Search

Background: Specific tolerance after combined kidney and bone marrow transplantation for multiple myeloma with end-stage renal disease through mixed lymphohematopoietic chimerism has been achieved, as evidenced by prolonged normal renal function without ongoing immunosuppression., Methods: To achieve potent antimyeloma responses and induce tolerance for the renal allograft, seven patients (median age: 48 years [range: 34-55 years]) with multiple myeloma and end-stage renal disease underwent a combined human leukocyte antigen-matched kidney and bone marrow transplant with lead follow-up time of more than 12 years. Preparative therapy for the transplant consisted of high-dose cyclophosphamide, equine antithymocyte globulin and pretransplant thymic irradiation. Cyclosporine as the sole posttransplant immunosuppressive therapy was tapered and discontinued as early as day 73 posttransplant., Results: All seven patients achieved mixed chimerism. One patient developed acute graft-versus-host disease and two chronic graft-versus-host disease. Five of seven patients are alive, four with no evidence of myeloma from 4 to 12.1 years posttransplant. Three patients have normal or near-normal renal function without needing systemic immunosuppression. Two patients with normal renal function off immunosuppression were returned to immunosuppressive therapy without evidence of rejection because of the occurrence of chronic graft-versus-host disease., Conclusions: These long-term follow-up data show that sustained renal allograft tolerance and prolonged antimyeloma responses are achievable after human leukocyte antigen-matched kidney and bone marrow transplantation and the induction of mixed lymphohematopoietic chimerism.

Background: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients., Methods: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4)., Results: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR., Conclusion: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.

Background: CD56+ human natural killer (NK) cells are the principal anti-pig cytotoxic effectors in vitro. Expression of certain human leukocyte antigen (HLA) class I molecules in porcine cells can inhibit NK cell-mediated natural cytotoxicity in serum-free medium, but had not been shown to inhibit antibody-dependent cellular cytotoxicity (ADCC) by CD16+ NK cells in the presence of human xenoreactive immunoglobulin G. Moreover, expression of HLA molecules might amplify the previously weak CD8+ cytotoxic T-lymphocyte (CTL) response against porcine cells., Methods: A novel porcine B-lymphoblastoid cell line (13271) was stably transfected with HLA-Cw*0304 gene constructs encoding wild-type (wt) Cw3 or genetically modified Cw3 unable to interact with CD8 (Cw3-D227K). The Cw3 transfectants were used in limiting dilution assays to estimate the CTL precursor frequency in CD56-depleted human peripheral blood mononuclear cells (PBMC) obtained from eight unrelated donors. The 13271 transfectants were also used as targets for clonal and polyclonal NK cells in the presence and absence of human serum, to measure inhibition of ADCC., Results: Expression of Cw3-wt in 13271 cells significantly increased the human CTL response compared with the empty-vector control transfectant, whereas no significant increase resulted from expression of CD8-interaction mutant Cw3-D227K molecules. The Cw3-D227K mutant was indistinguishable from Cw3-wt in its ability to inhibit both natural cytotoxicity and ADCC mediated by human NK clones that have the appropriate CD158b inhibitory receptor., Conclusions: Transgenic expression of HLA molecules in pig cells will likely amplify the CD8+ CTL response against the xenograft. Disruption of HLA-CD8 interaction could minimize this amplification without compromising NK-cell inhibition.

Background: In renal transplantation, chronic rejection is a major cause of late allograft loss. Recent studies indicate that a subset of chronic rejection is associated with anti-HLA donor specific antibodies (DSA) and complement C4d deposition in peritubular capillaries (PTC). Since rescue therapy with tacrolimus and mycophenolate mofetil has been found to limit antidonor B-cell responses in recipients with acute humoral rejection, we sought to determine whether a similar immunosuppressive regimen might be effective in patients with 'chronic humoral rejection'., Methods: Four renal allograft recipients with 'chronic humoral rejection' were prospectively identified. The diagnosis was based on: (1) progressive rise in serum creatinine over 12 months; (2) typical pathologic features by light microscopy (transplant arteriopathy and glomerulopathy); (3) widespread C4d deposits in PTC by immunofluorescence; (4) detection of 'de novo' DSA at the time of biopsy. Maintenance immunosuppression was CsA, prednisone and azathioprine (n=3) or prednisone and azathioprine (n=1). Rescue therapy with tacrolimus and mycophenolate mofetil was initiated in all patients, 12 hr after cyclosporine and azathioprine discontinuation., Results: At diagnosis, the mean serum creatinine was 3.9 mg/dl (range: 3.3 to 5.4 mg/dl). DSA was an IgG directed against HLA class II (n=3) or class I (n=2), that is one patient had both anti-HLA class I and class II antibodies. Pretreatment antibody titers varied between 1:8 and 1:128. Rescue therapy was associated with a rapid and sustained decrease in antibody titers. In two patients, DSA became undetectable after 9 months and a repeat biopsy performed after 12 months revealed a decrease in C4d deposition in PTC., Conclusion: These results suggest that a decrease in DSA production can be induced in renal allograft recipients with 'chronic humoral rejection' by using an immunosuppressive regimen that combines tacrolimus and mycophenolate mofetil. Limitation of antidonor antibody synthesis may be important for the treatment or the prevention of chronic rejection in organ transplantation.

Background: Acute rejection (AR) associated with de novo production of donor-specific antibodies (DSA) is a clinicopathological entity that carries a poor prognosis (acute humoral rejection, AHR). The aim of this study was to determine the incidence and clinical characteristics of AHR in renal allograft recipients, and to further analyze the antibodies involved., Methods: During a 4-year period, 232 renal transplants (Tx) were performed at our institution. Assays for DSA included T and B cell cytotoxic and/or flow cytometric cross-matches and cytotoxic antibody screens (PRA). C4d complement staining was performed on frozen biopsy tissue., Results: A total of 81 patients (35%) suffered at least one episode of AR within the first 3 months: 51 had steroid-insensitive AR whereas the remaining 30 had steroid-sensitive AR. No DSA were found in patients with steroid-sensitive AR. In contrast, circulating DSA were found in 19/51 patients (37%) with steroid-insensitive AR, and widespread C4d deposits in peritubular capillaries were present in 18 of these 19 (95%). In at least three cases, antibodies were against donor HLA class II antigens. DSA were not found in the remaining 32 patients but C4d staining was positive in 2 of 32. The DSA/C4d positive (n=18) and DSA/C4d negative (n=30) groups differed in pre-Tx PRA levels, percentage of re-Tx patients, refractoriness to antilymphocyte therapy, and outcome. Plasmapheresis and tacrolimus-mycophenolate mofetil rescue reversed rejection in 9 of 10 recipients with refractory AHR., Conclusion: More than one-third of the patients with steroid-insensitive AR had evidence of AHR, often resistant to antilymphocyte therapy. Most cases (95%) with DSA at the time of rejection had widespread C4d deposits in peritubular capillaries, suggesting a pathogenic role of the circulating alloantibody. Combined DSA testing and C4d staining provides a useful approach for the early diagnosis of AHR, a condition that often necessitates a more intensive therapeutic rescue regimen.

Background: Experimental and clinical evidence has demonstrated that the establishment of allogeneic chimerism after bone marrow transplantation may provide donor-specific tolerance for solid organ allografts., Methods: Based on the preliminary results of a clinical trial using nonmyeloablative preparative therapy for the induction of mixed lymphohematopoietic chimerism, we treated a 55-year-old woman with end stage renal disease secondary to multiple myeloma with a combined histocompatibility leukocyte antigen-matched bone marrow and renal transplant after conditioning with cyclophosphamide, antithymocyte globulin, and thymic irradiation., Results: The posttransplant course was notable for early normalization of renal function, the absence of acute graft-versus-host disease, and the establishment of mixed lymphohematopoietic chimerism. Cyclosporine, which was the only posttransplant immunosuppressive therapy, was tapered and discontinued on day +73 posttransplant. No rejection episodes occurred, and renal function remains normal on day + 170 posttransplant (14 weeks after discontinuing cyclosporine). Although there is presently no evidence of donor hematopoiesis, there is evidence of an ongoing antitumor response with a recent staging evaluation showing no measurable urine kappa light chains. The patient remains clinically well and is off all immunosuppressive therapy., Conclusion: This is the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen to treat a hematological malignancy and to provide allotolerance for a solid organ transplant.

We report a case of accelerated acute rejection of a renal allograft from a presumed ABO histo-blood group A2 donor in an O recipient, in which all of the published criteria for compatibility had been met. Flow cytometric analysis of the A and H antigen expression on the kidney donor's erythrocytes suggested that this donor did not have an A2 phenotype, but rather another subgroup of A. Some of the reported cases of accelerated acute rejection of A2 renal allografts in O recipients may have resulted from misapplication of the results of standard lectin agglutination to the transplant setting. The current case suggests that a more sophisticated method of ABO phenotyping, such as erythrocyte flow cytometric analysis, may be necessary in the transplant setting.

In this study, we have investigated whether lymphocyte growth from 196 liver transplant biopsies is correlated with the histologic diagnosis of graft rejection. One fragment of each biopsy was cultured in IL-2 to expand activated T cells, and the remainder of the biopsy was analyzed histologically. A significantly higher number of biopsies with rejection grew lymphocytes when compared to those biopsies showing no evidence of rejection (P = 0.009). Lower growth rates were observed with biopsies taken from patients on OKT3 therapy (12% vs. 29% from patients not on OKT3), so when the data were reanalyzed after omitting those biopsies we observed an even stronger correlation of growth with rejection (P = 0.001). In spite of the fact that hepatitis biopsies are also infiltrated by lymphoid cells, the frequency of lymphocyte growth (16%) was similar to that observed for the biopsies with no rejection or hepatitis (19%) and much lower than for biopsies with rejection (42%). Over all, 82% of the cultures tested were positive for donor PLT reactivity, suggesting that current culture conditions favor alloreactive lymphocyte proliferation. The size of the biopsy cultured was found to be an important factor in the propagation of biopsy-infiltrating lymphocytes. Only 25% of smaller (less than 3 mm long) biopsies with rejection grew lymphocytes compared to 91% of the large-sized biopsies (greater than 5 mm long). It is likely that culture techniques will need to be modified in order to successfully propagate infiltrating lymphocytes that recognize antigens other than alloantigens.