This conclusion is drawn from two experiments. autoreactive thymic B cells play important role in CD4 T cell tolerance. Introduction Thymic B cells are a unique and highly effective antigen presenting cell population within the thymic medulla (Klein et al., 2014; Perera and Huang, 2015). Together with conventional thymic dendritic cells, plasmacytoid dendritic cells, medullary thymic epithelial cells (mTECs), and macrophages they constitute a network of antigen presenting cells (APCs) in the medulla that is responsible for removing autoreactive T cell specificities from the developing repertoire (Klein et al., 2014). B cells are unique antigen presenting cells because their antigen presentation machinery is usually closely tied to the B cell receptor (BCR). Antigens that are bound by the BCR are internalized and presented much more efficiently than by other APCs (reviewed in (Lanzavecchia, 1990; Yuseff et al., 2013)). Therefore, the specificity of a B cell greatly influences the antigens that it presents. We have shown that autoreactive thymic B Rabbit Polyclonal to PIGX cells can mediate T cell unfavorable selection efficiently (Perera et al., 2013). While skewing the B cell repertoire towards a self antigen greatly enhances unfavorable selection, even the normal repertoire of thymic B cells is usually capable of presenting self antigens for unfavorable selection, suggesting that this thymic B cell repertoire may naturally contain autoreactive specificities. Still little is known about how the thymic B cell repertoire is usually selected and VX-787 (Pimodivir) regulated. Phenotypically, thymic B cells express a number of costimulatory molecules such as CD80, CD86, CD40, and increased levels of MHC Class II, which may facilitate their conversation with thymocytes (Ferrero et al., 1999; Perera et al., 2013). In the periphery, it is well established that cognate T-B interactions provide activating signals to the B cell partner, most notably through CD40 which, combined with BCR signals, results in proliferation, class switching, and antibody secretion (reviewed in (Stavnezer et al., 2008; Xu et al., 2012)). Thymic B cells respond poorly to mitogens like LPS or anti-IgM and tested their reactivity to nuclear antigens by staining Hep2 cells. Examples of such specific BCRs from the IgM+IgD+ and IgM?IgD? thymic B cell repertoires and corresponding ANA staining were shown in Fig. 6A and Fig. 6B respectively. Altogether we expressed 19 BCRs from the IgM+IgD+ repertoire and 19 from the IgM?IgD? repertoire that covered 26.2% and 42.6% of the total Ig reads for each population respectively. Quantification of the relative intensity of nuclear staining showed no positive anti-nuclear staining from any of the IgM+IgD+ BCRs, but 8 of 19 IgM?IgD? BCRs displayed significant ANA staining over background (Fig. 6C). These ANA positive BCRs were predominantly from the V5 family (6/7), with one being derived from the V6 family, and the contribution of these 8 VX-787 (Pimodivir) autoreactive BCRs accounted for 23.9% of the Ig reads from IgM?IgD? thymic B cells. VX-787 (Pimodivir) Thymic B cell class switching regulates the T cell repertoire We have shown that autoreactive thymic B cells are excellent APCs for T cell unfavorable selection (Perera et al., 2013). Because class-switched thymic B cells are enriched with autoreactivity, we hypothesized that they contributed to T cell unfavorable selection. Furthermore, we would predict increased T cell autoreactivity in mice where class switching was absent. To determine how T cell autoreactivity is usually affected, we adapted a CD4 T cell transfer protocol that has previously been used to measure autoreactivity within the T cell repertoire (Yamano et al., 2015). In this approach, polyclonal T cells are adoptively transferred into congenic hosts and their proliferation and differentiation is usually measured. WT CD4 VX-787 (Pimodivir) T cells transferred into B6 congenic hosts should proliferate minimally since they will have already be tolerant of all antigens in the new host. However T cells from a host with defective central tolerance should proliferate more extensively, as they will be encountering antigens to which they have not been previously tolerized. We purified CD4+ T cells from the spleens.