Bootcongres

Highly sensitive and specific solid phase assays enable the clear definition of anti-HLA antibody specificities in sera from transplant recipients. However, this provides limited information if the antibodies are very low titer or are absorbed by the graft. Furthermore, none of the currently available methods provide information on the HLA-specific memory B cell compartment. Our group has recently developed an ELISPOT assay that allows for quantification of HLA class I specific memory B cells using recombinant monomeric HLA class I molecules as capture matrix. However, since the predominance of post-transplant donor specific antibody (DSA) is formed against mismatched HLA class II antigens, we aimed at establishing an ELISPOT assay for the detection of HLA class II specific B memory B cells. Availability of the anti-DR11 antibody-producing human B cell hybridoma RTLK10E12 enabled us to perform proof of principle experiments for the detection of HLA class II specific memory B cells. We coated wells of ELISPOT plates with anti-IgG monoclonal antibodies and used purified biotinylated HLA class II molecules as the detection matrix. We tested the specificity of the ELISPOT system using HLA-DRB1*11:01 and HLA-DRB1*13:03 molecules to detect HLA-DR11 specific hybridoma cells. Spot formation was observed when HLA-DRB1*11:01 was used as the detection matrix whereas DRB1*13:03 did not lead to any spot formation. In order to assure that HLA-class II specific ELISPOT assay was detecting all hybridoma cells producing anti-HLA-DR11, we compared the number of total IgG spot forming units (SFU) to the number of SFU detected by the HLA-class II specific ELISPOT assay within the same experiments. Comparable numbers of SFU in total IgG ELISPOT (mean ± SD: 82 ± 5) versus the number of SFU (mean ± SD: 84± 9) in HLA class II specific ELISPOT using DRB1*11:01 molecule were observed showing that the ELISPOT was detecting all hybridoma cells.

The current data clearly shows that it is possible to detect cells that produce anti-HLA class II in an ELISPOT system. We are currently extending our observations to HLA class II specific memory B cells in HLA-sensitized kidney transplant recipients. The development of an HLA class II specific ELISPOT assay will be of value for risk assessment in patients with HLA class II DSA.