CD11c+ T-bet+ B Cells Require IL-21 and IFN-γ from Type 1 T Follicular Helper Cells and Intrinsic Bcl-6 Expression but Develop Normally in the Absence of T-bet

Abstract

CD11c⁺ T-bet⁺ B cells generated during ehrlichial infection require CD4⁺ T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (T_FH1) cells provide help to CD11c⁺ T-bet⁺ B cells via the dual secretion of IL-21 and IFN-γ in a CD40/CD40L-dependent manner. T_FH1 cell help was delivered in two phases: IFN-γ signals were provided early in infection, whereas CD40/CD40L help was provided late in infection. In contrast to T-bet⁺ T cells, T-bet⁺ B cells did not develop in the absence of B cell-intrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c⁺ memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing T_FH1 cells.

Figure 1:. IFN-γ IL-21 double-producing T_FH1 cells provide help to CD11c+ T-bet+ B cells

(A) Splenocytes from E. muris-infected CD40L-deficient or wild-type mice were analyzed on day 30 post-infection. The dot plots show the percentages of CD11c+ CD19+ B cells among total B cells. Graphs represent aggregate data from two independent experiments. Statistical significance was determined using a two-tailed un-paired t test (p < 0.0001, df = 9). (B) Splenocytes from E. muris-infected CD4-cre x Bcl-6^fl/fl or Bcl-6^fl/fl control mice were analyzed on days 30 (top plots) and 16 (bottom plots) post-infection. Representative dot plots show the percentages of CD11c+ CD19+ B cells among total B cells and the percentages of PD-1+ CXCR5+ cells among CD3+ CD4+ T cells. Graphs represent aggregate data from three independent experiments. Statistical significance was determined using two-tailed un-paired t tests (top: p < 0.4559, df =13; bottom: p = 0.0001, df = 15). (C) CD3+ CD4+ CXCR3+ CCR6-negative T cells from E. muris-infected, female, IFN-γ-reporter mice were analyzed for the expression of ICOS, CD44 and IFN-γ 16 days post-infection. Plots are representative of 4 mice. (D) Splenocytes from E. muris-infected, female, IFN-γ-deficient or wild-type mice were analyzed on day 18 post-infection. Representative dot plots show the percentages of CD11c+ B220+ B cells among total lymphocytes. Graphs represent aggregate data from two independent experiments. Statistical significance was determined using two-tailed un-paired t tests (left: p = 0.0023, df = 6; right: p = 0.0014, df = 6). (E) Splenocytes from E. muris-infected CD4-cre x T-bet^fl/fl or T-bet^fl/fl control mice were analyzed on days 30 (left plot) and 16 (right plot) post-infection. Representative dot plots show the percentages of CD11c+ CD19+ B cells among total B cells (top left), the percentages of T-bet^high and T-bet^low CD19+ B cells among total B cells (bottom left), and the percentages of T-bet+ CCR6-negative cells among CD3+ CD4+ T cells (right). Graphs represent aggregate data from three independent experiments. Statistical significance was determined using two-tailed un-paired t tests (top left: p < 0.0030, df =12; right: p = 0.0042, df = 7) and an ordinary one-way ANOVA (p < 0.0001, F = 28.36, df = 27) with Sidak’s multiple comparisons test (T-bet^high: p = 0.0015, df = 24; T-bet^low: p = 0.7372, df = 24). (F) CD3+ CD4+ CXCR3+ CCR6-negative PD-1+ CXCR5+ T cells from E. muris-infected, female, IL-21-reporter mice were analyzed for the expression of IL-21 and CD44 16 days post-infection. Plots are representative of 4 mice. (G) Splenocytes from E. muris-infected, female, wild-type mice on day 16 post-infection were cultured with a cell activation cocktail containing Brefeldin A for 4 hours at 37°C. CD3+ CD4+ CXCR3+ CD44+ T cells were analyzed for IFN-γ, ICOS, and IL-21 expression. Plots are representative of 5 mice. Not significant (n.s.) > 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.

Figure 2:. CD11c+ T-bet+ B cells have biphasic requirements for T cell help

(A) E. muris-infected, female, wild-type mice were treated with either anti-CD40L antibody or an isotype-matched irrelevant antibody every other day from days 16 to 30 post-infection and splenocytes were analyzed on day 30 post-infection. Representative dot plots show the numbers and percentages of CD11c+ CD19+ B cells among total B cells. Graphs represent aggregate data from two independent experiments. Statistical significance was determined using two-tailed un-paired t tests (left: p < 0.0001, df = 18; right: p < 0.001, df = df = 18). (B) E. muris-infected, female, wild-type mice were treated with either anti-CD40L antibody or an isotype-matched irrelevant antibody every other day from days 30 to 37 post-infection and splenocytes were analyzed on day 37 post-infection. Representative dot plots show the numbers and percentages of CD11c+ CD19+ B cells among total B cells. Aggregate data are shown in the plots on the right. Statistical significance was determined using two-tailed un-paired t tests (left: p = 0.0346, df = 8; right: p = 0.4484, df = 7). (C) E. muris-infected, female, wild-type mice were treated with either anti-IFN-γ antibody or vehicle control once every three days from days 16 to 30 post-infection and splenocytes were analyzed on day 30 post-infection. Representative dot plots show the numbers and percentages of CD11c+ CD19+ B cells among total B cells. Graphs represent aggregate data from two independent experiments. Statistical significance was determined using two-tailed un-paired t tests (left: p = 0.4008, df = 7; right: p = 0.8405, df = 7). (D) Sera from E. muris-infected, female, wild-type mice harvested on days 0, 8, 16, 21, and 30 post-infection were analyzed by ELISA for IFN-γ. Dots represent the arithmetic mean of five mice and the upper and lower bounds represent the standard deviation. n.s.> 0.05, ****p < 0.0001.

Figure 3:. Bcl-6 is required for the development of CD11c+ T-bet+ B cells

(A) T-bet+ CD19+ B cells from E. muris-infected, female, wild-type (black circles) or Bcl-6^fl/fl control (open circles) mice were analyzed for expression of Bcl-6 on days 16 and 30 post-infection. Aggregate data are shown in the plots on the right. Statistical significance was determined using two-tailed un-paired t tests (left: p < 0.0001, df = 7; right: p < 0.0048, df = 7). (B) Splenocytes from E. muris-infected CD19-cre x Bcl-6^fl/fl or Bcl-6^fl/fl control mice were analyzed on days 43 (top plot) and 16 (bottom plot) post-infection. Representative dot plots show the percentages of CD11c+ CD19+ B cells and the percentages of T-bet+ CD19+ B cells among total B cells. Aggregate data are shown in the plots on the right. Statistical significance was determined using two-tailed un-paired t tests (top left: p = 0.0010, df = 6; top right: p < 0.0001, df = 7; bottom left: p = 0.0334, df = 6; bottom right: p < 0.0001, df = 7). (C) Sera from E. muris-infected CD19-cre x Bcl6^fl/fl or wild-type mice on day 30 post-infection was analyzed by ELISA for IgM, pan IgG, IgG2b, IgG2c, and IgG3. Sera was collected in two independent experiments. Statistical significance was determined using an ordinary one-way ANOVA (p < 0.0001, F = 18.21, df = 11) with Sidak’s multiple comparisons test (IgM: p < 0.0001, IgG: p = 0.0008, IgG2b: p = 0.0653, IgG2c: p < 0.0003, IgG3: p = 0.4925, df = 40). n.s.> 0.05, *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 4:. CD11c+ B cells develop in the absence of T-bet

(A) Splenocytes from E. muris-infected Mb1-cre x T-bet^fl/fl (black circles), Mb1-cre x T-bet^fl/+ control mice (black circles), or T-bet^fl/fl control mice (open circles) were analyzed on day 30 post-infection. Representative dot plots show the percentages of CD11c+ B220+ B cells among total B cells. The graphs represent aggregate data from two independent experiments. Statistical significance was determined using a two-tailed un-paired t test (left: p = 0.0081, df = 16; right: p = 0.1754, df = 16). (B) CD11c+ B220+ B cells from E. muris-infected Mb1-cre x T-bet^fl/fl or T-bet^fl/fl mice were analyzed for expression of CD86, CD38, CD73, CD80, CD95, and PD-L2, thirty days post-infection. (C) Sera from E. muris-infected Mb1-cre x T-bet^fl/fl or T-bet^fl/fl mice on day 30 post-infection were analyzed by ELISA for IgM, pan IgG, IgG1, IgG2b, IgG2c, and IgG3. Sera were collected in two independent experiments. Statistical significance was determined using an ordinary one-way ANOVA (p < 0.0001, F = 18.21, df = 11) with Sidak’s multiple comparisons test (IgM: p > 0.9999, IgG: p = 0.5553, IgG1: p = 0.0380, IgG2b: p = 0.2164, IgG2c: p < 0.0001, IgG3: p = 0.9537, df = 78). n.s. > 0.05, *p < 0.05, ****p < 0.0001.