Editorial: Masters of fate: the APC cytokine milieu as a key regulator of distinct Tfh cell subsets

The development of protective antibody responses largely depends on CD4⁺ T cells that provide help to B cells in the form of cell-cell contacts and secretion of cytokines. CD4⁺ T cells are essential for the formation of GCs, distinct microanatomical structures within secondary lymphoid organs where antibody class-switching, affinity maturation, and the differentiation of high-affinity B cells into memory cells occur [1]. A specialized subset of CD4⁺ T cells, named Tfh cells, found near B cell follicles and within GCs, is exceptionally proficient at inducing GC B cell responses. Tfh cells are defined phenotypically by expression of PD-1, ICOS, and IL-21 and by expression of the chemokine receptor CXCR5 that is required for the migration of Tfh cells into follicles [2]. The transcriptional repressor Bcl-6 is essential for the generation of Tfh cells, and its expression distinguishes Tfh cells as a separate lineage of Th cells distinct from other CD4⁺ T cell subsets, such as Th1 and Th2 cells [3]. Studies in mice have demonstrated that Tfh cells are required for the formation of GCs, and in the absence of Tfh cells, the generation of memory B cells and long-lived plasma cells that produce high-affinity antibodies is impaired [2]. Although numerous studies have demonstrated that Tfh responses are essential for humoral immunity, there is also evidence that both deficient and excessive Tfh responses might contribute to the development of disease. Patients with primary immune-deficiencies, humoral autoimmunity, and certain cancers are associated with defects in genes that regulate Tfh differentiation and GC formation [4]. These associations suggest that maintaining a balance in Tfh responses is critical for host immune protection. Therefore, many investigators are actively pursuing the developmental regulation of Tfh cells and how they shape antibody responses.

Recent studies in humans have described coexpression of Bcl-6 and transcription factors associated with traditional Th cell subsets [3, 5]. These findings have led to the identification of Tfh-1 (T-bet⁺IFN-γ⁺), Tfh-2 (GATA3⁺IL-4⁺), and Tfh-17 (retinoic acid-related orphan receptor γt⁺IL-17⁺) subsets, highlighting the complexity of Tfh cells and their potential to impact differentially the antibody response through distinct cytokine profiles. Recognition of functionally distinct Tfh subsets in mice is less clear, but there is evidence that mouse Tfh cells comprise heterogeneous subsets that secrete cytokine profiles similar to Th1, Th2, and Th17 cells [6, 7]. Signals derived from APCs and the microenvironment of secondary lymphoid organs induce the early Tfh differentiation process and include multiple cytokines. The major cytokines that support Tfh differentiation in humans are IL-12, IL-23, and TGF-β, but IL-6, IL-21, and IL-1P can further help this differentiation process. How and whether these cytokines influence the differentiation of discrete Tfh subsets in humans and in mice are not well understood. The study by Hercor and colleagues [8] in this issue begins to elucidate the signals derived from DCs that fine-tune the balance of murine Tfh subsets, identifying the STAT3-activating cytokine IL-6 as an important mechanism for curtailing Tfh-2 responses and consequently, the production of IgE.

Most of our knowledge of the developmental mechanisms of Tfh cells has come from analyses of circulating human blood Tfh cells and from studies in mouse models. Through this work, it has become increasingly clear that the Tfh cell compartment is heterogeneous in phenotype and function. Human blood Tfh cells express CD4 and CXCR5 but lack expression of CD45RA, a characteristic of memory cells, and can be divided further into 3 major subsets based on the differential expression of specific chemokine receptors and cytokines. These include CXCR3⁺CCR6⁻ cells that secrete IFN-g and thus, are designated Tfh-1 cells; CXCR3⁻CCR6⁻ cells that secrete IL-4 and thus, are called Tfh-2 cells; and CXCR3⁻CCR6⁺ cells that secrete IL-17 and are called Tfh-17 cells [5]. Functional analysis of Tfh-1 cells to provide help to naive and memory B cells has demonstrated that Tfh-1 cells are poor helpers, as they failed to induce antibody secretion. In contrast, Tfh-2 cells potently stimulate B cells to produce IgG and induce antibody class-switching to IgE. Tfh-17 cells can also stimulate B cells to produce IgG and in addition, promote class-switching to IgA. How these functionally distinct blood Tfh subsets relate to lymphoid organ-resident Tfh cell subsets remains unclear and is an important research area for future work that should advance our understanding of the developmental mechanisms of human Tfh cells. Mouse Tfh cells are also heterogeneous, with evidence supporting the presence of Tfh subsets that share cytokine profiles with Th1, Th2, and Th17 cells. In particular, 2 studies using IL-4 reporter mice elegantly described the formation of GATA3⁺IL-4⁺ Tfh-2 cells that drive IgG1 and IgE antibody responses in reaction to helminth infection [6, 7]. An interesting aspect of this work was the finding that although IgG1 and IgE production was compromised in the absence of IL-4 signaling, the generation of Tfh cells remained intact [7]. This study provides an important clue that certain cytokines in secondary lymphoid organs are dispensable for Tfh development but might be important for instructing the cytokine profile of early Tfh precursors to generate an appropriate protective antibody response.

Despite these advances demonstrating phenotypically and functionally diverse Tfh cell subsets, relatively little is known about the mechanisms that control Tfh differentiation into discrete subsets. Given that activated myeloid APCs promote the early development of Tfh cells, APCs are an attractive candidate for supplying cytokines that influence the fate of Tfh subsets. Resolving the APC-derived cytokines involved in Tfh generation from those that alter Tfh subset differentiation has been a challenging task. In particular, the role of APC-derived IL-6 in these 2 processes has been a matter of debate. Work by Chakarov and Fazilleau [9] demonstrated that IL-6 produced by DCs is important for Tfh generation, as mice with DCs deficient of IL-6 exhibited substantially reduced Tfh cell numbers and GC responses after immunization. In agreement with these findings, IL-6 was also found to be a critical signal for CD4+ T cells to produce IL-21 that induces a beneficial IgG antibody response to influenza virus [10]. However, this study did not distinguish whether IL-6 affected the generation of Tfh cells or the differentiation of specific Tfh subsets. Curiously, IL-6 has also been shown to play a role in restricting Th2 cell responses. Earlier work, carried out by the Andris laboratory [11], identified that IL-6 secretion by DCs restrains Th2 responses, as measured by reduced differentiation of GATA3+CD4+T cells and IL-4 production, which is reversed when DCs are IL-6 deficient. This dichotomy of how APC-derived IL-6 influences the quality of T cell help could be attributed to differences in the experimental systems used in the above studies. However, delineating the role of IL-6 in the generation of Tfh cells versus effects on the differentiation of Tfh subsets is an important question to address.

Hercor and colleagues [8] examined whether the effects of DC-derived IL-6 to restrain Th2 responses includes regulation of Tfh-2 subset differentiation. To determine if IL-6, derived from APCs, plays a role in the generation of Tfh cells, WT mice were immunized with IL-6-deficient DCs that were pulsed with the antigen KLH. The resulting immune response exhibited an increased population of IL-4⁺ Tfh cells and reduced IL-21⁺ Tfh cells compared with mice immunized with WT KLH-pulsed DCs. These findings suggest that in the absence of IL-6 signaling, APCs drive Tfh-2 differentiation. Correspondingly, this skewing toward a Tfh-2 phenotype resulted in significantly increased serum antigen-specific IgE levels. An increase in GATA3 expression levels in Tfh cells from mice immunized with IL-6-deficient DCs further confirmed that the balance of functionally distinct Tfh subsets was shifted toward a Tfh-2 phenotype when CD4⁺ T cells were primed in the absence of IL-6. Interestingly, IL-6 production by DCs was found to be dispensable for Tfh development, as mice immunized with IL-6-deficient and WT DCs generated equivalent numbers that expressed normal Bcl-6 levels. The work by Hercor et al. [8] also determined that the effect of DC-derived cytokines to inhibit Tfh-2 responses was specific to IL-6. IL-12 has also been identified as an important cytokine for Tfh differentiation [2]. Tfh cells from mice immunized with IL-12-deficient, KLH-pulsed DCs expressed low levels of GATA3 and IL-4 that were similar to controls. Finally, Hercor et al. [8] assessed whether the transcription factor STAT3, the major signaling pathway downstream of IL-6R, played a role in Tfh subset differentiation. Mice with T cell-specific deletion of STAT3 generated significantly greater numbers of GATA3⁺ Tfh cells and increased IgE serum levels when immunized with antigen compared with WT controls. Collectively, these studies suggest that CD4⁺ T cell priming by DCs does not require IL-6 for Tfh lineage development but that IL-6 modulation of the STAT3 signaling pathway instructs early Tfh precursors or possibly pre-existing Tfh cells to down-regulate a Th2 phenotype (Fig. 1). These findings have important implications, as they suggest the plasticity of Tfh cells to respond to certain antigens, such as Th1-, Th2-, or Th17-polarizing pathogens, which in turn, fine tune an appropriate antibody response.

Figure 1. DCs modulate Tfh-2 responses through the IL-6/STAT3 signaling pathway.

(A) DC-derived IL-6 induces STAT3 activation in Tfh cells, causing down-regulation of GATA3 expression and the production of IL-4. This results in the lack of B cell helper activity to drive IgE production. (B) In the absence of IL-6 signaling from DCs, GATA3 is expressed, resulting in the development of IL-4-secreting Tfh-2 cells that promote IgE production.

The understanding of the mechanisms that skew Tfh subset differentiation could provide novel strategies to increase the amount, quality, and durability of protective antibody responses important for vaccine design. How different adjuvants affect APCs to drive the generation of Tfh-1, Tfh-2, and Tfh-17 cells is unknown and is important to address for the design of effective vaccines. In addition, knowledge of how Tfh subsets are regulated may help identify new targets for the treatment of diseases where Tfh cells play a role in the production of pathogenic antibodies. These include autoimmune and allergic diseases that involve both aberrant Tfh cell accumulation and alterations in the balance among Tfh-1, Tfh-2, and Tfh-17 cells. Increased ratios of blood Tfh-2 and Tfh-17 cells to Tfh-1 cells have been observed in patients with systemic lupus erythematosus, juvenile dermatomyositis, and Sjögren’s syndrome [4]. Such alterations in circulating Tfh subsets might reflect uncontrolled GC responses in secondary lymphoid organs and in inflamed tissues of patients with autoimmune disease that if restrained through modulating APC-derived cytokines, could reduce autoantibody production and disease activity. Strategies to target the IL-6/STAT3 axis might also be an effective approach for the treatment of atopic asthma and allergy to skew Tfh cells away from a Tfh-2 phenotype, thereby reducing pathogenic IgE production. Interestingly, mutations in STAT3 are tightly linked with hyper-IgE syndrome, and patients with this disease often have reduced frequencies of blood Tfh cells [12]. Finally, the signals that regulate Tfh-2 cells might be attractive therapeutic targets for certain cancers by reducing cytokines in the tumor microenvironment important for tumor survival. IL-4⁺ Th cells that express characteristics of Tfh cells have been found within tumors of follicular B cell lymphoma and may contribute to tumorigenesis by creating a prosurvival cytokine environment [4].

Abbreviations:

Bcl-6

B cell lymphoma 6

DC

dendritic cell

GC

germinal center

KLH

keyhole limpet hemocyanin

PD-1

pro-grammed cell death 1

Tfh

T follicular helper

WT

wild-type