Peripheral follicular helper T cells in acute viral diseases: a perspective on dengue

Abstract

Follicular helper T cells (T_FH) are a predominant subset of CD4⁺ T cells specialized in providing help to B cells in germinal centers and necessary to generate T cell-dependent antibody responses. Peripheral T_FH (pT_FH) are the counterpart of T_FH found in the circulation, which resemble T_FH in many aspects of their phenotype and function. The CD4⁺ pT_FH subset has received a lot of interest recently because they are easy to access and have the potential to serve as a biomarker for long-lasting humoral immunity. This review will discuss recent findings of pT_FH in human acute viral diseases with a focus on dengue infection.

Four serotypes of dengue virus (DENV1–4) cause an estimated 100 million symptomatic infections each year resulting in a spectrum of disease, ranging from a nonspecific febrile syndrome to dengue fever to more severe illness, dengue hemorrhagic fever (DHF) and dengue shock syndrome [1]. There are many risk factors for developing severe disease, which include viral factors, host genetics, nutritional status and background immunity [2]. Infection with any serotype of DENV for the first time (primary infection) is believed to confer life-long homotypic immunity [3]. Epidemiologic evidence clearly indicates that sequential infection with a heterologous serotype of DENV is a major risk factor for developing DHF [4]. DHF can occur in primary dengue infection of infants born to dengue immune mothers [5]. Cross-reactive antibodies with low avidity and altered T cell immune responses to the current infecting serotype are hypothesized to contribute to severe dengue disease during a second infection with a heterologous serotype [6,7]. A goal of many researchers, therefore, is to develop effective strategies that can induce potent neutralizing antibody and cross-reactive T cell responses to all four DENV serotypes.

T helper subsets

CD4⁺ T cells can contribute to protection against a number of pathogens. Naive CD4⁺ T cells activated by dendritic cells (DCs) presenting pathogen-derived peptides can differentiate into memory and effector T cells through a complex process [8]. There are different subsets of antigen-primed CD4⁺ T helper (T_H) cells classified as T_H1, T_H2, T_H9, T_H17, T_H22, T follicular helper cells (T_FH) and T regulatory cells (Tregs) [8]. T_H subsets are defined based on the expression of surface molecules, production of cytokines and expression of transcription factors [8]. T_FH are a predominant subset of CD4⁺ T cells specialized in providing help to B cells and necessary to generate T cell-dependent antibody responses in germinal centers (GCs) [9–11]. Peripheral T_FH (pT_FH) are the counterpart of T_FH found in circulation [12]. In this review, we will discuss recent findings of pT_FH following influenza and Ebola virus vaccination and natural DENV infection.

Features of T_FH

T_FH were identified for the first time in human tonsils and are characterized by high expression of the B cell zone-homing chemokine receptor CXCR5 that drives T_FH to B cell follicles, including GCs in response to chemokine ligand CXCL13 [9–11]. In addition to CXCR5 expression, T_FH express other surface markers such as PD-1, ICOS, OX40 and CD40L [9,10,13,14]. T_FH express low levels of the T cell zone-homing chemokine receptor CCR7 [9,10] and the IL-7Rα [15]. DCs initiate the differentiation of T_FH in the T cell zone in secondary lymphoid organs. After priming, T_H cells first interact with cognate B cells in the T-B cell border (pre-T_FH) and then T_FH position in the B cell follicles completing T_FH cell differentiation [16]. Furthermore, T_FH can form after prolonged antigen presentation by DCs in the absence of antigen presentation by B cells [17]. Already differentiated T_H1, T_H2 or T_H17 cells can also reprogram to T_FH in GCs [14]. The master regulator of T_FH differentiation is the transcription factor BCL-6 [18]. Other transcription factors that regulate T_FH generation include IRF-4, BATF, c-MAF and the STAT3 [19–22]. BLIMP-1 can modulate T_FH generation by suppressing BCL-6 expression [23]. IL-21 is considered the hallmark cytokine secreted by T_FH. IL-21 helps B cells secrete antibodies and can also act in an autocrine manner to generate T_FH [24,25]. In addition to IL-21, T_FH can secrete IL-4, IL-10 and IFN-γ [26,27].

T_FH function

The principal function of T_FH is to provide help to B cells in a cell-to-cell contact manner through ligands/receptors such as CD40L and ICOS and cytokines such as IL-4 and IL-21 in secondary lymphoid organs [13,24,26,28,29]. T_FH are critical for GC formation and maintenance of GC responses [23,30–32]. T_FH provides signals for B cell survival and proliferation [33,34], support memory B cell differentiation [35], Ig isotype class switching [36] and plasma cell differentiation [24]. T_FH can kill B cells that fail to present cognate antigen in the absence of prosurvival signals via Fas–FasL interactions [14].

T_FH in the circulation

Germinal center T_FH (GC T_FH) share many phenotypic and functional properties with CD4⁺ CXCR5⁺ T cells in the circulation (pT_FH) but there is considerable debate about cell surface markers that clearly define pT_FH. Given the ease of access to pT_FH and their potential to serve as a biomarker for monitoring antibody responses to vaccination or infection, several studies in humans have evaluated pT_FH in the last decade. The majority of pT_FH express CXCR5 and are in a quiescent state [37] with minimal to no expression of BCL-6 [12,14,27,38]. Memory GC T_FH can exit the GC, express markers similar to central memory T_H (CCR7⁺CD62L⁺) and circulate in different organs including the spleen, lymphoid nodes, bone marrow and blood [27,39]. Based on the expression of CCR7, it is possible that pT_FH are memory GC T_FH found in the circulation [9,10]. Some studies suggest that pT_FH may represent bonafide GC T_FH in the circulation while others suggest they are a distinct T cell subset [40,41]. Circulating pT_FH are further classified into three main subpopulations based on the expression of the chemokine receptors CXCR3 and CCR6, as pT_FH1 (CXCR3⁺CCR6^-), pT_FH2 (CXCR3^-CCR6^-) and pT_FH17 (CXCR3^-CCR6⁺) [12,37]. Expression profiling of transcription factors and cytokine production demonstrated that pT_FH1 express T-bet and produce IFN-γ; pT_FH2 express GATA3 and produce IL-4, IL-5, and IL-13 and pT_FH17 express RORγT and produce IL-17 and IL-22. The pT_FH17 subset is the main producer of IL-21 with lower production by other subsets [12]. This classification is not intended for GC T_FH since very few GC T_FH produce cytokines other than IL-21 and IL-4 [19,42]. In vitro studies have found varying capacity of pT_FH subsets to help naive B cells – pT_FH2 and pT_FH17 subsets efficiently help B cells while the pT_FH1 subset is less efficient at inducing antibody secretion from B cells [12,43,44]. Furthermore, pT_FH2 induce naive B cells to produce IgG and IgE isotype antibodies while the pT_FH17 subset induces IgG and IgA isotype secretion from naive B cells [12].

Other follicular T cells

The heterogeneity of T_FH has been evaluated in the last 5 years and new subsets have been identified. T follicular regulatory cells (T_FR) express some Treg-related molecules including CD25, CTLA-4, IL-10, TGF-β and the transcription factor FOXP3 in addition to T_FH-related molecules CXCR5, PD-1, ICOS and BCL-6 allowing the cells to migrate to the GCs [45]. T_FR however lack expression of CD40L, IL-4 and IL-21, do not provide help to B cells and restrain humoral immune responses [46–48]. T_FR form after infection or immunization and can be derived from thymus-derived (natural) FOXP3⁺ Tregs [46–48] and from naive T cells [49]. The ratio between T_FH and T_FR is important in the GC reaction [50,51]. A potential role proposed for T_FR is to control the magnitude of the GC reaction [46–48]. T_FR can be detected in blood (pT_FR) but these cells are not fully competent [52,53]. Other human follicular T cell populations that participate in the regulation of antibody responses include follicular CD8 T cells [54], follicular helper natural killer T (NKT_FH) cells [55,56], and follicular gamma delta (γδ) T cells [57]. Follicular CD8 T cells, follicular helper natural killer T and follicular γδ T cells have not been studied during human acute viral infections and are therefore not covered in this review.

T_FH have been implicated in different immune system disorders [58]. An increase in the frequency of T_FH is associated with autoimmunity and T cell lymphoma while a decrease is associated with immunodeficiency [58]. In human chronic viral diseases, pT_FH have been evaluated following Hepatitis B [59], Hepatitis C [60] and HIV infections [44,61]. Circulating T_FH have recently been studied after influenza and Ebola vaccination and natural DENV infection.

Peripheral follicular helper T cell responses following vaccination

Influenza vaccination

Protection against influenza viral disease can be mediated by neutralizing antibodies specific to the hemagglutinin (HA) protein induced by natural infection or vaccination [62] although cellular immunity may also contribute to protection. To prevent seasonal influenza, an annual vaccination is recommended for all persons older than 6 months of age [63]. However, there are still some gaps in eliciting vaccine efficacy in pregnant women, children between 6 months to 5 years and older adults [64]. Many studies have reported increased pT_FH [65–68] and pT_FR (FOXP3⁺ and CD25⁺) frequencies postvaccination [69,70]. After vaccination with the standard dose of influenza vaccine, reduced frequencies of pT_FH were found in the peripheral blood of elderly individuals [71,72], with a decreased ability to provide B cell help [71]. However, when a high dose influenza vaccine was administered in elderly individuals, an increased frequency and activation of pT_FH was observed. The frequency of pT_FH correlated with the frequency of plasmablasts in this study [73]. In the elderly, therefore, modulation of the dose of influenza vaccine could improve the induction and activation of pT_FH responses. Some studies suggest that CD4⁺ T cells or pT_FH do not express BCL-6 [65,74] while others suggest pT_FH (ICOS⁺CD38⁺) express mRNA and/or protein for BCL-6 [69,71]. Administration of an inactivated influenza vaccine (TIV) increased frequencies of the pT_FH1 subset [65,74] with no increase in the frequencies of pT_FH2 and pT_FH17 T cells [65]. Although pT_FH have reactivity to multiple influenza antigens, the majority of pT_FH are specific to HA antigens [66,69,75]. The frequency of pT_FH specific for influenza antigens was associated with circulating plasmablast frequencies, the frequency of CD21^loCD27⁺ and CD21^hiCD27⁺ memory B cells and protective antibody responses [66,67,69]. The frequency of pT_FH (ICOS⁺PD-1⁺CXCR3⁺) contributed to the generation of high-avidity antibodies after the administration of inactivated influenza vaccine [66]. However, pT_FH frequencies correlated with pre-existing antibody titers and not with the induction of primary antibody responses. In this study, purified CD4⁺ICOS⁺CXCR5⁺CXCR3⁺ T cells were most efficient at inducing memory B cells to differentiate into plasma cells to produce influenza-specific antibodies [65].

Ebola vaccination

Preclinical studies in nonhuman primates and mouse models have suggested that antibodies and CD4⁺ T cells mediated by a vaccine candidate based on recombinant vesicular stomatitis virus (rVSV) expressing the Zaire Ebola virus (ZEBOV) glycoprotein are involved in protection against Zaire Ebolavirus [76,77]. A single dose of the recombinant vesicular stomatitis virus-ZEBOV vaccine candidate induced ZEVOB-GP-specific pT_FH at day 28 and pT_FH frequencies were dependent on the vaccine dose and maintained at day 56. Interestingly, in this study, the pT_FH17 subset was the predominant subset induced followed by pT_FH2 and pT_FH1 cell subsets. The frequency of pT_FH17 but not pT_FH2 or pT_FH1 subsets correlated with antibody titers at day 28 postvaccination [78].

Peripheral follicular helper T cell responses following DENV infection

Fewer published studies have evaluated pT_FH during DENV infection. Rivino et al. identified a small population of DENV-specific CD4⁺CD45RA^-CXCR5⁺ memory T cells that secreted hallmark T_FH cytokine IL-21 by stimulating convalescent peripheral blood mononuclear cells (PBMC) with dengue peptides. IL-21 was only produced by CXCR5⁺CD4⁺ T cells while both CXCR5⁺ and CXCR5^- CD4⁺ T cells produced IFN-γ. The ability of DENV-specific pT_FH to provide B cell help was not assessed in this study; however, the results support other in vitro studies, which clearly demonstrate that CD4⁺CXCR5⁺CD45RA^- T cells in the circulation can help B cells to produce antibody [79]. This study differs from recently published work which suggest that CXCR5^-PD1^hiCD4⁺ T cells express factors including IL-21, CXCL13, ICOS and MAF and can provide help to B cells in patients with rheumatoid arthritis [80].

Vivanco-Cid et al. measured IL-21 levels in the sera of patients undergoing acute DENV infections (1–7 days after disease onset) and in early convalescence (8–10 days after disease onset) and found elevated levels in acute sera compared with healthy controls. Interestingly, they found the highest levels of IL-21 in sera of patients obtained during the early convalescent phase of primary DENV infection. A positive correlation between IL-21 levels and production of DENV-specific IgM and IgG antibodies was reported. In addition, significant differences in IL-21 levels were detected based on the clinical phase and the severity of DENV infection. Their findings suggest that IL-21 may have a protective role supporting B cell antibody production since T_FH are the main producers of IL-21 [81].

Our group recently evaluated frequencies of pT_FH in PBMC from Thai children. PBMC were collected during and after acute DENV infection at febrile (fever days -5 to -1), critical (fever days 0 to +1), early convalescence (fever days +3 to +8), and healthy (6-months to 2-years postenrollment) time points. We detected elevated frequencies of activated pT_FH (PD-1^hi pT_FH and PD-1⁺CD38⁺ pT_FH) in PBMC obtained during acute DENV infection with the highest frequencies of pT_FH activation detected during the critical phase of illness. The numbers of activated pT_FH were higher in PBMC obtained from patients with secondary compared with primary infections and in PBMC from patients with more severe disease [82]. We further phenotyped pT_FH in a subset of PBMC based on their expression of CXCR3 and CCR6 and the majority of pT_FH expressed CXCR3 but not CCR6. To determine whether there was a correlation between pT_FH frequencies and B-cell activation in vivo, we measured the frequency of plasmablasts at acute time points and found a positive correlation between the frequency of pT_FH activation and the frequency of plasmablasts. The clinical studies using samples from dengue patients are encouraging and warrant larger studies to evaluate the function of pT_FH in more detail.

DENV induces the generation of T_FH

A recent in vitro study found that DENV could drive the formation of pT_FH and subsequent antibody production [83]. DENV replication in human monocyte-derived DCs (mdDCs) and dermal DCs activated RIG-I and MDA-5 cytoplasmic RNA sensors leading to IFN-α/β transcription and IFN-α/βR activation. DENV infection specifically phosphorylated STAT1 to modulate IFN-α/βR signaling and drive IL-27 production. Co-culture of DENV-infected mdDCs with naive CD4⁺ T cells lead to increased expression of CXCR5, PD-1 and BCL-6, and the formation of IL-21 secreting T_FH (Figure 1). T_FH formation was abrogated by neutralizing antibodies against IL-27, indicating that T_FH polarization was dependent on IL-27. Co-culture of the differentiated T cells with CD19⁺ B cells induced the secretion of IgM and IgG antibodies [83]. This study concluded that the infection of DCs with DENV drives T_FH polarization, and subsequently supports antibody production.

Figure 1. . Differentiation of follicular helper T cells during dengue virus infection.

Infection of dermal DCs by DENV leads to the activation of RIG-1 and MDA-5 with type 1 IFN (IFN-α/β) and IL-27 production. DCs that present dengue peptides on MHC class II complexes prime naive DENV-specific CD4 T cells to proliferate and differentiate into T_FH which express CXCR5, PD-1, BCL-6 and IL-21. Cross-talk between T_FH and cognate B cells requires IL-21 and helps to stimulate IgM and IgG antibody secretion.

DC: Dendritic cell; DENV: Dengue virus; ICOS: Inducible T cell co-stimulator; ICOSL: ICOS ligand; TCR: T cell receptor.

Conclusion & future perspective

The data to date suggest that pT_FH subsets and the key hallmark cytokine IL-21 are detected in the circulation during acute natural DENV infections (Figure 2). Our data found the majority of pT_FH detected in the peripheral blood during the critical phase of acute dengue infection express markers associated with the inefficient helper pT_FH1 subset while other pT_FH subsets (pT_FH2 and pT_FH17) more efficient at inducing naive B cells to secrete Ig and isotype switch were less dominant [12,37]. These results are similar to findings in patients with acute malaria [84]. It is tempting to suggest that vaccination strategies should focus on generating more efficient helper T cells, namely pT_FH2 or pT_FH17 subsets. Next generation adjuvants that enhance antibody responses are associated with increased frequencies of T_FH in animal models [85–88]. A number of questions, however, still need to be first answered as T_FH have also been implicated as key drivers in many autoimmune diseases [89]. Specifically, in response to DENV infection or vaccination we need to know:

Figure 2. . Peripheral follicular helper T cells subsets during dengue virus infection.

The expression of chemokine receptors CXCR3 and CCR6 defines three subsets of pT_FH: pT_FH1 (CXCR3⁺CCR6^-), pT_FH2 (CXCR3^-CCR6^-) and pT_FH17 (CXCR3^-CCR6⁺). The main subset produced during the critical phase of DENV infection is pT_FH1, which has been associated with inducing nonspecific B-cell activation. Frequencies of pT_FH are higher in PBMC obtained from patients with secondary (2°) compared with primary (1°) dengue infection as well as in patients with DHF compared with DF during the critical phase of infection. IL-21 is elevated during clinical course of DENV infections.

DF: Dengue fever; DHF: Dengue hemorrhagic fever; PBMC: Peripheral blood mononuclear cell.

• Does the increased frequency of pT_FH1 subsets detected during acute dengue impact the quality of DENV-specific neutralizing antibodies generated during primary as well as secondary DENV infections?

• Do pT_FH frequencies correlate with the ability to generate DENV-specific memory B-cells?

• What about the specificity of the T_FH? Is recognition limited to specific nonstructural proteins? Are pT_FH capable of producing IL-21 during natural DENV infection?

• Which immunoglobulin isotypes are induced by DENV-specific pT_FH subsets?

• What is the role of T_FR in DENV infection?

• Are pT_FH and pT_FR detected following vaccination with live attenuated, protein-based, inactivated vaccine candidates? Do pT_FH frequencies correlate with vaccine-induced antibody responses? Which pT_FH subsets are induced after DENV vaccination?

Considerable progress has been made with phenotyping and characterizing pT_FH during acute viral infections. The number of studies on dengue and pT_FH are limited, but the initial findings are encouraging and highlight the potential for pT_FH to serve as a biomarker for eliciting strong antibody responses in response to natural dengue infection. Given the number of ongoing Phase II and III dengue vaccine trials, using pT_FH subsets as a biomarker of vaccine efficacy is an attractive possibility.

Executive summary.

• Follicular helper T cells (T_FH) are a distinct subset of effector T cells characterized by the expression of chemokine receptor CXCR5, the transcription factor BCL-6 and secretion of the cytokine IL-21 that are predominantly located in germinal centers in secondary lymphoid organs.

• Peripheral T_FH (pT_FH) characterized by CXCR5 and PD-1 expression are the counterpart of T_FH found in circulation.

• T_FH and pT_FH provide help to B cells to generate T cell-dependent antibody responses.

• Three pT_FH subsets can be distinguished based on the expression of chemokine receptors CXCR3 and CCR6 as pT_FH1 (CXCR3⁺CCR6^-), pT_FH2 (CXCR3^-CCR6^-), and pT_FH17 (CXCR3^-CCR6⁺). pT_FH2 and pT_FH17 subsets efficiently help B cells.

• T_FR are a recently described cell population that can regulate the antibody production. Influenza vaccines induce pT_FR and pT_FH. Influenza hemagglutinin-specific pT_FH produce IL-21 and express markers associated with the pT_FH1 subset.

• Ebola vaccine induces glycoprotein-specific pT_FH, and expresses markers associated with pT_FH17 which correlate with antibody responses.

• DENV induces the differentiation of naive CD4 T cells into T_FH and stimulates antibody production from naive B cells.

• High frequencies of pT_FH detected during the critical phase of dengue contribute to disease evolution.

• The majority of pT_FH found during the critical phase of dengue illness express markers associated with the pT_FH1 subset.

• IL-21 serum levels are elevated during the early convalescent phase of primary DENV infections and correlate with the production of DENV-specific IgM and IgG antibodies.