) target cells. These results suggest that CD56(+) NKT cells are aggressive cytotoxic cells to beta cells and that CD56 expression might be associated with the aggressiveness of effector T cells and the susceptibility of target cells.     

AsialoGM1+CD8+ central memory-type T cells in unimmunized mice as novel immunomodulator of IFN-gamma-dependent type 1 immunity

In unimmunized specific pathogen-free mice, there are unique memory-type CD8(+) T cell populations expressing asialoGM1 (ASGM1). These cells were classified into central memory-type T cells (T(CMT)) judging from their expression profile of CD44, IL-2Rbeta, CD62L and CCR7 cell-surface molecules. Among CD44(high)CD8(+) so-called memory CD8(+) T cell population, ASGM1(+)CD44(high)CD8(+) T(CMT), but not ASGM1(-)CD44(high)CD8(+) memory T cells, produced IFN-gamma by stimulation with anti-CD3 mAb. The physiological significance of ASGM1(+)CD8(+) T(CMT) as early source of IFN-gamma was also demonstrated in vivo. Namely, intravenous injection of anti-CD3 mAb (2 microg) resulted in early activation of IFN-gamma-producing ASGM1(+)CD8(+) T(CMT) cells as well as NKT and NK cells. Unexpectedly, however, few IFN-gamma-producing CD4(+) T cells were detected until 4 h after anti-CD3 mAb administration. Thus, ASGM1(+)CD8(+) T(CMT) were demonstrated to be early IFN-gamma producer, which may be crucial for T(h)1-dependent cellular immunity. Indeed, co-culture of naive CD4(+) T cells with ASGM1(+)CD8(+) T(CMT) but not ASGM1(-)CD8(+) T cells caused a great acceleration of IFN-gamma-producing T(h)1 cells in vitro. Finally, we found that T(h)1-prone C57BL/6 mice possessed higher percentage (10%) of ASGM1(+)CD8(+) T(CMT) in CD8(+) T cells compared with that (3%) of T(h)2-prone BALB/c mice. Moreover, ASGM1(+)CD8(+) T(CMT) derived from C57BL/6 mice produced higher levels of IFN-gamma compared with those from BALB/c mice. Thus, ASGM1(+)CD8(+) T(CMT), whose differentiation in vivo is genetically controlled, appear to play a critical role in the control of type 1 immunity, which is essential for therapy of tumors and infectious diseases.     

Human CD4(+)CD25(+) thymocytes and peripheral T cells have immune suppressive activity in vitro

CD4(+)CD25(+) T cells in mice and rats are capable of transferring protection against organ-specific autoimmune disease and colitis and suppressing the proliferation of other T cells after polyclonal stimulation in vitro. Here we describe the existence in humans of CD4(+)CD25(+) T cells with the same in vitro characteristics. CD4(+)CD8(-)CD25(+) T cells are present in both the thymus and peripheral blood of humans ( approximately 10 % of CD4(+)CD8(-) T cells), proliferate poorly in response to mitogenic stimulation and suppress the proliferation of CD4(+)CD25(-) cells in co-culture. This suppression requires cell contact and can be overcome by the addition of exogenous IL-2. CD4(+)CD25(+) cells from thymus and blood were poor producers of IL-2 and IFN-gamma, and suppressed the levels of these cytokines produced by CD4(+)CD25(-) cells. However, CD4(+)CD25(+) PBL produced higher levels of IL-4 and similar amounts of IL-10 as CD4(+)CD25(-) cells. Regulatory CD4(+)CD25(+) T cells have an activated phenotype in the thymus with expression of CTLA-4 and CD122 (IL-2Rbeta). The fact that CD4(+)CD25(+) regulatory T cells are present with a similar frequency in the thymus of humans, rats and mice, suggests that the role of these cells in the maintenance of immunological tolerance is an evolutionarily conserved mechanism.     

Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice

Through an imbalance in Th1 and Th2 cytokine profiles, diesel exhaust particles (DEP) are thought to induce Th2-dominated IgE and IgG1 production. However, the roles of CD4+ and CD8+ T-cell subtypes in the increased immune responses to antigen in mice exposed to DEP are unclear. In the present study, we investigated whether treatment with anti-CD4 or anti-CD8 mAb abrogated the adjuvant activity of DEP. On day -1 and day 1, each group of mice was injected intraperitoneally with anti-CD4, anti-CD8, or rat IgG (vehicle). On day 0, the mice were immunized with ovalbumin (OVA) or OVA plus DEP. After 3 weeks, each mouse was boosted with 10 microg of OVA alone. On day 7 after the first injection with OVA+DEP or OVA alone, the numbers of total, IA+, CD80+/IA+ and CD86+/IA+ cells in peritoneal exudate cells (PEC) were higher in OVA+DEP-immunized mice than in OVA-immunized mice. Depletion of CD8+ cells resulted in a modulation of the production of granulocyte-macrophage colony-stimulating factor, IL-12 and PGE(2) in peritoneal exudate fluid from OVA+DEP-immunized mice. On day 28, DEP injection markedly increased IL-4 production in the culture supernatants of spleen cells from CD4+ or CD8+-depleted mice. Depletion of CD8+ cells in OVA+DEP-immunized mice resulted in a decrease in IFN-gamma production compared with that in OVA-immunized mice. Adjuvant activity of DEP was observed in anti-OVA IgE, anti-OVA IgG1, anti-OVA IgG3, and total IgE production. Depletion of CD4+ T cells abrogated the adjuvant effect of DEP on anti-OVA IgE, and anti-OVA IgG1 production in plasma. However, depletion of CD8+ T cell inhibited the upregulated anti-OVA IgG3 production. These findings suggest that DEP injection may affect not only the function of CD4+ cells but also that of CD8+ T-cell subsets to modulate the synthesis of proinflammatory cytokine in PEC and type-1 and type-2 cytokine production in spleens.     

Contribution of CD8(+) T cells to gamma interferon production in human tuberculosis

The proportions of peripheral blood mononuclear cells (PBMC), CD4(+) T cells, and CD8(+) T cells that produce gamma interferon (IFN-gamma) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4(+) but not CD8(+) cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-gamma production. These results show that (i) IFN-gamma production by CD8(+) and CD4(+) cells correlates with the clinical manifestations of M. tuberculosis infection and (ii) IFN-gamma production by CD8(+) cells depends on CD4(+) cells.     

CD8(+) T cells regulate immune responses in a murine model of allergen-induced sensitization and airway inflammation

The role of CD8(+) T cells in the development of allergic airway disease is controversial. On the one hand, CD8(+) T cells are known to inhibit the development of airway hyperreactivity (AHR) in murine models of asthma. In humans, IL-10-producing CD8(+) T cells were shown to act as regulatory cells, inhibiting both proliferation and cytokine secretion of T cells. On the other hand, CD8(+) T cells can promote IL-5-mediated eosinophilic airway inflammation and the development of AHR in animal models. To examine this, we investigated the role of CD8(+) T cells during the induction of allergen-induced AHR and demonstrated a protective effect of CD8(+) T cells. Depletion of CD8(+) T cells prior to the immunization led to increased Th2 responses and increased allergic airway disease. However, after development of AHR, CD8(+) T cells that infiltrated the lungs secreted high levels of IL-4, IL-5 and IL-10, but little IFN-gamma, whereas CD8(+) T cells in the peribronchial lymph nodes or spleen produced high levels of IFN-gamma, but little or no Th2 cytokines. These data demonstrate protective effects of CD8(+)T cells against the induction of immune responses and show a functional diversity of CD8(+) T cells in different compartments of sensitized mice.     

CD2-CD48 interactions promote cytotoxic T lymphocyte induction and function: anti-CD2 and anti-CD48 antibodies impair cytokine synthesis, proliferation, target recognition/adhesion, and cytotoxicity.

The role of CD2 signaling in cytotoxic T lymphocyte (CTL) development was examined by stimulating mouse T cells with anti-CD3 monoclonal antibody (mAb) in the absence or presence of anti-CD2 mAb or anti-CD48 mAb or both. Induction of nonspecific CTL and interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis were impaired in the absence of CD2-CD48 interactions. Anti-CD2 mAb also inhibited activation-induced expression of the high-affinity IL-2 receptor (IL-2R). In contrast, IFN-gamma receptor (IFNGR) expression was increased in the presence of anti-CD2 mAb. Reduced cytotoxicity by CTL induced in the absence of CD2-CD48 interactions was associated with a diminished ability of CTL to conjugate with target cells and reduced expression of granzyme B and perforin. Anti-CD2 mAb did not affect expression of Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by anti-CD3-activated T cells. Cytotoxic effector function and granzyme B and perforin expression were rescued when exogenous IL-2 and IFN-gamma were added in combination with anti-CD2 mAb to anti-CD3-activated T cells at initiation of culture. We conclude that CD2-CD48 interactions during T cell activation are critical for the synthesis of sufficient IL-2 and IFN-gamma to drive CD8(+) T cells to differentiate into functional cytotoxic effector cells.     

Decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and elevation of CD19(+)BAFF-R(+) B cells and soluble ICAM-1 in myasthenia gravis

Myasthenia gravis (MG) is caused by T-cell-dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokine and chemokine sICAM-1 and to analyze the phenotypes of CD4(+) and CD8(+) regulatory cells as well as the expression of BAFF-R on CD19(+) B cells in peripheral blood from 75 MG patients and 50 healthy controls. There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-gamma, TNF-alpha, TGF-beta and sCTLA-4 in both sera and culture supernatants between MG patients and healthy controls. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Although the populations of CD8(+)CD28(-) and CD8(+)CD122(+) regulatory T cells were not different between MG patients and healthy controls, MG patients exhibited the decrease of CD4(+)CD25(high)Foxp3(+) regulatory T cells and the increase of CD19(+)BAFF-R(+) B cells, revealing that MG patients should display the dysfunction of T cell balance and the activation of B cell maturation.     

Role of mRNA stability in the different patterns of cytokine production by CD4+ cells from young and old mice.

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

IL-10 is involved in the suppression of experimental autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells

CD25(+)CD4(+) regulatory T cells inhibit the activation of autoreactive T cells in vitro and in vivo, and suppress organ-specific autoimmune diseases. The mechanism of CD25(+)CD4(+) T cells in the regulation of experimental autoimmune encephalomyelitis (EAE) is poorly understood. To assess the role of CD25(+)CD4(+) T cells in EAE, SJL mice were immunized with myelin proteolipid protein (PLP)(139-151) to develop EAE and were treated with anti-CD25 mAb. Treatment with anti-CD25 antibody following immunization resulted in a significant enhancement of EAE disease severity and mortality. There was increased inflammation in the central nervous system (CNS) of anti-CD25 mAb-treated mice. Anti-CD25 antibody treatment caused a decrease in the percentage of CD25(+)CD4(+) T cells in blood, peripheral lymph node (LN) and spleen associated with increased production of IFN-gamma and a decrease in IL-10 production by LN cells stimulated with PLP(130-151) in vitro. In addition, transfer of CD25(+)CD4(+) regulatory T cells from naive SJL mice decreased the severity of active EAE. In vitro, anti-CD3-stimulated CD25(+)CD4(+) T cells from naive SJL mice secreted IL-10 and IL-10 soluble receptor (sR) partially reversed the in vitro suppressive activity of CD25(+)CD4(+) T cells. CD25(+)CD4(+) T cells from IL-10-deficient mice were unable to suppress active EAE. These findings demonstrate that CD25(+)CD4(+) T cells suppress pathogenic autoreactive T cells in actively induced EAE and suggest they may play an important natural regulatory function in controlling CNS autoimmune disease through a mechanism that involves IL-10.     

PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2

Programmed death-1 (PD-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor expressed upon T cell activation. PD-1(-/-) animals develop autoimmune diseases, suggesting an inhibitory role for PD-1 in immune responses. Members of the B7 family, PD-L1 and PD-L2, are ligands for PD-1. This study examines the functional consequences of PD-1:PD-L engagement on murine CD4 and CD8 T cells and shows that these interactions result in inhibition of proliferation and cytokine production. T cells stimulated with anti-CD3/PD-L1.Fc-coated beads display dramatically decreased proliferation and IL-2 production, while CSFE analysis shows fewer cells cycling and a slower division rate. Costimulation with soluble anti-CD28 mAb can overcome PD-1-mediated inhibition by augmenting IL-2 production. However, PD-1:PD-L interactions inhibit IL-2 production even in the presence of costimulation and, thus, after prolonged activation, the PD-1:PD-L inhibitory pathway dominates. Exogenous IL-2 is able to overcome PD-L1-mediated inhibition at all times, indicating that cells maintain IL-2 responsiveness. Experiments using TCR transgenic CD4(+) or CD8(+) T cells stimulated with antigen-presenting cells expressing PD-L1 show that both T cell subsets are susceptible to this inhibitory pathway. However, CD8(+) T cells may be more sensitive to modulation by the PD-1:PD-L pathway because of their intrinsic inability to produce significant levels of IL-2.     

Differential requirement of CD28 for IL-12 receptor expression and function in CD4(+) and CD8(+) T cells

IL-12 is a potent inducer of IFN-gamma production and Th1 responses. Co-stimulation mediated by B7 has been shown to synergize with IL-12 for optimal IFN-gamma production and proliferation in vitro. In this study, we examined the requirement of CD28/B7 interactions for optimal induction of IL-12 receptor(R) beta1 and beta2 expression and IFN-gamma. IL-12-induced IFN-gamma production and STAT4 nuclear translocation were markedly reduced in CD28(-/-) splenocytes compared to that of wild-type (WT) splenocytes. Analysis of IL-12R expression revealed that IL-12 induced similar levels of IL-12R beta2 mRNA expression in WT and CD28(-/-) cells. In contrast, IL-12R beta1 expression was impaired in CD28(-/-) cells, indicating that expression of IL-12R beta1 and beta2 is differentially regulated by CD28. CD28(-/-) CD4(+) but not CD8(+) cells exhibited a defect in IL-12Rbeta1 expression that was associated with a marked decrease in IL-12 binding as well as IL-12-induced IFN-gamma production. IL-2 could restore IL-12R expression to CD28(-/-) CD4(+) cells, however, this occurred independently of IL-2-induced proliferation. Thus, these findings identify distinct requirements for CD28 in the capacity of CD4(+) and CD8(+) cells to respond maximally to IL-12.     

Interleukin-12 differentially regulates expression of IFN-gamma and interleukin-2 in human T lymphoblasts.

Interleukin-12 (IL-12) is known to upregulate expression of interferon-gamma (IFN-gamma) by activated T cells. However, the effects of IL-12 on production of other Th1-type cytokines are less well defined. In this study, we examined the effects of IL-12 on expression of several cytokines, including IFN-gamma, IL-2, tumor necrosis factor-alpha (TNF-alpha), and IL-10, by primary human CD3(+) T cells. Although purified resting T cells were largely nonresponsive to IL-12 stimulation, anti-CD3-activated T cell blasts were strongly responsive, as demonstrated by the ability of IL-12 to induce Stat4 DNA-binding activity. Restimulation of T lymphoblasts on immobilized anti-CD3 monoclonal antibodies (mAb) induced rapid expression of TNF-alpha mRNA and more gradual increases in mRNA levels for IL-2, IFN-gamma, and IL-10. IL-12 markedly upregulated expression of IFN-gamma and IL-10 but downregulated expression of IL-2 in a dose-dependent and time-dependent manner. The levels of IL-2 produced by IL-12-treated T cells correlated inversely with the levels of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production by IL-12-treated T cells, confirming that the inhibition of IL-2 production by IL-12 was IL-10 mediated. Thus, IL-12 amplified expression of IFN-gamma and IL-10 and, via its ability to upregulate production of IL-10, inhibited expression of IL-2. These findings demonstrate that IL-12 differentially regulates expression of the Th1-type lymphokines, IFN-gamma and IL-2, in T lymphoblasts.