Taking the HLA-specific memory B cell elispot to the next level: assaying the full donor HLA repertoire
G.E. Karahan, Y.J.H. de Vaal, J. Krop, D.L. Roelen, F.H.J. Claas, S. Heidt
Chair(s): Dr. Junior M. Lardy
Thursday 9 march 2017
13:00 - 13:15h
Categories: Poster - Basaal
Parallel session: Postersessie XV - Basaal 3
Pre-existing or de-novo donor-specific antibodies (DSA) represent an important risk factor affecting transplant outcome. Patients with a history of immunization, but lacking serum DSA may harbor dormant memory B cells which can rapidly produce DSA upon antigen re-encounter. Current methods to detect memory B cells mainly utilize synthetic monomeric/tetrameric HLA molecules which generally do not represent the complete HLA repertoire of an individual. Here, we present a donor-specific HLA-ELISPOT assay enabling the screening for HLA-specific memory B cells in peripheral blood of immunized individuals using cell lysates as a natural source of both HLA class I and II antigens. Peripheral blood mononuclear cells (PBMC) or splenocytes were treated with non-ionic detergents to obtain HLA-containing lysates. Human B cell hybridomas producing monoclonal HLA antibodies were tested against these lysates for validation purposes. Next, polyclonally activated peripheral blood B cells from women with a history of pregnancy were tested for the presence of HLA-specific memory B cells against paternal PBMC or autologous lysates as the source of HLA (women with serum anti-HLA n=10; women without serum anti-HLA n=10). Non-immunized individuals served as negative controls (n=10). For all hybridomas tested, we detected spot formation against the lysates that contain the corresponding HLA antigens whereas no spots were observed against lysates with irrelevant HLA, indicating the specificity of the assay. Comparable number of spots in total IgG and HLA-ELISPOT assays assured that all antibody-secreting cells were detected. Using this ELISPOT assay, we found significantly higher HLA class I-specific memory B cells (median frequency: 202, range: 0-802) in women with serum HLA class I antibodies compared to those without serum HLA class I antibodies (median frequency:0, range:0-8) and non-immunized males (median: 0, range: 0-25) (p<0.0001). Similarly, HLA class II-specific memory B cell frequencies were significantly higher in group of women with serum HLA class II antibodies (median frequency: 91, range: 22-768) compared to women without serum antibodies (median frequency: 3, range: 0-25) and non-immunized males (median frequency: 0, range: 0-22) (p<0.0001). HLA-specific memory B cell frequencies did not differ between women without serum HLA antibodies and non-immunized males (p>0.05). This novel lysate-based ELISPOT assay allows for the first time to quantify all donor HLA class I and II-specific memory B cells and may serve as a memory B cell crossmatch assay.
