HLA-specific B cells - I. a method for their detection, quantification, and isolation using HLA tetramers

Background. The development of highly sensitive and specific assays for detecting and characterizing HLA-specific antibodies has contributed to an appreciation of the extensive involvement of those antibodies in graft injury and dysfunction. However, understanding the regulatory processes of the humoral response to transplantation and the mechanisms underlying therapeutic agents and protocols for preventing and treating sensitization requires a way to study HLA-specific B cells. Methods. Lymphocyte preparations enriched for B cells were stained with one or more of three different HLA tetramers. Tetramer-positive (tet+) B cells were enumerated and evaluated for an association of their frequencies with known sensitization. In some cases, tet+ B cells were isolated and placed in culture with supplements known to activate B cells in a nonspecific fashion. Results. For all tetramers used, the frequencies of tet+ B cells were significantly higher (4.1%-5.5%) among sensitized patients than among nonsensitized patients (1.6%-3.2%, P < 0.001). Binding of the tetramers occurred by the surface immunoglobulin antigen receptor with little or no binding to antibody captured in the Fc receptor. Cultured tet+ B cells produced antibodies specific for epitopes of the tetramer antigen. There appeared to be a certain amount of crossreactivity in the binding of tetramers. The frequency of CD27+ cells among tet+ B cells was higher, on average (34.4%-38.8%) than among all B cells (26.2%) whereas the frequencies of CD38 were comparable in the two groups. Conclusions. Staining with HLA tetramers provides a means for identifying, quantifying, and isolating HLA-specific B cells.