Histologic and immunophenotypic evaluation of pretreatment renal biopsies in OKT3-treated allograft rejections

The histologic features immunophenotype of intragraft mononuclear populations, and HLA-Dr expression in pretreatment formalin-fixed renal allograft biopsies were correlated with outcome of OKT3 therapy in 35 steroid resistant renal transplant rejections. Therapeutic response (63% overall) was better in pure acute cellular rejections (ACR) (13/15, 87%) than ACR with interstitial fibrosis (4/12, 33%) or with vascular injury (5/8, 62%). Intragraft T lymphocytes were more numerous in vascular rejection (mean 566/mm2) compared with pure ACR (mean 265/mm2, P = .049), and macrophages were greater in pure ACR (203/mm2) compared with ACR with interstitial fibrosis (83/mm2, P = .051). Distribution of T cells, B cells, plasma cells, and macrophages among various histologic categories was otherwise statistically similar. There was no correlation between therapeutic response to OKT3 and intragraft concentrations of individual mononuclear cell subsets. Vascular and/or epithelial HLA-Dr expression was present in 17/25 (68%) cases and was not associated with histologic features or treatment response. Follow-up graft function (median 7 months) correlated significantly with therapeutic response to OKT3 (P = .0004) and histologic presence of interstitial fibrosis (P = .031), but was not related to concentration of individual mononuclear subsets or HLA-Dr expression. We conclude that intragraft concentrations of major mononuclear cell types may relate to histology, but that these do not predict treatment response or graft outcome, and thus poorly reflect intensity or possible heterogeneity of host immunologic rejection mechanisms.     

Immunohistological analysis of renal allograft biopsies from cyclosporin-treated patients

Differentiation of cyclosporin nephrotoxicity from renal allograft rejection is often difficult. Induction of tubular HLA-class II antigens and elevated levels of leucocyte infiltration are associated with allograft rejection but their association with cyclosporin nephrotoxicity is unclear. In order to determine these relationships, transplant biopsies (n=32) from patients considered to have cyclosporin nephrotoxicity, allograft rejection or stable graft function were stained with monoclonal antibodies specific for HLA-class II antigens and infiltrating leucocytes. Leucocyte infiltration was elevated during rejection but not in cyclosporin nephrotoxicity or stable graft function. While HLA-class II antigen expression was induced in 71% of the biopsies obtained during clinical rejection, no increased expression was found in the other 29%. Induced antigens were detected in five of the nine biopsies obtained in the presence of cyclosporin nephrotoxicity 90 days after transplantation. In four of these, induction was attributed to prolongation of increased class II expression following previous rejection episodes. Thus, the presence of induced class II antigens in the renal allograft does not exclude a diagnosis of cyclosporin nephrotoxicity.     

Reexposure to OKT3 in renal allograft recipients

Between 40% and 80% of patients treated with the monoclonal antibody OKT3 develop blocking antibody against its idiotypic region. Thus a major concern with the use of OKT3 as part of a baseline immunosuppressive regimen is that formation of blocking antibodies might preclude its subsequent use. Between 7/86 and 2/87, 32 patients received prophylactic OKT3 in addition to low-dose prednisone, azathioprine, and cyclosporine. Prophylactic OKT3 did not prevent rejection, as 21 of 32 patients studied developed rejection. Retreatment of 13 patients with OKT3 successfully reversed 12 rejections and lowered the number of T3-positive cells in spite of a low level of blocking antibody in two patients in this group. Of the patients analyzed, 38% developed blocking antibody on initial exposure to OKT3, but OKT3 reuse was denied only 4 patients due to the presence of these antibodies. Three of these had rejections reversed with steroids alone; the other patient lost the allograft. A high frequency of infectious complications occurred in the retreatment group, with viral infections predominating. Only one patient in the retreated group developed antibodies after the second use. Appearance of blocking antibodies after use of OKT3 as part of a base-line prophylactic immunosuppressive regimen did not significantly compromise access to OKT3 for treatment of subsequent rejection episodes, but multiple exposures to OKT3 did increase the frequency of infectious complications.     

Alterations in T lymphocyte subpopulations associated with renal allograft rejection

The peripheral blood OKT3 (total T), OKT4 (T helper/inducer), and OKT8 (T suppressor/cytotoxic) cells were determined by flow cytometry on twenty consecutive recipients of HLA-nonidentical cadaveric renal allografts. The absolute number of cells in all three populations decreased significantly posttransplantation, but no differences were found between patients experiencing rejection and those in quiescence. An OKT4/OKT8 ratio of greater than or equal to 1.7, either pretransplant or posttransplant, uniformly identified patients who subsequently experienced rejection. However, an OKT4/OKT8 ratio of less than 1.7 did not identify patients with a low risk of rejection. Pretransplant splenectomy was performed in 6 of 7 patients who rejected despite a low ratio. Serial monitoring of the OKT4/OKT8 ratio posttransplantation determined that an increase in the ratio of greater than or equal to 0.5 was a sensitive (81%) and specific (91%) indicator of a rejection episode. Graft survival was improved in patients with a high posttransplant OKT4/OKT8 ratio. These results indicate that the balance of helper and suppressor cell function may be of critical importance to the fate of an allograft, and that the alterations in this balance can be used to assist in the clinical management of allograft recipients.     

Detection of anti-HLA antibodies with flow cytometry in needle core biopsies of renal transplants recipients with chronic allograft nephropathy

The aim of this study was to assess the feasibility of detecting anti-HLA antibodies in eluates from needle core biopsies of renal transplants with chronic allograft nephropathy. Two methods of screening, the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FlowPRA) were compared. Twenty renal transplants with CAN were removed after irreversible graft failure. To assess the feasibility of detecting anti-HLA antibodies in small samples, needle core biopsies were sampled at the same place as surgical samples and at a second cortical area. Antibodies were eluted with an acid elution kit and anti-class I and class II IgG HLA antibodies detected using ELISA and flow cytometry. Flow cytometry was found to be more sensitive than ELISA for detecting anti-HLA antibodies in eluates from renal transplants with CAN (95% vs. 75% of positive cases). Detection of anti-HLA antibodies showed good agreement between surgical samples and needle core biopsies performed at the same place for anti-class I (80% vs. 65%, r=0.724 P<0.01) and anti-class II HLA antibodies (70% vs. 55%, r=0.827 P<0.01). In addition, differences in the detection of anti-class I HLA antibodies in needle core biopsies sampled at different sites suggests that immunization to class I donor antigen could be underestimated in needle core biopsy samples. These data indicate that anti-HLA antibodies can be detected in needle core biopsies from renal transplants. Provided further evaluation is done, elution might be a complementary method to detect anti-HLA antibodies when they are bound to the transplant.     

Protocol biopsies in the management of renal allograft recipients

The safety of the renal allograft biopsy and the standardization of allograft histopathology interpretation have renewed interest in the protocol biopsy. Recent studies in the areas of 'marginal' donors, surveillance of acute rejection, molecular biology and chronic rejection are discussed.     

Beta 3 integrin expression on T cells from renal allograft recipients

Recent studies emphasize the paramount significance of beta 3 integrin in cell adhesion and homing, which may be particularly relevant in cancer progression and metastasis. In contrast, the presence and potential role of beta 3 on human T cells is practically unknown. We show that T cells can express significant amounts of alpha-beta 3 integrin (CD41/CD61), and the expression of alpha(v)-beta 3 (CD51/CD61) remains very low. T-cell beta 3 integrin is probably transferred by platelet-derived microparticles.     

Treatment of acute cellular rejection with T10B9.1A-31 or OKT3 in renal allograft recipients.

T10B9.1A-31, a nonmitogenic immunoglobulin Mk monoclonal antibody that detects an epitope on the alpha/beta chains of the T cell antigen receptor (TCR alpha/beta), or OKT3, an anti-CD3 mAb, was employed in a randomized double-blind phase II clinical trial to treat biopsy-proven acute cellular renal allograft rejection. Two of the 40 patients initially selected for the protocol were considered to be nonevaluable. Analysis of the remaining 38 patients receiving both living related and cadaveric donor allografts revealed a patient survival of 100% and a graft survival of 97%. Primary rejection reversal was achieved in 18/19 (95%) patients treated with T10B9.1A-31 and in 20/21 (95%) of patients receiving OKT3. The two patients who did not respond to the first mAb responded to the crossover mAb. Rerejection occurred in 3/18 (17%) of patients treated with T10B9.1A-31 and in 3/20 (15%) treated with OKT3. The mean day of rejection reversal was 1.9 +/- 0.7 with T10B9.1A-31 and 3.37 +/- 1.21 with OKT3 treatment. The rise in mean serum creatinine after mAb administration and the mean creatinine on days 1 through 6 were significantly less in patients treated with T10B9.1A-31. Biopsy specimens analyzed for rejection revealed no significant difference between the T10B9.1A-31 and OKT3 cohorts. The mean serum creatinines at 30, 60, 180, and 360 days posttransplantation were the same for both groups. Significantly fewer febrile, respiratory, and untoward effects followed the first dose (day 0) and fewer febrile, gastrointestinal, and neurological side effects occurred with subsequent doses (days 1-9) in patients treated with T10B9.1A-31. Infectious complications occurred in 3/13 patients treated only with T10B9.1A-31, in 9/17 OKT3-treated patients, and in 4/8 patients treated with both mAb. Analysis of human antimouse antibody (HAMA) revealed that the development of HAMA with T10B9.1A-31 was similar to that of OKT3.     

The role of the protocol biopsies in renal allograft recipients

Subclinical rejection and long-term cyclosporine nephrotoxicity are well-known risk factors of chronic allograft nephropathy. In a prospective study 32 low-risk patients were randomized to either a reduced CsA dose (5 mg/kg/d) and daclizumab (group A, n = 16) for 7 months posttransplant with subsequent CsA tapering/withdrawal, or to a normal CsA dose (10 mg/kg/day) without daclizumab (group B, n = 16). Both groups received MMF and prednisone. Protocol biopsies were obtained at engraftment and 3 and 12 months after Tx. The number of rejection episodes was the primary endpoint. The secondary endpoints were: renal function, histological parameters related to CsA, and serum levels of TGF-beta and PDGF-BB. A low incidence of clinically suspected rejection episodes was observed (19% in group A and 12.4% in group B; P = NS). Although protocol biopsies showed 12 subclinical rejection episodes (six in group A, six in group B), serum creatinine levels were not different between the examined groups at 3 months. However, at 12 months, there was a statistically improved mean creatinine level in group A patients (1.2 mg/dL +/- 0.5 in group A vs 1.54 mg/dL in group B; P <.05). Chronic histopathologic changes were significant for biopsies at 3 and 12 months in both groups compared to the baseline findings for protocol biopsies (with no differences between groups, or between 3 and 12 months in both groups). Serum TGF-beta and PDGF-BB did not differ between the groups. Protocol biopsies may be useful to monitor safety and efficiency of new immunosuppressive protocols. Immunosuppressive regimens with low CsA doses followed by the drug's complete withdrawal seem to be efficient and safe in low-risk kidney allograft recipients.     

OKT3 treatment of steroid-resistant renal allograft rejection

The monoclonal antibody, Orthoclone OKT3 (OKT3), has been used with great efficacy in a prospective multicenter trial as therapy for first rejection episodes in cadaveric donor (CD) renal allograft recipients treated with azathioprine (AZA) and prednisone (P). However, although almost all rejection episodes were reversed, recurrent rejection occurred in approximately two-thirds of OKT3-treated patients in this earlier trial; infections also occurred in about two-thirds of patients, often related to the additional immunosuppression necessary to reverse the rerejection episodes. In the current series of patients, OKT3 was used to treat rejection in CD renal graft recipients in a protocol differing from the multicenter trial in two respects: baseline immunosuppression was cyclosporine (CsA) and P or CsA, AZA, and P (probably more potent immunosuppressive combinations than the AZA and P in the multicenter trial); and OKT3 treatment was reserved for rejection episodes resistant to 3 bolus infusions of methylprednisolone (MP), 5-10 mg/kg, rather than as primary therapy for first rejection episodes. Using this protocol, 46 of 74 rejection episodes (62%) diagnosed between 3/85 and 3/86 in CD renal allograft recipients were treated successfully with MP. Of the remaining 28 steroid-resistant rejection episodes, 27 (96%) were reversed with a 7-14-day course of OKT3, 5 mg/day. Only 5 recurrent rejection episodes (19%) have been observed in the 2-14-month follow-up period after OKT3 treatment; infections have occurred in 10 patients (36%), and three grafts (11%) have been lost in OKT3 treated patients. These results suggest that recurrent rejection and subsequent infection after OKT3 is used to treat rejection may be reduced in a protocol where CD renal allograft recipients are treated with baseline immunosuppression regimens including CsA and where OKT3 is reserved for steroid-resistant rejection. This approach appears to be both more cost-effective than, and as effective therapeutically as, treating all first rejection episodes with the monoclonal antibody.     

OKT3治疗移植肾难治性排斥反应

为了解ＯＫＴ３对移植肾难治性排斥反应的治疗效果，１９９３年１月至１９９６年６月，我们对４４例肾移植术后移植肾难治性排斥反应（其中加速性排斥反应７例，急性排斥反应３７例）应用ＯＫＴ３治疗。结果：６例加速性排斥反应及３１例急性排斥反应逆转，总逆转率为８４．６％。２５例对ＯＫＴ３治疗产生迅速反应，平均逆转时间为７±４天，１２例发生延迟性反应，平均逆转时间为３４±３天（Ｐ＜０．０１）。作者认为：ＯＫＴ３治疗难治性排斥反应效果显著。巨细胞病毒感染及细胞因子释放综合征是ＯＫＴ３治疗的主要副作用。患者对ＯＫＴ３治疗的延迟性反应与细胞因子释放综合征及排斥损害有关。产生低滴度抗ＯＫＴ３抗体的患者可再次接受ＯＫＴ３治疗。