The cytotoxic potential of CD8+ T cells and NK cells plays a crucial role in the immune response to pathogens. Although in vitro studies have reported that CD4+ T cells are also able to mediate perforin-mediated killing, the in vivo existence and relevance of cytotoxic CD4+ T cells have been the subject of debate. Here we show that a population of CD4+ perforin+ T cells is present in the circulation at low numbers in healthy donors and is markedly expanded in donors with chronic viral infections, in particular HIV infection, at all stages of the disease, including early primary infection. Ex vivo analysis shows that these cells have cytotoxic potential mediated through the release of perforin. In comparison with more classical CD4+ T cells, this subset displays a distinct surface phenotype and functional profile most consistent with end-stage differentiated T cells and include Ag experienced CD4+ T cells. The existence of CD4+ cytotoxic T cells in vivo at relatively high levels in chronic viral infection suggests a role in the immune response.

SummaryBeginning at the time of insulitis (7 wk of age), CD4 + and CD8 + mature thymocytes from nonobese diabetic (NOD) mice exhibit a proliferative unresponsiveness in vitro after T cell receptor (TCR) crosslinking. This unresponsiveness does not result from either insulitis or thymic involution and is long lasting, i.e., persists until diabetes onset (24 wk of age). We previously proposed that it represents a form of thymic T cell anergy that predisposes to diabetes onset. This hypothesis was tested in the present study by further investigating the mechanism responsible for NOD thymic T cell proliferative unresponsiveness and determining whether reversal of this unresponsiveness protects NOD mice from diabetes. Interleukin 4 (IL-4) secretion by thymocytes from >7-wk-old NOD mice was virtually undetectable after treatment with either anti-TCR c~/B, anti-CD3, or Concanavalin A (Con A) compared with those by thymocytes from age-and sexmatched control BALB/c mice stimulated under identical conditions. NOD thymocytes stimulated by anti-TCR o~/B or anti-CD3 secreted less IL-2 than did similarly activated BALB/c thymocytes. However, since equivalent levels of IL-2 were secreted by Con A-activated NOD and BALB/c thymocytes, the unresponsiveness of NOD thymic T cells does not appear to be dependent on reduced IL-2 secretion. The surface density and dissociation constant of the high affinity IL-2 receptor of Con A-activated thymocytes from both strains are also similar. The patterns of unresponsiveness and lymphokine secretion seen in anti-TCR/CD3-activated NOD thymic T cells were also observed in activated NOD peripheral spleen T cells. Exogenous recombinant (r)IL-2 only partially reverses NOD thymocyte proliferative unresponsiveness to anti-CD3, and this is mediated by the inability of IL-2 to stimulate a complete IL-4 secretion response. In contrast, exogenous rlL-4 reverses the unresponsiveness of both NOD thymic and peripheral T cells completely, and this is associated with the complete restoration of an IL-2 secretion response. Furthermore, the in vivo administration of rlL-4 to prediabetic NOD mice protects them from diabetes. Thus, the ability of rlL-4 to reverse completely the NOD thymic and peripheral T cell proliferative defect in vitro and protect against diabetes in vivo provides further support for a causal relationship between this T cell proliferative unresponsiveness and susceptibility to diabetes in NOD mice.

Our study indicates that anti-HLA class I antibodies play an important role in the pathogenesis of BOS and that monitoring of anti-HLA class I antibody development by a highly sensitive assay such as the PRA-STAT ELISA after LT can provide an early identification of an important subset of LT patients with an increased risk of developing BOS.

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A correlation between indirect allorecognition of mismatched donor HLA class I peptides and development of bronchiolitis obliterans syndrome (BOS) after lung transplantation has been previously observed. The aim of this study was to determine whether there was a correlation between indirect allorecognition of mismatched donor HLA class II peptides and development of BOS after lung transplantation. Peripheral blood mononuclear cells from nine BOSπ and nine BOSlung transplant recipients were cultured with synthetic peptides corresponding to the b-chain hypervariable region of a mismatched donor HLA-DR molecule. Then, proliferative alloreactivity as well as frequency of alloreactive T cells were determined. In addition, the immunodominant epitopes from the donor HLA-DR molecules were identified in selected patients. T cells from BOSπ patients showed a dose-dependent proliferative alloreactivity against donor HLA-DR peptides that was significantly higher than that observed in BOS-patients (pΩ0.001). Similarly, the frequency of HLA-DR alloreactive T cells was significantly higher in BOSπ patients than in BOS-patients (pΩ0.001). This T-cell alloreactivity was directed against a single immunodominant HLA-DR peptide. These results suggest that indirect alloreactivity to donor HLA class II molecules may play a role in the pathogenesis of BOS after lung transplantation.

SummaryThymic T cell anergy, as manifested by thymocyte proliferative unresponsiveness to antigens expressed in the thymic environment, is commonly believed to mediate the acquisition of immunological self-tolerance. However, we previously found that thymic T cell anergy may lead to the breakdown of tolerance and predispose to autoimmunity in nonobese diabetic (NOD) mice. Here, we show that NOD thymic T cell anergy, as revealed by proliferative unresponsiveness in vitro after stimulation through the T cell receptor (TCR), is associated with defective TCRmediated signal transduction along the PKC/p21~'qp42m~p k pathway of T cell activation. PKC activity is reduced in NOD thymocytes. Activation of p21 ~' * is deficient in quiescent and stimulated NOD T cells, and this is correlated with a significant reduction in the tyrosine phosphorylation of p42maP k, a serine/threonine kinase active downstream of p21 ~'s. Treatment of NOD T cells with a phorbol ester not only enhances their p21 r~s activity and p42m~p k tyrosine phosphorylation but also restores their proliferative responsiveness. Since p42m'p k activity is required for progression through to S phase of the cell cycle, our data suggest that reduced tyrosine phosphorylation of p42m'v k in stimulated NOD T cells may abrogate its activity and elicit the proliferative unresponsiveness of these cells. Functional inactivation or anergy of a T cell is manifested by a long-lasting proliferative unresponsiveness, and may occur as a consequence of an interaction between an Ag or mitogen with the CD3-TCR complex in the absence of a second non-Ag-specific costimulator signal provided by an APC (1). T cell anergy in the thymus and/or periphery generally results in immunological self-tolerance (2-4). In contrast, we have found that beginning at the time of insulitis (7 wk of age), mature CD4 * 8-and CD4-8 + thymic T cells from prediabetic NOD mice are anergic as assessed by stimulation of proliferation in vitro after TCR cross-linking with either an anti-TCR mAb, anti-CD3 mAb, or Con A. This anergy is not due to the inability of thymic APCs to provide costimulation (5, 6, and Rapoport, M., A. Jaramillo, D. Zipris, A. Lazarus, D. Serreze, E. Leiter, P. Cyopick, and T. Delovitch, manuscript submitted for publication), but rather arises predominantly from the inability of these NOD thymic T cells to be stimulated to produce sufficient amounts of IL-4 to support their proliferation (6, and Rapoport, M., et al., manuscript submitted for publication). IL-4 completely reverses this thymic T cell anergy in vitro and when administered in vivo, prevents the onset of diabetes in NOD mice (Rapoport, M. et al., manuscript submitted for publication). These studies raise the possibility that thymic T cell anergy might influence susceptibility to diabetes in NOD mice. Elucidation of the biochemical mechanisms of this NOD thymic T cell anergy may further unravel the basis of the polygenic control of susceptibility to type I diabetes.In many types of cells, interactions between membrane rece...

Allografts transplanted across HLA-sensitization results in an antibody-mediated rejection known as hyperacute rejection. Depleting anti-graft antibodies from the recipient by plasmapheresis prior to transplantation can prevent this rejection. We developed an in vitro model using polyclonal HLA class I antibodies obtained from highly sensitized patients awaiting transplantation, and analyzed their ability to provide signals following binding to human aortic endothelial cells (EC). Using this model, we show that EC undergo caspase 3-dependent cell death by apoptosis upon exposure to saturating concentrations of HLA class I antibodies and complement accompanied by loss of Akt activation and phosphorylation of Bad. In contrast, exposure of EC to sub-saturating concentrations of HLA class I antibodies conferred resistance towards antibody/complement-mediated lysis termed accommodation. Accommodated EC exhibited reduction in the expression of the adhesion molecules ICAM-1 and VCAM-1 and a significant increase in the expression of anti-apoptotic genes Bcl-xL, Bcl-2 and heme oxygenase-1. Further, induction of phosphatidylinositol 3-kinase (PI3K) and Akt activities that facilitate the phosphorylation of Bad were also noted. In conclusion, exposure of sub-saturating concentrations of HLA class I antibodies results in the induction of PI3K/Akt pathway that confers resistance to endothelial cells against antibody/complementmediated cell death.

Lung transplantation is recognized as the only viable treatment option in a variety of end-stage pulmonary diseases. However, the long-term survival after lung transplantation is limited by the development of obliterative bronchiolitis, and its clinical correlate bronchiolitis obliterans syndrome (BOS), which is considered to represent chronic lung allograft rejection. Histopathologically, BOS is an inflammatory process that leads to fibrous scarring of the terminal and respiratory bronchioles and subsequent total occlusion of the airways. The specific etiology and pathogenesis of BOS are not well understood. The current premise is that BOS represents a common lesion in which different inflammatory insults such as ischemia-reperfusion, rejection, and infection can lead to a similar histological and clinical outcome. However, the low incidence of BOS in non-transplanted individuals and the observation that early development of BOS is predicted by the frequency and severity of acute rejection episodes indicate that alloimmune-dependent mechanisms play a crucial role in the pathogenesis of BOS. The evidence presented in this review indicates that BOS is the result of humoral and cellular immune responses developed against major histocompatibility complex molecules expressed by airway epithelial cells of the lung allograft. This process is aggravated by alloimmune-independent mechanisms such as ischemia-reperfusion and infection. Currently, treatment of BOS is frequently unsuccessful. Therefore, a better understanding of the immunopathogenesis of BOS is of paramount importance toward improving long-term patient and graft survival after lung transplantation.