Detection of plasma cells, C4d deposits and donor-specific antibodies on sequential graft biopsies of renal transplant recipients with chronic dysfunction.

BACKGROUND: In order to look for a relationship between humoral mechanisms of rejection and chronic allograft dysfunction, plasma cells, C4d deposits and donor-specific antibodies (DSA) were simultaneously sought on serial biopsies of kidney allograft recipients. PATIENTS AND METHODS: Ten recipients with chronic dysfunction (G1) and 8 recipients with long-term normal graft function (G2) were included. Biopsies and serums were sampled at early graft dysfunction (T1), between 8months and 2years (T2) and after the third year following transplantation (T3). RESULTS: In G1, plasma cells represented 12.3% (T1), 8.2% (T2) and 14.1% (T3) of mononuclear cells. The mean percentage of plasma cells was 11.6% in G1 versus 0.4% in G2 (p<0.05). A progressive rise in C4d deposits was seen in G1, from 25% at T1 to 80% at T3. Donor-specific antibodies were identified in at least one serum sample of 60% of the patients in G1 and 12.5% of the patients in G2 (p=0.012), whereas donor-specific antibodies were eluted from at least one biopsy of 50% of the patients in G1 and 12.5% of the patients in G2 (p=0.03). In G1, C4d deposits were significantly associated with plasma cells (p=0.0012) and anti-HLA Abs in serum samples and/or eluates (p=0.026). CONCLUSION: This study shows that plasma cells, DSA and C4d are associated in renal transplants developing chronic rejection.

Distribution of donor-specific antibodies in the cortex and the medulla of renal transplants with chronic allograft nephropathy.

BACKGROUND: Emerging data suggest that donor-specific HLA antibodies should be more frequently found onto the transplant itself than in the bloodstream. It is now possible to detect such antibodies in kidney transplant needle biopsy samples by flow cytometry. In order to know if the detection of antibodies into such blind biopsy samples depends of the site of the biopsy, we have studied the distribution of antibodies in both the cortex and medulla of 12 transplants removed after graft loss due to chronic allograft nephropathy, and in 10 controls. METHODS: Donor-specific HLA antibodies were extracted from the cortex and the medulla of each removed transplant by an acid elution and characterized by Luminex assays. RESULTS: They were found in 58.3% of transplants with chronic allograft nephropathy and never in other kidney samples. The same antibodies were found in the bloodstream at the time of transplantectomy in only 16.6% of the recipients. The distribution between the cortex and medulla was concordant in 75% of cases. However, we observed 2 discrepancies: one in favor of the cortex and one in favor of the medulla. A majority (5/7) transplants with CAN and intra-graft donor-specific antibodies had also C4d deposits along peritubular capillaries. CONCLUSION: Testing for donor-specific HLA antibodies in kidney transplant needle biopsies can be of value provided the biopsy includes both the cortex and the medulla.

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.

Donor-specific antibodies in allograft rejection: clinical and experimental data.

The ability of donor-specific major histocompatibility complex alloantibodies to destroy a transplanted organ within minutes, the so-called hyperacute rejection phenomenon, has been known for a long time. It is a clear demonstration of the possible cytotoxic effect of antibodies. Apart from this particular situation, the role of antibodies in inducing acute or chronic allograft rejection remains controversial. Many clinical data have shown that transplant recipients capable of developing class I or class II anti-HLA antibodies experienced shorter survival periods than those who were not. This fact, in accordance with experimental data, only demonstrates that high antibody responders reject a transplant more easily than low responders. More interestingly, there is now increasing evidence that posttransplant appearance of donor-specific alloantibodies, and probably of alloreactive-induced autoantibodies, is strongly correlated with reduced graft survival rate, especially from chronic rejection. We demonstrated that donor-specific HLA antibodies can be found in more than 70% of transplanted kidneys with chronic allograft nephropathy, and that the intragraft presence of such antibodies is significantly correlated with high numbers of plasma cells on early biopsies and C4d deposits, a recognized marker of humoral rejection. It is likely that non-HLA antibodies also play a deleterious role in organ transplant outcome, particularly the heterogeneous group of anti-endothelial cells antibodies, anti-MIC antibodies, autoantibodies and some others with no recognized target. Convincing experimental data, especially using B cell and T cell deficient mice, strongly suggest that B cells and donor-specific antibodies are required for fully developed chronic allograft rejection. The role of antibodies in inducing the cascade of cytokines and growth factors leading to tissue lesions is of increasing interest since it is now possible to control B cell proliferation and antibody production.

Detection of donor-specific anti-HLA antibodies with flow cytometry in eluates and sera from renal transplant recipients with chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN), which remains the main cause of graft loss after kidney transplantation, is still poorly understood. Because anti-HLA antibodies may be involved in the pathogenesis of CAN, this study was performed to look for donor-specific antibodies (DSA) fixed onto renal transplants with CAN. METHODS: DSA were identified after elution with flow cytometric assay and/or flow cytometric crossmatches in 20 transplants removed after irreversible graft failure caused by CAN and in control samples from 2 transplants with relapsing glomerulopathy, 2 transplants lost after vascular thrombosis, and 4 normal kidneys. The results were compared with those obtained in the serum samples 1 year after grafting, at the time of transplantectomy, and within 2 months after transplantectomy. RESULTS: IgG anti-class I, anti-class II, or both DSA were identified in 70.6% of eluates versus 73.6% of posttransplantectomy serum samples (NS), 42.1% of 1-year postgrafting serum samples (P<0.05), and 31.6% of serum samples at the time of transplantectomy (P<0.05). Our data show a good correlation between the target of anti-HLA antibodies found in both eluates and posttransplantectomy serum samples, but the precise specificity of anti-HLA antibodies is more often assigned in posttransplantectomy serum samples than in eluates. This problem needs further evaluation. CONCLUSION: This study shows that testing for anti-HLA DSA in eluates from removed kidney transplants using flow cytometry can be achieved and is highly efficient. It already suggests that both anti-class I and anti-class II HLA antibodies can be involved in CAN. Further studies are now needed to evaluate the possibility of identifying such antibodies in the eluates of transplant biopsy specimens from recipients experiencing CAN.