A Novel Multiplex Approach to Define Donor Specific and Poly-Reactive B-Cell Receptor HLA Antigen Recognition in Clinical Transplantation.

Abstract

Study Purpose The role of anti-donor HLA specific B cells which are crucial effectors for generation of donor specific antibodies associated with chronic graft rejection is still unknown. Quantifying the frequency of HLA Ag-specific B cells is of great importance for better understanding of the mechanisms of graft rejection, earlier diagnosis of rejection and to guide specific B cell-targeted therapy. Aim: To quantify and characterize HLA antigen (Ag)-committed B cells identified by single antigen HLA-coated multiplexed beads (SAB) post-transplantation (PT). Methods PBMC from kidney transplant recipients (TR n=6) and healthy volunteers (HV n=6) were analyzed using SAB. Bead-B-lymphocyte Rosette (BBR) frequency was enumerated and their HLA specificities were characterized by flow cytometry. TR serum was tested for circulating anti-HLA antibodies using SAB. Donor specific rosette (DSR) polyreactivity, defined by the percentage of total SAB forming rosettes (Fig 1), were compared with presence or not of DSA of same specificities and their MFI. Results Significantly higher frequency of BBRs were identified in TR with circulating anti-HLA Abs compared to HV ((2.49±0.74 vs 0.27±0.09; p<0.02). Both DSR and Non-DSR were present in higher frequency (1.15 % vs 0.17% of B cells; P<0.05) and correlated with poor graft outcome. Not all DSR were associated with DSA at 12 months PT. Patients with poor graft outcome had wide breadth of B cell polyreactivity compared to recipients with good graft outcome (HLA class I 76.97± 28.3 vs 13.1 ± 3.15; p<0.05. HLA class II 91.9 ± 10.5 vs 13.69 + 10.3; p<0.05). Conclusions This new approach using multiplex beads highlights for the first time a pattern of HLA Ag-specific B cell polyreactivity. The breadth of HLA reactivity may be a determinant of an aggressive clinical course after transplantation. The wide array of available HLA Ags in a single test will help in defining B cell receptor poly & cross-reactivity. In addition it enables phenotyping, functional and gene expression analysis of HLA specific B cells.Figure: No Caption available.