*Purpose: Therapies involving CD4 regulatory T cells (Tregs) are promising approaches to mitigate graft rejection and autoimmunity. Understanding of the activation mechanisms that initiate Treg development and potentiates suppression will facilitate Treg application. Recent studies indicate that B cells from the thymic medulla express incomparably high levels of MHC class II (MHC-II) molecules, suggesting a prominent role in antigen presentation. Medullary B cells also cluster around Treg precursors going under final developmental steps. We thus hypothesized that thymic B cells preferentially present peptides on MHC-II molecules to maturing Treg precursors.

*Methods: We first derived the MHC-II IAα ^fl/fl mouse on the C57BL/6 background (IAαβ^b, IE⁰). This mouse was then crossed to mouse lines expressing Cre recombinase at early pro-B stage (Mb1-Cre) or at the pro-pre B transition (CD19-Cre). IAα gene disruption was examined in the resulting mb1 and CD19 lines by DNA-PCR and MHC-II IA^b expression was followed by flowcytometry (FCM). We measured the frequencies and numbers of effector and regulatory CD4 T cells, CD8 T cells and B cells isolated from blood and thymus. Lymphocyte activation was evaluated by FCM for expression of MHC-II, CD69, CD44 and CD86. Phosflow was also performed to document lymphocyte activation via kinase signaling (pS6, pAKT and pERK).

*Results: MHC-II gene extinction in B cells at early (mb1 mutant) or later (CD19 mutant) developmental stages showed differential phenotypes revealing the importance of timing and dosage of B cell MHC-II expression on the development and homeostasis of B and CD4⁺ T lymphocytes. Disruption of MHC-II gene expression at the pro-B cell stage (mb1 mutant) led to normal counts of blood B cells which were all MHC-II^neg. This phenotype remained stable over time. Thymic B cells exhibited an activated phenotype (CD69^hi, CD44^hi, CD86^hi), similar to that of wildtype B cells. Conversely, lack of MHC-II expression at later pro-pre B cell transition stage (CD19 mutant) led to low counts of MHC-II^neg B cells that recovered MHC-II expression over time. Thymic B cells from CD19 mutants harbored a resting phenotype (CD69^low, CD44^low, CD86^low). Together, these data indicate that early MHC-II expression has qualitative and quantitative impacts on B cell development and fitness. Interestingly, both mutants showed lower frequencies of circulating B cells among CD45⁺ lymphocytes. This defect was compensated by a spike in CD4⁺ T production, suggesting that MHC-II⁺ B cells impact on CD4⁺ T cell homeostasis. Accordingly, pilot studies on thymic CD4⁺ Tregs from MHC-II deficient mice showed significant reduction in TCR fitness during activation.

*Conclusions: In sum, we here provide evidence in support of a critical role of antigen-presentation by B cells in both B cell development and Treg maturation.

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