CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses.

This study documents a striking dichotomy between CD4 and CD8 T cells in terms of their requirements for CD40-CD40 ligand (CD40L) costimulation. CD40L-deficient (-/-) mice made potent virus-specific CD8 T cell responses to dominant as well as subdominant epitopes following infection with lymphocytic choriomeningitis virus. In contrast, in the very same mice, virus-specific CD4 T cell responses were severely compromised. There were 10-fold fewer virus-specific CD4 T cells in CD40L-/- mice compared with those in CD40L+/+ mice, and this inhibition was seen for both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) responses. An in vivo functional consequence of this Th cell defect was the inability of CD40L-/- mice to control a chronic lymphocytic choriomeningitis virus infection. This study highlights the importance of CD40-CD40L interactions in generating virus-specific CD4 T cell responses and in resolving chronic viral infection.     

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.     

CD40 engagement on dendritic cells, but not on B or T cells, is required for long-term control of murine gammaherpesvirus 68

CD4 T cells are not essential for primary clearance of replicating murine gammaherpesvirus 68 (MHV-68) but are required for effective long-term control. The virus reactivates in the lungs of major histocompatibility complex class II-deficient (CII-/-) mice that lack functional CD4 T cells. CD40 ligand (CD40L) is upregulated on activated CD4 T cells, and it is thought that CD40-CD40L interactions are an important component of CD4 T-cell help. Our previous studies have shown that agonistic antibodies to CD40 can substitute for CD4 T-cell function in the long-term control of MHV-68. In the present study, we sought to identify the CD40-positive cell type mediating this effect. To address this question, we adoptively transferred MHV-68 peptide-pulsed CII(-/-) dendritic cells (DC) that had been treated with an agonistic antibody to CD40 into MHV-68-infected CII(-/-) recipients. Viral reactivation was significantly lower in mice injected with anti-CD40-treated DC than in those injected with control DC or in mice that did not receive any DC. However, in similar experiments with B cells, anti-CD40 treatment had no effect. We also investigated the requirement for CD40 expression on T cells by adoptive transfer of T cells from CD40(+/+) or CD40(-/-) mice into T-cell-deficient recipients that were subsequently infected with MHV-68. The results showed that CD40 expression on T cells is not necessary for preventing viral reactivation. Taken together, our data suggest that CD40 engagement on DC, but not on T or B cells, is essential for effective long-term control of MHV-68.     

Estrogen receptor alpha, but not beta, is required for optimal dendritic cell differentiation and [corrected] CD40-induced cytokine production

Dendritic cells (DC) are critical actors in the initiation of primary immune responses and regulation of self-tolerance. The steroid sex hormone 17beta-estradiol (E(2)) has been shown to promote the differentiation of DCs from bone marrow (BM) precursors in vitro. However, the estrogen receptor (ER) involved in this effect has not yet been characterized. Using recently generated ERalpha- or ERbeta-deficient mice, we investigated the role of ER isotypes in DC differentiation and acquisition of effector functions. We report that estrogen-dependent activation of ERalpha, but not ERbeta, is required for normal DC development from BM precursors cultured with GM-CSF. We show that reduced numbers of DCs were generated in the absence of ERalpha activation and provide evidence for a cell-autonomous function of ERalpha signaling in DC differentiation. ERalpha-deficient DCs were phenotypically and functionally distinct from wild-type DCs generated in the presence of estrogens. In response to microbial components, ERalpha-deficient DCs failed to up-regulate MHC class II and CD86 molecules, which could account for their reduced capacity to prime naive CD4(+) T lymphocytes. Although they retained the ability to express CD40 and to produce proinflammatory cytokines (e.g., IL-12, IL-6) upon TLR engagement, ERalpha-deficient DCs were defective in their ability to secrete such cytokines in response to CD40-CD40L interactions. Taken together, these results provide the first genetic evidence that ERalpha is the main receptor regulating estrogen-dependent DC differentiation in vitro and acquisition of their effector functions.     

The CD40-CD154 interaction in B cell-T cell liaisons

The CD40 receptor is expressed constitutively on B lymphocytes, for which it provides important signals regulating clonal expansion, antibody production and isotype switching, as well as the development of humoral memory. The major source of CD154, the ligand for CD40, is activated T lymphocytes. Interactions between CD40 and CD154 provide a number of signals that play important roles in regulating the complex and multifactorial interactions between these two major cell types of the adaptive immune response. Understanding both the biological effects of this receptor-ligand interaction, as well as how CD40 signaling pathways are controlled, adds to our detailed picture of the complex interplay between B and T cells.     

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.     

Fas expression on antigen-specific T cells has costimulatory, helper, and down-regulatory functions in vivo for cytotoxic T cell responses but not for T cell-dependent B cell responses

Fas-mediated apoptosis is an important contributor to contraction of Ag-driven T cell responses acting only on activated Ag-specific T cells. The effects of targeted Fas deletion on selected cell populations are well described however little is known regarding the consequences of Fas deletion on only activated Ag-specific T cells. We addressed this question using the parent-into-F(1) (P-->F(1)) model of acute or chronic (lupus-like) graft-vs-host disease (GVHD) as a model of either a CTL-mediated or T-dependent B cell-mediated response, respectively. By transferring Fas-deficient lpr donor T cells into Fas-intact F(1) hosts, the in vivo role of Ag-specific T cell Fas can be determined. Our results demonstrate a novel dichotomy of Ag-specific T cell Fas function in that: 1) Fas expression on Ag-activated T cells has costimulatory, helper, and down-regulatory roles in vivo and 2) these roles were observed only in a CTL response (acute GVHD) and not in a T-dependent B cell response (chronic GVHD). Specifically, CD4 T cell Fas expression is important for optimal CD4 initial expansion and absolutely required for help for CD8 effector CTL. Donor CD8 T cell Fas expression played an important but not exclusive role in apoptosis and down-regulation. By contrast, CD4 Fas expression played no detectable role in modulating chronic GVHD induction or disease expression. These results demonstrate a novel role for Ag-specific T cell Fas expression in in vivo CTL responses and support a review of the paradigm by which Fas deficiency accelerates lupus in MRL/lpr lupus-prone mice.     

Phagocytosis of Fonsecaea pedrosoi conidia, but not sclerotic cells caused by Langerhans cells, inhibits CD40 and B7-2 expression

Fonsecaea pedrosoi is the major etiological agent of chromoblastomycosis, a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues, presenting a worldwide distribution. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. Langerhans cells (LC) are bone-marrow-derived, dendritic antigen-presenting cells of the epidermis, which constitutively express major histocompatibility complex (MHC) class II, and comprise 1-3% of total epidermal cells. LC are localized in suprabasal layers of the epidermis and in mucosa, where they play important roles in skin immune responses. The purpose of the present study was to evaluate the interaction of F. pedrosoi conidia or sclerotic cells with LC purified from BALB/c mice skin. We demonstrate here that LC phagocytose F. pedrosoi conidia but not sclerotic cells in the first 3 h of interaction, inhibiting hyphae formation during 12-hour coculture from both forms, internalized or not. Also, LC maturation, analyzed using CD40 and B7-2 expression, was inhibited by conidia, but not by sclerotic cells, indicating an important innate immunity function of LC against F. pedrosoi infection in these mice.     

CD134L engagement enhances human B cell Ig production: CD154/CD40, CD70/CD27, and CD134/CD134L interactions coordinately regulate T cell-dependent B cell responses

CD134 is a member of the TNFR family expressed on activated T cells, whose ligand, CD134L, is found preferentially on activated B cells. We have previously reported that the CD70/CD27 interaction may be more important in the induction of plasma cell differentiation after the expansion phase induced by the CD154/CD40 interaction has occurred. When CD134-transfected cells were added to PBMCs stimulated with pokeweed mitogen, IgG production was enhanced in a dose-dependent fashion. Addition of CD134-transfected cells to B cells stimulated with Staphylococcus aureus Cowan I strain/IL-2 resulted in little if any enhancement of B cell IgG production and proliferation. We found that while CD134-transfected cells induced no IgG production by themselves, it greatly enhanced IgG production in the presence of CD40 stimulation or T cell cytokines such as IL-4 and IL-10. The addition of CD134-transfected cells showed only a slight increase in the number of plasma cells compared with that in the culture without them, indicating that an increased Ig production rate per cell is responsible for the observed enhancing effect of CD134L engagement rather than increase in plasma cell generation. These results strongly suggest different and sequential roles of the TNF/TNFR family molecules in human T cell-dependent B cell responses through cell-cell contacts and the cytokine network.     

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.     

Repeated antigen exposure is necessary for the differentiation, but not the initial proliferation, of naive CD4(+) T cells

The mechanisms that regulate CD4(+) T cells responses in vivo are still poorly understood. We show here that initial Ag stimulation induces in CD4(+) T cells a program of proliferation that can develop, for at least seven cycles of division, in the absence of subsequent Ag or cytokine requirement. Thereafter, proliferation stops but can be reinitiated by novel Ag stimulation. This initial Ag stimulation does not however suffice to induce the differentiation of naive CD4(+) T cells into effector Th1 cells which requires multiple contacts with Ag-loaded APC. Thus, recurrent exposure to both Ag and polarizing cytokines appears to be essential for the differentiation of IFN-gamma-producing cells. Ag and cytokine availability therefore greatly limits the differentiation, but not the initial proliferation, of CD4(+) T cells into IFN-gamma-producing cells.     

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.     

Following direct CD40 activation, human primary microglial cells produce IL-12 p40 but not bioactive IL-12 p70

There is accumulating evidence that interleukin 12 (IL-12) is involved in the pathogenesis of multiple sclerosis. In the periphery, this cytokine is produced by antigen-presenting cells (APCs) following interaction with activated T cells. CD40 ligation plays a crucial role in this production. Microglial cells are thought to play a major role in antigen presentation in the central nervous system. In this work, we examined IL-12 production by human primary microglial cells after CD40 ligation. These cells expressed CD40 and MHC class II following interferon-gamma activation. IL-12 p40 mRNA and protein, but not bioactive IL-12 p70, were detected in response to direct CD40 activation. Microglial cells co-cultured with activated allogenic CD4+ T lymphocytes also produced IL-12 p40 but not IL-12 p70. This IL-12 p40 production was inhibited by anti-CD40 ligand. Altogether, these results suggest that CD40-CD40-ligand interaction provides a signal that triggers IL-12 p40 expression. However, other interaction(s) may be required during antigen presentation for bioactive heterodimeric IL-12 p70 to be produced by microglial cells.     

Cutting edge: depletion of CD4+CD25+ regulatory T cells is necessary,but not sufficient,for induction of organ-specific autoimmune disease

Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4(+)CD25(+) regulatory T cells. However, depletion of CD4(+)CD25(+) T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4(+)CD25(-) T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25(+) T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25(+) regulatory T cells.