Effective B cell-mediated immunity and antibody responses often require help from CD4 + T cells. It is thought that a distinct CD4 + effector T cell subset, called T follicular helper cells (T FH ), provides this help; however, the molecular requirements for T FH differentiation are unknown. We found that expression of the transcription factor Bcl6 in CD4 + T cells is both necessary and sufficient for in vivo T FH differentiation and T cell help to B cells in mice. In contrast, the transcription factor Blimp-1, an antagonist of Bcl6, inhibits T FH differentiation and help, thereby preventing B cell germinal center and antibody responses. These findings demonstrate that T FH cells are required for proper B cell responses in vivo and that Bcl6 and Blimp-1 play central but opposing roles in T FH differentiation.Each lineage of effector CD4 + T cells (T H 1, T H 2, T H 17, and T reg ) is defined and controlled by a unique master regulator transcription factor (T-bet, GATA3, RORγt, and Foxp3, respectively) (1). A proposed fifth effector subset, T follicular helper (T FH ) cells, is thought to provide help for the generation of B cell-mediated immune responses, including class switch recombination, germinal center differentiation, and affinity maturation (2). Here, we identified Bcl6 as a T FH master regulator and found that germinal center formation does not occur in the absence of T FH cells.

Chronic infections strain the regenerative capacity of antiviral T lymphocyte populations, leading to failure in long-term immunity. The cellular and molecular events controlling this regenerative capacity, however, are unknown. We found that two distinct states of virus-specific CD8+ T cells exist in chronically infected mice and humans. Differential expression of the T-box transcription factors T-bet and Eomesodermin (Eomes) facilitated the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection. T-bethi cells displayed low intrinsic turnover but proliferated in response to persisting antigen, giving rise to Eomeshi terminal progeny. Genetic elimination of either subset resulted in failure to control chronic infection, which suggests that an imbalance in differentiation and renewal could underlie the collapse of immunity in humans with chronic infections.

Summary T cell exhaustion is common during chronic infections. While CD4+ T cells are critical for controlling viral load during chronic viral infections, less is known about their differentiation and transcriptional program. We defined the phenotypic, functional and molecular profiles of exhausted CD4+ T cells. Global transcriptional analysis demonstrated a molecular profile distinct from effector or memory CD4+ T cells and also from exhausted CD8+ T cells, though some common features of CD4+ and CD8+ T cell exhaustion were revealed. We have demonstrated unappreciated roles for transcription factors (TFs) including Helios, type I interferon (IFN-I) signaling and a diverse set of co-inhibitory and costimulatory molecules during CD4+ T cell exhaustion. Moreover, the signature of CD4+ T cell exhaustion was found to be distinct from that of other CD4+ T cell lineage subsets and was associated with TF heterogeneity. This study provides a framework for therapeutic interventions targeting exhausted CD4+ T cells.

Cytokines are important modulators of lymphocytes, and both interleukin-21 (IL-21) and IL-6 have proposed roles in T follicular helper (Tfh) differentiation, and directly act on B cells. Here we investigated the absence of IL-6 alone, IL-21 alone, or the combined lack of IL-6 and IL-21 on Tfh differentiation and the development of B cell immunity in vivo. C57BL/6 or IL-21−/− mice were treated with a neutralizing monoclonal antibody against IL-6 throughout the course of an acute viral infection (lymphocytic choriomeningitis virus, LCMV). The combined absence of IL-6 and IL-21 resulted in reduced Tfh differentiation and reduced Bcl6 protein expression. In addition, we observed that these cytokines had a large impact on antigen-specific B cell responses. IL-6 and IL-21 collaborate in the acute T-dependent antiviral antibody response (90% loss of circulating antiviral IgG in the absence of both cytokines). In contrast, we observed reduced germinal center formation only in the absence of IL-21. Absence of IL-6 had no impact on germinal centers, and combined absence of both IL-21 and IL-6 revealed no synergistic effect on germinal center B cell development. Studying CD4 T cells in vitro, we found that high IL-21 production was not associated with high Bcl6 or CXCR5 expression. TCR stimulation of purified naïve CD4 T cells in the presence of IL-6 also did not result in Tfh differentiation, as determined by Bcl6 or CXCR5 protein expression. Cumulatively, our data indicates that optimal Tfh formation requires IL-21 and IL-6, and that cytokines alone are insufficient to drive Tfh differentiation.

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