Normal Th1 development following long-term therapeutic blockade of CD154-CD40 in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a Th1-mediated demyelinating disease of the CNS with similarities to multiple sclerosis. We and others have shown that a short-term course of anti-CD154 mAb treatment to block CD154-CD40 interactions can be used to prevent or even treat ongoing PLP139-151-induced relapsing EAE. However, little is known of the long-term effects of CD154 blockade on the development of antigen-specific T cell function. Here, we show that short-term treatment with anti-CD154 at the time of PLP139-151/CFA immunization inhibits clinical disease for up to 100 days after immunization. At this point, comparable numbers of Th1 cells are observed in anti-CD154 and control Ig-treated mice, as assessed by antigen-specific ELISPOT assays. Thus, the long-term Th1/Th2 balance is largely unaffected. Inflammatory responses are diminished in anti-CD154-treated mice, as indicated by reduced in vivo delayed-type hypersensitivity and reduced levels of splenic IFN-gamma secretion in vitro. However, upon adoptive transfer of T cells isolated from the spleens of anti-CD154-treated mice, these cells contributed as effectively to clinical disease as those obtained from control-treated mice. Thus, anti-CD154 therapy leads to long-term therapeutic efficacy without exerting a long-term influence on Th1 development.     

CD80 Costimulation Is Essential for the Induction of Airway Eosinophilia

CD80 and CD86 (B7-1 and B7-2) are the ligands on antigen-presenting cells (APCs) which bind CD28 and deliver the costimulatory signals necessary for T cell activation. The reasons for the existence of two CD28 binding molecules are not well understood. We created a mutant version of CTLA4-Ig that could selectively bind CD80 and block CD28-CD80 interaction but leave CD28-CD86 binding intact. CD80 blockade prevented antigen-induced accumulation of eosinophils and lymphocytes in the lung of immunized mice, but did not block antigen induced systemic blood eosinophilia or IgE antibody production. No preferential expression of CD80 could be demonstrated on a population of lung APC consisting mainly of macrophages. These results indicate that CD80 costimulation is not necessary for the induction of Th2 immune responses but rather for the maintenance or amplification of lung inflammatory responses.     

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2

Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig- treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig- treated animals was associated with increased staining for the Th2- related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig- treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig- treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.     

Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis

Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T cells specific for endogenous myelin epitopes released during acute disease, reflecting a critical role for epitope spreading in the perpetuation of chronic central CNS pathology. We asked whether blockade of the CD40–CD154 (CD40L) costimulatory pathway could suppress relapses in mice with established R-EAE. Anti-CD154 antibody treatment at either the peak of acute disease or during remission effectively blocked clinical disease progression and CNS inflammation. This treatment blocked Th1 differentiation and effector function rather than expansion of myelin-specific T cells. Although T-cell proliferation and production of interleukin (IL)-2, IL-4, IL-5, and IL-10 were normal, antibody treatment severely inhibited interferon-γ production, myelin peptide–specific delayed-type hypersensitivity responses, and induction of encephalitogenic effector cells. Anti-CD154 antibody treatment also impaired the expression of clinical disease in adoptive recipients of encephalitogenic T cells, suggesting that CD40–CD154 interactions may be involved in directing the CNS migration of these cells and/or in their effector ability to activate CNS macrophages/microglia. Thus, blockade of CD154–CD40 interactions is a promising immunotherapeutic strategy for treatment of ongoing T cell–mediated autoimmune diseases.     

Distinct regulatory roles of lymphocyte costimulatory pathways on T helper type-2 mediated autoimmune disease

We assessed the role of CD40-CD40L, cytotoxic T lymphocyte (CTL)A4/CD28- B7s, and CD2-CD48/CD58 lymphocyte costimulatory pathways in the development of mercury chloride (HgCl2)-induced autoimmune disease in mice, which is believed to be mediated by T helper (Th) subset Th2. Inhibition of CD40-CD40-L and CTLA4/CD28-B7s interactions by anti-CD40- L antibody and soluble CTLA4-immunoglobulin (Ig) fusion protein, respectively, abrogated the autoimmune disease without affecting interleukin 4 (IL-4) production, showing the importance of physical contact between T and B lymphocytes in the Th2-mediated process. In contrast, two anti-CD2 antibodies that have been shown to induce immunosuppression of Th1-mediated events exacerbated the autoantibody response and augmented IgG1, IgE, and IL-4 production, transforming a mild mesangial glomerulopathy into a severe systemic immune complex disease. These observations demonstrate that manipulation of lymphocyte accessory counterreceptor interactions may affect the course of Th2- associated autoimmune disease and suggest that signals resulting from CD2 engagement play an essential role in the regulation of the Th1-Th2 effector equilibrium.     

Th1 cytokines,programmed cell death,and alloreactive T cell clone size in transplant tolerance

The Th1 cytokines IL-2 and IFN-gamma, which inhibit T cell proliferation and promote activation induced cell death, may be required to diminish alloreactive T cell numbers and to foster tolerance across full allogeneic barriers. However, we hypothesized that these cytokines might be dispensable when the alloreactive T cell clone size is relatively small, as is seen in recipients of minor-mismatched grafts. We show that alloreactive T cell clone size of C57BL/6 mice against multiple minor-mismatched 129X1/sv mice was approximately 4-9-fold smaller than that against MHC-mismatched BALB/c mice. In the MHC-mismatched combination, CD28-B7 blockade by CTLA4Ig induced long-term graft survival in wild-type recipients, but this treatment was ineffective in IFNgamma(-/-) or IL-2(-/-) recipients. In contrast, in the minor-mismatched combination, CTLA4Ig induced long-term allograft survival in wild-type, IFNgamma(-/-), and IL-2(-/-) recipients. Bcl-x(L) transgenic animals, which are defective in "passive" T cell death, are likewise sensitive to the effects of CTLA4Ig only in the setting of the minor-mismatch grafts. Therefore, the alloreactive T cell clone size is an important determinant affecting the need for Th1 cytokines and T cell death in tolerance induction. These data have implications for the design of tolerance strategies in transplant recipients with varying degrees of MHC mismatching.     

Limited role of CD28-mediated signals in T helper subset differentiation

The role of CD28-mediated signals in T helper cell maturation is not fully understood. We tested the requirement for costimulation through CD28 in several systems of CD4+, T cell differentiation. In vivo priming of mice with genetic disruption of CD28 (CD28-/-) yielded normal levels of antigen-specific interferon gamma production but markedly diminished levels of interleukin 4 (IL-4) after in vitro restimulation. In response to the pathogenic microbe, Leishman a major, C57BL6 CD28-/- mice were fully capable of controlling infection and exhibited a normal T helper 1 response. BALB/c CD28-/- mice unexpectedly exhibited normal susceptibility to L. major. BALB/c CD28-/- mice developed high levels of IL-4 mRNA and protein induction in the draining lymph nodes. In addition, susceptibility of BALB/c CD28-/- mice was reversed by neutralization of IL-4 in vivo. We also activated transgenic CD28-bearing T cells from the BALB and C57BL background in vitro in the presence of CTLA4Ig. BALB cells had greater IL-4 producing capacity than C57BL cells in the absence of costimulation. Diverse factors including costimulatory signals, genetic polymorphism, and the nature of the immunogen all influence T helper phenotype commitment, but these results provide evidence that CD28 is not an absolute requirement for generating either Th1 or Th2 responses.     

CD28-independent induction of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated disease initiated by antigen-specific CD4(+) T cells. Signaling through CD28 is a critical second signal for activation of T cells, and CD28 knockout (CD28KO) mice have been reported to be resistant to induction of EAE. We now report that CD28KO mice have no intrinsic defect in mediating disease, because they developed EAE after passive transfer of primed T cells. After immunization, peripheral T cells from CD28KO mice were primed and developed memory phenotype, but had decreased antigen-specific IFN-gamma production as compared with cells from wild-type (WT) animals. Reimmunization of CD28KO mice brought out clinical disease and increased IFN-gamma production in vitro. Pathologically, there were cellular infiltrates in the central nervous system, in contrast to single-immunized mice. We show furthermore that blocking B7-1 or CTLA4, but not B7-2, in CD28KO mice induces disease after a single immunization. Thus, EAE can be induced in animals lacking CD28-dependent costimulation, suggesting that alternative costimulatory pathways were used. Blocking the OX40-OX40L costimulatory pathway differentially affected disease induction in CD28KO mice as compared with WT controls. Our data show that EAE may develop in the absence of CD28 T-cell costimulation. These findings have implications for therapies aimed at blocking costimulatory signals in autoimmune diseases.     

Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection

Simultaneous blockade of the CD40 and CD28 costimulatory pathways is an effective treatment strategy to promote allograft acceptance but does not lead to indefinite allograft survival. The immune mechanisms responsible for costimulation-independent rejection are not defined. Here we have studied the rejection responses of murine C57BL/6 recipients, which we show to be relatively resistant to inhibition by combined CD40/CD28 blockade. We demonstrate that asialo GM1(+) CD8(+) cells play a critical role in this costimulation blockade-resistant rejection. These results provide new insights into the costimulatory requirements for T-cell subsets and demonstrate for the first time that combined blockade of the CD40 and CD28 pathways does not adequately inhibit CD8-mediated skin allograft rejection. Furthermore, we provide evidence that asialo GM1 is a potentially important therapeutic target for CD8-dependent immune responses.     

Regulatory functions of CD8+CD28- T cells in an autoimmune disease model

CD8+ T cell depletion renders CD28-deficient mice susceptible to experimental autoimmune encephalomyelitis (EAE). In addition, CD8-/-CD28-/- double-knockout mice are susceptible to EAE. These findings suggest a role for CD8+ T cells in the resistance of CD28-deficient mice to disease. Adoptive transfer of CD8+CD28- T cells into CD8-/- mice results in significant suppression of disease, while CD8+CD28+ T cells demonstrate no similar effect on the clinical course of EAE in the same recipients. In vitro, CD8+CD28- but not CD8+CD28+ T cells suppress IFN-gamma production of myelin oligodendrocyte glycoprotein-specific CD4+ T cells. This suppression requires cell-to-cell contact and is dependent on the presence of APCs. APCs cocultured with CD8+CD28- T cells become less efficient in inducing a T cell-dependent immune response. Such interaction prevents upregulation of costimulatory molecules by APCs, hence decreasing the delivery of these signals to CD4+ T cells. These are the first data establishing that regulatory CD8+CD28- T cells occur in normal mice and play a critical role in disease resistance in CD28-/- animals.     

Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40-CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector

Blockade of the CD40-CD40L and CD80/CD86-CD28 costimulatory pathways represents a strategy to inhibit the immune response against Ad vectors designed for gene therapy applications. Since most previous studies have used a CTLA4-Ig fusion molecule binding to both CD80 and CD86, the respective roles of these B7 molecules remained undefined. We have studied the effect of blocking monoclonal Abs (mAbs) directed against the costimulatory molecules CD40L, CD80 and CD86, alone or in different combinations, on the humoral and cellular immune responses against Ad. Groups of mice were transiently treated with each combination of blocking mAbs upon systemic injection of a first Ad vector. Combinations of anti-CD80 + anti-CD86 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition of the immune response against Ad. Using either of these mAb pairs, a second vector could be administered 1 month after the first injection but with lower efficiency than in naive animals. Thus, CD86 stands as the pivotal B7 molecule involved in the development of the immune response against Ad. However, only the blockade of both CD80 and CD86 in addition to CD40L fully inhibited the humoral and cellular responses against the Ad vector, such that readministration after 1 month was as efficient as in naive animals. At the time of readministration, treated animals had regained their ability to mount a normal immune response to the second Ad vector, showing that tolerance was not induced.     

Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas

Abdominal aortic aneurysms (AAAs) cause death due to complications related to expansion and rupture. The underlying mechanisms that drive AAA development remain largely unknown. We recently described evidence for a shift toward T helper type 2 (Th2) cell responses in human AAAs compared with stenotic atheromas. To evaluate putative pathways in AAA formation, we induced Th1- or Th2-predominant cytokine environments in an inflammatory aortic lesion using murine aortic transplantation into WT hosts or those lacking the receptors for the hallmark Th1 cytokine IFN-gamma, respectively. Allografts in WT recipients developed intimal hyperplasia, whereas allografts in IFN-gamma receptor-deficient (GRKO) hosts developed severe AAA formation associated with markedly increased levels of MMP-9 and MMP-12. Allografts in GRKO recipients treated with anti-IL-4 antibody to block the characteristic IL-4 Th2 cytokine or allografts in GRKO hosts also congenitally deficient in IL-4 did not develop AAA and likewise exhibited attenuated collagenolytic and elastolytic activities. These observations demonstrate an important dichotomy between cellular immune responses that induce IFN-gamma- or IL-4-dominated cytokine environments. The findings establish important regulatory roles for a Th1/Th2 cytokine balance in modulating matrix remodeling and have important implications for the pathophysiology of AAAs and arteriosclerosis.     

T cell costimulatory pathways: blockade for autoimmunity

The activation of T lymphocytes is thought to require at least two signals, one delivered by the T cell receptor (TCR) complex after antigen recognition, and one provided on engagement of costimulatory receptors. The B7-1/B7-2-CD28/cytotoxic T lymphocyte antigen-4 (CTLA-4) and CD154-CD40 pathways have been shown to be crucial in regulating T cell activation and tolerance. Novel members of the B7-CD28 superfamily have recently been discovered and they seem to be particularly important for regulating the responses of previously activated T cells. Superimposition of inhibitory signals like those delivered by CTLA-4 and programmed death (PD)-1-PD-1-ligand (PD-L1) pathway leads to a complex network of positive and negative costimulatory signals, the integration of which modulates immune responses. Furthermore, expression of several B7 homologues on cells other than professional antigen-presenting cells (APCs), indicate new mechanisms for regulating T cell responses in peripheral tissues. This review focuses on our current understanding of the members of the B7-CD28 superfamily and discusses their therapeutic potential.     

Costimulation via OX40L expressed by B cells is sufficient to determine the extent of primary CD4 cell expansion and Th2 cytokine secretion in vivo

The development of effector and memory CD4 cell populations depends upon both T cell receptor (TCR) engagement of peptide/major histocompatibility complex (MHC) class II complexes and ligation of costimulatory molecules with counter receptors on antigen-presenting cells (APCs). We showed previously that sustained interactions with APCs could be crucial for optimal expansion of CD4 cells and for development of effectors that secrete cytokines associated with Th2 cells. Using an adoptive transfer model with TCR transgenic CD4 cells, we now show that responses of CD4 cells primed in B cell-deficient mice become aborted, but are fully restored upon the transfer of activated B cells. Although B cells have the capacity to secrete multiple cytokines that could affect CD4 priming, including IL-4, we were unable to distinguish a role for cytokines that are secreted by B cells. However, B cell costimulation via the OX40L/OX40 pathway that has been implicated in CD4 cell expansion, survival, and Th2 development was required. Th2 but not Th1 responses were impaired in OX40L-deficient recipients and normal responses were restored with OX40L sufficient B cells. The results suggest that without engagement of OX40L on B cells, CD4 cell responses to many protein Ag would be dominated by Th1 cytokines. These data have important implications for strategies to achieve optimal priming of CD4 subsets.     

Cd40 but not CD154 knockout mice have reduced inflammatory response in polymicrobial sepsis: a potential role for Escherichia coli heat shock protein 70 in CD40-mediated inflammation in vivo

The CD40-CD154 system controls various aspects of the host inflammatory response in models of cellular and humoral immunity. Recently, we described a role for CD40 in the innate immune response in polymicrobial sepsis. However, recent data suggests that CD40 maybe activated by CD154 or directly via bacterial heat shock protein (HSP) 70. Therefore, we decided to test the mechanism of CD40 activation in murine polymicrobial sepsis. Wild-type (WT), CD40, and CD154 underwent cecal ligation and puncture (CLP). Compared with WT mice, CD40 had improved survival in association with attenuated production of IL-12, TNF-alpha, and IL-6. In contrast, CD154 mice behaved similar to WT mice with regard to mortality and cytokine production. The differential response of CD40 and CD154 mice to CLP was not due to a general attenuated response to inflammatory stimuli, as all three strains had similar survival after LPS administration, and CD40 macrophages had normal production of IL-12 in response to lipopolysaccharide. In contrast, CD40 macrophages had attenuated IL-12 production in response to Escherichia coli HSP70 (DnaK). Furthermore, intraperitoneal administration of DnaK resulted in a 4-fold increase in IL-12 in WT mice, which was absent in CD40 mice. This data demonstrates CD154-independent CD40 activation in polymicrobial sepsis and suggests that bacterial HSP70 is capable of stimulating CD40 in vitro and in vivo.     

Disease-associated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell responses against MAGE-6 in HLA-DRB10401(+) patients with renal cell carcinoma or melanoma

T helper type 1 (Th1)-type CD4(+) antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4(+) T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4(+) T cells from human histocompatibility leukocyte antigens (HLA)-DRbeta1*0401(+) patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-gamma and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6-derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus- or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4(+) T cell secretion of IL-10 and transforming growth factor (TGF)-beta1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4(+) subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4(+) T cell responses to provide optimal clinical benefit.     

CD28 costimulation is required for in vivo induction of peripheral tolerance in CD8 T cells

Whereas ligation of CD28 is known to provide a critical costimulatory signal for activation of CD4 T cells, the requirement for CD28 as a costimulatory signal during activation of CD8 cells is less well defined. Even less is known about the involvement of CD28 signals during peripheral tolerance induction in CD8 T cells. In this study, comparison of T cell responses from CD28-deficient and CD28 wild-type H-Y-specific T cell receptor transgenic mice reveals that CD8 cells can proliferate, secrete cytokines, and generate cytotoxic T lymphocytes efficiently in the absence of CD28 costimulation in vitro. Surprisingly, using pregnancy as a model to study the H-Y-specific response of maternal T cells in the presence or absence of CD28 costimulation in vivo, it was found that peripheral tolerance does not occur in CD28KO pregnants in contrast to the partial clonal deletion and hyporesponsiveness of remaining T cells observed in CD28WT pregnants. These data demonstrate for the first time that CD28 is critical for tolerance induction of CD8 T cells, contrasting markedly with CD28 independence of in vitro activation, and suggest that the role of CD28/B7 interactions in peripheral tolerance of CD8 T cells may differ significantly from that of CD4 T cells.     

Costimulation controls diabetes by altering the balance of pathogenic and regulatory T cells

The development of autoimmune diabetes in the nonobese diabetic (NOD) mouse results from a breakdown in tolerance to pancreatic islet antigens. CD28-B7 and CD40 ligand-CD40 (CD40L-CD40) costimulatory pathways affect the development of disease and are promising therapeutic targets. Indeed, it was shown previously that diabetes fails to develop in NOD-B7-2-/- and NOD-CD40L-/- mice. In this study, we examined the relative role of these 2 costimulatory pathways in the balance of autoimmunity versus regulation in NOD mice. We demonstrate that initiation but not effector function of autoreactive T cells was defective in NOD-B7-2-/- mice. Moreover, the residual proliferation of the autoreactive cells was effectively controlled by CD28-dependent CD4+CD25+ regulatory T cells (Treg's), as depletion of Treg's partially restored proliferation of autoreactive T cells and resulted in diabetes in an adoptive-transfer model. Similarly, disruption of the CD28-B7 pathway and subsequent Treg deletion restored autoimmunity in NOD-CD40L-/- mice. These results demonstrate that development of diabetes is dependent on a balance of pathogenic and regulatory T cells that is controlled by costimulatory signals. Thus, elimination of Treg's results in diabetes even in the absence of costimulation, which suggests a need for alternative strategies for immunotherapeutic approaches.