TCF-1 and LEF-1 help launch the T_FH program

Follicular helper T cells (T_FH cells) differentiate from naive T cells, but the picture of this differentiation process remains incomplete. Two studies now identify the related transcriptional regulators TCF-1 and LEF-1 as important early participants in this process.

Follicular helper T cells (T_FH cells) are a specialized subset of T cells that provide signals necessary for antigen-specific B cells to generate the germinal center (GC). This structure is required for class-switch recombination, somatic hypermutation and the selection of B cells that produce high-affinity antibodies, as well as for the generation of long-lived antibody-secreting plasma cells and memory B cells¹. A distinguishing marker of T_FH cells is the chemokine receptor CXCR5, which is required for their entry into B cell follicles. T_FH cells access the follicle by upregulating CXCR5 and by downregulating CCR7 and P-selectin glycoprotein ligand 1 (CD162). The T_FH cells with the highest expression of CXCR5 and another marker, PD-1, seem to 'preferentially' accumulate in GCs by sensing the CXCR5 ligand CXCL13 (refs. 2,3) (Fig. 1). T_FH cells also express several costimulatory molecules, including CD40L, ICOS, OX40 and members of the SLAM family, all of which have important roles in T cell–dependent B cell responses driven by cognate T cell–B cell interactions^2,3. T_FH cells are also the source of interleukin 21 (IL-21) and IL-4, cytokines that are necessary for class-switch recombination. In this issue of Nature Immunology, Lifan Xu et al.⁴ and Youn Soo Choi et al.⁵ identify the transcriptional regulators LEF1-1 (encoded by Lef1) and TCF-1 (encoded by Tcf7) as important participants early in the T_FH developmental process (Fig. 1).

Figure 1: LEF-1 and TCF-1 control early steps in the T_FH developmental process.

Marina Corral Spence/Nature Publishing Group

LEF-1 and TCF-1 are positive regulators that control the expression of Bcl6, Il6ra, Il6st and Icos, which encode products known to be required for T_FH differentiation. IgG1, immunoglobulin G1; CCL19 and CCL21, chemokines; TCR, T cell antigen receptor; DC, dendritic cell.

The lineage commitment of T_FH cells begins with the initial priming of naive CD4⁺ T cells by dendritic cells or by other myeloid cell–derived antigen-presenting cells in the T cell zone (Fig. 1). After a few rounds of cell division, these T cells express Bcl-6, a transcriptional repressor that belongs to the BTB-POZ family, whose function is critical for T_FH differentiation, as indicated by the complete absence of T_FH cells among Bcl6^−/− CD4⁺ T cells^6,7,8. Moreover, ectopic Bcl6 expression in CD4⁺ T cells reconstitutes the generation T_FH cells. Bcl-6 expression in B cells is also critical for T_FH development, because T_FH cells are again abrogated in mice with conditional deficiency of Bcl-6 in B cells⁸. Those observations indicate that the transcriptional program of Bcl-6 seems to be controlled via priming by antigen-presenting cells.

It is important both for basic knowledge and for vaccine development to understand how the T_FH program, including regulation of Bcl6 expression, is controlled. Cytokine pathways, such as IL-6–STAT1, IL-12–STAT4 and IL-21–STAT3, have been linked to the induction of T_FH cells and Bcl6 expression². Batf and Ascl2 are critical transcription factors that support T_FH differentiation by controlling the expression of Bcl6 and Cxcr5, respectively, which suggests that Bcl-6 and CXCR5 are regulated by independent molecular mechanisms. Despite such advances, the gap between the regulation of Bcl6 expression and the T_FH program is still puzzling, because there are no obvious mechanisms by which Bcl-6 controls transcriptional programs for T_FH differentiation, and this makes it likely that other unknown factors are involved.

Choi et al.⁵ and Xu et al.⁴ identify two additional participants in the T_FH program: LEF1-1 and TCF-1. These are members of the TCF-LEF subfamily and belong to a family of high-mobility-group proteins that work as downstream repressors of the canonical Wnt signaling pathway. TCF-1 controls mainly the proliferation and population expansion of thymocytes; thus, lack of TCF-1 causes a substantial reduction in thymic cellularity and a partial block in thymocyte differentiation at the transition from the CD8⁺ immature single-positive stage to the CD4⁺CD8⁺ double-positive stage. Although LEF1-1-deficient mice have normal T cell development in the thymus, deficiency in both LEF1-1 and TCF-1 leads to a complete block in differentiation^9,10. TCF-1 and LEF-1 are also involved in the function and formation of memory CD8⁺ T cells¹¹. In CD4⁺ T cells, TCF-1 is reported to be a positive regulator of the expression of genes encoding the transcription factor GATA-3 and IL-17A and a negative regulator of expression of the gene encoding interferon-γ¹².

Choi et al. perform an unbiased transcriptome analysis of early T_FH cells and the T_H1 subset of helper T cells to identify transcription factors that control the T_FH program⁵. They identify Lef1 because it satisfies two further criteria: its 'preferential' expression in early T_FH cells in vivo, and the large effect on differentiation into fully committed GC T_FH cells and the generation of GC B cells when it is ablated. These authors further demonstrate high expression of Tcf7 by early T_FH cells and that synergistic regulation by LEF-1 and TCF-1 is essential for the full T_FH cell program. Studies using a green fluorescent protein (GFP) reporter system (TCF-1–GFP) further support the proposal of the functional importance of TCF-1 in T_FH cells. The expression of TCF-1–GFP is highest in naive T cells but is lower in antigen-primed T cells and effector T cells. After infection with lymphocytic choriomeningitis virus, TCF-1–GFP expression is greatly diminished in T_H1 cells, while high expression is maintained in T_FH cells. Mature T cells with conditional deletion of Lef1 and Tcf7, which lack both Lef-1 and TCF-1, generate considerably fewer fully committed GC T_FH cells than do their wild-type counterparts, which suggests that coordination of LEF-1 and TCF-1 is required for T_FH differentiation through the regulation of circuits upstream of Bcl6. The binding of LEF-1 and TCF-1 to several different genes consequently results in the promotion of Bcl6 expression, sustained expression of the cytokine receptor subunits IL-6Rα and gp130 (IL-6st), and enhanced expression of ICOS to accomplish T_FH differentiation.

Xu et al. also find higher expression of TCF-1 in early committed T_FH cells after infection with lymphocytic choriomeningitis virus, in contrast to its decreased expression in T_H1 cells⁴. They also show diminished development of fully committed T_FH cells resulting from TCF-1 deficiency in two independent conditional deletion systems (T cell–specific deletion (Cd4-Cre) and tamoxifen-induced deletion (ERT2-Cre)), in system of chimeras reconstituted with a mixture of Tcf7^−/− bone marrow and wild-type bone marrow, and in a system of short hairpin RNA–mediated knockdown. However, unlike Choi et al.⁵, they find that TCF-1 needs no cooperation with LEF-1 in the T_FH program. Moreover, T_FH cells lacking TCF-1 show lower expression than TCF-1-sufficient cells of genes encoding several T_FH markers (including Bcl6, Icos, Ascl2, Cxcr5, Il6ra, Il6st, Il21 and Il4) and instead show increased expression of non-T_FH cell signature genes (Tbx21, Gzmb, Gata3, Rorc and Foxp3); this suggests that TCF-1 is a cell-fate regulator that functions by suppressing the differentiation programs of non-T_FH effector cells.

Xu et al. further demonstrate that expression of TCF-1 is critical for the axis of Bcl-6 and the transcription factor Blimp-1 in T_H1-versus-T_FH cell-fate determination⁴. Studies of an in vitro 293T human embryonic kidney cell overexpression system indicate that co-expression of the p33 isoform of TCF-1, Bcl-6 and TLE3 (the dominant member of the TLE corepressor complex) in CD4⁺ T cells enhances formation of the p33–Bcl-6 complex. These in vitro data indicate that TCF-1 binds both directly and indirectly to the Bcl6 promoter to upregulate its expression, while indirect binding of the p33–Bcl-6 complex to 5′ regulatory regions of the gene encoding Blimp-1 represses Blimp-1 expression. Given these results, Xu et al. propose that TCF-1 is an upstream regulator of the Bcl-6–Blimp-1 axis required for T_FH differentiation⁴.

These two studies raise some interesting questions about how the activity of TCF-1 and/or LEF-1 is able to influence the differentiation program of T_FH cells and other helper T cell subsets. Both studies agree on the point that TCF-1 is a positive regulator of Bcl-6 and a negative regulator of Blimp-1, but there is clear discrepancy in the proposed activity of LEF-1. Choi et al. suggest that LEF-1 is needed in a synergistic way with TCF-1 to regulate commitment to the T_FH lineage⁵, while Xu et al. raise the conflicting possibility that LEF-1 may not necessarily lead to full commitment⁴. TCF-1 directly binds to the Bcl6 promoter to control its expression regardless of LEF-1's binding, whereas TCF-1 can repress Lef1 expression. TCF-1 is needed to inhibit Blimp-1 activity to stop differentiation into other helper T cell subsets. In the same context, TCF-1 is known to be positive regulator for the functions of T_H2 cells and T_H17 cells¹¹, which indicates that TCF-1 must have a complicated role to be able to provide positive differentiation signals for both T_FH cell subsets and non-T_FH cell subsets. Indeed, a published study has indicated that T_FH cells can differentiate from T_H2 cells able to express several T_FH cell signature genes¹³. Since both T_H2 cells and T_FH cells need TCF-1 activity for their differentiation, TCF-1 may provide the mechanism by which T_H2 cells have the potential to differentiate into T_FH cells. Choi et al.⁵ and Xu et al.⁴ propose several interesting molecular mechanisms downstream of TCF-1 and LEF-1, but it remains unclear whether the T_FH program is regulated by a single mainstream pathway or by the coordination of multiple pathways. Further research should focus on determining how molecules downstream of TCF-1 and LEF-1 control the T_FH-differentiation program.