SUMMARY The nature of follicular helper CD4+ T (Tfh) cell differentiation remains controversial, including the minimal signals required for Tfh differentiation, and the time at which Tfh differentiation occurs. Here we determine that Tfh development initiates immediately during dendritic cell (DC) priming in vivo. We demonstrate that inducible costimulator (ICOS) provides a critical early signal to induce the transcription factor Bcl6, and Bcl6 then induces CXCR5, the canonical feature of Tfh cells. Strikingly, a bifurcation between Tfh and effector Th cells was measurable by the second cell division of CD4+ T cells, at day 2 after an acute viral infection: IL2Rαint cells expressed Bcl6 and CXCR5 (Tfh cell program), whereas IL2Rαhi cells exhibited strong Blimp1 expression that repressed Bcl6 (effector Th cell program). Virtually complete polarization between Bcl6+ Tfh cells and Blimp1+ effector Th cell populations developed by 72 hours, even without B cells. Tfh cells were subsequently lost in the absence of B cells, demonstrating a B cell requirement for maintenance of Bcl6 and Tfh cell commitment via sequential ICOS signals.

Interleukin 2, STAT5, and Blimp-1 work together to suppress differentiation of follicular helper T cells in mice.

T follicular helper (TFH) cells are specialized effector CD4+ T cells that help B cells develop germinal centers and memory. However, the transcription factors that regulate TFH differentiation remain incompletely understood. Here we report that selective loss of either Lef1 (LEF-1) or Tcf7 (TCF-1) resulted in TFH defects, while deletion of Lef1 and Tcf7 severely impaired TFH differentiation and germinal centers. Forced expression of LEF-1 enhanced TFH differentiation. LEF-1 and TCF-1 coordinated TFH differentiation by two general mechanisms. First, they established the responsiveness of naïve CD4+ T cells to TFH signals. Second, they promoted early TFH differentiation via the multipronged approach of sustaining expression of IL-6Rα and gp130, enhancing ICOS expression, and promoting expression of Bcl6.

Bcl6 is required for CD4 T cell differentiation into follicular helper cells (Tfh). Here, we examined the role of IL-6 in early processes of in vivo Tfh differentiation, as the timing and mechanism of action of IL-6 in Tfh cell differentiation has been controversial in vivo. We found that early Bcl6+CXCR5+ Tfh cell differentiation was severely impaired in the absence of IL-6; however, STAT3 deficiency failed to recapitulate that defect. IL-6 receptor signaling activates the transcription factor STAT1 specifically in CD4 T cells. Strikingly, we found that STAT1 activity was required for Bcl6 induction and early Tfh differentiation in vivo. IL-6 mediated STAT3 activation is important for downregulation of IL-2Rα to limit Th1 differentiation in an acute viral infection. Thus, IL-6 signaling is a major early inducer of the Tfh differentiation program unexpectedly mediated by both STAT3 and STAT1 transcription factors.

Follicular helper CD4 T cells (Tfh) are a distinct type of differentiated CD4 T cells uniquely specialized for B cell help. Here we examined Tfh cell fate commitment, including distinguishing features of Tfh versus Th1 proliferation and survival. Using cell transfer approaches at early time points after an acute viral infection, we demonstrate that early Tfh cells and Th1 cells are already strongly cell fate committed by day three. Nevertheless, Tfh cell proliferation was tightly regulated in a TCR-dependent manner. The Tfh cells still depend on extrinsic cell fate cues from B cells in their physiological in vivo environment. Unexpectedly, we found that Tfh cells share a number of phenotypic parallels with memory precursor CD8 T cells, including selective upregulation of IL7Rα and a collection of co-regulated genes. As a consequence, the early Tfh cells can progress to robustly form memory cells. These data support the hypothesis that CD4 and CD8 T cells share core aspects of a memory cell precursor gene expression program involving Bcl6, and a strong relationship exists between Tfh cells and memory CD4 T cell development.

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Summary Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies had identified bone marrow and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM secreting cells in spleen, and for the first time in the bone marrow as IgM+ IgDlo/−CD19hi CD43+ CD5+/− B-1 cells. The newly identified population of bone marrow B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and bone marrow B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Together the study identifies populations of non-terminally differentiated B-1 cells in spleen and bone marrow as the most significant producers of natural IgM.

CD4+ follicular helper T cells (TFH cells) are essential for germinal center (GC) responses and long-lived antibody responses. Here we report that naive CD4+ T cells deficient in the transcription factor Foxp1 ‘preferentially’ differentiated into TFH cells, which resulted in substantially enhanced GC and antibody responses. We found that Foxp1 used both constitutive Foxp1A and Foxp1D induced by stimulation of the T cell antigen receptor (TCR) to inhibit the generation of TFH cells. Mechanistically, Foxp1 directly and negatively regulated interleukin 21 (IL-21); Foxp1 also dampened expression of the costimulatory molecule ICOS and its downstream signaling at early stages of T cell activation, which rendered Foxp1-deficient CD4+ T cells partially resistant to blockade of the ICOS ligand (ICOSL) during TFH cell development. Our findings demonstrate that Foxp1 is a critical negative regulator of TFH cell differentiation.