T. Mongkolsuk

Significance of C1q-fixing Donor-Specific Antibodies After Kidney Transplantation

OBJECTIVES De novo donor-specific HLA antibodies (DSA) are associated with allograft rejection and allograft loss. However, not all DSA are equally detrimental to allograft function. The ability to activate complement may be an important factor differentiating clinically relevant DSA from nonrelevant DSA. The C1q assay detects a subset of HLA antibodies that can fix complement. This study aimed to investigate the correlation between C1q-fixing de novo DSA (dnDSA) and clinical outcomes posttransplant. METHODS This retrospective study included 193 sera from kidney transplant recipients who underwent posttransplant DSA testing and/or kidney biopsy for clinical causes. Thirty-five of the 193 (18.1%) had immunoglobulin G DSA. Seventeen of the 35 patients were excluded owing to the presence of pretransplant HLA antibodies. We then analyzed C1q DSA at the time of biopsy in 18 recipients who developed dnDSA. The clinical outcomes of patients with C1q-positive DSA and C1q-negative DSA were compared. RESULTS C1q-positive DSA were detected in 10 of 18 patients (55.6%). The incidences of transplant glomerulopathy were significantly higher among patients with C1q-positive DSA than patients with C1q-negative DSA (80% vs 0%; P = .001). Although patients with C1q-positive DSA experienced more chronic antibody-mediated rejection and graft loss (80% vs 37.5% [P = .145]; 60% vs 25% [P = .188]), the differences were not significant. The receiver operating characteristic curve analysis showed that the C1q assay was an excellent predictor of transplant glomerulopathy with area under the curve of 0.9 (95% CI, 0.769-1.000). CONCLUSION The presence of C1q-positive dnDSA was associated with an increased risk of transplant glomerulopathy. The C1q assay is potentially a powerful method for identifying patients at risk for transplant glomerulopathy.

Cytotoxic Flow Cytometric Crossmatch in Renal Transplantation: A Single Assay to Simultaneously Detect Antibody Binding and Cytotoxicity

OBJECTIVES The complement-dependent lymphocytotoxicity crossmatch (CDC-XM) detects cytotoxic parameters of preformed antibodies. The flow cytometric crossmatch (FCXM) is used to detect the binding of recipient antibodies to donor cells. Because these two assays provide different information, both methods are often performed to assess the compatibility of donor-recipient pairs. The aim of this study was to develop a single assay that can simultaneously detect antibody binding and cytotoxicity. METHODS A procedure called cytotoxic flow cytometric crossmatch (cFCXM) that determines cell death and antibody binding simultaneously was developed. The assay was validated in parallel with extended incubation CDC-XM. Receiver operating characteristic analysis was used to determine the cut-off level. Furthermore, pretransplantation sera from seven recipients with pretransplantation donor-specific antibodies (DSA) and negative CDC-XM were retrospectively tested for cFCXM (4 without antibody-mediated rejection (AMR) and three with AMR). RESULTS The optimal method for the simultaneous detection of antibody binding and cytotoxicity in a single assay has been determined. Four of four patients (100%) with pretransplantation DSA and without AMR had negative cFCXM in both parameters. Of three patients with pretransplantation DSA who developed AMR, two patients (66.7%) had positive B-cell cFCXM in both parameters, and 1 patient (33.3%) had positive T-cell cFCXM in a binding parameter only. The first patient had anti-DR9, DR53, DQ9, the second patient had anti-A11, DR12 and the last one had an anti-B46 in their pretransplantation sera. These 3 cases experienced biopsy-proven AMR after living-donor kidney transplantation. CONCLUSION The newly developed assay, cFCXM, can simultaneously determine cytoxicity and antibody binding using a single platform. Furthermore, this assay can detect clinically significant HLA alloantibodies undetectable by conventional crossmatches. The cFCXM could serve as a new tool for the detection of a recipients alloantibodies.

Shared Molecular Eplet Stimulates Acute Antibody-Mediated Rejection in a Kidney Transplant Recipient With Low-Level Donor-Specific Antibodies: A Case Report

HLA antibodies usually recognize epitopes rather than antigens. This case report reveals that acute antibody-mediated rejection (AMR) that occurred in a kidney transplant recipient with low-level donor-specific antibodies (DSAs) could be explained by shared epitope. A 39-year-old woman received a first kidney transplant from a deceased donor (HLA-DRB1 11:06, 12:02, DRB3 02:02, 03:01). She developed acute AMR confirmed by kidney biopsy on day 4 after transplantation. Antibody testing with pretransplant serum showed anti-DR11 DSA below cutoff level (mean fluorescence intensity [MFI], 702; cutoff >1,000). However, high-level DSAs were detected on day 5 after transplantation (anti-DR11 MFI, 8,531; anti-DR12 MFI, 3,146). We hypothesized that the sharp rise in DSA levels was a result of anamnestic response with donor-antigen sensitization that occurred during pregnancy. High-resolution HLA-DR typing of her husband showed HLA-DRB1 03:01, 15:02:01, DRB3 02:02, DRB5 01:02. No sharing between donor HLAs eliciting reactive antibodies and her husbands HLAs was detected. Nevertheless, we speculated that shared epitope, not antigen, was the cause of allosensitization. To identify the shared epitope recognized by patients antibodies, we used HLAmatchmaker, a computer algorithm that considers small configurations of polymorphic residues referred to as eplets as essential components of HLA epitopes for analysis. The results showed that 149H, which was the eplet shared by HLA-DRB1 03:01 (from her husband) and DRB1 11:06, DRB1 12:02, DRB3 03:01 (from donor), was the most prevalent eplet on DRB1 reactive alleles in Luminex assay. In conclusion, pretransplant low-level DSAs can induce AMR early after transplantation as a result of shared epitopes with a previous immunizer.

Genetic profiles of killer-cell immunoglobulin-like receptors and HLA ligands in Thai blood donors.

Killer-cell immunoglobulin-like receptors (KIRs) play an important role in natural killer (NK) cell regulation. Interaction of KIRs with human leukocyte antigen (HLA) class I molecules can transmit signals to regulate the function of NK cells. In this study, the diversities of KIR genes and their ligands in 500 Thai blood donors were investigated. The coexistence of inhibitory KIRs (iKIR), activating KIRs (aKIR) and their ligands in the same individuals were also analyzed. Overall, 36 KIR genotypes were identified. The most common genotype was genotype AA1 (40.8%). All individuals carried at least one iKIR-HLA pair whereas 18% of the individuals lacked aKIR-HLA pair. The most common compound KIR-HLA profile was the presence of 3 iKIR-HLA pairs with 1 aKIR-HLA pair (21.4%). The most common compound gene profile of KIR-HLA pairs was the combined presence of KIR2DL3-C1, 3DL1-Bw4, 3DL2-A3/A11 and the full length KIR2DS4-its ligands (8%). This study provided a comprehensive analysis of the KIR-HLA profiles in Thai blood donors in regards to KIR genotypes, HLA ligands, KIR-HLA ligand pairs and compound gene profiles of both iKIRs and aKIRs and their ligands. These findings will be useful as baseline information for further studies in the associations of KIR genes and various diseases.

Association of soluble human leukocyte antigen-G with acute tubular necrosis in kidney transplant recipients.

BACKGROUND Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule that displays strong immune-inhibitory properties and has been associated with allograft acceptance. However, there are conflicting data on the correlation of soluble HLA-G (sHLA-G) and acute rejection and no data on the correlation with acute tubular necrosis in kidney transplantation. OBJECTIVE To evaluate the association of sHLA-G level in early post-transplant period and allograft rejection/ and acute tubular necrosis (ATN) in kidney transplant recipients. METHODS The sera procured before transplantation and serially on day 3 and day 7 after transplantation from 76 kidney transplant recipients were analyzed for the level of sHLA-G by enzyme-linked immunosorbent assay. RESULTS The levels of sHLA-G from three serial sera did not differ between patients with acute rejection and patients without rejection. However, the sHLA-G levels on day 3 post-transplant and day 7 post-transplant in patients with ATN were significantly higher than that in patients without ATN (16.3 vs 9.85 U/ml, p = 0.018, for day 3 post-transplant and 12.47 vs 5.42 U/ml, p = 0.044, for day 7 post-transplant). In addition, the ROC analysis of sHLA-G for identifying patients with ATN showed that the area under curve was 0.67 (95% confidence interval 0.54-0.80). CONCLUSIONS There was no significant difference for sHLA-G levels between patients with acute rejection and without rejection. Interestingly, high levels of sHLA-G in day 3 and day 7 after transplantation were associated with acute tubular necrosis. Our findings raise the question whether the increased levels of sHLA-G in patients with acute tubular necrosis after transplantation might be a result of ischemia and reperfusion injury.

Acute Antibody-Mediated Rejection by De Novo Anti-HLA-DP beta and -DP alpha Antibodies after Kidney Transplantation: A Case Report

The presence of isolated de novo anti-DP antibodies is uncommon, making it difficult to determine the impact of anti-DP antibodies on graft outcome. We describe a case of acute antibody-mediated rejection mediated by de novo donor-specific anti-HLA-DP antibodies. Furthermore, the generation of non-donor-specific anti-DP antibodies (NDSAs) detected in the patients sera was investigated. An 18-year-old woman with pretransplant 0% panel-reactive antibody received kidney transplantation from a living donor. She experienced combined acute T-cell-mediated and antibody-mediated rejection at 15 months after transplantation. High resolution HLA typing of the donor and the patient revealed that they were mismatched at both DPB1 (DPB1*31:01) and DPA1 (DPA1*02:02) loci. The single antigen bead (SAB) testing of patients sera revealed antibodies against donors DPB1*31:01 and DPA1*02:02 alleles. Antibodies against several non-donor-specific DP antigens were also detected. No antibodies against other HLA class I and II antigens were detected. In order to explain the reactivity pattern of NDSAs, HLAMatchmaker program was used to identify immunizing eplets shared between donor alleles and reactive beads. The analysis showed 84DEAV, a DPB1 eplet, as a shared eplet found on DPB1*31:01 (mismatched donor allele) and on DPB1-reactive alleles in SAB assay. Additionally, 50RA, a DPA1 eplet, was identified as a shared eplet found on DPA1*02:02 (mismatched donor allele) and on DPA1-reactive alleles in SAB assay. This case highlights the clinical significance of HLA-DP antibodies. Furthermore, the generation of NDSA anti-DP antibodies by epitope sharing underscores the importance of HLA-DP epitope matching in kidney transplantation.