Background. HLA-specific memory B cells may contribute to the serum HLA antibody pool upon antigen reexposure. The aim of this pilot study was to investigate the presence of concurrent donor-specific memory B cell–derived HLA antibodies (DSA-M) in renal allograft recipients with pretransplant donor-specific HLA antibodies (DSA) and its association with occurrence of antibody-mediated rejection (AMR) using a recently developed method. Methods. Twenty patients with Luminex single antigen bead (SAB) assay-defined DSA but negative complement-dependent cytotoxicity crossmatches were enrolled. Plasma samples and peripheral blood mononuclear cells were collected at 3 timepoints (pretransplant, mo 6, mo 12). We analyzed IgG-purified and concentrated culture supernatants from polyclonally activated peripheral blood mononuclear cells using SAB assays and compared HLA antibody profiles with same day plasma results. Results. Plasma SAB analysis revealed 35 DSA in 20 patients pretransplant. DSA-M were detected in 9 of 20 (45%) patients and for 10 of 35 specificities (29%). While median mean fluorescence intensity values of DSA with concurrent DSA-M (5877) were higher than those of DSA without DSA-M (1476), 3 of 6 patients with AMR and low mean fluorescence intensity DSA (<3000) had DSA-M. Overall, pretransplant DSA/DSA-Mpos allograft recipients showed a higher incidence of biopsy-proven (sub)clinical AMR (P = 0.032) and a higher extent (g≥1 + ptc≥1) of microvascular inflammation (67% vs 9%, P = 0.02). In 17 patients (28 DSA) with posttransplant analyses, persisting DSA posttransplant had more often DSA-M (6/12; 50%) than nonpersisting DSA (2/16; 13%). Conclusions. Assessment of DSA-M might be a novel tool to supplement serum HLA antibody analysis for pretransplant risk stratification in patients with DSA.
Detection of HLA‐specific memory B cells can provide additional information on sensitization of alloantigen‐exposed individuals and refine immunological risk assessment. We have recently developed an assay enabling profiling of memory B cell‐derived HLA antibodies using luminex single antigen bead (SAB) assay. Here, we compared the performance of the SAB kits from two vendors for memory B cell‐derived HLA antibody detection. IgG was isolated from culture supernatants of polyclonally activated B cells from alloantigen‐exposed (n = 7) or nonexposed (n = 5) individuals, using our previously established method. Eluates containing isolated IgG from culture supernatants were tested for the presence of HLA antibodies using luminex SAB analysis from both One Lambda and Lifecodes (Immucor). In contrast to Lifecodes, high mean fluorescence intensity (MFI) signals were found for negative control beads in One Lambda (median MFI for class I:1730 and for class II:728), accompanied by high MFI values for self HLA‐coated beads, especially for HLA‐C. Despite high background in the One Lambda assays, 91% concordance for HLA class I and 85% concordance for HLA class II were found between the specificities detected using SAB kits from the two vendors. Our results show that HLA‐specific memory B cells can be profiled using kits from both vendors. However, when analyzing One Lambda results one should be aware of the restrictions related to nonspecific binding particularly in HLA‐C‐coated beads, and pay attention to self HLA‐coated beads in order to accurately identify the reactivities leading to the definition of the actual HLA antibody specificities.
For hematopoietic stem cell transplantation (HCT) HLA 10/10 (HLA-A, B, C, DRB1, DQB1) matched donors are optimal, but are not available for all patients. The identification of permissive/non-immunogenic mismatches may improve the outcome of HLA mismatched transplants. We hypothesize that HLA alleles identical within the antigen recognition domain (ARD), but mismatched outside the peptide binding groove or α-helices are often permissive mismatches. We evaluated the functional impact of non-ARD mismatches by performing in vitro functional T cell assays. Cytotoxic T Lymphocyte precursor assays were performed for 23 HLA class I mismatches and 96% (22 out of 23) were negative. Mixed lymphocyte reaction assays were conducted on 10 HLA class II mismatches and all were negative. However, 4 out of 10 combinations were positive in the Elispot and all involved one direction: a DRB1*14:01/DRB3*02:01 responder against a DRB1*14:54/DRB3*02:02 stimulator. These positive responses were confirmed by Primed Lymphocyte Testing and the DRB1* mismatch seemed to be responsible for the response. In conclusion, HLA mismatches with amino-acid differences outside the ARD are not very immunogenic. However, in some cases weak T cell reactivity in vitro can be observed. The impact of these responses on clinical outcome of HCT remains to be established.
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