ICOS costimulates invariant NKT cell activation

It has been reported that costimulatory molecules, CD80/86-CD28 and CD154-CD40, critically contribute to activation of CD1d-restricted invariant NKT (iNKT) cells. Here we have demonstrated that ICOS, a new member of the CD28 family, plays a substantial role in iNKT cell activation. iNKT cells constitutively expressed ICOS as well as CD28 independently, and ICOS expression was further up-regulated 2-3 days after alpha-galactosylceramide (alpha-GalCer) treatment. Blockade of ICOS-mediated costimulation by administration of anti-ICOS ligand (B7RP-1) mAb or by ICOS gene knockout substantially inhibited alpha-GalCer-induced IFN-gamma and IL-4 production, cytotoxic activity, and anti-metastatic effect. Moreover, blockade of both B7RP-1-ICOS and CD80/86-CD28 interactions mostly abolished the alpha-GalCer-induced immune responses. These findings indicate that iNKT cell activation is regulated by CD28 and IOCS independently.     

Regulation by Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 of alpha-galactosylceramide-induced antimetastatic activity and Th1 and Th2 responses of NKT cells

Interaction of alpha-galactosylceramide (alpha-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by alpha-GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of alpha-GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN-gamma and IL-4 as well as cell proliferation in response to alpha-GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4+ mononuclear cells incubated with alpha-GalCer and CD11c+ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN-gamma and IL-4 than did those incubated with alpha-GalCer and CD11c+ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for alpha-GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4+ T cells by DCs.     

Disruption of CD154:CD40 blocks generation of allograft immunity without affecting APC activation.

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-gamma, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154-/- mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.     

Differential requirement for CD28/CTLA-4-CD80/CD86 interactions in drug-induced type 1 and type 2 immune responses to trinitrophenyl-ovalbumin

The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-gamma, TNF-alpha, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.     

CD40/CD154 interactions are required for the optimal maturation of skin-derived APCs and the induction of helminth-specific IFN-gamma but not IL-4

The mechanisms through which Schistosoma mansoni larvae induce Th1 rather than Th2 immune responses are not well understood. In this study, using CD154-/- mice exposed to radiation-attenuated S. mansoni larvae, we demonstrate roles for CD154/CD40 in the activation of skin-derived APCs and the development of Th1 cells in the skin-draining lymph nodes (sdLN). The presence of CD154 was important for optimal IL-12p40 and essential for Ag-specific IFN-gamma, but CD154 expression by wild-type CD4- cells was insufficient to rescue recall responses of CD4+ cells from CD154-/- mice. This defect is probably due to impaired CD40-dependent IL-12 production in vivo, because administration of anti-CD40 Ab, or rIL-12, restored IFN-gamma production by sdLN cells from CD154-/- mice. CD154 ligation of CD40 was not required for the migration of skin-derived APCs, but did have a limited role in their maturation (increased MHC II and CD86). Unexpectedly, although CD4 cells from CD154-/- mice were deficient in their ability to produce IFN-gamma, they produced significant amounts of IL-4 and IL-5 in the presence of skin-derived APCs from wild-type and CD154-/- mice. Thus, in contrast to IFN-gamma, the production of Th2-associated cytokines is (in this model) independent of CD154. We conclude that whereas the priming of Th1 responses soon after exposure to schistosome larvae is completely CD40/CD154 dependent, IL-4, IL-5, and IL-13 are independent of CD154, suggesting a dichotomy in the specific mechanisms that induce these cytokines by CD4+ cells in the sdLN.     

Selective requirement for CD40-CD154 in drug-induced type 1 versus type 2 responses to trinitrophenyl-ovalbumin

CD154 is transiently expressed by activated T cells and interacts with CD40 on B cells, dendritic cells, macrophages, and monocytes. This costimulatory receptor-ligand couple seems decisive in Ag-driven immune responses but may be differentially involved in type 1 vs type 2 responses. We studied the importance of CD40-CD154 in both responses using the reporter Ag popliteal lymph node assay in which selectively acting drugs generate clearly polarized type 1 (streptozotocin) or type 2 (D-penicillamine, diphenylhydantoin) responses to a constant coinjected Ag in the same mouse strain. Treatment of mice with anti-CD154 reduced characteristic immunological parameters in type 2 responses (B and CD4(+) T cell proliferation, IgG1 and IgE Abs, and IL-4 secretion) and only slightly affected the type 1 response (small decrease in IFN-gamma production, influx of CD11c(+) and F4/80(+) cells, and prevention of architectural disruption of the lymph node, but no effect on IgG2a Ab and TNF-alpha secretion or B and CD4(+) T cell proliferation). The findings indicate that the CD40-CD154 costimulatory interaction is a prerequisite in drug-induced type 2 responses and is only marginally involved in type 1 responses. The observed expression patterns of CD80 and CD86 on different APC (B cells in type 2 and dendritic cells in type 1) may be responsible for this discrepancy.     

Differential requirements for expression of CD80/86 and CD40 on B cells for T-dependent antibody responses in vivo

The CD80/86-CD28 and CD40-CD40 ligand costimulatory pathways are essential for Th cell-dependent B cell responses that generate high-affinity, class-switched Ab in vivo. Disruption of either costimulatory pathway results in defective in vivo humoral immune responses, but it remains unclear to what extent this is due to deficient activation of Th cells and/or of B cells. To address this issue, we generated mixed chimeras in which CD80/86- or CD40-deficient bone marrow-derived cells coexist with wild-type (WT) cells, thereby providing the functional T cell help and accessory cell functions required for fully competent B cell responses. We were then able to assess the requirement for CD80/86 or CD40 expression on B cells producing class-switched Ig in response to a T-dependent Ag. In CD80/86 WT plus CD80/86 double-knockout mixed chimeras, both WT- and CD80/86-deficient B cells produced IgG1 and IgE responses, indicating that direct signaling by CD80/86 is not essential for efficient B cell activation. In marked contrast, only WT IgG1 and IgE responses were detected in the chimeras containing CD40-deficient cells, demonstrating that CD40 expression on B cells is essential for class switching by those B cells. Thus, while disrupting either the CD80/86-CD28 or the CD40-CD40 ligand costimulatory pathway abrogates T-dependent B cell immune responses, the two pathways are nonredundant and mediated by distinct mechanisms.     

Alpha-galactosylceramide-induced liver injury in mice is mediated by TNF-alpha but independent of Kupffer cells

NKT cells expressing phenotypic markers of both T and NK cells seem to be pivotal in murine models of immune-mediated liver injury, e.g., in Con A-induced hepatitis. Also alpha-galactosylceramide (alpha-GalCer), a specific ligand for invariant Valpha14 NKT cells, induces hepatic injury. To improve the comprehension of NKT-cell mediated liver injury, we investigated concomitants and prerequisites of alpha-GalCer-induced hepatitis in mice. Liver injury induced by alpha-GalCer injection into C57BL/6 mice was accompanied by intrahepatic caspase-3 activity but appeared independent thereof. alpha-GalCer injection also induces pronounced cytokine responses, including TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-6. We provide a detailed time course for the expression of these cytokines, both in liver and plasma. Cytokine neutralization revealed that, unlike Con A-induced hepatitis, IFN-gamma is not only dispensable for alpha-GalCer-induced hepatotoxicity but even appears to exert protective effects. In contrast, TNF-alpha was clearly identified as an important mediator for hepatic injury in this model that increased Fas ligand expression on NKT cells. Whereas intrahepatic Kupffer cells are known as a pivotal source for TNF-alpha in Con A-induced hepatitis, they were nonessential for alpha-GalCer-mediated hepatotoxicity. In alpha-GalCer-treated mice, TNF-alpha was produced by intrahepatic lymphocytes, in particular NKT cells. BALB/c mice were significantly less susceptible to alpha-GalCer-induced liver injury than C57BL/6 mice, in particular upon pretreatment with d-galactosamine, a hepatocyte-specific sensitizer to TNF-alpha-mediated injury. Finally, we demonstrate resemblance of murine alpha-GalCer-induced hepatitis to human autoimmune-like liver disorders. The particular features of this model compared with other immune-mediated hepatitis models may enhance comprehension of basic mechanisms in the etiopathogenesis of NKT cell-comprising liver disorders.     

Age-associated augmentation of the synthetic ligand- mediated function of mouse NK1.1 ag(+) T cells: their cytokine production and hepatotoxicity in vivo and in vitro

We recently reported that the direct antitumor effectors in the liver induced by alpha-galactosylceramide (alpha-GalCer) are NK cells that are activated by the IFN-gamma produced from NK1.1 Ag(+) T cells (NKT cells) specifically stimulated with alpha-GalCer, whereas NKT cells cause hepatocyte injury through the Fas-Fas ligand pathway. In the present study, we investigated how mouse age affects the alpha-GalCer-induced effect using young (6-wk-old), middle-aged (30-wk-old), and old (75-wk-old) mice. The serum IFN-gamma and IL-4 concentrations as well as alanine aminotransferase levels after the alpha-GalCer injection increased in an age-dependent manner. An alpha-GalCer injection also induced an age-dependent increase in the Fas ligand expression on liver NKT cells. Under the stimulus of alpha-GalCer in vitro, the liver mononuclear cells from old and middle-aged mice showed vigorous proliferation, remarkable antitumor cytotoxicity, and enhanced production of both IFN-gamma and IL-4 in comparison to those of young mice, all of which were mediated mainly by NK1.1(+) cells. Furthermore, liver mononuclear cells from old mice stimulated with alpha-GalCer showed a more potent Fas-Fas ligand-mediated cytotoxicity against primary cultured hepatocytes than did those from young mice. Most alpha-GalCer-injected old mice, but no young mice, died, while anti-IFN-gamma Ab pretreatment completely inhibited mouse mortality. However, alpha-GalCer-induced hepatic injury did not improve at all by anti-IFN-gamma Ab treatment, and the Fas-ligand expression of liver NKT cells did not change. Taken together, the synthetic ligand-mediated function of NKT cells is age-dependently up-regulated, and the produced IFN-gamma is responsible for alpha-GalCer-induced antitumor immunity and the mouse mortality, while hepatic injury was unexpectedly found to be independent of IFN-gamma.     

Essential role for both CD80 and CD86 costimulation, but not CD40 interactions, in allergen-induced Th2 cytokine production from asthmatic bronchial tissue: role for alphabeta, but not gammadelta, T cells

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-gamma, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-alphabeta but not TCR-gammadelta induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75. 3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific alphabeta T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 alphabeta T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.     

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Full development of Th2 immunity requires both innate and adaptive sources of CD154

The CD40-CD154 interaction is critical for Th2 response generation during helminth infection and following immunization with helminth-conditioned dendritic cells, yet the key cellular sources of these molecules have still to be defined in vivo. In this study, we demonstrate that the requirement for CD40 expression during murine Th2 response induction is restricted exclusively to the Ag-bearing dendritic cells. In contrast, development of full Th2 immunity required CD154 expression on multiple populations. In this respect, optimal production of IL-5, IL-10, and IL-13 was dependent upon CD154 expression by both CD4(+) T cells and non-lymphoid cells. IL-4 production had less stringent costimulatory requirements, with expression of CD154 on either non-lymphoid cells or T cells alone being sufficient to enable production of this archetypal Th2 cytokine. Disparities in CD154 requirements for T cell and B cell responses were revealed during experimental schistosomiasis where, even in the face of robust Th2 generation, B cell class-switching was entirely dependent upon expression of CD154 by the lymphoid compartment. These data help define the costimulatory interactions that occur during the generation of Th2 immunity, and challenge the widely held view that CD154 expressing T cells are the sole contributors in this process.     

IL-12 augments CD8+ T cell development for contact hypersensitivity responses and circumvents anti-CD154 antibody-mediated inhibition.

During sensitization with dinitrofluorobenzene for contact hypersensitivity (CHS) responses, hapten-specific CD8(+) T cells develop into IFN-gamma-producing cells, and CD4(+) T cells develop into IL-4/IL-5-producing cells. Administration of IL-12 during sensitization skews CD4(+) T cell development to IFN-gamma-producing cells, resulting in exaggerated CHS responses. In the current report we tested the role of IL-12 on CD8(+) T cell development during sensitization and elicitation of CHS to dinitrofluorobenzene. Administration of IL-12 during hapten sensitization induced the expression of IL-12Rbeta2 on both CD4(+) and CD8(+) T cells, augmented IFN-gamma production by these T cell populations, and increased the magnitude and duration of the CHS response to hapten challenge. CHS responses were virtually identical in wild-type and IL-12 p40(-/-) mice. Since engagement of CD40 on APC may stimulate IL-12 production, we also tested the role of CD40-CD154 interactions on the development of IFN-gamma-producing CD4(+) and CD8(+) T cells following hapten sensitization. Development of IFN-gamma-producing CD4(+) T cells during hapten sensitization was absent in wild-type mice treated with anti-CD154 mAb or in CD154(-/-) mice. In contrast, the absence of CD40-CD154 signaling had little or no impact on the development of IFN-gamma-producing CD8(+) T cells. These results demonstrate that the development of hapten-specific Th1 effector CD4(+) T cells in CHS requires both CD40-CD154 interactions and IL-12, whereas the development of IFN-gamma-producing effector CD8(+) T cells can occur independently of these pathways.     

Cutting edge: V alpha 14-J alpha 281 NKT cells naturally regulate experimental autoimmune encephalomyelitis in nonobese diabetic mice

Although deficiencies in the NKT cell population have been observed in multiple sclerosis and mouse strains susceptible to experimental autoimmune encephalomyelitis (EAE), little is known about the function of these cells in CNS autoimmunity. In this work we report that TCR Valpha14-Jalpha281 transgenic nonobese diabetic mice, which are enriched in CD1d-restricted NKT cells, are protected from EAE. The protection is associated with a striking inhibition of Ag-specific IFN-gamma production in the spleen, implying modulation of the encephalitogenic Th1 response. This modulation is independent of IL-4 because IL-4-deficient Valpha14-Jalpha281 mice are still protected against EAE and independent of NKT cell-driven Th1 to Th2 deviation, because no increased autoantigen-specific Th2 response was observed in immunized Valpha14-Jalpha281 transgenic mice. Our findings indicate that enrichment and/or stimulation of CD1d-dependent NKT cells may be used as a novel strategy to treat CNS autoimmunity.     

Differential requirement for the CD40-CD154 costimulatory pathway during Th cell priming by CD8 alpha+ and CD8 alpha- murine dendritic cell subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8 alpha(+) and CD8 alpha(-) murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8 alpha(+) DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8 alpha(-) DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8 alpha(+) DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8 alpha(+) DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8 alpha(+) DC-driven Th1 responses but not CD8 alpha(-) DC-driven Th2 responses to protein Ags.     

Critical role for CXC chemokine ligand 16 (SR-PSOX) in Th1 response mediated by NKT cells

The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-gamma and IL-4 by administration of alpha-galactosylceramide (alphaGalCer). Of note, the alphaGalCer-induced production of IFN-gamma was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to alphaGalCer-induced anti-tumor effect mediated by IFN-gamma from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-gamma, but not IL-4, after their culture with alphaGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-gamma by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.     

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.     

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.