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.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.     

CD4 help-independent induction of cytotoxic CD8 cells to allogeneic P815 tumor cells is absolutely dependent on costimulation

Mice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4-/- mice and MHC class II-/- mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.     

Immunobiology of allograft rejection in the absence of IFN-gamma: CD8+ effector cells develop independently of CD4+ cells and CD40-CD40 ligand interactions

Both wild-type (WT) and IFN-gamma-deficient (IFN-gamma(-/-)) C57BL/6 mice can rapidly reject BALB/c cardiac allografts. When depleted of CD8(+) cells, both WT and IFN-gamma(-/-) mice rejected their allografts, indicating that these mice share a common CD4-mediated, CD8-independent mechanism of rejection. However, when depleted of CD4(+) cells, WT mice accepted their allografts, while IFN-gamma(-/-) recipients rapidly rejected them. Hence, IFN-gamma(-/-), but not WT mice developed an unusual CD8-mediated, CD4-independent, mechanism of allograft rejection. Allograft rejection in IFN-gamma(-/-) mice was associated with intragraft accumulation of IL-4-producing cells, polymorphonuclear leukocytes, and eosinophils. Furthermore, this form of rejection was resistant to treatment with anti-CD40 ligand (CD40L) mAb, which markedly prolonged graft survival in WT mice. T cell depletion studies verified that anti-CD40L treatment failed to prevent CD8-mediated allograft rejection in IFN-gamma(-/-) mice. However, anti-CD40L treatment did prevent CD4-mediated rejection in IFN-gamma(-/-) mice, although grafts were eventually rejected when CD8(+) T cells repopulated the periphery. The IL-4 production and eosinophil influx into the graft that occurred during CD8-mediated rejection were apparently epiphenomenal, since treatment with anti-IL-4 mAb blocked intragraft accumulation of eosinophils, but did not interfere with allograft rejection. These studies demonstrate that a novel, CD8-mediated mechanism of allograft rejection, which is resistant to experimental immunosuppression, can develop when IFN-gamma is limiting. An understanding of this mechanism is confounded by its association with Th2-like immune events, which contribute unique histopathologic features to the graft but are apparently unnecessary for the process of allograft rejection.     

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.     

CD4+ and CD8+ T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-gamma-producing CD8(+) T cells, whereas CD4(+) T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8(+) and CD4(+) T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154(-/-) animals. DNFB sensitization of CD154(-/-) mice primed IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8(+), but not CD4(+), T cell development and the CHS response to challenge. F(ab')(2) of MR1 failed to inhibit CD8(+) T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8(+) T cell development and CHS responses in mice depleted of CD4(+) T cells or in CD4(-/-) mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4(+) T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-gamma-producing CD8(+) T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8(+) T cell development where anti-CD154 mAb acts indirectly through CD4(+) T cells to impair the ability of hpLC to prime CD8(+) T cells.     

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

Targeting LFA-1 and cd154 suppresses the in vivo activation and development of cytolytic (cd4-Independent) CD8+ T cells

Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4+ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8+ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8+ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8+ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8+ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8+CD25high cell population in the liver.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Treatment of allograft recipients with donor-specific transfusion and anti-CD154 antibody leads to deletion of alloreactive CD8+ T cells and prolonged graft survival in a CTLA4-dependent manner

A two-element protocol consisting of one donor-specific transfusion (DST) plus a brief course of anti-CD154 mAb greatly prolongs the survival of murine islet, skin, and cardiac allografts. To study the mechanism of allograft survival, we determined the fate of tracer populations of alloreactive transgenic CD8+ T cells in a normal microenvironment. We observed that DST plus anti-CD154 mAb prolonged allograft survival and deleted alloreactive transgenic CD8+ T cells. Neither component alone did so. Skin allograft survival was also prolonged in normal recipients treated with anti-CD154 mAb plus a depleting anti-CD8 mAb and in C57BL/6-CD8 knockout mice treated with anti-CD154 mAb monotherapy. We conclude that, in the presence of anti-CD154 mAb, DST leads to an allotolerant state, in part by deleting alloreactive CD8+ T cells. Consistent with this conclusion, blockade of CTLA4, which is known to abrogate the effects of DST and anti-CD154 mAb, prevented the deletion of alloreactive transgenic CD8+ T cells. These results document for the first time that peripheral deletion of alloantigen-specific CD8+ T cells is an important mechanism through which allograft survival can be prolonged by costimulatory blockade. We propose a unifying mechanism to explain allograft prolongation by DST and blockade of costimulation.     

Induction of CTL responses by simultaneous administration of liposomal peptide vaccine with anti-CD40 and anti-CTLA-4 mAb

Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257-264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257-264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RLmale symbol1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RLmale symbol1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

IL-4 modulation of CD4+CD25+ T regulatory cell-mediated suppression

Murine CD4(+)CD25(+) T regulatory (Treg) cells were cocultured with CD4(+)CD25(-) Th cells and APCs or purified B cells and stimulated by anti-CD3 mAb. Replacement of APCs by B cells did not significantly affect the suppression of CD4(+)CD25(-) Th cells. When IL-4 was added to separate cell populations, this cytokine promoted CD4(+)CD25(-) Th and CD4(+)CD25(+) Treg cell proliferation, whereas the suppressive competence of CD4(+)CD25(+) Treg cells was preserved. Conversely, IL-4 added to coculture of APCs, CD4(+)CD25(-) Th cells, and CD4(+)CD25(+) Treg cells inhibited the suppression of CD4(+)CD25(-) Th cells by favoring their survival through the induction of Bcl-2 expression. At variance, suppression was not affected by addition of IL-13, although this cytokine shares with IL-4 a receptor chain. When naive CD4(+)CD25(-) Th cells were replaced by Th1 and Th2 cells, cell proliferation of both subsets was equally suppressed, but suppression was less pronounced compared with that of CD4(+)CD25(-) Th cells. IL-4 production by Th2 cells was also inhibited. These results indicate that although CD4(+)CD25(+) Treg cells inhibit IL-4 production, the addition of IL-4 counteracts CD4(+)CD25(+) Treg cell-mediated suppression by promoting CD4(+)CD25(-) Th cell survival and proliferation.     

IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells.

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1alpha/beta-deficient (IL-1(-/-)) mice and enhanced in IL-1R antagonist(-/-) mice. The intrinsic functions of T, B, and APCs were normal in IL-1(-/-) mice. However, we showed that IL-1(-/-) APCs did not fully activate DO11.10 T cells, while IL-1R antagonist (-/-) APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1(-/-) mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.     

CD40 engagement enhances antigen-presenting langerhans cell priming of IFN-gamma-producing CD4+ and CD8+ T cells independently of IL-12

The delivery of CD40 signaling to APCs during T cell priming enhances many T cell-mediated immune responses. Although CD40 signaling up-regulates APC production of IL-12, the impact of this increased production on T cell priming is unclear. In this study an IL-12-independent T cell-mediated immune response, contact hypersensitivity (CHS), was used to further investigate the effect of CD40 ligation on the phenotypic development of Ag-specific CD4(+) and CD8(+) T cells. Normally, sensitization for CHS responses induces hapten-specific CD4(+) T cells producing type 2 cytokines and CD8(+) T cells producing IFN-gamma. Treatment of mice with agonist anti-CD40 mAb during sensitization with the hapten 2,4-dinitrofluorobenzene resulted in CHS responses of increased magnitude and duration. These augmented responses in anti-CD40 Ab-treated mice correlated with increased numbers of hapten-specific CD4(+) and CD8(+) T cells producing IFN-gamma in the skin draining lymph nodes. Identical results were observed using IL-12(-/-) mice, indicating that CD40 ligation promotes CHS responses and development of IFN-gamma-producing CD4(+) and CD8(+) T cells in the absence of IL-12. Engagement of CD40 on hapten-presenting Langerhans cells (hpLC) up-regulated the expression of both class I and class II MHC and promoted hpLC migration into the T cell priming site. These results indicate that hpLC stimulated by CD40 ligation use a mechanism distinct from increased IL-12 production to promote Ag-specific T cell development to IFN-gamma-producing cells.     

CD154 signaling regulates the Th1 response to herpes simplex virus-1 and inflammation in infected corneas

Approximately 7 days after HSV-1 corneal infection, BALB/c mice develop tissue-destructive inflammation in the cornea termed herpes stromal keratitis (HSK), as well as periocular skin lesions that are characterized by vesicles, edema, and fur loss. CD4(+) T cells and Th1 cytokines contribute to both the immunopathology in the cornea and the eradication of viral replication in the skin. We demonstrate that disruption of CD40/CD154 signaling does not impact the initial expansion of CD4(+) T cells in the draining lymph nodes, but dramatically reduces the persistence and Th1 polarization of these cells. Despite the reduced Th1 response, CD154(-/-) mice developed HSK and periocular skin disease with similar kinetics and severity (as assessed by clinical examination) as wild-type (WT) mice. However, when the composition of the inflammatory infiltrate was examined by flow cytometric analysis, CD154(-/-) mice exhibited significantly fewer CD4(+) and CD8(+) T cells and neutrophils than WT mice at the peak of HSK. Moreover, CD4(+) T cells from infected corneas of CD154(-/-) mice produced significantly less IFN-gamma than those of WT mice when stimulated with viral Ags in vitro. The IFN-gamma production of cells from infected corneas of WT mice was not affected by addition of anti-CD154 mAb to the stimulation cultures. This suggests that CD154 signaling is required at the inductive phase, but not at the effector phase, of the Th1 response within the infected cornea. We conclude that local disruption of CD40/CD154 signaling is not likely to be a useful therapy for HSK.     

CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.     

TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.