*Purpose: B cells in mice tolerized to fully mismatched heart allografts with anti-CD154+donor spleen cells (CoB) are intrinsically dysfunctional and unable to differentiate into germinal center B cells (GCBC) and antibody-secreting cells (ASCs). Remarkably, these B cells acquire the ability to suppress naïve B cell responses in a donor-specific manner. Here we tested whether dysfunction and ability to suppress are induced simultaneously, and due to the lack of sustained T:B interaction that is mediated by SLAM-associated protein (SAP) or absent CD40:CD154 interactions.

*Methods: SAP-KO and CD154-KO B/6 mice were transplanted with B/c hearts without CoB, and on post-operative day 30, enriched B cells were adoptively transferred (AdTr) to secondary MD4 BCR-Tg hosts, either alone or at a 1:1 ratio with naïve B cells, then challenged with B/c spleen cells to test for dysfunction and ability to suppress. Donor-specific IgG from MD4 hosts was quantified on day 14 post-AdTr.

*Results: A) In SAP-KO recipients, donor-specific IgG response was significantly reduced; following AdTr and B/c challenge, SAP-KO B cells were neither dysfunctional nor suppressive. B) In CD154-KO recipients, donor-specific IgG response was completely inhibited; following AdTr and B/c challenge, CD154-KO B cells were intrinsically dysfunctional but failed to suppress naïve B responses. In contrast, AdTr B cells from tolerant WT recipients of B/c hearts and CoB were dysfunctional and suppressed naïve B cell responses.

*Conclusions: B cell dysfunction and ability to suppress are independently induced; dysfunction was induced by the absence of CD40:CD154 interactions, but the ability to suppress was not induced even when CoB was administered to CD154-KO recipients. We are currently testing the hypothesis that low levels of CD40 signaling, as a result of incomplete blockade of CD40:CD154 interactions with CoB, is necessary to induce suppressive properties in donor-specific B cells in tolerant recipients.

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