Development and function of follicular helper T cells

Abstract

Most currently available vaccines rely on the induction of long-lasting protective humoral immune responses by memory B cells and plasma cells. Antibody responses against most antigens require interactions between antigen-specific B cells and CD4⁺ T cells. Follicular helper T cells (T_FH cells) are specialized subset of T cells that provide help to B cells and are essential for germinal center formation, affinity maturation, and the development of high-affinity antibodies. T_FH-cell differentiation is a multistage process involving B-cell lymphoma 6 and other transcription factors, cytokines, and costimulation through inducible costimulator (ICOS) and several other molecules. This article reviews recent advances in our understanding of T_FH cell biology, including their differentiation, transcriptional regulation, and function.

Graphic Abstract

Development of follicular helper T cells is controlled by TCR, cytokine, and ICOS-mediated signals, and downstream transcription factors.

Key words:

• helper T cell
• follicular helper T cells
• germinal center
• transcription factors
• antibody production

Cognate interactions between antigen (Ag)-specific B cells, CD4⁺ T cells, and dendritic cells (DCs) in response to foreign Ags lead to the formation of germinal centers (GCs). GCs are specialized structures in B-cell follicles of secondary lymphoid organs. In GCs, somatic hypermutation of immunoglobulin variable genes is induced in B cells and selection of high-affinity B cells takes place¹⁾, followed by the generation of long-lived memory B cells or plasma cells. This process ensures the development of long-lived humoral immunity after infection or vaccination with T cell-dependent Ag.

It has been known that CD4⁺ T cells are required for the formation of productive GC responses^1–3), as well as for generating Ag-specific memory B cells and plasma cells. However, the exact nature of the CD4⁺ T cells that provides help to B cells remained enigmatic. More than two decades ago, an in vitro study by Coffman and Mosman first discovered the heterogeneity of effector T cells, which were then named as T-helper 1 (T_H1) or T_H2 cells⁴⁾. It showed that T_H2 cells were the major T_H subset engaged in helping B cells by secreting interleukin 4 (IL-4), IL-5, IL-10, and IL-13. T_H1 cells also contributed to the regulation of antibody response by producing interferon-γ (IFN-γ) and inducing B-cell class switching toward IgG2a. The T_H1/T_H2 dichotomy has dominated the field of immune regulation until recently. However, it has been unclear whether those cells really represent the cells which engage in B cell help in lymphoid organs.

In recent years, T follicular helper (T_FH) cells have been recognized as the key cell type required for the formation of GCs and the generation of long-lived humoral memory⁵⁾. Similar to other CD4⁺ T-cell lineages (T_H1, T_H2, T_H17, and regulatory T cells), the generation of T_FH cells requires signaling pathways activated downstream of cytokines and cell surface molecules, and the subsequent activation of transcription factors. Here, I discuss recent advances in understanding the requirements for the generation and acquisition of effector function by T_FH cells.

I. T follicular helper cells

The presence of CD4⁺ T cells expressing CXCR5, a chemokine receptor required for the migration into B-cell follicles, was first reported in 1999⁶⁾. CXCR5 was up-regulated on antigen-specific CD4⁺ T cells in the lymph nodes in Ag-immunized mice. Subsequently in the early 2000, the presence and function of human CXCR5⁺CD4⁺ T cells have been revealed. CD4⁺ T cells in human tonsils were found to express CXCR5. Importantly, such CXCR5⁺CD4⁺ T cells were potent to induce antibody production by B cells in vitro, relative to CXCR5⁻ counterparts. On the basis of their localization and functions, tonsillar CXCR5⁺ CD4⁺ T cells were designated as T_FH cells^7–9). A superior capacity of CXCR5⁺ T_FH cells to induce B-cell activation was confirmed in mouse system¹⁰⁾. T_FH cells show distinct cytokine production and gene expression pattern from T_H1, T_H2, and T_H17 cells^11–13). T_FH cells utilize TNF family molecule CD40L and the cytokine IL-21 and IL-4 to help B cells^14–17). In 2009, the transcription factor Bcl6 was discovered to be an essential factor for T_FH-cell generation in vivo in mice^18–20) and since then T_FH cells have been recognized as a T_H subset distinct from T_H1, T_H2, and T_H17 cells (Fig. 1).

Fig. 1. Differentiation of naïve CD4⁺T cells into distinct helper T-cell subsets.

II. Development of T_FH cells

T_FH-cell differentiation has been proposed to be a multistage process involving serial interaction of CD4⁺ T cells, first with DCs and then with antigen-presenting B cells (Fig. 2). The presentation of antigen by DCs is required to initiate the T_FH-cell differentiation program²¹⁾. Bcl6 induction occurs quite early during the DC priming stage and inducible costimulator ICOS is a key upstream molecule for induction of Bcl6 and thus instruction of T_FH-cell differentiation²²⁾. Initial Bcl6 induction and subsequent CXCR5 expression occur in T-cell zone; the activated CD4⁺ T cells then relocate to the T–B border, where early T_FH cells interact with B cells to maintain Bcl6 expression for full differentiation and relocation into GCs as mature T_FH cells^18,21–24). The origin of T_FH cells may not be restricted to naïve CD4⁺ T cells. Some evidences have suggested that other T_H subsets including T_H1, T_H2, T_H17, and regulatory T cells have a potential to acquire T_FH-cell phenotype in GCs, although there is still controversy regarding this issue⁵⁾. Importantly, GC-T_FH cells show heterogeneity in cytokine expression patterns under different immunization protocols and by different types of infectious agents. They are capable of expressing not only T_FH signature cytokine IL-21, but also IL-4 or IFN-γ^25,26).

Fig. 2. Multiple steps involved in T_FH-cell development.

Notes: T_FH-cell development requires serial interaction with DCs and antigen-specific B cells. Upon priming with dendritic cells, some of the activated T cells up-regulate a chemokine receptor, CXCR5, and transcription factors, including Bcl6 (a). The activated CD4⁺ T cells migrate toward the border of T-cell zone and B-cell follicle, where the T cells interact with antigen-specific B cells (b). This interaction with B cells further up-regulates CXCR5 and Bcl6, while down-regulates CCR7, which allows the T cells relocate to B-cell follicle (TFH) (c). Some of TFH cells participate in germinal center (GC) reaction (GC-T_FH) (d).

Differentiation of CD4⁺ T cell into distinct effector T_H subset is mediated in large part by exposure to various cytokines. For example, Th1 cells develop in the presence of IL-12 and IFN-γ, whereas differentiation of T_H2 cells requires IL-4²⁷⁾. T_FH-cell differentiation is not exceptional. IL-6 appears to be the earliest cytokine signal involved in initiation of T_FH-cell differentiation in vivo. In the absence of IL-6, early Bcl6⁺CXCR5⁺ T_FH differentiation is severely impaired²⁸⁾. Nonetheless, in vitro culture of murine CD4⁺ T cells with IL-6 failed to generate the T cells with complete T_FH phenotypes. In vitro, IL-6 is an indeed potent inducer of IL-21^19,29). Although IL-6 was initially reported to drive CXCR5 and Bcl6 expression in vitro¹⁹⁾, neither Bcl6 nor CXCR5 was induced by IL-6 in vitro in two other studies^29,30). These studies suggest that cytokines other than IL-6 may be involved in complete T_FH differentiation. Indeed, a recent paper reports that type I IFNs (IFN-α/IFN-β) are potent cytokines to induce T_FH-like cells in vitro³¹⁾. Type I IFNs promote sustained Bcl6 expression and induces CXCR5⁺PD1⁺ cells. Although type I IFNs fail to induce IL-21 by activated CD4 + T cells, addition of IL-6 compensates IL-21 production. Hence, type I IFNs + IL-6 appears to be the most successful conditions for murine T_FH-cell differentiation in vitro, with the robust induction of CXCR5, PD1, Bcl6, and IL-21. In contrast, IL-2 interferes with T_FH-cell development. It has been demonstrated that IL-2 administration impaired the differentiation of T_FH cells, thus resulting in a reduction of GCs and long-lived antibody responses³²⁾. IL-2 signaling directly inhibits T_FH-cell response, since IL-2Rα-deficient CD4⁺ T cells are more prone to become T_FH cells in vivo³²⁾.

In a past few years, significant progress has been made in understanding of human T_FH-cell development. It appears that the development of T_FH cells might differ between mice and humans. In humans, TGF-β acts with IL-12 or IL-23 to promote the expression of multiple T_FH signature molecules, including CXCR5, IL-21, and Bcl6³³⁾. This is in sharp contrast to murine CD4⁺ T cells, in which TGF-β inhibits the expression of T_FH molecules such as IL-21, ICOS, and Bcl6^19,34,35), implying a difference in T_FH differentiation mechanisms between species. In vitro human T_FH cells generated in the presence of TGF-β and IL-12 or IL-23 had enhanced B cell help activity, indicating that TGF-β signaling is important for human T_FH differentiation and function.

III. Transcriptional regulation of T_FH-cell development

Bcl6 is highly expressed in murine and human T_FH cells and is essential for T_FH differentiation. In the absence of Bcl6, T_FH cells are unable to form and subsequently GCs are not present^18–20). Like other lineage-defining factors for other T_H subsets, such as T-bet, GATA3, RORγt, or Foxp3, overexpression of Bcl6 not only enforces T_FH-cell differentiation but also attenuates differentiation to other T_H subsets^18–20). In addition to Bcl6, several transcription factors are demonstrated to be required for T_FH development. These include basic leucine zipper transcription factor (Batf), interferon regulatory factor 4 (IRF4), Maf, achaete-scute homologue 2 (Ascl2), Foxp1, and signal transducers and activators of transcription (STATs). Action of these transcription factors in T_FH development is summarized in Fig. 3.

Fig. 3. Transcription factors that regulate T_FH-cell development.

Notes: Many transcription factors are involved in T_FH-cell development. Transcription factors shown in boxes with straight lines are the positive regulators, whereas those in boxes with dashed lines are the negative regulators for T_FH development. Among them, Bcl6 plays a central role during T_FH-cell development. STAT1, STAT3, BATF, and IRF4 positively regulate Bcl6 induction, whereas a STAT5-Blimp1 axis inhibits Bcl6 induction. Ascl2 is involved in CXCR5 expression. c-Maf is required for optimal IL-21 expression. Foxp1 negatively regulates IL-21 or ICOS expression.

Batf and IRF4 are both essential for T_FH-cell development^36,37) but they are required for multiple different CD4⁺ T-cell programs, including T_H17^38,39), T_H2⁴⁰⁾, and T_H9^41,42). Batf and IRF4 are induced in non-polarized T cells receiving stimulation and are found to be bound to the target genes independently of polarizing conditions⁴³⁾. Thus, it can be considered that these factors act as “pioneering factors” that enable expression and function of downstream cell-fate-determining factors⁴⁴⁾, including Bcl6.

c-Maf is highly expressed in T_FH cells and c-Maf-deficient chimeric mice exhibited defected T_FH cells⁴⁵⁾. T_FH-cell impairment in the absence of c-Maf is due to defective induction or maintenance of IL-4 and IL-21 in an ICOS-dependent manner⁴⁵⁾. A recent study has demonstrated that Bcl6 and c-Maf cooperate to instruct human T_FH-cell differentiation and that c-Maf induces IL-21 and CXCR5 expression⁴⁶⁾.

A basic helix-loop-helix (bHLH) transcription factor, Ascl2, is highly expressed in T_FH cells³⁰⁾. Overexpression of Ascl2 specifically up-regulates CXCR5, but not Bcl6 or other T_FH signatures in T cell in vitro, as well as accelerating T-cell migration to the B-cell follicles and T_FH-cell development in vivo³⁰⁾. Acute deletion of Ascl2 results in impaired T_FH-cell development and GC responses³⁰⁾. Ascl2 directly regulates T_FH-related genes including CXCR5 or CXCR4, whereas it inhibits expression of T_H1 and T_H17 signature genes³⁰⁾.

The transcription factor Foxp1 is expressed in resting naïve CD4⁺ T cells and is required for quiescence and homing of naïve CD4⁺ T cells⁴⁷⁾. Naïve CD4⁺ T cells deficient in Foxp1 preferentially differentiate into T_FH cells, which results in substantial enhancement of GC and antibody responses⁴⁸⁾. Foxp1 directly and negatively regulates IL-21. Foxp1 also dampens expression of ICOS and its downstream signaling at early stages of T-cell activation. Thus, Foxp1 is a critical negative regulator of T_FH-cell differentiation.

In response to cytokine stimulation, STATs are also deeply involved in T_FH differentiation Function of STATs during T_FH differentiation is somewhat complex and overlapping. STAT3-deficient CD4⁺ T cells have a profound defect in T_FH-cell differentiation^49,50). It has been shown that STAT3 is involved in IL-21 expression by CD4⁺ T cells³⁵⁾ and in down-regulation of IL-2Rα to limit T_H1 cell differentiation²⁸⁾. STAT1 is required for IL-6-mediated Bcl6 induction for early T_FH differentiation²⁸⁾. STAT1 is also activated by type I IFN and activated STAT1 directly regulates key T_FH genes, including Bcl6 and CXCR5³¹⁾. Interestingly, IL-12-STAT4 axis positively regulates the differentiation of T_FH cells in mice and human, although STAT4 appears to be more important in human T_FH differentiation⁵¹⁾. In contrast, as mentioned above, IL-2 signals through STAT5 to limit T_FH-cell differentiation^32,52,53). STAT5 induces the transcriptional repressor Blimp-1⁵²⁾, which serves to repress Bcl6 and T_FH-cell differentiation¹⁸⁾. Thus, the IL-2/STAT5/Blimp1 axis limits T_FH-cell generation by suppressing Bcl6.

IV. Function of T_FH cell

T_FH cells are essentially required for the induction of GC. However, T_FH cells play important roles even after GCs are established (Fig. 4). Once T_FH cells are generated, some of them further up-regulate CXCR5 and Bcl6 and migrate from follicles to GC (GC-T_FH cells). As mentioned at the beginning, GC is the primary site of affinity maturation of B cells and recent evidences have shown that GC-T_FH cells are essential for achieving the goal of GC responses, which is to generate and select GC B cells with higher affinity for the antigens¹⁾. GC is divided into two anatomically distinct regions: the light zone (LZ) and the dark zone (DZ). GC B cells undergo proliferation and somatic hypermutation in the DZ and then migrate to the LZ. In the LZ, GC B cells capture antigen through surface immunoglobulin and present it. Selection of high-affinity GC B cells take place in LZ as a result of the interaction with cognate GC-T_FH cells. GC-T_FH cells discern the high-affinity GC B cells according to the level of presented antigenic peptide on their MHC class II and help high-affinity GC B cell clones to expand, hypermutate, or differentiate into long-lived plasma cells or memory B cells (Fig. 4). These selection events are controlled by T_FH-cell-derived signals, including ICOS, CD40L, and cytokines IL-4 and IL-⁵⁾. Recent intravital imaging revealed that GC-T_FH cells continually scan the surface of many GC B cells in the LZ and form short-lived contacts which results in the selection of high-affinity B-cell clones^54,55). Interestingly, GC-T_FH cells are not confined to GC. Unlike GC B cells, which are clonally restricted, T_FH cells distributed among all GCs and continually emigrated into the follicle and entered a different GC⁵⁵⁾ (Fig. 4). Moreover, newly activated T_FH cells invade preexisting GCs, where they contribute to B-cell selection. Conceivably, the dynamic exchange of T_FH cells between GCs ensures diversified and robust support for B-cell clonal expansion and affinity maturation.

Fig. 4. Function of T_FH cells in GCs.

Notes: (A) GC-T_FH cells localize in the light zone of GCs and scan the amount of antigenic peptide presented on GC B cells. GC B cells with high-affinity BCRs present high levels of antigenic peptide and get survival signals from GC-T_FH cells. Selected high-affinity B cells differentiate into plasma cells or memory B cells, or migrate back to the dark zone of GC to undertake further round of proliferation and somatic hypermutation. (B) GC-T_FH cells are not confined to GC. GC-T_FH cells can emigrate to the follicles and enter different GCs (a) or relocate to the T-cell zone (b). Further, newly generated T_FH cells can participate in preexisting GCs (c).

V. Memory T_FH

Although the existence of T_FH-cell memory was initially controversial, accumulating evidences now clearly show that T_FH cells can give rise to resting memory T cells (Memory T_FH cells) in both mice and human^24,56–60). Memory T_FH cells can be long-lived in the absence of the antigens⁶⁰⁾. Upon leaving a GC, the T_FH cells acquire a less activated and less polarized phenotype: Memory T_FH cells down-regulate most of the canonical T_FH markers, including PD1 and Bcl6, but up-regulate CCR7, IL-7Rα, and CD62L^57,58,60,61). Notably, memory T_FH cells maintain low, but significant levels of CXCR5 expression^57,58,62), which makes memory T_FH cells distinguishable from other memory T cells. Memory T_FH cells have the capacity to recirculate in blood^59,63). Circulating T_FH memory cells are a heterogeneous pool of cells having distinct functions which is broadly categorized according to the expression of several chemokine receptors. This issue is discussed in recent reviews⁶⁴⁾.

Memory T_FH cells preferentially become T_FH cells and GC-T_FH cells upon reactivation^24,58,60). Thus, it appears that T_FH memory cells are committed populations that are poised for the lineage-specific reexpression of effector molecules upon recall. How this could be achieved is currently unknown. But it has been demonstrated that distinct DNA methylation pattern of Gzmb locus between memory T_FH and memory T_H1 cells⁵⁸⁾, suggesting that epigenetic modification of lineage-specific gene locus reinforces the T_FH lineage commitment. Functionally, memory T_FH cells are potent in helping B-cell activation⁶⁵⁾. A recent study demonstrated that memory T_FH cells are required for efficient memory B-cell responses⁶²⁾. In the absence of memory T_FH cells, activation of memory B cells to differentiate into plasma cells is compromised. Importantly, antigen-specific memory B cells, but not DCs, function as antigen-presenting cells for memory T_FH cells⁶²⁾, suggesting that cognate interaction between memory T_FH cells and memory B cells elicit efficient activation of memory antibody responses.

VI. Conclusion

Since the identification of T_FH cells just over a decade ago, much has been discovered about the function of T_FH cells and the molecules required for their differentiation. T_FH cells are essential for the generation of affinity-matured antibodies; therefore, they have an obvious role not only in protective immunity against pathogen but also in the induction of a range of diseases, particularly autoimmune diseases. The progress in the understanding of T_FH biology will lead to improved vaccine designs and better management of autoimmune diseases or allergy.

Disclosure statement

No potential conflict of interest was reported by the authors.

Notes

This review was written in response to the author’s receipt of the Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for the Encouragement of Young Scientist in 2011.