Figure 3.

CD4+ T cell help is mandatory for memory donor-specific antibody (DSA) response. (A) Allogeneic heart from HLA A2 transgenic (A2) or BALB/c donor was transplanted to C57BL/6 recipient. Fifty days posttransplantation HLA A2-specific memory B cells were enumerated in the spleen of recipients by ELISpot. Representative wells are shown and the number of A2-specific memory B cells (mean ± SD) is plotted for the two groups (n = 4). (B) Graphical representation of the experimental setting used to evaluate the importance of CD4+ T cell help in memory DSA response. (C) Flow cytometry was used to quantify the proportion (mean ± SD) of B cells (B220+CD19+) and helper T cells (CD3+CD4+) in controls (C57BL/6; left column) and RAG2 KO recipients before memory B cell transfer (day 0, second column from the left), just prior heart transplantation (day 7, third column from the left), and 42 days after A2 heart transplantation (right column). (D) The purity of B cell suspension, obtained from the spleen of C57BL/6 mice sensitized with a A2 or a BALB/c heart transplantation, was evaluated by flow cytometry: before magnetic isolation (left panel), after magnetic isolation (middle panel) and after flow sorting (just prior transfer to RAG2KO mice, left panel). (E) HLA A2-specific memory B cells were enumerated by ELISpot in B cell suspensions before transfer (left histogram) and in the spleen of RAG2 KO animals transplanted with an A2 heart 100 days after transfer (right histogram). (F) Evolution of normalized donor-specific antibody titer (mean ± SD) was monitored in the circulation of 3 groups of recipients transplanted with an A2 heart: wild-type C57BL/6 (positive controls, dotted line), RAG2 KO transferred with anti-BALB/c memory B cells (negative controls, dashed line), and RAG2 KO transferred with anti-A2 memory B cells (experimental group, black line).