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#33-OR
MICROHETEROGENEITY WITHIN THE HUMORAL RESPONSE AGAINST HLA-A2 AS DETECTED AMONG THE ANTIBODIES PRODUCED BY SINGLE, TETRAMERIC HLA-a2 ISOLATED B-CELLS.
Arend Mulder, PhD, Chantal Eijsink, MT, Marrie J. Kardol, MT, Marry EI Franke, MT, Michel Kester, MT, Ilias I.N. Doxiadis, PhD and Frans H.J. Claas, PhD. Leiden Netherlands, Leiden University Medical Center, 2300RC, Immunohaematology and Bloodtransfusion.
Characterizing the individual B-cells that participate in the production of anti HLA antibodies (Abs) requires isolation and culture of these cells and a suitable assay for detection of antibodies produced in these B-cell cultures.
By flow sorting of immunomagnetically purified B-cells that had been labeled with HLA-A2/PE tetrameric complexes and by culturing them in the CD40 driven EL4.B5 system, we succesfully established monoclonal B-cell lines, producing HLA-A2 Abs, from an individual with a high HLA-A2 specific B-cell frequency. The tetramer guided flow sort step effected a 170-fold increase in the HLA-A2 specific B-cell frequency, thus generating a considerable enrichment. More importantly, heterogeneity with respect to HLA-A2 epitope reactivity was observed when immunoglobulins (n=62, both IgG and IgM) produced by these monoclonal B-cell cultures were assayed by CDC on a cell panel informative for epitopes shared by HLA-A2,A28,A24,B17 in various combinations. An additional level of heterogeneity within the HLA-A2 response was discovered when the same immunoglobulins were assayed by solid phase on HLA-A2 monomers that were loaded with a random panel of 9 peptides. Here, the Abs demonstrated differential monomer panel reactivity with many patterns.
Given the numbers of B-cells stainable with HLA-A2 tetramers, the study of individual alloreactive B-cells in transplant patients is now within reach, and will allow a delineation of the degree of complexity of the humoral response towards a single HLA class I antigen.