SummaryBlocking 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 donormatched 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-% 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 Ibl0. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of riD2 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 Thl but spares Th2 cytokines in vivo.

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The renin-angiotensin system (RAS) plays an important role in the regulation of inflammation and in the progression of chronic kidney disease. Accumulation of inflammatory cells into the renal parenchyma has been a hallmark of chronic kidney disease; however, little is known concerning the presence and the function of RAS elements in T and natural killer (NK) cells. Here is reported a co-stimulatory effect of angiotensin II (AngII) by showing an augmentation of mitogen and anti-CD3-stimulated T and NK cell proliferation with AngII treatment. Angiotensinogen and AngI also generated the same effect, suggesting that NK and T cells have functional renin and angiotensin-converting enzyme activity. Indeed, they express renin, the renin receptor, angiotensinogen, and angiotensin-converting enzyme by mRNA analysis. Flow cytometric analysis and Western blot revealed angiotensin receptor 2 (AT 2 ) expression in T and NK cells, whereas AT 1 expression was found in T and NK cells and monocytes by Western blot. These receptors were shown to be functional in calcium signaling, chemotaxis, and proliferation. However, AT 1 and AT 2 antagonists alone or in combination were unable to abrogate completely the effects of AngII, suggesting that another AngII receptor may also be functional in leukocytes. This is the first study to show that T and NK cells are fully equipped with RAS elements and are potentially capable of producing and delivering AngII to sites of inflammation. Because their chemotaxis is enhanced by AngII, this creates a potential inflammatory amplification system. B ecause of its hemodynamic effects, angiotensin II (AngII) plays a central role in the progression of chronic kidney diseases (CKD) and ischemic heart disease (1,2). AngII has been shown to be a potent proinflammatory molecule, and the beneficial effects of renin-angiotensin system (RAS) blockade are due not only to lowering BP but also to a reduction in inflammation (3). One of the main features of CKD is the accumulation of inflammatory cells, which plays a crucial role in disease progression (4), and recruitment of macrophages to the kidney through AngII infusion has been reported in various rodent models (5,6). In both diabetic nephropathy and atherosclerosis, monocytes/macrophages have been reported to play a key role (7-9). Monocytes have also been the primary focus of studies that have examined the interaction of AngII and inflammatory cells (10). However, the importance of T, natural killer (NK), and dendritic cells (DC) in inflammation and vascular disease has only recently begun to be appreciated. DC have been shown to present oxidized LDL to T cells, generating autoreactive T cells and promoting arterial injury (11). NK cells participate through the production of proatherogenic cytokines such as IFN-␥ (12). Previous studies on AngII-induced inflammation and its role in kidney disease primarily focused on the induction of inflammatory molecules and the paracrine effects of AngII in vascular remodeling and tissue fibrosis (13,14). Despite these ...

These data demonstrate for the first time that T cells of patients with chronic graft dysfunction are primed to recognize and respond to specific donor-derived major histocompatibility complex allopeptides. Our results support the hypothesis that T cells primed via the indirect pathway of allorecognition may be important mediators of chronic rejection and provide the rationale to develop specific therapeutic strategies to prevent or interrupt this process.

Blocking CD28-B7 T-cell costimulation by systemic administration of CTLA4Ig, a fusion protein which binds B7 molecules on the surface of antigen-presenting cells, prevents rejection and induces tolerance in experimental acute allograft rejection models. We tested the effect of CTLA4Ig therapy on the process of chronic renal allograft rejection using an established experimental transplantation model. F344 kidneys were transplanted orthotopically into bilaterally nephrectomized LEW recipients. Control These data confirm that early blockade of the CD28-B7 T-cell costimulatory pathway prevents later development and evolution of chronic renal allograft rejection. Our results indicate that T-cell recognition ofalloantigen is a central event in initiating the process of chronic rejection, and that strategies targeted at blocking T-cell costimulation may prove to be a valuable clinical approach to preventing development of the process.The most common cause of human allograft failure after the first year posttransplant is the incompletely understood clinicopathological entity of chronic rejection (1-3). This process affects all types of solid organ grafts and is characterized morphologically by obliterative vasculopathy, interstitial fibrosis with variable degrees of mononuclear cell infiltration, and in the case of the kidney, glomerulosclerosis. Risk factors for its development include HLA mismatching, acute rejection, early graft ischemia, differences in donor and recipient size, adequacy of immunosuppression, and viral infections. The exact mechanisms responsible for its pathogenesis are unknown, although there is evidence that chronic graft dysfunction is mediated by both alloantigendependent as well as alloantigen-independent mechanisms (4). At this moment no specific therapies are available.T-cell recognition of alloantigens is the key initial event which ultimately results in allograft rejection (5, 6). T cells require two signals for full activation. The first signal is produced by engagement of the T-cell receptor with the foreign antigen presented as a peptide by antigen-presenting cells, thus providing antigenspecificity to the immune response. The second is a "costimulatory" signal mediated, among other interactions, by the T-cell accessory molecule CD28 interacting with the B7 family (B7-1 and B7-2) of molecules on antigen-presenting cells (7-9). In vitro, blockade of costimulatory signals inhibits T-cell activation and induces a state of antigen-specific unresponsiveness. In vivo, agents which block CD28-B7 costimulation, including the fusion protein CTLA4Ig or anti-B7 monoclonal antibodies, have been shown to inhibit the immune response in experimental models of acute transplant rejection and autoimmunity (10). In this study we have investigated the role of T-cell costimulatory blockade in an established model of chronic renal allograft rejection. MATERIALS AND METHODSAnimals. Inbred 200-250 g male LEW rats (RT11) were used as graft recipients and F344 (RT1lvl) rats served as donors (Harlan-Spra...

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Early blockade of T cell-costimulatory activation pathways prevents development of experimental chronic allograft rejection. Ongoing T cell recognition of alloantigen and activation may also play an important role in progression of chronic rejection, but definitive evidence is lacking. We Our data are the first to demonstrate that blocking T cell-costimulatory activation late after transplantation, after initial graft injury, prevents progression of chronic allograft rejection supporting the hypothesis that ongoing T cell recognition of alloantigen and activation are key mediators of ongoing chronic allograft rejection. ( J. Clin. Invest.