Zoe Hartmann (Hannover/ DE), Eline Gall (Hannover/ DE), Murielle Verboom (Hannover/ DE), Dr. Susann Placzko (Hannover/ DE), Wiebke Hiemisch (Hannover/ DE), Prof. Dr. Rainer Blasczyk (Hannover/ DE), Dr. Christina Bade-Döding (Hannover/ DE)
Background
HLA specific antibodies in peripheral blood leads to the exclusion of donors carrying the corresponding HLAs while the absence of DSAs leads to the acceptance of donors, even in the presence of repeated HLA mismatches. As a secondary immune response might remain unseen the need to provide a robust method for immune monitoring before SOT becomes obvious. The existence of circulating antibodies cannot be translated into reliable information on the memory B cell compartment.
Methods
We generated an artificial secondary immune response by re-expose recipient B cells to repeated HLA mismatches in vitro. Utilizing soluble HLA technology, recombinant HLA class I or II molecules were expressed and affinity purified. From whole blood of recipients polyclonal B cell activation was performed using the TLR7/8 agonist Resiquimod and IL-2 to generate memory B cell-derived plasma cells. A sophisticated ELISPOT assay has been developed using recombinant HLA molecules as targets for secreted IgGs from CD19+/27+/138+/38+ B cells. Autologous recombinant HLA antigens served as negative controls.
Results
This artificial secondary immune response allowed unequivocal detection of memory B cells against a specific HLA antigen. We could detect HLA specific memory B cells in recipients where no DSAs following a documented immunization record have been specified. In other cases, the emerge of a secondary immune response following acceptance of a repeated mismatch could be virtually excluded, when no HLA specific memory B cells could be detected.
Conclusion
The ELISPOT enables the quantitative and qualitative detection of HLA-specific memory B cells and to dissect donor/recipient incompatibilities that would remain unseen with accepted screening methods. In living kidney Tx, this method has been applied to adjust donor selection to prevent humoral immune responses. This detection of a secondary immune response has the potential to be used in routine diagnostics, as it provides robust and incontrovertible data on the individual immunization status.
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