Transplantation is the treatment of choice for most end-stage diseases of the kidney, liver, and heart. Although patients benefit in terms of improved quality of life and longevity, they must take immunosuppressive drugs to prevent organ rejection. Side effects lead to significant problems including an increased incidence of cancer, infection, diabetes, osteoporosis, and renal failure. Thus, transplantation remains a trade of a fatal disease for a chronic condition, and minimization of immunosuppression has become an increasingly investigated clinical strategy. Sadly, there are no tests that adequately determine one's need for immunosuppression, nor are there tests that determine excessive immunosuppression. This group designs and implements therapy allowing for organ transplantation without long-term drug requirements or consequences of acquired immunodeficiency - a state defined as tolerance. We test tolerance regimens and immunosuppressive minimization strategies in human kidney recipients. Assays are also developed to more precisely assess immune interventions. Experimental evidence suggests that aggressive perioperative T-cell depletion can reduce the risk of allograft rejection. Studies in primate models have also suggested that depletion is pro-tolerant. Clinical trials have thus been initiated with the humanized CD52-specific monoclonal antibody (Mab) alemtuzumab, a depleting agent approved for use against leukemia. In prior studies, alemtuzumab alone did not induce tolerance. Rather, we observed a reversible, lymphopenic form of rejection. We have characterized this previously un-described form of rejection as being rich in macrophages and strongly associated with TNF-alpha transcription. Allograft dysfunction occurs in the absence of large T-cell infiltrates or evidence of cellular cytotoxicity, and we proposed rejection to be largely a result of renal toxicity from cytokines. To specifically target the predominant cytokine seen, additional patients were treated with the TNF-alpha specific Mab infliximab. While infliximab delayed, and lessened the clinical severity of the rejection episodes for up to 108 days, it did not prevent monocytic infiltration into allograft or clinical eventual rejection. Since blocking TNF did not prevent the infiltration of cells responsible for rejection, we have focused our attention on more proximal elements of rejection, specifically monocyte activation and recruitment. A subsequent trial was initiated specifically targeting monocyte activation by supplementing alemtuzumab induction with the monocyte inhibitory drug deoxyspergualin (DSG). This drug specifically inhibits NFkB nuclear translocation, a transcription factor strongly associated with lymphopenic rejections. Despite myelosuppressive doses of DSG and exceptionally good T-cell depletion lymphopenic rejection occurred in all patients without a significant delay compared to patients treated with alemtuzumab alone. An additional cohort of 4 patients was treated with rapamycin added at the time of surgery. Two early rejection episodes were observed in this group. This is similar to other's experience with sirolimus, but different from subsequent studies by others using calcineurin inhibitor (CNI) based immunosuppression suggesting that rejection may be mediated via a CNI sensitive mechanism: perhaps depletion refractory cells. Additionally, we have studied patients in a trial involving aggressive depletion with rabbit anti-thymocyte globulin (RATG) in combination with monotherapy sirolimus. Most patients were able to have rejection-free survival with sirolimus and there was no graft or patient loss, although the induction appears logistically difficult to implement and reversible rejection rates remain unacceptably high (38%). To more completely investigate the nature of rejections in the alemtuzumab and RATG trials, we have adapted the polychromatic (12-color) flow cytometry technique of Roederer to evaluate depletion resistant populations. We have studied patients undergoing depletion with alemtuzumab or RATG evaluating the phenotype of the residual cells and their sensitivities to immunosuppressants. Surprisingly, post-depletion T-cells are of a single effector memory phenotype, which expand in the first month and are uniquely prevalent at the time of rejection. They are resistant to antibody-mediated depletion in vitro despite expressing the targeted ligands. Furthermore, they proliferate and express cytokines in vitro following polyclonal stimulation despite coincubation with steroids, DSG, or sirolimus, but are effectively inhibited by CNIs. These data demonstrate that following T-cell depletion transplant patients' residual cells, although easy to immunosuppress, particularly with a CNI, are not any more tolerant than other patients requiring immunosuppression. In fact, given the marked depletion of regulatory cells in depleted patients, the use of depletion may in fact be antagonistic to other tolerance therapies like costimulation blockade. In other studies, 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 (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. All findings have been referenced to normal kidney. We have extensively characterized posttransplant reperfusion. Our data show that macrophages are the initial cells migrating into the allograft (not T cells targeted by most immunosuppressants) and that other factors not addressed with immunosuppression are over expressed including complement, mCSF, and adhesion molecules; each of which may constitute targets for future intervention. To improve upon the methods available to quantitate renal reperfusion, we have developed a novel infrared-based intraoperative camera and shown it to be a more accurate monitor of ischemic injury than just cold ischemic time. We subsequently studied biopsies from patients with SCR or ACR . Biopsies were studied by histologically and using a panel of 72 genes known to be associated with inflammation. No histological parameter distinguished SCR and ACR. However, transcripts associated with effector T-cell activation, particularly CD3, CD28, RANTES, Fas ligand, granzyme B and the TH1 transcription factor Tbet clearly distinguish the patient categories. Transcripts from genes associated with monocyte costimulation or chemotaxis were equivalently elevated in SCR and ACR, indicating the SCR infiltrate was arrested prior to effector activation. Thus, SCR is distinguished from ACR not by the magnitude or composition of the infiltrate, but rather by the activation state of T-cells within the allograft. This strongly suggests that immunosuppression withdrawal on the basis of clinical stability can risk rejection or indolent damage. We favor unrecognized SCR as an underlying etiological factor for chronic allograft nephropathy (CAN).
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