T_FH cells are well described phenotypically in humans, and more recently in mice, as expressing high levels of the chemokine receptor CXCR5 and molecules such as ICOS, PD1, interleukin-21 (IL-21), and BTLA (2–9). Given that CD4⁺ T cells can up-regulate CXCR5 and/or ICOS after activation (2, 10), it is important to phenotypically distinguish T_FH from highly activated CD4⁺ T cells. We identified T_FH cells in mice in the context of acute infection with lymphocytic choriomeningitis virus (LCMV) by adoptively transferring T cell receptor (TCR) transgenic T cells specific for the LCMV epitope gp66-77 in the context of major histocompatibility complex (MHC) class II molecule I-A^b (SMtg). T_FH cells were CXCR5^high ICOS^high PD1^high BTLA^high CD200^high SLAM^low (Fig. 1, A and B, and fig. S1) and capable of producing IL-21 (Fig. 1C). We confirmed these results for polyclonal LCMV-specific CD4⁺ T cell responses (fig. S2). CXCR5 is the receptor for the B cell follicle chemokine CXCL13 (11), and T_FH cells were selectively able to migrate in response to CXCL13 in vitro (Fig. 1D), consistent with the importance of CXCR5 for T_FH (6, 12).

To understand how T_FH differentiation is transcriptionally regulated, we performed gene expression microarray analysis of virus-specific T_FH and non-T_FH effector CD4⁺ T cells (Fig. 1E and figs. S3 and S4). Notably, the transcription factor B cell CLL/lymphoma 6 (Bcl6) was strongly up-regulated in T_FH (Fig. 1E). This is in agreement with previous reports of elevated Bcl6 expression in murine and human T_FH cells (3–5, 8). Furthermore, Blimp-1 (prdm1) was the most down-regulated transcription factor in T_FH cells (fig. S4), consistent with a recent report (13). Bcl6 is essential for germinal center B cell differentiation (14–16), and Blimp-1 is well characterized as an antagonist of Bcl6 that can also be directly repressed by Bcl6 (16–20). Up-regulation of Bcl6 mRNA (Fig. 1F) and down-regulation of Blimp-1 mRNA (Fig. 1G) were confirmed by quantitative polymerase chain reaction (qPCR). Bcl6 protein expression was detected in germinal center CD4⁺ T cells (fig. S2), consistent with previous observations in human lymphoid tissue (4, 21).

Although Bcl6 mRNA expression has been correlated with T_FH, no experimental data supporting a specific role for Bcl6 in T_FH differentiation have been reported. We expressed Bcl6 in SMtg CD4⁺ T cells via a retroviral vector (RV) with a bicistronic mRNA coexpressing green fluorescent protein (GFP) (fig. S5). Transduced Bcl6-RV⁺ SMtg and control untransduced SMtg CD4⁺ T cells were transferred into naïve C57BL/6 hosts, which were subsequently infected with LCMV, and T_FH differentiation was examined (Fig. 2, A to E). Bcl6 expression drove nearly absolute T_FH differentiation in vivo (80 to 90%; Fig. 2, B and C), in contrast to T_FH differentiation in control untransduced (GFP^–) SMtg cells in the same mice (Fig. 2B) or mice that received SMtg transduced with a control retrovirus expressing only GFP (GFP-RV⁺) and untransduced SMtg in equal proportions (Fig. 2C). Comparably striking results were seen in studies where only Bcl6-RV⁺ or GFP-RV⁺ SMtg CD4⁺ T cells were transferred into host mice (fig. S6). Bcl6 overexpression did not affect T cell expansion in vivo (fig. S5). Constitutive expression of Bcl6 drove up-regulation of CXCR5, PD-1, ICOS, CD200, and BTLA expression (Fig. 2, D and E, and fig. S6), as well as the inhibition of SLAM and Blimp-1 (fig. S6; see below). These results indicate that Bcl6 expression drives full T_FH differentiation in vivo.

T_FH differentiation is known to require the presence of B cells and is thought to require the presence of antigen-specific B cells (6). We thus hypothesized that Bcl6 expression induced by interaction with antigen-specific B cells could be the event that commits a T cell to T_FH differentiation. To test this, we examined whether Bcl6 expression in CD4⁺ T cells was sufficient to drive T_FH differentiation in μMT B cell–deficient mice and in B cell receptor (BCR) transgenic mice of an irrelevant specificity (MD4, specific for hen egg lysozyme). GFP-RV⁺ SMtg CD4⁺ T cells failed to differentiate into T_FH in μMT or MD4 mice infected with LCMV (Fig. 2, F and G), which demonstrates that T_FH differentiation in the context of a viral infection is dependent on the presence of antigen-specific B cells. In contrast, Bcl6-RV⁺ SMtg cells differentiated into T_FH in the absence of antigen-specific B cells or even in the total absence of B cells (Fig. 2, F and G). These results indicate that cognate T-B interactions induce Bcl6 expression in CD4⁺ T cells and that Bcl6 is sufficient to drive T_FH differentiation, even in the absence of such interactions.

T_FH cells are thought to provide B cell help in vivo (2, 22). We assessed the capacity of Bcl6-RV⁺ SMtg CD4⁺ T cells to help B cells in vivo by examining germinal center development in LCMV-infected mice. Overexpression of Bcl6 increased the already robust frequency of germinal center B cells after LCMV infection (Fig. 3, A and B). As an additional measure of B cell help, we also examined the role of Bcl6 in T cell–dependent antibody production. Constitutive expression of Bcl6 in OT-II CD4⁺ T cells enhanced NP-Ova serum immunoglobulin G (IgG) responses (Fig. 3C and fig. S7), which were sustained (Fig. 3C). Our results suggest that Bcl6 was specifically enhancing T_FH differentiation and not skewing the T_H1/T_H2 profile of the CD4⁺ T cells, because all IgG isotypes were enhanced in the mice receiving Bcl6-expressing OT-II CD4⁺ T cells, with the strong IgG1 dominance maintained (fig. S7).

The results of these experiments showed that Bcl6 expression was sufficient to drive the differentiation of functional T_FH. To test whether Bcl6 was also necessary for T_FH differentiation, we examined Bcl6^–/– CD4⁺ T cells. Bcl6^–/– mice have an abundance of highly activated CD4⁺ T cells (fig. S8) and succumb to early mortality (14, 15). To circumvent these issues, we transferred Bcl6^+/+ or Bcl6^–/– OT-II bone marrow into irradiated C57BL/6 recipients (fig. S8). Bcl6^–/– OT-II CD4⁺ T cells obtained from chimeric mice did not exhibit lymphoproliferation or spontaneous activation upon transfer into C57BL/6 mice (fig. S8, D to F). Bcl6^–/– or Bcl6^+/+ OT-II recipient mice were subsequently immunized with Ova in alum. Strikingly, Bcl6^–/– OT-II CD4⁺ T cells did not differentiate into T_FH cells (Fig. 3D). We hypothesized that if T_FH cells are necessary for B cell help in vivo, a cell-intrinsic CD4⁺ T cell block in T_FH differentiation should result in a failure to generate antigen-specific B cell responses such as germinal center formation. To test this hypothesis, we transferred Bcl6^–/– or Bcl6^+/+ OT-II CD4⁺ T cells into Icos^–/– mice, which have ineffective B cell help (2, 23). After NP-Ova immunization, Icos^–/– mice that received Bcl6^–/– OT-II CD4⁺ T cells were unable to form germinal centers, in contrast to mice that received wild-type OT-II CD4⁺ T cells (Fig. 3, E to G, and fig. S9). These data demonstrate that Bcl6 is necessary for T_FH differentiation and that T_FH cells are necessary for germinal center formation. Together, these results indicate that Bcl6 is a bona fide master regulator of T_FH differentiation in vivo.

Blimp-1 is a known antagonist of Bcl6, capable of directly inhibiting Bcl6 expression in B and T cells (17, 18). Conversely, Blimp-1 expression can be inhibited by Bcl6 (16–18, 20). On the basis of our observations that Bcl6 drives T_FH cell differentiation and function, and because Blimp-1 was the single most down-regulated transcription factor in T_FH cells by gene expression array analysis (Fig. 1E and fig. S4) and qPCR (Fig. 1G), we hypothesized a role for Blimp-1 in blocking T_FH differentiation in vivo. We constructed a Blimp-1 retroviral expression vector, Blimp1-RV (fig. S5), designed to express physiological levels of Blimp-1. Only CD4⁺ T cells expressing low levels of the GFP reporter were used for in vivo experiments (fig. S10A). Blimp-1 blocked Bcl6 protein expression in activated antigen-specific CD4⁺ T cells in vivo (Fig. 4A). To determine the effects of Blimp-1 on T_FH differentiation, we mixed Blimp1-RV⁺ SMtg CD4⁺ T cells and untransduced control SMtg cells in equal proportions and transferred them into host mice subsequently infected with LCMV. We observed normal proliferation of Blimp-1–expressing SMtg CD4⁺ T cells (fig. S10); however, T_FH differentiation was severely abrogated, with an 80% reduction in T_FH frequency (Fig. 4, B and C). Blockade of T_FH differentiation by Blimp-1 was also observed when mice separately received Blimp1-RV⁺ versus GFP-RV⁺ SMtg cells (fig. S10, F and G). Constitutive expression of Blimp-1 inhibited acquisition of the T_FH phenotype: SLAM expression was increased (Fig. 4D), whereas CXCR5, ICOS, and PD-1 expression were all decreased (Fig. 4D and fig. S10). Inhibition of T_FH differentiation by Blimp-1 was physiological and specific, because the expression levels of SLAM, ICOS, and PD-1 by Blimp1-RV⁺ SMtg CD4⁺ T cells were equivalent to the expression levels seen in wild-type activated non-T_FH SMtg CD4⁺ T cells, and not naïve cells (fig. S10I). Blimp1-RV⁺ and wild-type non-T_FH SMtg cells also expressed comparable amounts of the cytokines interferon-γ (IFN-γ) and IL-2 (fig. S11). High amounts of Blimp-1 expression can inhibit proliferation in B and T cells (17, 24, 25). The moderate level of Blimp-1 expression used in our experiments (fig. S10E) did not affect proliferation in vivo (fig. S10, C, D, and H), in agreement with previous in vitro studies (26) and our observation that non-T_FH CD4⁺ T cells express 20 times as much Blimp-1 as do T_FH cells and are still proliferative. Blimp-1 expression did not affect expression of the T helper lineage–specific transcription factors Foxp3, GATA3, and RORγt (fig. S11), which indicates that Blimp-1 did not induce differentiation into other helper lineages. Collectively, these data suggest that Blimp-1 acts specifically to repress Bcl6 and thus blocks T_FH differentiation.

Given that Blimp-1 is a physiological inhibitor of Bcl6 expression and T_FH differentiation in vivo, we performed an additional test of the necessity of T_FH for B cell help by transferring Blimp1-RV⁺ OT-II and GFP-RV⁺ OT-II CD4⁺ T cells into SAP-deficient (sh2d1a^–/–) mice [SAP-deficient mice exhibit a CD4⁺ T cell–intrinsic defect in germinal center formation (27–29)] subsequently immunized with NP-Ova. We observed germinal centers and anti–NP-Ova serum IgG in GFP-RV⁺ OT-II CD4⁺ T cell recipient mice after immunization (Fig. 4, E and F). Strikingly, although OT-II cell numbers were normal in Blimp1-RV⁺ OT-II recipient mice (fig. S12), germinal centers were reduced by 90% (Fig. 4E). Constitutive Blimp-1 expression also inhibited the NP-Ova–specific IgG response, reducing the serum antibody concentration to only 16% of normal levels (Fig. 4F). All IgG isotypes were reduced (fig. S12), confirming that Blimp-1 was specifically inhibiting T_FH differentiation. These results demonstrate both that Blimp-1 inhibits CD4⁺ T cell help to B cells and that T_FH cells are required for B cell help in vivo.

To confirm the biological role of Blimp-1 in inhibiting T_FH differentiation in vivo, we tested the ability of Blimp-1–deficient CD4⁺ T cells to differentiate into T_FH. To avoid autoimmunity complications (30, 31), we deleted Blimp-1 (prdm1) in vitro in mature prdm1^fl/fl CD4⁺ T cells (32) by means of a Cre-expressing RV. We transferred Cre⁺ SMtg⁺ prdm1^fl/fl and control Cre^– SMtg⁺ prdm1^fl/fl CD4⁺ T cells into mice subsequently infected with LCMV. Deletion of prdm1 substantially enhanced T_FH differentiation in vivo (Fig. 4G) without altering proliferation (fig. S13). These data indicate that Blimp-1 expression in vivo normally restricts Bcl6 expression and T_FH differentiation. In sum, our results reveal that Bcl6 and Blimp-1 are reciprocal master regulators of T_FH differentiation, with T_FH differentiation in vivo requiring the presence of Bcl6 and the absence of Blimp-1.

There has been extensive speculation about a role for Bcl6 in T_FH differentiation, based on gene expression data from human (2, 4) and murine T_FH studies (3, 8, 9, 13). Our data directly show that Bcl6 specifically drives T_FH differentiation and is a bona fide master regulator. The relationship between T_FH and other CD4⁺ T cell lineages has been a long-standing problem. The predominant CD4⁺ T cell response to LCMV is T_H1 (fig. S14), and it is notable that T-bet and IFN-γ were still expressed in the T_FH in vivo, although at lower levels than in T_H1/non-T_FH LCMV-specific CD4⁺ T cells (fig. S14). These observations are consistent with a model in which T_FH cells follow their own differentiation pathway but are not an isolated lineage and can exhibit partial characteristics of T_H1/T_H2 polarization depending on environmental conditions. This overlapping differentiation model would resolve the conundrum in the literature that neither T_H1, T_H2, nor T_H17 are required for B cell help in vivo (8, 33, 34), but that cells with T_H1, T_H2, or T_H17 phenotypes can provide B cell help in vivo (9, 35–39).

The capacity for B cell help is a central attribute of CD4⁺ T cells and is a cornerstone of protective immunity. It is well known that in B cells, Bcl6 and Blimp-1 are powerful antagonistic master regulators of germinal center B cell differentiation and plasma cell differentiation. Our findings that Bcl6 and Blimp-1 also control T_FH differentiation illustrate the elegant use of the same antagonistic transcription factors to drive different functions in two lymphocyte populations differentiating in parallel: antigen-specific B cells and the T_FH cells that provide their help. Manipulation of these signaling pathways in vivo may have substantial therapeutic benefit for enhancing vaccines or, conversely, blocking auto-antibody responses.

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