In vitro studies have revealed several pathways by which T cells can respond to alloantigens, including CD4+ direct responses to allogeneic class 11 antigens, CD8+ direct responses to allogeneic class I antigens, and CD4+ "indirect" responses to peptides of alloantigens presented in association with responder class 11 molecules. In vivo studies of skin graft rejection, however, have so far provided dear evidence for the contribution of only the two direct pathways and not for indirect recognition. We have used major histocompatibility complex class 11-deficient mice as donors to test the role of indirect recognition in rejection of skin grafts. Class 11-deficient skin was always rejected without delay by normal recipients. Removal of recipient CD8+ cells (to leave the animals dependent on CD4+ function) or depletion of recipient CD4+ cells revealed that CD4+ cells were usually involved and sometimes absolutely required in this rapid rejection. Since the donor grafts lacked class II antigens, the CD4+ cells must have recognized donor antigens presented in association with recipient class 11 molecules. These results therefore indicate that indirect recognition can initiate rapid skin graft rejection.Theories to explain the special importance of major histocompatibility complex (MHC) antigens in graft rejection and the extraordinary strength of the immune response to these antigens are all based on the capacity of T cells to recognize allogeneic MHC antigens directly without the usual requirement that their peptides be processed and presented by recipient antigen presenting cells (APCs) (1-3). As a result, studies of the mechanisms of graft rejection have been dominated by concern with the direct pathways of alloreactivity. A few investigators, however, have considered the possibility that graft rejection might also occur through more classical immunologic mechanisms in which peptides of donor antigens are presented in association with recipient MHC molecules. La Rosa and Talmage (4) described this possibility as "indirect" recognition, but the actual role of this pathway in graft rejection has been difficult to determine.Recently, "knockout" mice lacking MHC class II antigens have been produced by the technique of targeted gene disruption by homologous recombination in embryonic stem cells (5, 6). Since these mice lack the MHC class II antigens responsible for stimulating CD4+ T cells, grafts from these mice can be used to examine rejection in the absence of direct stimulation of these helper T cells. The experiments described in this report suggest that in the absence of donor class II antigens, indirect recognition remains an effective pathway of alloreactivity leading to rapid graft rejection. MATERIALS AND METHODSClass II-Deficient Mice. The development and initial characterization of the class II-deficient mice used in these experiments have been described (5). Briefly, the Apb gene was disrupted in the D3 embryonic stem cell line of 129/Sv origin by the technique of homologous recombination. Cloned e...

I schemia-reperfusion (I/R) injury could lead to ischemic renal failure, which is one of the most common forms of acute renal failure of the native kidneys as well as of the renal allograft (1-5). Despite major advances in understanding the pathogenesis of I/R, no satisfactory therapy is available, and ischemic acute renal failure remains a major cause of morbidity and mortality (6). I/R injury begins with tissue hypoperfusion/hypoxia and leads to depletion of cellular ATP and cytoskeletal damage (7,8). Restoration of blood flow is accompanied by reperfusion injury, which is characterized by production of a number of inflammatory molecules such as cytokines, reactive oxygen species (ROS), activation of leukocyte and endothelial cells, and upregulation of adhesion molecules/chemokines. The interactions of activated leukocytes with injured endothelial cells play a crucial role in the pathogenesis of the disease (6,9,10). There is a growing body of evidence indicating that ischemic renal failure is an inflammatory disease (6,11). Antineutrophil strategies, T cell depletion, and blockade of T cell CD28-B7 co-stimulation have been reported to be beneficial in protection against I/R injury (12-15). Chemokines are 8-to 10-kd proteins that play a central role in inflammation. They regulate all of the steps that are necessary for the recruitment of leukocytes to the sites of inflammation, including leukocyte activation, adhesion, chemoattraction, and transmigration across the endothelial barrier (16 -18). Chemokines are also important regulators of T helper 1 (Th1)/Th2 responses as Th1 chemokines selectively recruit Th1 cells to sites of inflammation (19 -23). A Th1 shift has been shown to have a deleterious effect in the pathogenesis of I/R (24). The CXC chemokine receptor 3 (CXCR3) is expressed predominantly on a majority of activated inflammatory (16). A high expression of CXCR3 receptor and its ligands has been reported in different models of inflammation, including rheumatoid arthritis, multiple sclerosis lesions, type 1 diabetes, and allograft rejection (25-28). Anti-CXCR3 strategies to reduce inflammation have yielded promising results in these disease models (24,25,29 -33). The role of CXCR3 in the pathogenesis of I/R injury remains to be elucidated. Therefore, we tested the hypothesis that CXCR3 signaling plays a role in the induction of inflammation through Th1 cell infiltration, mediating renal I/R injury. Materials and Methods Animals CXCR3Ϫ/Ϫ mice were generated in Craig Gerald's laboratory (30).C57BL/6 mice, purchased from Jackson Laboratory (Bar Harbor, ME),

Previous studies have shown that CD4+ T cells are responsible for the great strength of cell-mediated xenograft rejection in the mouse. In vitro studies have suggested that this CD4+ response is to xenogeneic antigens that are presented indirectly. The present studies were carried out in order to determine whether the strength of cell-mediated xenograft rejection in vivo is dependent on the CD4+ indirect response. We grafted pig skin onto mice that express class II MHC antigens only on their thymic epithelial cells (II-4+ mice). These mice have normal numbers of functional peripheral CD4+ T cells; however they lack class II MHC expression on their antigen presenting cells and are thus incapable of mounting a CD4+ T cell-mediated indirect response. Xenograft survival was prolonged on these mice. Furthermore, administration of cyclosporine and anti-CD8 monoclonal antibodies to II-4+ recipients prolonged xenograft survival to at least the same extent as allograft survival, demonstrating that the strength of cell-mediated xenograft rejection resides in the CD4+ indirect response. Despite the increased survival time, xenograft rejection still occurred in the absence of the indirect pathway. Depletion of the II-4+ recipients of their CD4+ T cell population prolonged xenograft survival even further, suggesting the presence of a weaker CD4+ direct mechanism which was virtually undetectable in vitro.

We investigated the influence of allograft primary vascularization on alloimmunity, rejection and tolerance in mice. First, we showed that fully allogeneic primarily vascularized and conventional skin transplants were rejected at the same pace. Remarkably, however, short-term treatment of mice with anti-CD40L antibodies achieved long-term survival of vascularized skin and cardiac transplants but not conventional skin grafts. Non-vascularized skin transplants triggered vigorous direct and indirect pro-inflammatory type 1 T cell responses (IL-2 and γIFN) while primarily-vascularized skin allografts failed to trigger a significant indirect alloresponse. Similar lack of indirect alloreactivity was also observed after placement of different vascularized organ transplants including hearts and kidneys while hearts placed under the skin (non-vascularized) triggers potent indirect alloresponses. Altogether, these results suggest that primary vascularization of allografts is associated with lack of indirect T cell alloreactivity. Finally, we show that long-term survival of vascularized skin allografts induced by anti-CD40L antibodies was associated with a combined lack of indirect alloresponse and a shift of the direct alloresponse towards a type 2 cytokine (IL-4, IL-10) secretion pattern but no activation/expansion of regulatory T cells. Therefore, primary vascularization of allografts governs their immunogenicity and tolerogenicity.