OR07: A NOVEL MULTIPLEX APPROACH TO DEFINE PERIPHERAL BLOOD HLA-SPECIFIC B-CELL SUBSETS IN CLINICAL TRANSPLANTATION

Abstract

T o quantify and characterize HLA-specific B cells subsets identified by single antigen HLA-coated multiplexed beads (SAB) in kidney transplant recipients. PBMC from kidney transplant recipients ( n = 8) and healthy volunteers ( n = 10) were incubated with SAB (One lambda) for HLA class I and II. HLA Bead-B-cell Rosette (BBR) frequency & specificity were analyzed by flow cytometry. HLA-specific B cells specificities and polyreactivity was defined by the percentage of total SAB forming BBR. Transplant recipients serum was tested for circulating HLA antibodies using SAB & MFI were estimated. Significantly higher frequency of HLA-specific B cells were identified in transplant recipients with circulating anti-HLA antibodies compared to healthy volunteers. Circulating HLA-specific B cells (donor specific and non-specific) were in higher frequency (1.15% of B cells vs 0.17% of B cells; p < 0.05), wide breadth of polyreactivity (HLA class I 76.97 ± 28.3; HLA class II 91.9 ± 10.5) vs (HLA class I 13.1 ± 3.15; HLA class II 13.69 ± 10.3); p < 0.05 among recipients with poor graft outcome compared with those with good graft outcome. Not all HLA-specific B cells were associated with IgG HLA antibodies detected above the cutoff values. The frequency & breadth of HLA-specific B cells reactivity may be a determinant of an aggressive clinical course after transplantation. This novel approach has the advantage of the wide array of available HLA antigens in a single test which will help in defining HLA B cell receptor poly & cross-reactivity. In addition to enable phenotyping, functional and gene expression analysis of HLA specific B cells.