Follicular Helper T Cells and Autoimmune Diseases

Abstract

Follicular helper T (Tfh) cells can control the antibody affinity maturation and memory by supporting the formation of germinal center (GC) and regulating clonal selection and differentiation of memory and antibody-secreting B cells. Therefore, Tfh cells play an important role in the development of some autoimmune diseases, such as rheumatoid arthritis and autoimmune hepatitis. The generation and function of Tfh cells are determined by T-cell antigen receptor (TCR), co-stimulation, and cytokine signals, together with specific mechanisms. In this part, the specialization, development, and regulation of metabolic and differentiation mechanisms on Tfh cells will be summarized, which is crucial to understanding pathogenesis and informing the development of emerging therapies for autoimmune diseases.

1. Introduction

The human immune response can be divided into innate immunity and adaptive immunity. Many types of cells of the myeloid lineage are involved in innate immunity, including dendritic cells (DCs), natural killer (NK) cells, macrophages, monocytes, mast cells, and so on. The adaptive immune response is related to the participation of T and B lymphocytes, which will be activated in a specific manner upon recognizing the antigens. The former can be further differentiated into two basic phenotypes according to the surface marker of cluster of differentiation (CD): cytotoxic T cells, which can be distinguished by the CD8, and T helper cells (Th), which are characterized by CD4. As one subpopulation of CD4⁺ T lymphocytes, follicular helper T cells (Tfh) are usually but not exclusively found in the lymph nodes and spleen, while the other Th cells are predominantly found in lymphoid tissues and blood [1, 2]. They can provide signals directed to B cells located in the germinal center (GC) to promote differentiation and antibody generation [3]. The Tfh-B-cell interaction occurring in the inter-follicular regions of GC is crucial for inducing immune responses efficiently [4].

Autoimmune disease is a category of disease with a prevalence of 7–9% globally that seriously affects human health and results in appreciable mortality [5]. Our knowledge of the roles of Tfh cells in autoimmune diseases has significantly increased during the past decades, and it has been demonstrated that Tfh cells play critical roles in the pathogenesis of many autoimmune diseases, such as rheumatoid arthritis (RA), autoimmune hepatitis (AIH), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Primary Sjogren’s Syndrome (pSS), myasthenia gravis (MG), inflammatory bowel disease (IBD), immunoglobin G4-related disease (IgG4-RD), etc. [6, 7]. The tissue damage and organ dysfunction of autoimmune diseases are initiated by immune responses mistakenly targeting an individual’s cellular, especially by the autoantibody in the humoral immune response. Help to B cells and the formation of DC provided by Tfh allows the affinity maturation of antibody responses and promotes disease pathogenesis by forming immune complexes to mediate inflammation and tissue damage via activating the complement and effector cells in a tissue-specific manner. Although usually located in secondary lymphoid organs, Tfh cells can be identified in human blood, and their frequency and phenotype are often altered in different autoimmune diseases [8].

Herein we will review the recent progress on the specialization and differentiation of Tfh cells, as well as the significance and functionality of these cells in GC formation and the mutation of high-affinity antibody-producing B cells in this chapter. Moreover, how these subsets of Tfh cells engaged in diverse autoimmunity illnesses will also be explored to provide us with novel targets for therapeutic intervention in these disorders.

2. Specialization and development of Tfh

As the unique subset of T lymphocytes with the ability to migrate into or out of follicles, Tfh cells co-localize with B cells in secondary lymphoid organs (SLOs) and deliver contact-dependent and soluble signals to support the survival and differentiation of the latter [9]. Tfh cells can be found in the T-cell zone (pre-Tfh cells phenotype), at the T-B border, and within the GC (the most mature stage), and express different markers in line with the location [10]. The differentiation of Tfh cells is initiated in the T-cell zone of SLOs when naïve CD4⁺ T cells encounter activated antigen-presenting dendritic cells (DCs). The transcription and translation level of Bcl6 in early pre-Tfh cells elevates within hours at first cell division and then they rapidly gain expression of the canonical surface markers CXC chemokine receptor (CXCR) 5, programmed death receptor (PD)-1, and inducible costimulator (ICOS). The differentiation of early pre-Tfh cell phenotype is also regulated by the transcription factor achaete-scute homolog (ASCL) 2 in a Bcl6-independent manner. ASCL2 can directly induce the upregulation of CXCR5 and downregulation of C chemokine receptor (CCR7) and P-selectin glycoprotein ligand (PSGL)-1 via binding to the Cxcr5 locus [11]. Thereafter, the Tfh cells produce pro-Tfh cytokines, including IL-6 and IL-21, to promote the release of XC chemokine ligand (CXCL)13 by follicular dendric cells (FDCs) and CXCL12 by CXCL12-producing reticular cells (CRCs) in the B-cell zone. Recognition of CXCR5 by CXCL13 leads to recruiting of the immature Tfh into the B-cell zone and to interacting with bystander B cells, which line the follicular parenchyma. Resembling DCs, bystander B cells express inducible costimulator ligand (ICOSL) constitutively to induce ICOS signaling in Tfh cells, which is crucial for follicular retention of Tfh cells and the occurrence of GC reaction [12]. ICOS signaling in Tfh cells further induces CXCR5 and suppresses CCR7, PSGL-1, and CD62L expression through the Kruppel-like factor (KLF) 2, and promotes the motility of Tfh cells in a PI3K-dependent manner to migrate further into the follicle by responding to CXCL13 more robustly [13]. Bystander B cells also express PD-L1 to provide the suppressive signal to follicular entry by activated T cells, which is why continuous ICOS signaling is necessary to maintain the Tfh cells phenotype and follicular localization, as well as the recruitment of exclusively ICOS-high expressing cells into the follicle. Both CXCR5 and ICOS signaling promote the homing of the now-mature Tfh cells into the GC. GC Tfh cells express higher levels of surface makers, including CXCR5 and PD-1, and more canonical Tfh cell-associated molecules, such as Bcl6, IL-21, IL-4, and Maf, at the transcript level [14]. The localization of Tfh cells to the GC is guided by Epstein-Barr virus-induced molecule (EBI) 2 and sphingosine-1-phosphate receptor (S1PR) 2, and the former (also known as Gpr183) is downregulated in GC Tfh cells and GC B cells while the latter is upregulated [15]. The spatial segregation of Tfh cells in the follicle may also be regulated by surrounding B cells-associated factors, such as plexin B (PlxnB), which can interact with semaphorin 4C (Sema4C) expressed on GC Tfh cells [16]. At last, interactions between Tfh cells and B cells in the GC result in the maturation of B cells into plasma cells to produce antibodies.

3. Subsets and features of Tfh

Human circulating Tfh (cTfh) cells can be subdivided into three main populations according to the expression of surface makers CXCR3 and CCR6: (1) CXCR3⁺CCR6⁻ Tfh1 cells, that share properties with Th1 cells by secreting interferon (IFN)-γ; (2) CXCR3⁻ CCR6⁻ Tfh2 cells, that resemble Th2 cells by expressing interleukin (IL)-4; (3) CXCR3⁻CCR6⁺ Tfh17 cells, which are similar to Th17 cells in the production of IL-17. The capacity of Tfh1 cells to assist B cells is not well-defined, while the cTfh2 and Tfh17 cells are considered the most efficient B-cell helpers by inducing naïve B cells to produce immunoglobulins (Tfh2 cells promoting IgG and IgE production and Tfh17 cells promoting IgG and IgA secretion) and promote isotype switching [17].

Human cTfh cells can be further subdivided based on ICOS, PD-1, and CCR7 expression. ICOS⁻ and/or PD-1⁻ Tfh cells are considered quiescent subsets, whereas PD-1⁺ICOS+CCR7^lo subsets are recently activated memory Tfh cells with a high capacity to differentiate B cells into IgG-producing cells. This latter population is also described in human autoimmunity, not being abundant in healthy individuals, and positively correlates with disease activity and serum antibody titers of some autoimmune diseases, indicating that it may be a useful biomarker in screening autoimmune patients [18].

4. Regulation of Tfh cells differentiation

The transcriptional environment also changes during these three stages of Tfh cell differentiation. In its primary stage, the expression of Bcl6 is low and the expression of BATF, IRF1, pSTAT5, and TCF-1 is enhanced. During the second stage, Bcl6 expression remains low while pSTAT3, Tbet, and RORγt are upregulated. In the final stage of maturation, Bcl6 is now highly expressed, while the expression of Tbet, GATA3, RORγt, pSTAT5, and Blimp1 is suppressed.

Furthermore, it is increasingly clear that metabolic regulation plays an important role in the differentiation of Tfh cells, including glycolysis, ATP and fatty acid metabolism.

4.1 Glycolysis

Much available data indicates that Tfh cells are distinguished by higher levels of glycolysis and lower levels of oxidative phosphorylation than other subsets of T cells [19]. The process of regulating the glucose metabolism in Tfh cells is very intricate because too low or too high level of glycolysis is not conducive to the differentiation of Tfh cells. Activation of mTORC1 and mTORC2 caused by ICOS can induce the increase in anabolism, high expression of glucose transporter 1 (Glut1) and the promotion of Tfh cell development, while the expression of Bcl-6 inhibits the expression of glycolysis-related enzymes. It is indicated that glycolysis, which is required during early T-cell activation, is not mandatory during the differentiation process of mature Tfh cells. Moreover, IL-2 elevates the activation of Akt and mTORC1 to result in the conversion of low-glycolytic Tfh cells to high-glycolytic Th1 cells [20].

4.2 ATP synthesis and metabolism

ATP-gated ionotropic P2X7, one of the purinergic receptors, is selectively and abundantly expressed in the plasma membranes of Tfh cells and can mediate apoptosis in these cells. Knockouting of P2rx7 can promote Tfh cells to resist apoptosis, elevate the absolute number of Tfh cells in Peyer’s patches, and enhance the GC responses significantly [21].

The A2a adenosine receptor (A2aR) is a stimulatory G protein (Gs) protein-coupled G protein-coupled receptor (GPCR) that increases cyclic adenosine monophosphate (AMP) levels. The loss of A2aR leads to an increase in relative Tfh cell differentiation, while the stimulation of A2aR reduces Tfh and GC B-cell numbers. Furthermore, the hypoxic environment inside GC can induce the production of extracellular adenosine to regulate the differentiation of Tfh cells via A2aR [22].

4.3 Fatty acid metabolism

As the proteins that can catalyze the synthesis of monounsaturated fatty acids from saturated fatty acids (SFAs) localized in the endoplasmic reticulum, stearoyl-CoA desaturases (SCDs) are capable of promoting the differentiation of Tfh [23]. It was indicated that fatty acid metabolism is also involved in the development of Tfh cells. However, the mechanism whereby fatty acid metabolism regulates Tfh differentiation remains unclear.

5. Tfh and autoimmune and autoinflammatory diseases

5.1 Tfh and rheumatoid arthritis

Rheumatoid arthritis (RA) is a prevalent chronic autoimmune disease characterized by synovial inflammation and joint cartilage destruction, resulting in joint deformity and disability [24]. It was found that the proportion of cTfh cells in RA patients was significantly higher than that in healthy people [25] and the Tfh cells are increased in the synovium of patients with RA compared with patients with inflammatory osteoarthritis [26]. The frequencies of circulating CD4⁺CXCR5⁺CD40L⁺Tfh cells, a Tfh cell subpopulation, are positively correlated with disease activity score-28 with erythrocyte sedimentation rate (DAS28-ESR), rheumatoid factors (RFs), and anti-cyclic citrullinated peptide (anti-CCP) autoantibodies, which are of great significance in the pathogenesis of RA [27]. In addition, the levels of BCL-6 in Tfh cells and IL-21 in the serum of RA patients are higher than those of healthy people, and the expression of IL-21 was positively correlated with disease activity and serum autoantibodies [28]. Deng’s research found that in CD4⁺ T cells of RA patients, excessive activation of the IL-6-pSTAT3 signal leads to abnormal activation of Tfh cells [29]^.

5.2 Tfh and autoimmune hepatitis

Autoimmune hepatitis (AIH) is a chronic immune-mediated liver disease, featured by circulating autoantibodies, elevated serum IgG levels, and histologic interface hepatitis [30]. Compared with healthy individuals, AIH patients have an increase in Tfh cells and a decrease in follicular regulatory T (Tfr) cells. Moreover, the number of Tfr cells is negatively correlated with the number of Tfh cells, so the imbalance between Tfr and Tfh cells may lead to excessive production of autoantibodies [31]. Ma et al. found that compared with healthy controls, the number of circulating CD38⁺, CD86⁺, or CD95⁺ B cells, ICOS⁺, and PD-1⁺ Tfh cells significantly increased, and serum IL-21 levels increased. The number of ICOS⁺ or PD-1⁺ Tfh cells is positively correlated with CD86⁺ and CD95⁺ B cells, respectively. The number of CD38⁺ B cells, ICOS⁺, or PD-1⁺ Tfh cells is positively correlated with the patient’s serum IgG or IgM concentration, respectively. Therefore, it was indicated that circulating activated Tfh and plasma cells may be associated with hypergammaglobulinemia in the pathogenesis of human AIH [32]. However, there is a study reporting that in the experimental autoimmune hepatitis (EAH) model, Tfh cells were lower than in the healthy control group, while Tfr cells were much higher [33]. Besides, the liver injury of EAH mice can be improved by inhibiting Tfh cells or adjusting the imbalance between Tfr cells and Tfh cells [7, 34].

5.3 Tfh and systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease marked by impaired immune tolerance and the production of high-affinity autoantibodies. Abnormal production of type I IFN in SLE patients promotes Tfh cells to secrete IL-21 and IFN-γ by activating STAT4, leading to pathogenic B-cell response [35]. In SLE patients, the increased Tfh cells in SLE patients were mostly T-cell factor-1 (TCF1) negative subsets with weakened function, and the changes in these subgroups can reflect the progression of SLE patients [36]. Compared with healthy people, the proportion of cTfh17 cells in SLE patients is increased [37]. However, Szabó et al. found that the frequency of cTfh17 cells decreased in patients with lupus nephritis, which is related to higher disease activity scores [38]. These different results may be due to the use of different markers to detect cTfh17 cells. The frequency of cTfh2 cells was positively correlated with the level of anti-dsDNA autoantibodies and the frequency of plasma cells. Research has also found that interferon γ (IFN-γ) expressing cTfh cells in the MRL/lpr mice are increased [39]. The decrease of Tfr cells and Tfr/Tfh cell ratio in renal lymph nodes are related to the increase of GC-B cells and the onset of lupus nephritis in a mouse model [40].

5.4 Tfh cells and other autoimmune diseases

Primary Sjogren’s Syndrome (pSS) is an autoimmune disease with persistent lymphocyte infiltration in exocrine glands, including salivary and lacrimal glands, leading to Sicca syndrome [41]. The percentage and absolute number of PD-1⁺ICOS⁺Tfh cells in pSS patients are significantly higher than those in healthy controls, and the number of PD-1⁺ICOS⁺Tfh cells is related to B cells. The PD-1⁺ICOS⁺Tfh cells are positively correlated with autoantibodies, erythrocyte sedimentation rate, IgG, IL-2, IL-4, IL-10, IL-17, IFN-γ, TNF-α, IL-21, and disease activity [42].

Systemic sclerosis (SSc) is a connective tissue disease with high mortality, which is characterized by fibrosis of the skin and internal organs [43]. Tfh cell infiltration can be observed in the skin lesions of SSc patients [44]. Compared with healthy controls, the number of cTfh cells in SSc patients increased, the expression level of BCL-6 in cTfh cells was higher, and the ability to secrete IL-21, induce B-cell differentiation and produce IgG and IgM was enhanced [45, 46]. The levels of cTfh1 and cTfh17 in SSc patients increased, and the proportion of cTfh1 cells was positively correlated with autoantibody titer and IL-21 concentration [45].

Inflammatory bowel disease (IBD) is a non-infectious, chronic inflammatory, multifactorial condition encompassing Crohn’s disease, ulcerative colitis (UC), and indeterminate colitis [47]. Studies have found that Tfh cells in intestinal GC of IBD patients increase and Tfr cells decrease. In addition, the levels of BCL-6 and IL-21 in the peripheral blood of IBD patients were significantly higher than those of healthy controls [48].

6. Taking Tfh cells into therapy for autoimmune diseases

Increasingly, evidence has demonstrated that the elevation in the number and function of Tfh cells plays an important role in the occurrence and progress of various autoimmune diseases. The study on the mechanism of Tfh cells in autoimmune diseases not only reveals the pathogenesis of autoimmune diseases but also provides a new idea for targeting Tfh cells to treat autoimmune diseases. In this section, we summarize some drugs and methods for Tfh cells to treat autoimmune diseases.

7. Exhausting B cells

Tfh cells can be reduced or eliminated by exhausting B cells that continuously present antigens to Tfh cells, such as using anti-CD20 antibodies. By detecting Tfh cells in the spleen of patients with immune thrombocytopenia treated with rituximab, it was found that all CD19⁺ B cells in the spleen were missing, and the mature Tfh cells were eliminated, which indicated that B cells might be necessary to maintain Tfh cells in secondary lymphoid organs [49].

7.1 Regulate Tfh cell differentiation

The differentiation of Tfh cells is mainly regulated by the signals of costimulatory molecules and cytokine receptors. CD28, CD40L, ICOS and OX40 are necessary to produce Tfh cells and their auxiliary B cells [3]. By blocking this surface co-receptor, the differentiation of Tfh cells can be inhibited, thus treating autoimmune diseases. For example, CTLA4 fusion protein can block CD28 costimulatory signal and inhibit Tfh cell production by binding CD80 and CD86, thus reducing the number of PD-1⁺Tfh cells in RA patients [50] and cTfh cells in pSS patients [51].

7.2 Regulate cytokines and cytokine signals

Targeting cytokines is the most direct and successful treatment strategy for autoimmune diseases. The function of Tfh cells can be regulated by blocking cytokines or cytokine downstream pathways, such as inhibiting IL-6 or Janus kinases (JAKs) [52].

7.3 Regulate intestinal microecology

Intestinal probiotics may reduce the number of pathogenic microorganisms in the intestine and the differentiation of related autoreactive Tfh cells, thus inhibiting the development of autoimmune diseases. Transplantation of fecal microflora can alleviate liver injury in EAH mice by regulating the balance between Tfr cells and Tfh cells [7].

8. Challenge, bottleneck and prospect

Although targeting Tfh cells has shown enormous therapeutic potential in animal models of several autoimmune diseases, more rigorously designed studies are still needed to ensure the biological safety of these therapies. Because Tfh cells play a key role in antibody production, targeting Tfh cells for autoimmune diseases may lead to a decrease of various antibodies, including antibodies that protect us from viruses, bacteria, parasites, and fungi, thus causing serious infections and no response to vaccines [53]. Therefore, more efforts are needed to fully elucidate the role of Tfh cells in autoimmune diseases. We need to further use cutting-edge single-cell technology to analyze the heterogeneity within the Tfh cell population and understand the specificity, plasticity, and stability of specific Tfh cell subsets so that we can better understand which Tfh cells or molecules in Tfh are the most pathogenic in autoimmune diseases. Therefore, fine-tuning the specific subpopulation of Tfh cells or the unique molecules differentially expressed in Tfh cells related to the pathogenesis of autoimmune diseases, and inhibiting pathogenic Tfh cells without affecting the protection against infection, may be the development trend of targeted Tfh cell therapy in the future. In addition, it is also necessary to further implement more precise individualized treatment in targeted Tfh cell therapy, and study how to use Tfh cell characteristics, such as Tfh cell frequency and functional-related molecular testing to stratify patients and guide the treatment plan and prognosis evaluation of targeted immunotherapy.

In this paper, a large number of evidence of Tfh cells and the rapid growth of autoimmune diseases are reviewed, and the incidence and progress of Tfh cells in autoimmune diseases and the research status of targeted therapy are introduced. Although there are still some challenges and bottlenecks, the prospect of using Tfh cells to treat autoimmune diseases is still very optimistic. We hope that through further research and technology development, we can deeply understand the specific mechanism of Tfh cell subsets and specific molecules in autoimmune diseases, and develop safer, more effective, and more accurate treatment strategies for Tfh cells, further transfer targeted therapy from laboratory to clinical application, and bring better health and quality of life to patients.

Acknowledgments

This study was funded by Special Project of Scientific And Technological Cooperation and Exchange in Shanxi Province (Grant Number: 202104041101013, Grant Recipient: Yang Liu); Science and Technology Innovation Talent Team of Shanxi Province (Grant Number: 202204051002033, Grant Recipient: Huiqin Hao); High-Level Key Disciplines Of Traditional Chinese Medicine Project (Grant Number: CZ2023012, Grant Recipient: Huiqin Hao); Integrated Traditional Chinese and Western Medicine Basic Discipline Construction Project of Shanxi University of Chinese Medicine (Grant Number: 2023XKJS-03, Grant Recipient: Huiqin Hao); Innovative Team of Prevention and treatment of autoimmune hepatitis with integrated Chinese and Western medicine (Grant Number: 2022TD2003, Grant Recipient: Yang Liu).