Figure 4.

Oct2 influences and Slamf1 expression in B cells. (A) Analysis of Slamf1 expression in Oct2-null and Oct2^+/+ B cells activated in vitro. Cells were cultured for 48 h in the presence of CpG or aCD40 before RNA was prepared for RNA sequencing. Filled bars, Oct2^+/+ gray bars, Oct2^−/−. (B) Slamf1/CD150 protein expression in cells of the indicated genotypes. Cells were assessed directly ex vivo (resting, top panels), or were activated in vitro for 48 h with either LPS (B cells) or anti-CD3 (T cells). Macrophages were expanded from fetal liver as described in Section “Materials and Methods.” In the histograms, WT cells are represented with heavy black lines and Oct2^−/− as red. Unstained controls are indicated by thin black lines. (C) WEHI231 cells (Oct2^+/+) and OM1 cells (Oct2^−/−), either uninfected or transduced with an Oct-ER expression vector, were cultured in the presence or absence of estradiol (Es) for 24 h and CD36 and Slamf1 levels were determined by flow cytometry. Thin lines indicate background fluorescence of unstained controls, blue lines represent CD36 and Slamf1 levels in uninduced cultures, and heavy black lines, the levels after Es induction. (D) Slamf1 expression during B cell maturation. Colors indicate the populations represented in each histogram. For bone marrow, recirculating B cells were B220⁺⁺ and IgM⁺, immature B were B220⁺ and IgM⁺⁺, and precursor B were B220⁺ and IgM⁻. For spleen, immature B cells were IgM^hi, IgD^lo, MZ B cells were IgM^hi, CD21^hi, and Fo B cells were IgM^lo, IgD^hi, CD23⁺, and CD21⁺. Lymph node B cells were IgM^lo, IgD^hi, and CD23⁺. (E) Flow cytometric analysis of splenic B cells from naïve and immunized mice (9 days after SRBC immunization). Top panels show the percentage of GL7⁺Fas⁺ GC B cells (gated) among total B220⁺ cells in spleens from mice reconstituted with WT or Oct2^−/− fetal liver. Bottom panels show the Slamf1 levels on non-GC (gray lines) and GC (black line) B cells for each animal, gated as shown in the upper panels.