A Novel ELISPOT Assay to Quantify HLA-Specific B Cells in HLA-Immunized Individuals

Abstract

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. Enriched peripheral B cells were pre-activated in a CD40L-driven fashion before being assayed by the HLA-specific B cell ELISPOT assay. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B cell hybridomas producing HLA antibodies of defined specificity. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non HLA-immunized individuals and transplant patients with serum HLA antibodies. B cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while non-immunized individuals had none (Figure 1). Specificities that can currently be tested in the HLA-specific ELISPOT include HLA-A1, HLA-A2, HLA-B7 and HLA-B44. The assay is highly reproducible, as determined by repeat testing of B cells from a single bleeding date.[Figure 1]: Figure 1. HLA-specific B cells are detected in HLA-immunized individuals, but not in non-immunized individuals. (A) Raw ELISPOT data of 2 HLA-A2 immunized and 2 non-immunized individuals. Frequency is the number of spots per million B cells. Enriched B cells were activated for 7 days and seeded into ELISPOT plates coated with monomeric HLA molecules (B). Frequencies of HLA-A2 specific antibody producing B cells of HLA-A2 immunized and non-immunized healthy individuals.Transplant recipients who were immunized by an HLA-mismatched graft had up to 143 HLA-specific B cells per million total B cells. Moreover, preliminary data suggest that immunosuppressive drugs do not interfere with the detection of HLA-specific B cells. In conclusion, we have developed and validated a highly specific, reproducible and sensitive HLA-specific B cell ELISPOT assay, can be used to determine the frequency of peripheral HLA-specific B cells in transplant recipients. This technique constitutes a new tool for quantifying humoral immune responses.