Reversing endogenous alloreactive B cell GC responses with anti-CD154 or CTLA-4Ig

Alloantibodies mediate acute antibody-mediated rejection as well as chronic allograft rejection in clinical transplantation. To better understand the cellular dynamics driving antibody production, we focused on the activation and differentiation of alloreactive B cells in the draining lymph nodes and spleen following sensitization to allogeneic cells or hearts. We used a modified staining approach with a single MHC Class I tetramer (K^d) bound to two different fluorochromes to discriminate between the Class I-binding and fluorochrome-streptavidin-binding B cells with a high degree of specificity and binding efficiency. By Day 7-8 postsensitization, there was a 1.5- to 3.2-fold increase in the total numbers of K^d-binding B cells. Within this K^d-binding B cell population, approximately half were IgD ^low, MHC Class II^high and CD86⁺, 30-45% expressed a germinal center (Fas⁺GL7⁺) phenotype and 3-12% were IRF4^hi plasma cells. Remarkably, blockade with anti-CD40 or CTLA-4Ig, starting on Day 7 postimmunization for 1 or 4 weeks, completely dissolved established GCs and halted further development of the alloantibody response. Thus MHC Class I tetramers can specifically track the in vivo fate of endogenous, Class I-specific B cells and was used to demonstrate the ability of delayed treatment with anti-CD154 or CTLA-4Ig to halt established allo-B cell responses. This study describes the use of MHC Class I tetramers to specifically track the in vivo fate of endogenous, Class I-specific B cells, and the use of this approach to demonstrate the ability of delayed treatment with anti-CD154 or CTLA-4Ig to halt established allospecific B cell responses.