1. Our prior clinical studies with the humanized CD52-specific monoclonal antibody alemtuzumab indicate that aggressive perioperative T-cell depletion does not reduce the risk of early allograft rejection, and treatment must be accompanied by other immunosuppressive agents. Unlike the pro-tolerant effect seen in animal models, alemtuzumab treatment is associated with a novel form of rejection that is macrophage rich and associated with the presence of a specific T-cell type, the effector memory T cell, which is resistant to depletion. In vitro studies have shown that calcineurin inhibitors uniquely prevent the activity of this cell type and we have now shown in a pilot clinical trial that the combination of depletion with a single low dose of the calcineurin inhibitor tacrolimus prevents rejection in humans without the need for steroids or multi drug immunosuppression. In addition to attenuating the activity of effector memory T cells, calcineurin inhibition also appears to limit the liberation of critical chemokines from the allograft, and thus tacrolimus may have unrecognized effects of immune chemotaxis as well as T cell activation. Understanding the role of these effector memory T cells, their resistance to depletion, and the role of homeostatic proliferation in limiting tolerance induction will provide additional mechanistic insights into the development of tolerance in humans.? 2. Theoretically, tacrolimus appears to limit adaptive immune processes like activation induced cell death that may be required for the development of allograft tolerance. It is also nephrotoxic, a significant issue when considering renal transplantation. The mTOR inhibitor sirolimus has been suggested to be a better long term agent as it is less nephrotoxic, and may foster specific elimination of graft specific T cells. However, we have shown that sirolimus as a sole agent is a sub-optimal drug for preventing post-depletional rejection. We have therefore initiated a clinical trial that combines tacrolimus and sirolimus until the risk of early rejection has decreased (approximately 6 months). Patients are then randomized to wean to either tacrolimus or sirolimus. We are now accruing patients to this trial to determine if the early use of dual therapy followed by weaning to monotherapy will favor adaptive tolerance without risking early rejection. We are specifically comparing patients that have been successfully weaned to monotherapy tacrolimus or sirolimus to determine if the predicted benefits and drawbacks of each agent are seen in humans as predicted by prior animal study. Initial results suggest equal efficacy in preventing rejection in both arms but a trend toward increasing donor-specific hyporesponsiveness in the sirolimus arm, as detected by ELISPOT and mixed lymphocyte responses.? 3. We have investigated the use of quantitative PCR-based technologies to more precisely delineate immune activity in renal allografts compared to histology and serve as a tool to improve the diagnostic evaluation of allograft recipients. The diagnosis of acute cellular rejection (ACR) is typically made histologically. However, many patients with histological rejection have normal renal function. The significance of this so-called sub-clinical rejection (SCR) has not been defined. The treatment of SCR is particularly controversial in tolerance trials, as treatment might disrupt potentially salutary regulation. We have developed a PCR-based platform that allows for quantitative quadruplicate analysis of 96 transcripts in quadruplicate (384 wells) from 100ng of total RNA template in approximately 3 hours. To evaluate the utility of this method, and establish a baseline for standard transplant pathology to which tolerance trials can be compared, we have characterized most states relevant to renal transplantation in patients undergoing standard immunosuppression, and correlated findings to histology. We have now identified 3 genes that have strong correlation with the functional significance of a rejection episode. These are Fas ligand, the TH1 transcription factor Tbet and CD152. We are now establishing a prospective trial to investigate the use of molecular monitoring to improve post transplant immune management and are extending the use of this transcriptional technique to other graft pathologies. We have demonstrated that BK virus nephropathy is associated with a strong, cytotoxic T cell response, not unlike that of acute rejection, but of larger magnitude. Moreover, biopsies with BK virus have strong induction of genes associated with renal graft fibrosis and epithelial mesenchymal transformation. These findings have supported additional mechanistic studies, reported in Z01 DK062008-05. Our studies in transplant glomerulopathy also indicate marked immunologic activation, well in advance of clinical disease, and we are in the process of identifying if there are unique pathways that can be disrupted to ameliorate graft disease. Transcriptome patterning will also be studied in the coming year in native kidneys of recipients of other solid organs as well as in pancreas and small bowel transplants.? 4. We have also initiated studies investigating the utility of quantitative viral replication assessment in determining ones overall degree of immunosuppression. We have determined that most transplant recipients have active replication for the Epstein Barr Virus (EBV) and that this exceeds that seen in non-transplanted individuals both qualitatively and quantitatively. By establishing a norm for EBV replication we have been able to determine parameters for over and under immunosuppression and have been able to correlate this with the risk for rejection of over immunosuppression. Similarly, we have shown that utilizing BK viruria as a marker of over-immunosuppression, patients have been assessed prospectively and immunosuppression withdrawn under close surveillance. Resolution of significant viruria is almost universal and acute rejection in these circumstances is less likely. ? 5. Our collaborative trial with NHGRI 04-DK-0057 (Renal transplantation in recipients with nephropathic cystinosis) has enrolled 10 patients. We have transplanted four patients, and the remaining are being followed with progressive chronic kidney disease. These patients demonstrate reduced T cell memory to common antigens, perhaps in part due to the fact that most are younger recipients (< 18 yo). We also demonstrate the relative safety and efficacy of steroid free immunosuppression in a young adult and pediatric population that is not sensitized. Cystinosis, however, requires continued treatment after transplantation, and there appears to be no significant drug interaction between cystagon and other post-transplant medications.? 6. In addition to cellular assays such as mixed lymphocyte response, peripheral leukocyte transcription, flow cytometry to assess the presence of memory cell subpopulations and regulatory T cells, ELISPOT detected IFN-gamma to alloantigen, and measurement of donor specific antibody by luminex, we have prospectively analyzed PHA-stimulated ATP production in CD4+ T cells, as well as memory antigen stimulated responses. This work demonstrates that it is 12 times more likely to develop infection in recipients with low values for ATP production ( 25 ng/ml). Similarly, high levels of stimulated ATP production ( 700 ng/ml) are 30 times more likely to reject. Memory stimulated ATP responses are significantly lower and are lost during the first 6 months of induction. Repopulated T cells are quite active as demonstrated by the relatively high per cell production of ATP. We are continuing to analyze our single center data to address the impact of immunosuppression and to monitor repopulation activity after depletion.
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