Locally produced and activated complement as a mediator of alloreactive T cells.

Immune-mediated rejection remains a significant obstacle preventing long term survival of transplanted organs. Emerging information derived from multiple groups has recently shown that the complement system, traditionally considered a central arm of innate immunity and a primary effector arm of antibody-mediated immunity, plays an additional key role as a regulator of adaptive alloreactive T cell immunity. Complement components produced by immune cells are activated locally and the resultant activation products guide the development of effector T cell immune responses. In the context of organ transplantation, manipulation of local complement activation influences the strength and effector functions of alloreactive T cells which are central mediators of immune mediated rejection. Further definition of the molecular basis underlying complement's effects on cellular alloimmunity has the potential to provide novel targets for the prevention and treatment of injury to solid organ transplants.

Donor deficiency of decay-accelerating factor accelerates murine T cell-mediated cardiac allograft rejection.

Decay-accelerating factor (DAF) is a cell surface regulator that accelerates the dissociation of C3/C5 convertases and thereby prevents the amplification of complement activation on self cells. In the context of transplantation, DAF has been thought to primarily regulate antibody-mediated allograft injury, which is in part serum complement-dependent. Based on our previously delineated link between DAF and CD4 T cell responses, we evaluated the effects of donor Daf1 (the murine homolog of human DAF) deficiency on CD8 T cell-mediated cardiac allograft rejection. MHC-disparate Daf1(-/-) allografts were rejected with accelerated kinetics compared with wild-type grafts. The accelerated rejection predominantly tracked with DAF's absence on bone marrow-derived cells in the graft and required allograft production of C3. Transplantation of Daf1(-/-) hearts into wild-type allogeneic hosts augmented the strength of the anti-donor (direct pathway) T cell response, in part through complement-dependent proliferative and pro-survival effects on alloreactive CD8 T cells. The accelerated allograft rejection of Daf1(-/-) hearts occurred in recipients lacking anti-donor Abs. The results reveal that donor DAF expression, by controlling local complement activation on interacting T cell APC partners, regulates the strength of the direct alloreactive CD8(+) T cell response. The findings provide new insights into links between innate and adaptive immunity that could be exploited to limit T cell-mediated injury to an allograft following transplantation.

Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis.

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell-expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.

Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells.

Costimulatory signals are critical to T cell activation, but how their effects are mediated remains incompletely characterized. Here, we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs), C5aR and C3aR, on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly, impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation, providing a link between GPCR signaling, CD28 costimulation, and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability, and their amplification by cognate APC partners thus is critical to T cell costimulation.

Decay accelerating factor can control T cell differentiation into IFN-gamma-producing effector cells via regulating local C5a-induced IL-12 production.

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF), a cell surface C3/C5 convertase regulator, exerts control over T cell responses. Extending these results, we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1(-/-)) APCs produce significantly more IL-12, C5a, and IFN-gamma compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-gamma-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC, demonstrating a link between DAF, local complement activation, IL-12, and T cell-produced IFN-gamma. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-gamma-producing effector T cells. Overall, our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production, this in turn, contributes to directing T cell differentiation toward an IFN-gamma-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity.

Mouse endothelial cells cross-present lymphocyte-derived antigen on class I MHC via a TAP1- and proteasome-dependent pathway.

In vivo studies suggest that vascular endothelial cells (ECs) can acquire and cross-present exogenous Ag on MHC-I but the cellular mechanisms underlying this observation remain unknown. We tested whether primary female mouse aortic ECs could cross-present exogenous male Ag to the T cell hybridoma, MHH, specific for HYUty plus D(b). MHC-I-deficient male spleen cells provided a source of male Ag that could not directly stimulate the MHH cells. Addition of male but not female MHC-I-deficient spleen cells to wild-type syngeneic female EC induced MHH stimulation, demonstrating EC cross-presentation. Lactacystin treatment of the donor male MHC-I-deficient spleen cells, to inhibit proteasome function, markedly enhanced EC cross-presentation showing that the process is most efficient for intact proteins rather than degraded peptide fragments. Additional experiments revealed that this EC Ag-processing pathway is both proteasome and TAP1 dependent. These studies demonstrate that cultured murine aortic ECs can process and present MHC-I-restricted Ag derived from a separate, live cell, and they offer insight into the molecular requirements involved in this EC Ag presentation process. Through this pathway, ECs expressing cross-presented peptides can participate in the effector phase of T cell-mediated inflammatory responses such as autoimmunity, anti-tumor immunity, and transplant rejection.

Decay-accelerating factor modulates induction of T cell immunity.

Decay-accelerating factor (Daf) dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. We demonstrate that during primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. We show that cognate T cell-APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important.

Skewed Th1/Th2 immune response to Sarcoptes scabiei.

Scabies is a contagious skin disease of humans and many other species of mammals. Previous studies suggested that the balance between the Th1 and Th2 immune responses may influence the outcome of a scabies infestation in a sensitized host. Therefore, in this study, we examined the T-helper cell cytokine profiles of splenocytes and lymph node cells in BALB/c mice that were immunized with scabies extract (primary response), infested with scabies mites (primary response), or immunized and then infested (secondary response). Lymphocyte cytokine expression was analyzed by flow cytometry after staining for intracellular cytokines. Immunization with scabies extract induced production of interferon-gamma (IFNgamma) (Th1 response) by both spleen and lymph node cells. Mice that were infested with scabies increased production of interleukin-4 by lymph node cells and of IFNgamma by splenocytes. Mice that were first immunized and then infested with mites increased production of IFNgamma by both spleen and lymph node cells. However, this increased level of IFNgamma was only about half of that induced by immunization alone. These results suggest that live scabies mites produced something that inhibited IFNgamma production in the lymph nodes of scabies-immunized mice. Our data also indicate that lymphocytes in the spleen and lymph nodes can present different cytokine response profiles.