Elvira Poggi

Impact Of Donor-specific Antibodies On Chronic Rejection Occurrence And Graft Loss In Renal Transplantation: Posttransplant Analysis Using Flow Cytometric Techniques1

Background. Improvements in immunosuppressive therapy have greatly reduced acute rejection (ARj) episodes, ensuring better short-term graft outcome, but have not modified long-term survival in renal transplantation. It is now well accepted that chronic rejection (CRj) can be determined by both immune and/or nonimmune mechanisms. The aim of this study was to evaluate the importance of the posttransplant humoral immune response towards mismatched HLA graft antigens in CRj occurrence and graft outcome. Methods. Serum samples from 120 nonpresensitized renal transplant recipients were prospectively screened for 1 year after surgery by means of flow cytometry cross-match (FCXM) and FlowPRA beads (microbeads coated with purified HLA class I and class II antigens) assays. All transplants were followed-up for 2 years or until graft removal. Results. FCXM monitoring identified donor-specific antibodies (DS-Abs) in 29 (24.2%) of 120 transplanted patients. Correlation with clinical data highlighted a higher incidence of ARj in DS-Abs–positive patients compared to negative patients (62% vs. 13%, P <0.00001). Furthermore, graft failure occurred more frequently among FCXM-positive patients than among negative patients (34% vs. 1%, P <0.00001). The deleterious effect of DS-Abs on graft function was confirmed by serum creatinine levels 2 years after transplantation. These were in fact higher in subjects producing DS-Abs than in subjects with only ARj (mean creatinine: 2.5±1.3 mg/dL vs.1.7±0.5 mg/dL, P =0.04). FlowPRA analysis of DS-Ab HLA specificity highlighted the presence of anti-HLA class I antibodies in 85% of FCXM-positive patients, who also presented with a higher incidence of HLA-B mismatches than FCXM-negative patients (1.23±0.66 vs. 0.92±0.59, P =0.02). Conclusions. Flow cytometric techniques are precious tools for investigating the activation of the humoral response against HLA antigens of the graft in renal transplantation. DS-Abs production has a worse impact on organ function and survival than ARj episodes. These findings represent further proof of the threat posed by DS-Abs on long-term graft function and draw attention to the need for a specific immunosuppressive therapy aimed at counteracting the different kinds of immune activation toward graft.

Public epitope specificity of HLA class I antibodies induced by a failed kidney transplant: alloantibody characterization by flow cytometric techniques.

Background. Patients whose kidney grafts fail develop alloantibodies that react with many HLA molecules. We analyzed the epitope specificity of HLA class I alloantibodies in the sera of 55 patients who had been sensitized by kidney grafts, and investigated the immunogenicity of various polymorphic epitopes. Methods. HLA class I alloantibodies were detected and characterized by flow cytometry (FlowPRA beads). Potential “immunizing epitopes” were identified by comparing the amino acid sequences of HLA class I antigens/alleles of the donor, recipient and the antibody-reactivity pattern. Results. In the 55 anti-HLA class I-positive patients, 82 different antibody reactivity patterns were identified; all but 5 (94%) were determined by a “public epitope” of donor HLA-A and/or -B molecules. Forty-five of 50 patients who showed HLA-A Res-MMs with their donors produced HLA-A antibodies, but only 31 of 51 subjects with HLA-B Res-MMs produced HLA-B antibodies (P=0.001; O.R.=5.81). The antibody patterns were specific for a “single” epitope of the mismatched donor molecules in 91% of patients. Forty-three of the 120 (36%) mismatched HLA-A and/or -B epitopes were positively correlated with antibody production. The polymorphic determinants of higher immunogenic capacity were b80N (Bw6-associated) and ab82–83LR (Bw4-associated) public epitopes. Conclusions. The humoral immune response against a kidney graft mainly produces HLA class I antibodies specific for “public epitopes” of mismatched donor molecules. A “single” donor-epitope may determine the production of a spread antibody pattern. In renal transplantation, epitope matching is better than HLA antigen matching for avoiding or minimizing development of HLA antibodies.

Posttransplant donor-specific antibody characterization and kidney graft survival.

Abstract This study was designed to investigate the clinical relevance of donor‐specific antibodies (DS‐Abs) and their influence on graft survival. Among 106 patients who underwent cadaveric kidney donor transplantation and were monitored by flow cytometry crossmatch (FCXM) during the 1st posttransplantation year, 25 (23.6%) resulted positive for DS‐Ab production. During a 2‐year follow up only 12 of the 81 FCXM‐negative patients (14.8%) suffered rejection vs 17 of 25 FCXM‐positive patients (68%; P = 0.00001). Correlating graft loss to DS‐Ab production, 9 FCXM‐positive patients lost the graft vs only 1 among the FCXM‐negative patients. A worse graft function was evidenced in FCXM‐positive subjects who had also suffered rejection episodes than in those which had acute rejection but did not produce DS‐Abs. A high incidence of HLA‐AB mismatches was found in FCXM‐positive subjects which produced anti‐class I antibodies. FCXM appears useful in estimating post‐transplant alloimmune response. Moreover our findings confirm the harmful effects of anti‐class IDS‐Abs on long‐term graft survival.

B-sides serologic markers of immunogenicity in kidney transplanted patients: report from 2012-2013 flu vaccination experience.

Background Safety and immunogenicity data of seasonal influenza vaccination in transplanted patients (Tps) are controversial. Preexisting cross-reactive antibodies generated by repeated vaccination with drift variant strains could bias interpretation of immunogenicity data in Tp. Methods The unadjuvanted 2012–2013 seasonal influenza vaccine was administered to 81 kidney Tps being routinely vaccinated against influenza and 23 healthy controls (HCs). Immunogenicity was evaluated by both strain-specific antibody responses with standard hemagglutination inhibition assay and by memory B-cell enzyme-linked immunosorbent spot. Safety was also evaluated by measuring anti–human leukocyte antigen (HLA) antibodies. Results The majority of Tps were seroprotected before vaccination (81.5%, 81.5%, and 43.2% vs. 47.8%, 34.8%, and 30.4% in HC for H1N1, H3N2, and B strain, respectively) resulting into lower seroconversion rates (P⩽0.01) as compared with HC (40.7%, 39.5%, and 54.3% vs. 73.9%, 82.6%, and 65.2% for H1N1, H3N2, and B strain, respectively). An inverse correlation was found between seroconversion rates and number of previous vaccinations in Tps. On the contrary, similar increase in the frequencies of strain-specific memory B cells were detected by B-cell enzyme-linked immunosorbent spot in both Tps and HCs after vaccination. No serious adverse events have been reported. Donor-specific HLA antibodies increased in two patients after vaccination, and de novo anti-HLA antibodies were identified in two additional patients (non–donor-specific HLA antibodies). Conclusion This report on safety and immunogenicity of the seasonal unadjuvanted 2012–2013 flu vaccination suggests that evaluating immunogenicity of influenza vaccination exclusively by hemagglutination inhibition assay may be misleading in individuals receiving yearly seasonal vaccines. Further investigations are required to understand the relation between vaccination and anti-HLA antibody development.

Acute kidney transplant rejection mediated by angiotensin II type 1 receptor antibodies in a pediatric hyperimmune patient

BackgroundSeveral cases of severe antibody-mediated rejection (AMR) secondary to antibodies against the angiotensin II type 1 receptor (AT1R-Ab) have been described with variable outcome.Case-Diagnosis/TreatmentWe report the case of a 13-year-old boy whose first kidney transplant failed due to steroid-resistant acute cellular rejection, with the subsequent development of sensitization. He received a second kidney transplant which was complicated by early humoral rejection, with weakly positive staining for the complement degradation product C4d. Test results were negative for donor-specific antibodies against human leukocyte antigens (HLA-DSA) and MHC class I-related chain A (MICA) but positive for AT1R-Ab. Retrospective testing of the sera collected during the first kidney transplant was also positive for AT1R-Ab. We therefore hypothesized that the failure of the first transplant was secondary to the same cause. Losartan was immediately introduced into the therapeutic regimen, and the patient showed an excellent clinical and histological recovery.ConclusionsTesting for AT1R-Ab in any hypertensive patient with acute rejection who tests negative or weakly positive for C4d and negative for HLA-DSA and who is refractory to therapy is highly advisable. Pre-transplant AT1R-Ab may be indicative of the outcome in patients whose first transplant failed. Prompt initiation of treatment with losartan—immediately after transplantation in patients with pre-existing AT1R-Ab—should be encouraged.

Kidney transplant monitoring by anti donor specific antibodies.

Donor-specific anti-HLA antibodies were studied by cytotoxicity crossmatching (CTXM) and flow cytometry crossmatching (FCXM) in 117 kidney transplant candidates; the same study was carried out in 33 cadaver-donor kidney recipients, during the first 3 post-transplant months, for which donor cells were available. Pre-transport evaluation showed that 82.9% of subjects were CTXM negative/FCXM negative, 6.8% of patients were positive in both tests, and 10.3% were CTXM negative/FCCM positive. Post-transplant monitoring for donor-specific antibodies (Abs-DS) showed that nine recipients (27.3%) were FCXM positive; six of them were IgG+ and three IgM+. In comparing these results with the clinical course, a significant association between FCXM IgG+ and rejection episodes was observed (P < 0.01).