Functional differentiation and regulation of follicular T helper cells in inflammation and autoimmunity

Summary

Follicular T helper (T_FH) cells are specialized T cells that support B cells, which are essential for humoral immunity. T_FH cells express the transcription factor B‐cell lymphoma 6 (Bcl‐6), chemokine (C‐X‐C motif) receptor (CXCR) 5, the surface receptors programmed cell death protein 1 (PD‐1) and inducible T‐cell costimulator (ICOS), the cytokine IL‐21 and other molecules. The activation, proliferation and differentiation of T_FH cells are closely related to dynamic changes in cellular metabolism. In this review, we summarize the progress made in understanding the development and functional differentiation of T_FH cells. Specifically, we focus on the regulatory mechanisms of T_FH cell functional differentiation, including regulatory signalling pathways and the metabolic regulatory mechanisms of T_FH cells. In addition, T_FH cells are closely related to immune‐associated diseases, including infections, autoimmune diseases and cancers.

The progress about the development and functional differentiation of T_FH cells. The regulatory mechanisms of T_FH cell functional differentiation, including regulatory signalling pathways and the metabolic regulatory mechanisms of T_FH cells. T_FH cells are closely related to immune‐associated diseases, including infections, autoimmune diseases, and cancers.

Abbreviations

AITD

autoimmune thyroid disease

AMP

adenosine 5’‐monophosphate

AMPK

activated protein kinase

APCs

antigen‐presenting cells

ApoAI

apolipoprotein AI

AS

atherosclerosis

BC

breast cancer

Bcl‐6

B cell lymphoma 6

BTLA

B‐ and T‐lymphocyte attenuator

CIA

collagen‐induced arthritis

CRC

colorectal cancer

CXCR 5

chemokine (C‐X‐C motif) receptor 5

DAMPs

damage‐associated molecular patterns

DCs

dendritic cells

FDCs

follicular dendritic cells

Foxo

forkhead box O

GATA‐3

GATA‐binding protein 3

GCs

germinal centres

GD

glucose deprivation

HIV

human immunodeficiency virus

IBD

inflammatory bowel disease

ICOS

inducible T cell co‐stimulator

IFN‐γ

interferon‐γ

IRF4

interferon regulatory factor 4

JDM

juvenile dermatomyositis

LCMV

lymphocytic choriomeningitis virus

LZ

light zone

MS

multiple sclerosis

PAMPs

pathogen‐associated molecular patterns

PD‐1

programmed cell death protein 1

PKC

protein kinase C

PPs

peyer’s patches

pSS

sjogren’s syndrome

RA

rheumatoid arthritis

RORγt

retinoid‐related orphan nuclear receptor γt

S1PR1

sphingosine‐1‐phosphate receptor

SAP

SLAM‐associated protein

SLE

systemic lupus erythaematosus

STATs

signal transducers and transcriptional activators

T1D

type 1 diabetes

TCR

T cell receptor

T_FH

follicular T helper

T_H1

type 1 T helper cells

TNF‐α

tumour necrosis factor‐α

Tox

thymocyte selection‐associated high mobility group box protein

WT

wild‐type

INTRODUCTION

Effector CD4⁺ T helper cells (T_H) include multiple cell subsets, such as type 1 T helper (T_H1) cells, T_H2 cells, T_H9 cells and T_H17 cells, regulatory T (T_reg) cells and follicular T helper (T_FH) cells. ¹ , ² , ³ , ⁴ (Figure 1). Th cells are divided into different subgroups according to the different effector cytokines secreted and the specific transcription factors expressed, which play decisive roles in each subgroup. ³ , ⁴ , ⁵ In response to stimulation by antigens and cytokines, naïve T cells differentiate into T_H1, T_H9, T_H17 and other cell subsets (Figure 1). The main effector factors secreted by T_H1 cells are interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), and T_H1 differentiation is mainly regulated by the transcription factor T‐Box transcription factor (T‐bet); T_H2 cells are regulated by the transcription factor GATA‐binding protein 3 (GATA‐3) and produce the cytokines interleukin (IL)‐4 and IL‐13; T_H17 cells express the transcription factor retinoid‐related orphan nuclear receptor γt (RORγt) and produce the cytokine IL‐17; and T_H9 cells express the transcription factors PU.1 and interferon regulatory factor (IRF) 4 and produce the cytokine IL‐9. ³ , ⁴ , ⁵ , ⁶ , ⁷ T_reg cells express the specific transcription factor Foxp3, produce the cytokine TGF‐β and play immunosuppressive and immunomodulatory roles. ⁸ , ⁹ The differentiation of T‐cell subsets is determined by the cytokines expressed during T‐cell activation. ¹⁰ For example, IL‐12 induces T‐bet in T_H1 cells, and IL‐6 or IL‐23 can induce RORγt activation during T_H17 cell differentiation. ⁸ , ¹¹ , ¹² , ¹³

Figure 1.

Different T helper cell subsets. Effector CD4⁺ T‐cell lineage differentiation and the expression of transcription factors.

T_FH cells develop differently from other subsets of effector CD4⁺ T helper cells, express the main regulatory transcription factor B‐cell lymphoma 6 (Bcl‐6) and produce the cytokine IL‐21. ¹ , ² , ³ , ⁴ T_FH cells are a newly discovered subset of T cells that play an important role in regulating B‐cell‐mediated humoral immunity (Figures 1 and 2). T_FH cells were originally characterized as unique activated CD4⁺ T cells in the germinal centres (GCs) of the human tonsil that express chemokine (C‐X‐C motif) receptor (CXCR) 5 and promote the differentiation and function of B cells. ¹⁴ Subsequent gene expression and phenotypic analysis showed the anatomical localization and upregulated expression of cell surface proteins, including inducible T‐cell costimulatory molecule (ICOS). ¹⁵ , ¹⁶ , programmed cell death (PD)‐1. ¹⁷ , B‐ and T‐lymphocyte attenuator (BTLA) and CD40L. ¹⁸ , ¹⁹ , which indicate that T_FH cells are different from other T helper cell populations. T_FH cells express the master transcription factor Bcl‐6 and the cytoplasmic adapter and signalling molecule SLAM‐associated protein (SAP), which are required for T_FH cell differentiation, and secrete the cytokine IL‐21. ¹⁸ , ²⁰

Figure 2.

Overview of T_FH cell differentiation and localization. The process of T_FH cell differentiation highlights the chemokines needed for CD4⁺ T‐cell trafficking into the T‐cell and B‐cell zones. The expression of chemokine receptors on the surface of T cells plays a decisive role in the migration of T_FH cells. CXCL13 is the ligand of CXCR5 and is secreted by B‐cell follicles and germinal centres. However, CCL19 and CCL21, the ligands of CCR7, are highly present in the T‐cell region.

T_FH cells, as a specific subset of CD4⁺ T cells, can be classified by chemokine receptor expression. CXCR3 and CCR6 are characteristic receptors used to define subgroups of T_FH cells. ²¹ , ²² , ²³ CXCR3 is a chemokine receptor that is highly expressed on the surface of T_H1 cells, while CCR6 is expressed in T_H17 cell subsets. ²⁴ , ²⁵ T_FH cells can also be further divided into subgroups according to the chemokines expressed on the cell surface. Studies have defined CXCR3⁺ CCR6⁻ T_FH cells as T_FH1 cells, which express the transcription factor T‐bet and secrete the cytokine IFN‐γ; CXCR3⁻ CCR6⁻ cells are termed T_FH2 cells, express the transcription factor GATA3 and secrete the cytokines IL‐4, IL‐5 and IL‐13; and CXCR3⁻ CCR6⁺ T_FH cells are termed T_FH17 cells, express the transcription factor RORγt and secrete the cytokines IL‐17A, IL‐17F and IL‐22. ²¹ , ²² , ²³ , ²⁴ , ²⁵

DEVELOPMENT AND FUNCTIONAL DIFFERENTIATION OF T_FH CELLS

T_FH cells are differentiated through three processes (Figures 2 and 3). Because of their tissue‐specific anatomical location, T_FH cells possess unique functions that differ from those of other T_H subsets. The three‐stage differentiation process is divided according to T_FH cell migration and localization. ¹⁸ , ²⁶ , ²⁷ , ²⁸

Figure 3.

Transcriptional regulation of T_FH cell differentiation. IL‐6R/IL‐21R‐STAT3 signalling functions in the differentiation of T_FH cells. Bcl‐6, c‐Maf, Batf and other transcription factors play regulatory roles in T_FH cell differentiation.

The first stage of T_FH cell differentiation is the migration of T_FH progenitor cells to B‐cell follicles. ²⁹ Naïve T cells receive antigen signals presented by dendritic cells (DCs) and activate T‐cell receptor (TCR) signalling. ²⁸ Stimulatory cytokines, such as IL‐6, induce the expression of the master transcription factor Bcl‐6 and the critical chemokine receptor CXCR5 in T_FH progenitor cells. ²⁹ , ³⁰ The first stage has been extensively studied and is vital in T_FH differentiation.

Currently, research on T_FH cells is mainly performed in mice. In humans, most studies on T_FH cells are focused on tonsillar and circulating T_FH cells in peripheral blood. ²⁹ The costimulatory receptor and cytokine signals transduced during the differentiation of T_FH cells are almost identical in mice and humans. During the first differentiation stage, naïve T‐cell interactions with DCs are mainly regulated by IL‐6, ICOS, IL‐2 and the TCR signalling pathways. ³¹ , ³² ICOS plays an important role in regulating the differentiation and migration of T_FH cells. ¹⁵ , ¹⁶ ICOSL signalling is necessary for antigen‐specific B cells in most cases, except when in the presence of a large number of antigens or during the migration of a large number of antigen‐specific B cells. ¹⁵ , ¹⁶ B cells act as not only antigen‐presenting cells (APCs) but also the ICOS ligand (ICOSL) in T_FH cell differentiation. ³³ During acute infection and immune responses, B cells are the main APCs. Antigen presentation is important for antigen‐specific CD4⁺ T‐cell‐required antigen recognition during each cell division. ³³ During the migration of activated CD4⁺ T cells from the T‐cell zone to the B‐cell zone, a series of changes take place in their surface molecules. CXCL13 is the ligand of CXCR5 and is highly expressed in the follicles and germinal centres of B cells. However, CCL19 and CCL21, the ligands of CCR7, are highly expressed in the T‐cell region. ¹⁸ , ³³ , ³⁴ , ³⁵ , ³⁶ CD4⁺ T cells that are sensitized by DCs upregulate CXCR5 and downregulate CCR7 to induce B‐cell migration to the T‐B cell junction. ¹⁷ , ³⁷ ICOS and CXCR5 promote PI3 K signalling, PD‐1 inhibits PI3 K activity, and PI3 K signalling determines whether T_FH cell precursors enter the follicle. The structural expression of bystander B‐cell ICOSL and PD‐1 mediates T_FH ICOS signalling. ICOS signalling promotes PI3 K activation, T‐cell pseudopodia formation and T‐cell migration. The interaction of CXCL13‐CXCR5 also promotes PI3 K signalling, which stimulates precursor T_FH cells to enter the follicular region. ¹⁸ miR19‐72 promotes the differentiation of T_FH cells. miR19‐72 downregulates the phosphatases PHLPP2 and PTEN, which inhibit the PI3 K‐ICOS signalling pathways. ³⁸ IL‐2 inhibits the differentiation of T_FH cells. Early T_FH differentiation is regulated by a variety of signalling pathways, such as the IL‐6, ICOS, IL‐2 and TCR pathways, mainly by DCs, and by the regulated expression of CXCR5 and Bcl‐6. ³⁹ IL‐6 promotes CD4⁺ T‐cell and B‐cell activation during Plasmodium infection. IL‐6 promotes CD4⁺ T‐cell activation and B‐cell responses during the blood stage of Plasmodium infection, which contributes to the production of parasite‐specific antibodies. The IL‐21 and IL‐21 receptors show increased expression and activity levels during the immunopathogenesis of multiple sclerosis. Thus, several related regulators play critical regulatory roles in the early stage of T_FH cell differentiation and function.

The second stage of T_FH cell differentiation occurs during the interaction between T_FH cells and antigen‐specific B cells at the junction of the T‐B cell border and in the follicles and interfollicular region. The chemokine environment of the T‐cell region and B‐cell region affects the migration of T cells. ¹⁴ , ¹⁸ , ³⁶ , ⁴⁰

The third stage of T_FH differentiation takes place in GCs. GCs are specialized structures found in the B‐cell follicles of secondary lymphoid tissues, where B cells produce high‐affinity antibodies and differentiate into memory B cells and long‐lived plasma cells. ⁴¹ , ⁴² GCs are special structures composed of GC T_FH cells, GC B cells, follicular dendritic cells (FDCs), macrophages and stroma. ⁴³ The B7 receptor PD‐1 is highly expressed on T_FH cells and is one of the main markers of T_FH cells located in GCs. T_FH cell precursors initially express PD‐1 during cell activation because of their interaction with antigen‐presenting DCs. The expression of PD‐1 is enhanced when precursor T_FH cells interact with B cells at the junction of the T‐B border. The steady interaction between T and B cells leads to the formation of GCs, and the expression of PD‐1 on T_FH cells peaks in GCs. ⁴¹ , ⁴² , ⁴³ Notably, the role of PD‐1 in the localization of T_FH cells varies with the location and maturation stage of T cells. The expression levels of the chemokine receptors CXCR5 and CXCR4 are high, and the expression levels of PSGL1 and sphingosine‐1‐phosphate receptor (S1PR1) are low in GC T_FH cells during this stage. ⁴⁴ , ⁴⁵ The expression of G protein‐coupled receptor 2 (EBI2), which is induced by the Epstein–Barr virus, requires special attention because EBI2 ligands are present in B‐cell follicles but not in GCs. The decreased expression of EBI2 in GC B cells and GC T_FH cells is important for their correct localization in GCs. ⁴⁶ , ⁴⁷ In addition, adhesion molecules play important roles in GC T_FH cells, regulating the interaction and localization with GC B cells. The signalling lymphocyte activation molecule (SLAM) family receptors SLAMF6, also known as Ly108 and NTB‐A; CD84 and SLAM, are self‐ligands that are differentially expressed in GC T_FH cells and GC B cells. ⁴⁸ The expression of SLAM‐associated protein (SAP) is necessary for the development of GC T_FH cells and the production of most memory B cells and memory plasma cells. The absence of SAP weakens the adhesion of T_FH cells to GC B cells, making T_FH cells unable to remain in GCs and impairing the maturation and function of B cells. The importance of SAP is largely due to its ability to block the powerful inhibitory signals of SLAMF6. After the competitive binding of SAP and the phosphatase SHP‐1 to SLAMF6 and SAP, respectively, SLAMF6 transmits a positive T_FH cell differentiation signal, which supports the adhesion and function of T_FH cells. In contrast, when SLAMF6 binds to SHP‐1, a negative regulatory T_FH cell differentiation signal is transmitted that inhibits the adhesion of T_FH cells and B cells. ⁴⁸ , ⁴⁹ , ⁵⁰

However, T_FH cell localization to GCs is not the end‐point of T_FH cell differentiation. T_FH cells migrate from one GC to another or migrate into the blood or lymph for circulation. ⁵¹ , ⁵² , ⁵³ On the other hand, the position of B cells in the GCs is fixed; that is, they cannot be removed. After moving out of the GC, T_FH cells become memory T_FH cells, and the expression of Bcl‐6 is downregulated. After T_FH cells leave the GC, the activity and polarization of T_FH cells decrease, IL‐7Rα is upregulated, and these cells differentiate into resting memory T_FH cells. ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ Memory T_FH cells have a central memory phenotype, are located mainly in the spleen, lymph nodes and bone marrow and can be recycled in the blood. Approximately 20% of human central memory CD4⁺ T cells are CXCR5⁺, indicating that memory T_FH cells are the main components of the human memory T‐cell population. Memory T_FH cells are less predominant than reactivated T_FH cells and GC T_FH cells. ¹⁸ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ In humans, memory T_FH cells are phenotypically heterogeneous, at least in the blood, and in this population, a considerable number are resting memory T_FH cells that express low levels of PD‐1. These PD‐1^low memory T_FH cells are the most polarized and powerful memory T_FH cells. The expression of Bcl‐6 in T_FH cells is unstable and requires continuous enhancement. ¹⁸ , ⁵¹ , ⁵² , ⁵³ , ⁵⁴ , ⁵⁵ Therefore, when GC T_FH cells leave GCs, the expression of Bcl‐6 is decreased, and as the cells transform into resting memory T_FH cells, the expression of Bcl‐6 decreases further (Figure 2).

REGULATION OF T_FH CELL FUNCTION AND DIFFERENTIATION

Regulatory roles of cytokines in T_FH cell differentiation

Cytokines play important roles in lymphocyte differentiation (Figure 3). IL‐6 and IL‐21 play roles in T_FH cell differentiation and act directly on B cells. IL‐6 transiently induces the expression of Bcl‐6 in the early stage of T_FH cell differentiation, and it is also an inducer of IL‐21 expression in inactivated mouse CD4⁺ T cells. IL‐6 is the most important cytokine involved in the early differentiation of T_FH cells. ³² IL‐6 is produced by DCs, macrophages, B cells and other types of cells in response to a series of external and internal pathogen‐associated molecular patterns (PAMPs) and damage‐associated molecular patterns (DAMPs). ¹⁸ In the absence of IL‐6, the early differentiation of mouse T_FH cells is inhibited, but differentiation is increased in response to supplementation with IL‐21 and IL‐27. Many kinds of DCs and monocytes regulate the differentiation of T_FH cells. ³² , ⁵⁶ , ⁵⁷ , ⁵⁸

IL‐2 negatively regulates T_FH cell differentiation. ⁵⁹ Furthermore, IL‐2‐STAT5 signalling inhibits the expression of the transcription factor Bcl‐6 in T_FH cells. STAT5 promotes the expression of BLIMP1, which inhibits the expression of Bcl‐6. The expression of PD‐1 in GC T_FH cells is high, and PD‐1 signalling inhibits the production of IL‐2, which suggests that IL‐2 inhibits the expression of PD‐1. ³⁹ However, during naïve T‐cell differentiation, IL‐2 signalling is enhanced to different degrees. ⁵⁹ Therefore, the regulatory effects of IL‐2 signalling on T_FH cell differentiation are different and depend on the intensity of IL‐2 signalling.

IL‐12 is an important cytokine that induces naïve CD4⁺ T cells to differentiate into T_H1 cells and promotes the expression of the specific transcription factor T‐bet. IL‐12 but not IFN‐γ is induced during Salmonella infection and represses T_FH cell differentiation. ⁶⁰ T_FH1 cells, a subgroup of T_FH cells, produce both the T_FH‐associated cytokine IL‐21 and the T_H1 cytokine interferon‐γ. IL‐12 promotes T_FH1 differentiation and function by increasing T‐bet and Bcl‐6 expression. ⁶¹

Type I IFN‐α/β participates in the incomplete differentiation of T_FH cells and induces the expression of Bcl‐6, CXCR5 and PD‐1 through STAT1 signalling without producing IL‐21. ⁶² , ⁶³ , ⁶⁴ Type II IFN‐γ plays an active role in the differentiation of T_FH cells. As shown in the Sanroque lupus model, the accumulation of T_FH is the result of excessive IFN‐γ. IFN‐γ blockade reduces the number of T_FH cells in lupus tissue, indicating that IFN‐γ plays an important and active role in the production of T_FH cells. In terms of mechanism, excessive IFN‐γ signalling leads to increased expression of Bcl‐6. In contrast, in the late stage of T_H1 cell differentiation, Bcl‐6 binds to the IFN‐γ gene locus and inhibits excessive Ifn mRNA expression. ⁶² Thus, Bcl‐6 and IFN‐γ regulate each other through a negative feedback mechanism.

Therefore, several cytokines show distinct regulatory roles in determining T_FH cell lineage differentiation. IL‐6, IL‐21, IL‐27 and IL‐12 have positive regulatory effects on T_FH cell differentiation, and IFN‐γ, IL‐2 and IL‐10 play inhibitory roles in T_FH cell differentiation.

Transcriptional regulation of T_FH cell function and differentiation

Multiple transcription factors synergistically promote the development and differentiation of T cells (Figure 3). T cells have different functional subsets, and their transcription factors are expressed in different forms and levels at different stages of development and differentiation. During T_FH differentiation, different transcription factors promote or inhibit the differentiation of T_FH cells.

Some transcription factors play positive roles in T_FH differentiation. ⁶⁵ Signal transducers and transcriptional activators (STATs) are also closely related to the differentiation of T_FH cells. STAT3 plays an important role in the differentiation of T_FH cells in the mouse CD4⁺ T‐cell population, and STAT1 and STAT4 are also very important in the differentiation of T_FH cells. ⁶⁶ , ⁶⁷ The expression of STAT3 and IL‐21 in mouse CD4⁺ T cells is very important. ⁶⁵ Mouse CXCR5 and IL‐6 drive STAT3 and STAT1 to interact with Maf and Batf, bind to the promoter and initiate the expression of CXCR5. In contrast to their effects in mice, STAT3 and STAT4 are equally important for the differentiation of T_FH cells in humans. ⁶⁵ , ⁶⁸ STAT5 inhibits the differentiation of T_FH cells, and the opposite effects of STAT3 and STAT5 on the differentiation of T_FH cells are similar to the effects of STAT3 and STAT5 on the differentiation of T_H17 cells. ⁶⁹ , ⁷⁰

The promoter of Bcl‐6 harbours binding sites for the transcription factors STAT and Forkhead box O (Foxo). ⁶⁵ , ⁷¹ Under low IL‐2 conditions, STAT3 binds to the promoter of Bcl‐6, and when IL‐2 increases, STAT5 binds to the promoter of Bcl‐6. Interestingly, STAT5 replaces the STAT3 complex and excessively recruits the inhibitory complex, which explains why IL‐2 inhibits the differentiation of T_FH cells. ⁷¹ Maf is highly expressed in T_FH cells and is related to the expression of CXCR5, IL‐21 and IL‐4. c‐Maf is a basic leucine zipper transcription factor that binds to the IL‐4 promoter and plays an important role in the differentiation of T_H2 cells. ¹⁵ , ⁷² The loss of c‐Maf leads to a reduction in IL‐17 production. ICOS‐mediated c‐Maf expression is necessary for IL‐21 production and T_FH cell expansion. ⁷²

IRF4 and basic leucine zipper transcription factor (Batf) also play important roles in regulating the differentiation of T_FH cells. ⁷³ , ⁷⁴ Batf is a positive regulator of Bcl‐6. In the absence of Batf, the coexpression of Bcl‐6 and Maf is required for the expression of CXCR5 in vivo. ⁷³ Although Batf is only moderately increased in T_FH cells, Batf ^−/− mice cannot produce T_FH cells. Batf directly binds to the promoters of Bcl‐6 and Maf. The E protein Ascl2 also promotes the differentiation of T_FH cells. Ascl2 is not the only regulatory factor of CXCR5. There is redundancy among several E proteins that are expressed in T cells. Multiple E proteins promote the expression of CXCR5 by binding to enhanced subregions. ⁷⁴ The function of E proteins is tightly regulated by DNA‐binding inhibitor 2 (ID2) and ID3 (Figure 3).

T helper cell‐inducing POZ/Kruppel‐like factor (Thpok) and thymocyte selection‐associated high mobility group box protein (Tox) have been reported to regulate other transcription factors that are crucial to T_FH cell differentiation, such as Bcl‐6 and c‐Maf. Thpok and Tox are critical for the development of T cells. ⁷⁵ , ⁷⁶ It has been shown that these factors bind to the promoters or other transcriptional regulatory regions of T_FH differentiation‐related transcription factors and regulators (such as CXCR5 and ICOS) to promote their expression. There may be other upstream transcription factors that promote T_FH cell development.

Negative regulatory transcription factors in T_FH cell differentiation have also been reported in other studies (Figure 3). Foxp1 and Foxo1 are expressed in resting immature CD4⁺ T cells and are necessary for immature CD4⁺ T cells to maintain resting and homing states. ⁷⁷ Downregulating the expression of Foxo1 and Foxp1 can positively regulate the differentiation of T_FH. For example, itchy E3 ubiquitin protein ligase (Itch) can bind to Foxo1 and accelerate Foxo1 degradation. ICOS and PI3 K‐Akt signalling promote the differentiation of T_FH cells, while Akt signalling leads to a decrease in Foxo1 transcriptional activity. ⁷⁸ Inhibiting Foxo1 and Foxp1 enhances the expression level of ICOS. ⁷⁹ E26 transformation‐specific sequence‐1 (Ets1) downregulates the expression of GATA3 and IL‐4. The specific loss of Ets1 in mouse CD4⁺ T cells leads to systemic lupus erythematosus (SLE) and the terminal differentiation of T_FH2 cells. ⁸⁰

Taken together, these data suggest that Bcl‐6 is the master transcription factor in T_FH cell differentiation; STAT3, STAT4 and STAT1 promote the expression of Bcl‐6 and T_FH differentiation; and transcription factors such as IRF4, MAF, BATF, Ascl2 and ThpoK also play positive roles in T_FH cell differentiation and function. Transcription factors such as Foxo1, Foxo3a and BLIMP1 negatively regulate the differentiation and function of T_FH cells.

METABOLIC REGULATION OF T_FH CELL FUNCTION AND DIFFERENTIATION

Metabolic regulation plays an important role in the differentiation and function of T_FH cells (Figure 4). Some studies of glucose metabolism in T_FH cells suggest that glycolysis, which is mediated by mTOR‐ and HIF1α‐related signalling, is positively regulated during the differentiation process. However, some studies suggest that T_FH cells have less robust metabolic functions than other Th cell subsets, such as T_H1 cells, and oxidative phosphorylation but not glycolysis plays a major role in differentiation.

Figure 4.

Regulation of metabolism in T_FH cell differentiation. Basic processes and correlations of glucose, lipid and amino acid metabolism. mTOR signalling and transcription factors such as Myc and HIF1α promote T_FH cell differentiation related to improvements in metabolism. The differentiation of T_FH cells is inhibited by the glycolysis inhibitors 2‐DG and succinate, a substrate of oxidative phosphorylation.

mTORC1 and mTORC2 are essential for the differentiation of T_FH cells and other T_H cell subsets. ⁸¹ , ⁸² Specifically, mTORC1 promotes T_H1 and T_H17 cell differentiation, whereas mTORC2 favours T_H2 cell differentiation by orchestrating metabolic reprogramming and lineage‐specific gene transcription. It has been reported that mTORC1 and mTORC2 are critical for T_FH differentiation based on studies of the phenotype acquired in Raptor‐ and Rictor‐knockout mice. ⁸² The loss of Rictor or Raptor results in a decrease in the T_FH cell population and attenuates the humoral immune response. ⁸² Aberrant activation of mTORC1 can lead to autoimmune diseases. The mTORC1‐4E‐BP‐eIF4E axis can promote Bcl‐6 protein synthesis. ⁸³ In addition, some studies suggest that mTORC1 inhibits the differentiation of T_FH cells through the IL‐2‐mTORC1 axis. ⁸⁴ The regulatory effects of mTORC1 on T_FH cell differentiation are contradictory, and the precise regulatory mechanisms need to be further studied. It was recently reported that mTORC2 plays a positive regulatory role in the differentiation and function of T_FH cells. Additionally, mTORC1 inhibits the differentiation and expansion of T_reg cells. However, some studies have reported that mTORC1 is essential for the differentiation and function of T_reg cells by activating the STAT3‐TCF‐1‐Bcl‐6 axis. ⁵ mTORC1 participates in the regulation of multiple signal pathways, and its regulatory functions on the differentiation and function of T_FH cells and T_reg cells might differ due to different experimental conditions and external stimuli.

mTOR signalling‐related glucose metabolism is critical for T_FH cell differentiation. A deficiency in PTEN, a negative regulator of mTOR, promotes T_FH cell differentiation and GC formation. ⁸² , ⁸⁵ mTORC1 and mTORC2 signalling links ICOS to anabolic metabolism and T_FH cell‐associated transcriptional programmes in Peyer's patches (PPs). ⁸² This finding suggests that mTOR activation and glycolysis are required for T_FH cell differentiation. K/BxN mice are models of spontaneous rheumatoid arthritis and autoimmune sero‐positive arthritis. The production of high levels of anti‐GPI IgG requires CD4⁺ follicles to facilitate the expansion of T_FH cells. The metabolic activity of CD4⁺ T cells and B cells in K/BxN mice was higher than that in KRN control mice. Prophylactic inhibition of glycolysis with 2‐deoxy‐D‐glucose (2‐DG) significantly reduced joint inflammation, the activation of adaptive and innate immune cells, and the production of pathogenic autoantibodies. The self‐reactive T_FH cells of K/BxN mice exhibit a high level of glycolysis, and their function can be inhibited by downregulating glucose metabolism. The high expression of hexokinase HK2 in joint‐infiltrating lymphocytes in patients with rheumatoid arthritis (RA) indicates that the metabolism of these cells is highly glycolytic. ⁸⁶ These results suggest that glycolysis and/or mTOR signalling is critical for the differentiation of T_FH cells.

Roquin inhibits the PI3 K‐mTOR signalling pathway, spontaneous activation of T cells and abnormal differentiation of T_FH and T_H17 cells. ⁸⁷ The deletion of Roquin leads to an increase in the ratio of T_FR cells to T_FH cells, and the inhibition of PI3 K‐mTOR signalling restores the increase in T_FR and T_FH cells caused by Roquin deletion. ⁸⁸ The PI3 K‐mTOR signalling pathway has been suggested to play an important positive regulatory role in glycolysis; therefore, it is suggested that glycolysis signalling mediated by the PI3 K‐mTOR pathway promotes the differentiation of T_FH and T_FR cells. ⁸⁷ T_FR cells, similar to T_reg cells, play immunosuppressive roles. T_FR cells express CXCR5, Bcl‐6 and other characteristic factors that function in the germinal centre. ⁸⁸ mTORC1 promotes the differentiation of T_FR cells, suggesting that mTORC1 positively regulates the expression of CXCR5, ICOS and Bcl‐6 in T cells. However, other studies present opposite findings. For example, it was found that T_FH cells exhibit reduced glycolysis and mitochondrial respiration, accompanied by reduced mTOR activity compared with those of T_H1 cells in acute viral infection. ⁸⁴ It has been shown that overactivated PI3Kδ affects humoral immunity in APDS mouse model (Pik3cd ^{E1020  K/+}). ⁸⁹ In Pik3cd ^E1020 K/+ mice, T_FH cells and GC B cells are particularly abundant, but the efficiency of the B‐cell response to the immune‐based conversion of antigen‐specific B cells was decreased. T_FH cells in Pik3cd ^E1020 K/+ mice are typically limited to the light zone (LZ), which specifically targets high‐affinity B cells and induces survival and proliferation. ⁸⁹ APDS patients present with structural disorders in germinal centres, and the increased invasion of T_FH cells impairs humoural immune response.

Protein kinase C (PKC) plays important roles in cell metabolism and signal transduction. The function of mitochondria in B cells is downregulated in Pkc ^−/− mice, and the antigen presentation ability of B cells is inhibited. It was also found that the reaction of GCs in these mice can be decreased, and the production of plasma cells and immunoglobulin is decreased. The mitochondrial regulation mediated by PKC is partly regulated through the mTOR signalling pathway. Thus, mitochondria are considered to be particularly important for the GC response, which requires the participation of T_FH cells. ⁹⁰ These findings suggest that PKC also regulates the function of T_FH cells by regulating mitochondrial functions.

Bcl‐6 is an important transcription factor in the differentiation of T_FH cells. ⁹¹ The expression of Bcl‐6 is significantly upregulated in activated CD4⁺ T cells subjected to glucose deprivation (GD) or 2‐DG. Adenosine 5’‐monophosphate (AMP)‐activated protein kinase (AMPK), a metabolic sensor, is activated when glycolysis is decreased. The AMPK antagonist compound C inhibits the expression of Bcl‐6 induced by glucose deprivation. ⁹¹ A study showed that AMPK plays a critical role in the differentiation of T_FH cells. Mature T_FH cells have a lower metabolic state than T_H1 cells. ⁹¹ These data show that a decrease in cell metabolism is an inducer of T_FH differentiation, not merely the result of T_FH differentiation.

Resting T cells mainly rely on oxidative phosphorylation in mitochondria to produce ATP. Under antigen stimulation, the downstream AKT signalling pathway is mediated by the TCR pathway and costimulatory receptor CD28, and the AKT signalling pathway has been extensively studied with respect to the positive regulation of glycolysis. Under the metabolic conditions generated by glycolysis, naïve CD4⁺ T cells differentiate into T_H1, T_H17 and other cell subsets. DUSP6, a MAPK phosphatase, connects TCR signalling to activation‐induced metabolism that favours glycolysis and attenuates T_FH cell differentiation. ⁹² Bcl‐6 expression is highly upregulated in activated CD4⁺ T cells following glucose deprivation, and this pathway is insensitive to inhibition by IL‐2. In a manner similar to glucose deprivation, the glucose analogue 2‐DG inhibits glycolysis, and 2‐DG induces Bcl‐6 expression in activated CD4⁺ T cells. ⁹¹ Although these studies revealed that Bcl‐6 is induced when glycolysis is inhibited, the inhibition of glycolysis is also detrimental to T‐cell activation. Therefore, during robust T‐cell proliferation, glucose uptake and glycolysis are required, and Bcl‐6 is activated by low‐energy signals transmitted by AMPK during high T‐cell proliferation and activity. ⁸⁶ , ⁹¹ , ⁹³ These results suggest that the mTOR pathway positively regulates T_FH cell differentiation, but the mechanism by which the transcription programme is regulated by mTOR has not been clarified.

Ovalbumin (OVA) sensitization and influenza virus infection are considered to be models for the study of T_FH cells. T_FH cells are induced to differentiate during OVA sensitization and viral infection. Succinate is a substrate in oxidative phosphorylation. The injection of succinate into wild‐type (WT) mice sensitized by OVA inhibited T_FH cell differentiation and the GC response. In addition, 2‐DG is an inhibitor of glycolysis. Under OVA sensitization and viral infection, the injection of 2‐DG into the body inhibited the differentiation and function of T_FH cells. ⁹⁴ Therefore, the inhibition of glycolysis or the enhancement of oxidative phosphorylation inhibits the differentiation of T_FH cells in vivo, suggesting that glycolysis is the main metabolic mechanism regulating T_FH cell differentiation.

T_FH CELLS IN IMMUNE‐ASSOCIATED DISEASES

T_FH cells affect humoural immunity by regulating B‐cell development. In recent years, with continuous research on T_FH cell differentiation and functions, it has been revealed that T_FH cells are closely related to many immune‐associated diseases, including infectious diseases, autoimmune diseases and cancers (Figure 5 and Table 1).

Figure 5.

T_FH cell‐associated immune‐mediated diseases. T_FH cells play regulatory roles in immune‐associated diseases, including infections, autoimmune diseases and cancers.

Table 1.

Immunoregulation of T_FH cells in diseases.

Disease			Prognosis effects	Immune functions
Infection	LCMV	Mouse	Promote viral clearance	IL−6 and Bcl−6 upregulation. 96
				Production of late‐stage antibodies. 97
	HCV	Human	Anti‐infection	IL−21 and ICOS upregulation. 99
	HIV	Human	Anti‐infection	Plasma cell and autoantibody production decreases. 100 , 102 , 103
	GAS	Human	Promote bacterial clearance	Granzyme B expression. 128
Autoimmune diseases	SLE	Mouse; human	Hyperactive	Autoantibody production. 51 , 80 , 106 , 107
	pSS	Human	Hyperactive	Autoantibody production. 108 , 109
	JDM	Human	Hyperactive	Autoantibody production. 52
	AITD	Human	Hyperactive	Autoantibody production; IL−21 and Bcl−6 upregulation. 110 ; CXCR5 and CXCL13 expression. 111
	RA	Human	Hyperactive	Autoantibody production; IL−21 upregulation. 112 ;
		Mouse		Autoantibody production; CXCR5 expression. 113
Cancers	BC	Human	Positive correlation with good prognosis	8‐gene TFH cell signature. 123 ; CXCL13 expression. 124
	CRC	Mouse; human	Positive correlation with good prognosis	CXCL13 and IL−21 expression. 125
	Other cancers	Mouse	Downregulate antitumour immunity	IL−4 expression. 126
			Upregulate antitumour immunity	IL−21 expression. 127

Immunoregulatory roles of T_FH cells in infection

T_FH cells help B cells differentiate into plasma cells to produce antibodies, which are crucial for eliminating viruses and bacteria. In the context of viral and bacterial infection, some investigations have been performed on lymphocytic choriomeningitis virus (LCMV) infection, hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection and group A Streptococcus (GAS) bacterial infection.

In mice infected with LCMV Clone 13, a persistent variant of LCMV, persistent viral infection progressively drives CD4⁺ T‐cell development to disfavour T_H1 cells and favour T_FH cells. ⁹⁵ In other words, LCMV infection promotes T_FH cell differentiation. In addition, in persistent LCMV infection in vivo, T_FH cells are crucial for viral clearance. ⁹⁶ Cytokine production and other changes were analysed in mice infected with LCMV Clone 13. IL‐6 signalling in T_FH cells promoted the clearance of LCMV Clone 13 and enhanced the function of T_FH cells by upregulating Bcl‐6. A similar study also demonstrated that T_FH cells were essential in helping B cells produce late‐stage antibodies to neutralize LCMV in mice. ⁹⁷

T_FH cells are also essential in controlling HCV infection in a manner similar to the control of LCMV. During HCV infection, it has been observed that T_FH cells also significantly increase the percentage of cells coexpressing CXCR5 and PD‐1 in both acute infection and chronic infection. ⁹⁸ , ⁹⁹ In patients with acute HCV infection, T_FH cells express specific markers and secrete IL‐21 in response to HCV. Virus‐specific T_FH cells can be detected in blood samples, and ICOS expression is significantly increased in CXCR3‐expressing HCV‐specific T_FH cells. ⁹⁹ In the livers of patients with chronic HCV infection, virus‐specific T_FH cells are enriched compared with other types of cells. ⁹⁹

HIV causes defects in the human immune system, and an increasing number of people die from HIV infection. Research has shown that T_FH cells are severely dysregulated in HIV‐infected patients. ¹⁰⁰ , ¹⁰¹ CXCR5⁺PD‐1⁺Bcl‐6⁺ cells harbouring HIV DNA are significantly increased in infected patients. Deregulation of T_FH‐mediated B cells contributes to diminished B‐cell responses during HIV infection, which suggests that T_FH cells have a significant impact on the control of HIV infection. ¹⁰⁰ GC T_FH cells have a higher probability of harbouring HIV infection than other cells in vitro, as indicated by the higher frequency of infected cells. ¹⁰² , ¹⁰³

GAS bacterial infection is the main cause of recurrent tonsillitis (RT) in childhood. ¹⁰⁴ Aberrant pyrogenic exotoxin A‐activated GC T_FH cells express granzyme B to kill B cells in RT patients. ⁶³

Immunoregulatory roles of T_FH cells in autoimmunity

Autoimmunity is closely associated with excessive autoantibody production. The differentiation and function of T_FH cells are closely related to autoimmune diseases. ¹⁰⁵ Overactive T_FH cells were found in subsets of patients with systemic lupus erythematosus (SLE), Sjogren's syndrome (pSS), juvenile dermatomyositis (JDM), autoimmune thyroid disease (AITD) and rheumatoid arthritis (RA) (Figure 5 and Table 1).

SLE is an autoimmune disease that is currently well understood in the field of T_FH cell‐related diseases. The number of circulating T_FH cells is increased in mice and patients with the autoimmune disease SLE. ⁵¹ , ¹⁰⁶ , ¹⁰⁷ T_FH cells are closely associated with SLE, suggesting that disordered homeostasis of the T‐cell–B‐cell equilibrium is a major cause of SLE. ¹⁰⁶ , ¹⁰⁷ In Ets‐deficient mice, T_FH2 (Bcl6⁺GATA3^high) cells are the main T_FH cell subset, and these mice are highly likely to develop SLE. ⁸⁰ Similarly, there are more T_FH2 cells in SLE patients than in healthy individuals. ⁸⁰ It has been found that T_FH cells play an important role in the severity of pSS. ¹⁰⁸ , ¹⁰⁹ The proportion of peripheral T_FH cells is increased in patients. The percentage of T_FH cells is positively correlated with the numbers of both activated T cells and Th1 cells. ¹⁰⁸ In JDM patients, the T_FH cell subset proportions are severely unbalanced, with a profound skewing of the T_FH cell subset towards T_H2 and T_H17 cells. ⁵² Studies have shown that T_FH cells play important roles in AITD. ¹¹⁰ , ¹¹¹ In patients with AITD, the frequency of circling T_FH cells is increased, and the mRNA expression of IL‐21 and Bcl‐6 is also enhanced. ¹¹⁰ In addition, the chemokine receptor CXCR5 and its ligand CXCL13 are essential in regulating T‐cell and B‐cell compartmentalization in the thyroid tissue of patients with AITD. ¹¹¹ T_FH cell and serum IL‐21 levels are significantly increased in the RA patient group compared with those of the corresponding healthy control group. Moreover, B‐cell proliferation and antibody secretion are increased in RA patients. Research has also shown that IL‐21 promotes B‐cell activity. Thus, IL‐21 may be related to the pathogenesis of rheumatoid arthritis. ¹¹² In a collagen‐induced arthritis (CIA) mouse model, CXCR5‐deficient mice showed strong resistance to CIA, suggesting that CXCR5 is an indispensable factor for inducing autoimmune diseases. ¹¹³

T_FH cells contribute to metabolic‐associated autoimmune disorders, such as diabetes and atherosclerosis. Type 1 diabetes (T1D) is called insulin‐dependent diabetes mellitus (IDDM) and is associated with metabolic disorders. T_FR cells negatively regulate the function of T_FH cells during inflammation. It has been reported that T_FR cell deficiency is involved in the pathogenesis of T1D. Thus, it was hypothesized that T_FH cells participate in the onset of diabetes. ¹¹⁴ Type 2 diabetes (T2D) mellitus is a chronic metabolic disease that is strongly associated with obesity. In mucosal biopsy samples, B cells and plasma blasts from T2D patients exhibited a slight but significant increase in the frequency of cells with surface IgG expression compared with those from healthy individuals. To a large extent, the pathogenesis of T2D is related to obesity, but some patients with T2D have a body mass index (BMI) that is inconsistent with obesity. It was found that non‐obese T2D patients exhibited significantly higher levels of faecal IgG and higher frequencies of CD4⁺CXCR5⁺ T (T_FH) cells than BMI‐matched healthy controls. ¹¹⁵ Atherosclerosis (AS) is associated with lipid metabolism disorders and is the main cause of coronary heart disease, cerebral infarction and peripheral vascular disease. T_reg cells function in antiatherosclerosis. T_reg cells lose Foxp3 expression during inflammation and are then converted into other CD4⁺ T‐cell subsets. During atherosclerosis development, T_reg cells can switch their phenotype and become pro‐atherogenic T_FH cells and apolipoprotein AI (ApoAI), this conversion can be achieved by reducing the expression of IL‐2Rα and p‐STAT5 levels. ¹¹⁶ There is cross‐regulation between atherosclerosis and other autoimmune diseases, such as SLE. Dyslipidaemia in atherosclerotic diseases significantly increases the severity of SLE, with enhanced production of autoantibodies, such as IgG2c, and the T_FH cell response via IL‐27. ¹¹⁷ STAT4 suppresses Foxp3 expression, regulates T_FH cell functions and promotes atherogenesis in insulin‐resistant low‐density lipoprotein receptor‐deficient (Ldlr ^−/−) mice. ¹¹⁸ Pre‐B‐cell leukaemia transcription factor 1 isoform d (Pbx1d) can regulate T_FH cell expansion and T_reg cell homeostasis as a lupus susceptibility gene. Cholesterol favours T_FH cell differentiation or maintenance, and the interaction between Pbx1d and dyslipidaemia alters T_FH cell and T_reg cell fates and functions. ¹¹⁹

Furthermore, different subsets of T_FH cells (T_FH2 or T_FH13 cells) are specifically involved in autoimmune diseases. Dysfunction or imbalance in CD4⁺ T cells leads to the pathogenesis of autoimmune diseases. For example, T_H1 cells play critical roles in insulin‐dependent T1D, inflammatory bowel disease (IBD) and RA; T_H17 cells participate in the pathogenesis of autoimmune encephalomyelitis (EAE); and T_H22 cells are critical in psoriasis, obsessive–compulsive spondylitis and multiple sclerosis (MS). T_FH cells are critical for antibody production and humoural immunity, and their aberrant activity is closely related to the pathogenesis of autoimmune diseases. Among the T_FH cell subsets, T_FH2 and T_FH13 cells are recognized as crucial participants in the onset and exacerbation of autoimmune diseases. Asthma causes airflow obstruction and is induced by inflammatory disorders. Circulating T_FH2 cells are polarized, and the ratio of T_FH2 cells to T_FH1 cells is increased in the peripheral blood of patients with asthma. ¹²⁰ T_FH2 cells are polarized in both allergic rhinitis (AR) and AR +asthma cases. In one study, it was found that peripheral blood lymphocytes of patients with AR or AR +asthma diseases were preferentially polarized to the T_FH2 phenotype. T_FH2 cells can produce IgE‐related cytokines, such as IL‐4, IL‐5 and IL‐13, similar to T_H2 cells, leading to allergic inflammation. ¹²¹ It was reported that the expression of CD23 was enhanced on CD19⁺CD20⁺CD27⁺IgD⁻ switching memory B cells and positively correlated with antigen‐specific IgE levels and the T_FH2 cell ratio in AR patients. T_FH2 cells have the capacity to promote CD23 expression on switching memory B cells by secreting the cytokine IL‐4. ¹²²

Immunomodulatory roles of T_FH cells in cancer

An increasing number of studies have shown that T_FH cells have an indispensable role in a variety of cancers, including breast cancer (BC) and colorectal cancer (CRC) (Figure 5 and Table 1).

It has been observed in breast cancer patients an 8‐gene T_FH cell signature in tumour tissue who has a significant positive correlation with organized antitumour immunity, long‐term patient survival and preoperative response to chemotherapy. ¹²³ In addition, researchers have also shown that T_FH cells promote local memory B‐cell differentiation, thus enhancing antitumour immunity. ¹²⁴ In human CRC, T_FH cells also have a positive effect on the survival of cancer patients. A study showed that the density of T_FH cells increased, whereas the levels of most T cells decreased during tumour progression. CXCL13 and IL‐21 expression are also associated with cancer progression. ¹²⁵

However, studies have shown that T_FH cells expressing IL‐4 are associated with downregulated antitumour immunity in other cancers. ¹²⁶ CNS2‐deleted mice were injected subcutaneously with various tumour cells and exhibited stronger antitumour immunity than control WT mice. In addition to IL‐4, IL‐21 is another major cytokine that is expressed by T_FH cells. However, in contrast to the effect of IL‐4, IL‐21 enhances antitumour immunity in mouse models. ¹²⁷ When PD‐1 or CTLA‐4 blockade was combined with IL‐21, the mice showed improved treatment outcomes. ¹²⁷

CONCLUDING REMARKS

T_FH cells have been extensively investigated in mouse and human studies. These studies have demonstrated that T_FH cells contribute to both humoural immunity and immune‐related diseases. However, despite in‐depth study, the regulatory factors and mechanisms of T_FH cell differentiation, especially in the pathogenesis of various important immune‐related diseases, remain unclear and need further study. Further study of T_FH cell subgroups, including the T_FH1, T_FH2 and T_FR cell subgroups, and other subpopulations may be helpful to further clarify the roles of these cells in immune‐related diseases, which will provide the basis for the diagnosis and treatment of diseases in the future.

AUTHOR CONTRIBUTIONS

L.D., Y.H. and Y.C. consulted the references; Y.X.W., A.J., Y.F.W., Q.Y. and W.L. participated in discussions, L.D., Y.B. and G.L. contributed to writing the manuscript and participated in discussions.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

DATA AVAILABILITY STATEMENT

Not applicable.