Several divergent and often competing programmes of CD4+ T cell differentiation are now well recognized, leading to the development of distinct functional subsets, including T helper (Th) 1, 2, or 17 cells, follicular helper (Tfh) cells, and regulatory T (Treg) cells (1–5). The relative balance of CD4+ T cell differentiation to one or more of these functional subsets largely depends on a multitude of T cell-extrinsic factors, with the cytokine milieu naïve T cells encounter during the priming phase playing a major role (1–5). However, CD4+ T cell differentiation can also be shaped by T cell-intrinsic properties, such as the relative affinity of the T cell receptor (TCR), favoring development of particu lar subsets (6–8). The combined effect of such T cell-extrinsic and T cell-intrinsic factors can result in considerable diversity of functional responses, allowing adaptive immunity to modify its response according to the nature of the antigenic stimulus.

Acute viral infections typically induce a CD4+ T cell response that is almost exclusively composed of Tfh and Th1 cells, in approximately equal proportion. Indeed, the ratio of Tfh to Th1 cells in the CD4+ T cell response to acute lymphocytic choriomeningitis virus (LCMV) has been amply reported

#### *Edited by:*

*Georgia Fousteri, San Raffaele Hospital (IRCCS), Italy*

#### *Reviewed by:*

*Hu Zeng, Mayo Clinic, United States Thomas Ciucci, National Cancer Institute (NCI), United States*

#### *\*Correspondence:*

*George Kassiotis<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 28 March 2018 Accepted: 18 May 2018 Published: 05 June 2018*

#### *Citation:*

*Danelli L, Donnarumma T and Kassiotis G (2018) Correlates of Follicular Helper Bias in the CD4 T Cell Response to a Retroviral Antigen. Front. Immunol. 9:1260. doi: 10.3389/fimmu.2018.01260*

**39**

close to 1 and 2 for LCMV Armstrong (9–14) and clone 13 (Cl13) (15–19), respectively, and similar results were also reported for influenza A virus infection (20–23).

Several well-defined factors have been demonstrated to influence the balance of Th1 and Tfh cells in response to viruses, as well as other challenges. One of these is the availability of IL-2, determined both by the rate of production by effector CD4+ T cells and the rate of consumption by Treg cells or dendritic cells (20, 24–26). IL-2 has been reported to negatively affect Tfh differentiation, in favor of Th1 differentiation (20, 24–26). The balance of Th1 and Tfh differentiations in response to infection with viruses, as well as other classes of pathogens, is also strongly influenced by the nature of the dominant antigen-presenting cell (APC) type (2, 3, 5, 6). For example, antigen presentation by B cells is considered critical for consolidating Tfh differentiation, whereas presentation by macrophages is thought to promote Th1 differentiation (2, 3, 5, 6).

How T cells integrate the multitude of intrinsic and extrinsic factors regulating the balance of their differentiation is not currently completely understood. It is possible that these factors do not operate independently, but are linked at the level of APC-T cell interaction. Indeed, the type of dominant APC determines the cytokine milieu (e.g., IL-12 production or IL-2 consumption) (20, 24–26), the provision of costimulatory signals (e.g., ICOS-L) (5, 27), and the TCR signal strength, given that APC types differ in the potency of stimulation.

We studied the CD4+ T cell response to model retroviral antigen from the gp70 envelope glycoprotein of the Friend murine leukemia virus (F-MLV) (28, 29), to assess the relative contribution of individual intrinsic and extrinsic factors, previously linked to the balance of Th1 and Tfh differentiations. Here, we report an atypically strong bias toward Tfh differentiation in response to this retroviral antigen, when presented during natural infection, and describe the variables of the immune response that determine the balance of Th1 and Tfh differentiations in this setting.

### MATERIALS AND METHODS

### Mice

Inbred C57BL/6 (B6) and CD45.1+ congenic B6 (B6.SJL-*Ptprca Pep3b*/BoyJ) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME, USA). TCRβ-transgenic EF4.1 mice (30), Nur77-GFP transgenic mice (31), B cell receptordeficient (*Ighm*−/−) (32), Tcra-deficient (*Tcra*−/−) (33), Rag1-deficient (*Rag1*−/−) mice (34), and Rag2-deficient (*Rag2*−/−) mice (35) were on the B6 genetic background and were maintained at the Francis Crick Institute's animal facilities. All animal experiments were approved by the ethical committee of the Francis Crick Institute and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986 (ASPA).

### CD4**+** T Cell Adoptive Transfer

Single-cell suspensions were prepared from the spleens of donor CD45.1+ or CD45.2+ TCRβ-transgenic EF4.1 mice or CD45.2+ Nur77-GFP EF4.1 doubly transgenic mice, and CD4+ T cells were enriched using an immunomagnetic positive selection kit (StemCell Technologies, Vancouver, BC, Canada), at >90% purity. Donor transgenic CD4+ T cells (1 × 106 per recipient) were injected into recipient mice intravenously.

### Retroviral Infection and Immunization

The Friend virus (FV) used in this study was a retroviral complex of a replication-competent B-tropic F-MLV (F-MLV-B) and a replication-defective spleen focus-forming virus (SFFV). Stocks were prepared as previously described (36). Mice were injected intravenously with 0.1 mL PBS containing ~50 (low dose), 1,000 (intermediate dose), or 5,000 (high dose) spleen focus-forming units of FV. The F-MLV-NB envL128I variant was generated by mutagenesis of the respective codon (CTC → ATT) in plasmid pLRB302, containing the complete NB-tropic F-MLV clone FB29. The resulting plasmid was then transfected into *Mus dunni* fibroblast cells (*M. dunni* cells; CRL-2017). Stocks of F-MLV-B, F-MLV-NB envL128I, or F-MLV-N helper virus were grown in *M. dunni* cells. Mice received an inoculum of ~104 infectious units of helper virus by intravenous injection. Ad5.pIX-gp70 stocks were prepared at a titer of 9 × 109 viral genomes per milliliter by infection of 293A cells as previously described (37). Approximately 5 × 108 Ad5.pIX-gp70 viral genomes per mouse were administered intravenously. Immunization with FBL-3 tumor cells was carried out by intravenous injection of 1.5 × 106 FBL-3 cells (38). For peptide immunization, mice received an intraperitoneal injection of a total of 12.5 nmol of synthetic env122-141 peptide mixed in Sigma Adjuvant System (Sigma-Aldrich, St. Louis, MO, USA). Where indicated, recipient mice also received blocking antibodies against PD-1 (10 mg/kg, clone RMP1-14) and LAG3 (10 mg/kg, clone C9B7W) (both from BioXCell, West Lebanon, NH, USA), injected intraperitoneally on days 0, 1, 3, and 5 post FV infection.

### Antibodies and Flow Cytometry

Spleen single-cell suspensions were stained for 20 min at room temperature or at 4°C with directly conjugated antibodies to surface markers. For detection of intracellular antigens, subsequent to surface staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. They were then incubated for 45 min at room temperature with directly conjugated antibodies to intracellular antigens. Zombie UV Fixable Viability Kit (BioLegend, San Diego, CA, USA) was used to label and exclude dead cells from analysis. The following anti-mouse antibodies were used: BV785- or BV711-anti-CD4 (clone GK1.5), PE/Cy7-anti-CD45.1 (clone A20), PE/Cy7 anti-CD279 (PD-1, clone 29F.1A12), BV785-anti-CD150 (SLAM, clone TC15-12F12.2) (from BioLegend); V500-anti-CD44 (clone IM7), BV421- or PerCPCy5.5-anti-CD162 (PSGL1, clone 2PH1), BV421-anti-Ly6C (clone AL-21), PE-anti-Bcl6 (clone K112-91), FITC-anti-Vα2 (clone B20.1) (from BD Biosciences, San Jose, CA, USA); PE-anti-CD25 (clone PC61.5), PE-Cyanine7-anti-CD45R (B220, clone RA3-6B2), APC-eFluor-780-anti-CD45.2 (clone 104), eFluor450-anti-CD45.1 (clone A20), PE-anti-CD223 (LAG3, clone eBioC9B7W), APC-anti-Ter119 (clone TER-119), APC-anti-Vα2 (clone B20.1), FITC- or APC-anti-TCRβ (clone H57-597) (from Thermo Fisher Scientific, Waltham, MA, USA); Alexa(R)488- or Alexa(R)647-anti-TCF1 (clone C63D9) (from Cell Signaling Technology, Danvers, MA, USA). For CXCR5 staining, splenocytes were incubated with biotin rat anti-mouse CXCR5 antibody (clone 2G8, BD Biosciences) at 37°C for 25 min, followed by incubation with APC- or PE-streptavidin (BioLegend) for 20 min at room temperature. FV-infected cells were detected by using surface staining for the glycosylated product of the viral gag gene (Glyco-Gag), using the matrix (MA)-specific monoclonal antibody 34 (mouse IgG2b), followed by an FITC-anti-mouse IgG2b secondary reagent (clone 12-3 from BD). Multi-color cytometry was performed on LSRFortessa flow cytometers (from BD Biosciences) and analyzed with FlowJo v10.1 (Tree Star Inc., Ashland, OR, USA).

### Fluorescence Microscopy

Frozen OCT (Dako)-embedded spleen sections were fixed in cold acetone, stained with fluorescein labeled peanut agglutinin (PNA, Vector Laboratories), and with directly conjugated antibodies against anti-mouse/human B220 (clone RA3-6B2, AlexaFluor 594, BioLegend) and anti-mouse CD45.1 (clone A20, Alexa Fluor 647, BioLegend). Stained sections were mounted in fluorescent mounting medium (Dako) and viewed with an Olympus IX83 inverted microscope system (Olympus Corporation, Shinjuku, Tokyo, Japan).

### Analysis of Single-Cell RNA-Sequencing Data

Gene transcription in env-reactive CD4+ T cells was assessed using publicly available single-cell RNA-sequencing data (European Nucleotide Archive accession number PRJEB14043) as previously described (39). These included the transcriptional profiles of single env-reactive donor CD4+ T cells isolated from the spleens of wild-type (WT) recipients infected with FV or immunized with Ad5.pIX-gp70, 7 days previously. They also included the transcriptional profiles of single env-reactive donor EF4.1 CD4+ T cells that carried a WT *Bcl6* allele (*Bcl6wt*) or a conditional *Bcl6* allele (*Bcl6fl* ), purified from the spleens of WT recipient mice, 7 days after FV infection. Expression values were analyzed using the Qlucore Omics Explorer 3.3 (Qlucore, Lund, Sweden), and pathway analyses were performed using The Database for Annotation, Visualization and Integrated Discovery v6.8 (https:// david.ncifcrf.gov/home.jsp).

### Cytokine Gene Transcription and Protein Production

Serum levels of IL-2 were measured on a Luminex system (Bio-Plex 100) using the mouse cytokine kits (Bioplex Mouse cytokine group II and Bioplex Mouse Cytokine Standard; Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions, as previously described (30, 40). Transcription of the indicted cytokine genes in env-reactive CD4+ T cells was assessed using publicly available single-cell RNA-sequencing data, as described above.

### Statistical Analyses

Statistical comparisons were made using SigmaPlot 13.0 (Systat Software Inc., Germany) or GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Parametric comparisons of normally distributed values that satisfied the variance criteria were made by unpaired Student's *t*-tests or One Way Analysis of variance (ANOVA) tests. Data that did not pass the variance test were compared with non-parametric two-tailed Mann–Whitney Rank Sum tests or ANOVA on Ranks tests. *p* Values are indicated by asterisks as follows: \**p* < 0.05; \*\**p* < 0.005; \*\*\**p* < 0.0005. Hierarchical clustering, principal component analysis, and heatmap production were performed with Qlucore Omics Explorer 3.3 (Qlucore).

### RESULTS

### The CD4**+** T Cell Response to F-MLV Env Is Heavily Dominated by Tfh Cells

To study CD4+ effector T cell development, we employed a well-described adoptive transfer system, where EF4.1 TCRβtransgenic CD4+ T cells, reactive with the dominant H2-Ab restricted env122-141 epitope within the F-MLV gp70 glycoprotein, were transferred into WT B6 recipients (37, 41). Transferred T cells were primed in recipient mice by infection with FV, a retroviral complex of F-MLV and SFFV that causes chronic infection in B6 mice (28, 29).

As previously reported (41, 42), a considerable proportion (~50%) of env-reactive EF4.1 CD4+ T cells developed a PD-1high CXCR5+ phenotype consistent with Tfh cells (**Figure 1A**). Further detailed phenotypic characterization confirmed the Tfh profile of these cells as CXCR5+ PD-1high Bcl6+ PSGL1− Ly6C− SLAMlow (**Figure 1B**). Consistent with their phenotype, a large proportion of donor CD4+ T cells localized within B cell follicles or germinal centers in the spleens of recipient mice (**Figure 1C**), further supporting strong Tfh differentiation of env-reactive EF4.1 CD4+ T cells.

Notable, however, were the relative paucity (~10%) of envreactive EF4.1 CD4+ T cells with a Th1 phenotype (PSGL1+ PD-1+ SLAM+ CXCR5− Bcl6− Ly6C−) (43) and the presence of a sizable population (~35%) of cells that lacked markers of Tfh or Th1 commitment (PSGL1− SLAM− CXCR5−) (**Figures 1A,B**). The latter population, referred here as Th0 to denote their uncommitted state (44), retained TCF-1 expression and expressed low levels of Bcl6 (**Figure 1B**).

As antigen availability can greatly influence CD4+ T cell differentiation, we compared the efficiency of Tfh development in response to different FV loads (**Figure 1D**). Surprisingly, the proportion of seemingly uncommitted Th0 cells correlated inversely with levels of FV infection, both in terms of absolute numbers and proportion within env-reactive donor CD4+ T cells (**Figures 1B,E**). Indeed, whereas the highest dose of FV primed increased absolute numbers of all three EF4.1 CD4+ T cell subsets, Th0 cells exhibited the highest increase (**Figure 1E**). In contrast, the lowest dose of FV elicited considerable reduced numbers of Th0 and Th1 cells, while favoring development of Tfh cells, which now comprised the overwhelming majority (**Figure 1E**).

Thus, FV infection induces primarily a Tfh phenotype in env-reactive EF4.1 CD4+ T cells and, to a much lesser extent, a Th1 phenotype (**Figure 1E**), whereas Th2, Th17, Treg, or CTL

FIGURE 1 | Follicular helper (Tfh) cells heavily dominate the CD4+ T cell response to F-MLV env. (A,B) CD4+ TCRβ-transgenic EF4.1 T cells were adoptively transferred into CD45.1+ CD45.2+ WT recipients infected with an intermediate dose of Friend virus (FV) (1,000 spleen focus-forming units) and analyzed by flow cytometry in the spleen 7 days later. (A) Gating strategy for the identification of env-reactive (CD4+ CD44+) and Tfh-phenotype (PD-1+ CXCR5+) donor cells in these recipients. (B) Flow cytometric characterization of Th1 (in blue), Th0 (in gray), and Tfh (in red) subsets in env-reactive donor CD4+ CD44+ T cells. (C) CD45.1+ CD4+ EF4.1 T cells were adoptively transferred into FV-infected CD45.2+ WT recipients and detected by immunofluorescence microscopy in spleen sections 7 days later (RP, red pulp; B, B cell zone; T, T cell zone; GC, germinal center). Donor CD4+ were identified as CD45.1+ cells and germinal centers were visualized by staining with peanut agglutinin (PNA). (D) Flow cytometric example of FV-infected Glygo-Gag+ Ter119+ cells (*left*) and frequency of Glygo-Gag+ cells in the Ter119+ population (*right*) in the spleens of WT B6 mice infected with different doses of FV. Mean frequency (±SEM, *n* = 3 mice per group) of one representative of two experiments is shown. (E) Profile of PSGL1 and CXCR5 expression (*left*), absolute number (*middle*), and mean frequency (±SEM) (*right*) of Th1, Th0, and Tfh cells in env-reactive donor CD4+ T cells from the spleen of recipient mice infected with the indicated doses of FV (data are from 4–6 experiments for each FV dose with at least three mice per experiment). (F) Tfh/Th1 ratio in env-reactive donor CD4+ T cells after FV infection (individual symbols denote the mean Tfh/Th1 ratio in independent experiments with at least 3 mice per experiment), compared the Tfh/Th1 ratio reported in the literature for virus-specific CD4+ T cells responding to infection with lymphocytic choriomeningitis virus (LCMV) Armstrong (Arm), LCMV clone 13 (Cl13) or influenza virus PR38 (IAV).

differentiation is not typically observed (37, 39, 42). This skewing in favor of Tfh differentiation was much more pronounced in FV infection, where there are on average 5.2 times more Tfh than Th1 cells, than in other acute viral infections, where this ratio is consistently reported to be closer to 1 (9–23) (**Figure 1F**).

### Distinguishable Transcriptional Activity of Uncommitted Th0 Cells

The strong bias in Tfh development following FV infection suggested that the seemingly uncommitted Th0 cells were primed env-reactive CD4+ T cell that had not successfully completed the program of Tfh differentiation. To better place them in the spectrum of Th differentiation, we compared the transcriptional profiles of Th0 cells using single-cell RNA-sequencing data obtained with env-specific EF4.1 CD4+ T cells primed by FV or a replication-defective human adenovirus serotype 5 vector expressing the F-MLV gp70 glycoprotein (Ad5.pIX-gp70) (39). Th1, Tfh, and Th0 cells were defined here according to their expression of *Cxcr5* and *Selplg* (the gene encoding PSGL1). We first selected the top 204 genes, whose expression best differentiated Th1 and Tfh cells (≥2-fold change, *p* ≤ 0.05) (**Figure 2A**).

Transcription of these genes in Th0 cells showed a profile that was intermediate between the Th1 and Tfh extremes (**Figure 2A**), consistent with their unpolarized phenotype. Direct comparison between Th0 cells and the other two subsets revealed an extensive set of genes that were differentially expressed (≥2-fold change, *p* ≤ 0.05), the majority of which were absent from Th0 cells (**Figure 2B**; Table S1 in Supplementary Material). Specific to Th0 cells was expression of 88 genes (Table S1 in Supplementary Material), involved in active metabolic pathways (**Figure 2C**), indicating a highly activated phenotype of Th0 cells, despite incomplete differentiation.

To further probe any subset commitment of Th0 cells, albeit incomplete, we examined their dependency on *Bcl6* expression. This was achieved by using single-cell RNA-sequencing data obtained with env-specific EF4.1 CD4+ T cells that carried a WT *Bcl6* allele (*Bcl6wt*) or a conditional *Bcl6* allele (*Bcl6fl* ) that was deleted by Cre-mediated recombination upon T cell activation (39). When primed by FV in WT hosts, 41% (25/61) of *Bcl6wt* EF4.1 CD4+ T cells displayed a Th0 phenotype (*Cxcr5*−*Selplg*−), whereas this proportion was reduced to 21% (9/42) in *Bcl6fl* EF4.1 CD4+ T cells (**Figure 2D**), despite comparable numerical priming of both types of T cell under these conditions (39). Together, these data suggested that at least a proportion of Th0 cells may have initiated but not completed Tfh differentiation, particularly in conditions of high viral loads.

### Inhibitory Receptors Restrain Full Tfh Maturation in Response to FV Infection

To further probe how FV loads might influence Tfh differentiation of env-reactive EF4.1 CD4+ T cells, we examined the potential effect of inhibitory receptors. At the peak of their response to FV infection, env-reactive EF4.1 CD4+ T cells have been previously shown to express high levels of multiple inhibitory receptors, including PD-1 and LAG3 (39). Analysis of the earliest time-points at which donor CD4+ T cell expansion can be reliably demonstrated (**Figure 3A**) revealed that PD-1 and LAG3 expression reached near maximum levels already by day 4 post FV infection (**Figure 3B**).

Also comparable between days 4 and 7, post infection was the relative ratio of Tfh and Th1 cells in env-reactive EF4.1 CD4+ T cells (**Figure 3C**), in agreement with commitment to Tfh or Th1 differentiation at this early time-point (45). Given that proliferation of donor CD4+ T cells is atypically slowing down between days 4 and 7 post FV infection (37), we reasoned that the early induction of inhibitory receptors during FV infection might restrict further differentiation. Consistent with this notion,

FIGURE 3 | Follicular helper (Tfh) cells maturation control by inhibitory receptor induction. CD4+ EF4.1 T cells were adoptively transferred into WT recipients infected with intermediate dose of Friend virus (FV) and analyzed by flow cytometry in the spleens on days 4 (*n* = 9) and 7 (*n* = 13) post-transfer and infection. (A) Absolute number of env-reactive CD4+ T cells were recovered. (B) Flow cytometry profile (*left*) and mean frequency (±SEM) (*right*) of PD-1+ LAG3+ cells within env-reactive donor CD4+ T cells. Each symbol is an individual mouse. (C) Mean frequency (±SEM) of T helper (Th) subsets, defined by PSGL1 and CXCR5 expression, in env-reactive donor CD4+ T cells. (D) Representative flow cytometric profile of PD-1 and LAG3 expression (*left*), mean frequency (±SEM) of PD-1+ LAG3+ cells, and (E) mean fluorescence intensity (MFI) of PD-1 staining in env-reactive CD4+ T cells isolated from the spleens of recipient mice on day 7 post transfer and infection with the indicated dose of FV. Each symbol is an individual mouse (*n* = 11, *n* = 14, and *n* = 9 for low, intermediate and high FV dose, respectively). (F) Correlation between the frequency of Th0 cells and the MFI of PD-1 expression in env-reactive donor CD4+ T cells from the same mice described in (D). (G) Flow cytometric analysis (*left*), absolute number (*middle*), and mean frequency (±SEM) (*right*) of Th subsets, defined by PSGL1 and CXCR5 expression, in env-reactive donor CD4+ T cells, 7 days after transfer into FV-infected recipients with or without additional treatment with PD-1 and LAG3 blocking antibodies (*n* = 4 from one of two representative experiments).

frequencies of env-reactive EF4.1 CD4+ T cells coexpressing PD-1 and LAG3, as well as the intensity of PD-1 expression, directly correlated with both FV loads and the proportion of the uncommitted Th0 subset of env-reactive EF4.1 CD4+ T cells (**Figures 3D–F**).

To test whether the early expression of PD-1 and LAG3 during FV infection was impeding Th differentiation of env-reactive EF4.1 CD4+ T cells, we treated recipient mice with PD-1 and LAG3 blocking antibodies. Such treatment during FV infection was previously demonstrated to promote CTL differentiation of donor CD4+ T cells, which is not typically observed in FV infection (39). However, the CD4+ CTL subset induced by PD-1 and LAG3 blockade during FV infection constituted only ~7% of env-reactive EF4.1 CD4+ T cells (39), and it was possible that Tfh differentiation was still favoured. Indeed, PD-1 and LAG3 blockade during FV infection drove efficient differentiation of uncommitted Th0 env-reactive EF4.1 CD4+ T cells into Tfh cells, which again formed the large majority, with a small numerical increase in Th1 cells and a small numerical loss of Th0 cells (**Figure 3G**).

Together, these results suggested that FV infection induces primarily Tfh differentiation of env-reactive EF4.1 CD4+ T cells, partly restrained by inhibitory receptor expression, in turn induced by strong TCR signaling.

### Multifactorial Contribution to Tfh Cell Development During FV Infection

The ability of FV infection to promote Tfh differentiation, especially under conditions when PD-1 and LAG3 were not maximally expressed or were blocked, seemed rather exceptional. A host of well-established intrinsic and extrinsic factors may help or hinder Tfh development and, alone or in combination, could account for the dominance of Tfh cells in the response to FV infection. Included among these factors are the avidity of the TCR, the form of antigen and duration of its presentation, the strength of interaction with B cells, and the cytokine environment, particularly the availability of IL-2.

We next investigated how modulation of one or more variables known to affect Tfh cell development, might shape differentiation of CD4+ T cells in response to FV. High TCR avidity has long been suggested as a contributor to Tfh cell differentiation in other systems and could contribute to Tfh bias also in response to FV infection (6–8). EF4.1 CD4+ T cells comprise a semi-polyclonal repertoire of env-reactive TCRs, differing in their avidity for the cognate antigen, according to the pairing of the transgenic TCRβ chain with endogenous TCRα chains (30, 41). Indeed, clonotypes with higher or lower TCR avidity for H2-Ab -env122-141 can be identified by the use of TCR Vα2 or Vα3 (non-Vα2) endogenous chains, respectively, and used to examine the effect of TCR avidity on Th differentiation (30, 41). Nevertheless, Tfh development was broadly comparable between Vα2+ and Vα3+ env-reactive CD4+ T cell clonotypes, albeit development of Th1 cells appeared more efficient in lower avidity Vα3+ clonotypes in response to FV infection (**Figure 4A**). Thus, the bias in Tfh differentiation of EF4.1 CD4+ T cells was observed across clonotypes with a range of TCR avidities.

As an independent way to assess the effect of TCR avidity, we introduced mutations that alter the potency of the F-MLV env122-141 epitope to stimulate particular clonotypes. Prior work highlighted the L residue at position 128 as an important contributor to recognition, particularly by higher avidity Vα2+ env-reactive CD4+ T cell clonotypes (46). Epitopes carrying an L128I mutation behave as strong agonists for all clonotypes (46). F-MLVs expressing variants of the env122-141 epitope were also compared with N-tropic F-MLV (F-MLV-N), whose replication in B6 mice is restricted by the product of the *Fv1*b allele, therefore reducing the amount of available antigen.

Friend murine leukemia virus carrying the envL128I epitope induced significantly higher numbers of env-reactive EF4.1 CD4+ T cells than WT F-MLV-B, whereas F-MLV-N was less immunogenic (**Figure 4B**). Consistent with its potency, F-MLV-NB envL128I infection primed Vα2+ as well as Vα3+

env-reactive CD4+ T cell clonotypes, in contrast to WT F-MLV infection, which favored Vα2+ clonotypes (**Figure 4B**). As a result, the frequency of Vα2+ clonotypes in env-reactive CD4+ T cells was higher following infection with WT F-MLV than with F-MLV-NB envL128I (**Figure 4B**). Moreover, expression of inhibitory receptors PD-1 and LAG3 was also regulated according to the potency of antigenic stimulation, reaching maximal levels in response to F-MLV envL128I infection (**Figure 4B**). However, despite notable differences in induced clonal expansion and potency of antigenic stimulation between the mutant F-MLV viruses, differentiation of primed EF4.1 CD4+ T cells was highly comparable and still heavily skewed toward the Tfh subset (**Figure 4C**).

Th differentiation in response to a given antigen is also influenced by the context in which it is presented and in particular by its vector. We, therefore, compared the degree of Tfh differentiation of EF4.1 CD4+ T cells induced by F-MLV env122-141 in different immunization regimens. These included vaccination with Ad5. pIX-gp70 (47), transient env124-138 peptide immunization in Sigma adjuvant and transplantation of the FV-induced FBL-3 tumor cell line (38).

Each immunization regimen induced a characteristic pattern of clonotypic composition and inhibitory receptor expression (**Figure 5A**). In comparison with FV infection, Ad5.pIX-gp70 immunization induced a sizeable Tfh, as well as Th1 population of env-reactive CD4+ T cells, as previously shown for CD4+ CTL development (39), without concomitant increases in the Th0 subset (**Figures 5A,B**). Peptide immunization induced lower numbers of env-reactive CD4+ T cells, but this reduction affected mostly Th0 and Tfh cells (**Figures 5A,B**). Finally, FBL-3 tumor cell immunization promoted expansion of Th1 cells, at the expense of Tfh cells, but also primed Th0 cells (**Figures 5A,B**). Thus, each immunization regimen elicited a somewhat distinct Th differentiation balance in env-reactive EF4.1 CD4+ T cells. Nevertheless, Tfh cells continued to be a prominent subset in response to all the regimens (**Figure 5B**).

Finally, the Tfh differentiation was examined in the context of altered lymphocyte interaction. To this end, EF4.1 CD4+ T cells were transferred into FV-infected hosts deficient in B cells (*Ighm*−/−), T cells (*Tcra*−/−), or both B and T cells (*Rag2*−/−). Again, clonotypic composition or inhibitory receptor expression in the env-reactive donor CD4+ T cells was characteristic of each type of recipient (**Figure 6A**). Consistent with the established role for B cells in consolidating Tfh differentiation, env-reactive EF4.1 CD4+ T cells produced a larger fraction of Th1 cells in *Ighm*−/− hosts, than in WT hosts (**Figures 6A,B**). Expectedly, enhanced Th1 differentiation in B cell-deficient hosts was at the expense of Tfh differentiation (**Figures 6A,B**). Despite this shift, however, Th1-phenotype env-reactive EF4.1 CD4+ T cells in *Ighm*−/− hosts did not outnumber those with a Tfh phenotype or those with an uncommitted Th0 phenotype, with all three subsets represented in almost equal proportions (**Figure 6B**). Surprisingly, in comparison with WT or *Ighm*−/− hosts, Th1 differentiation of env-reactive EF4.1 CD4+ T cells was significantly more pronounced in *Tcra*−/− hosts, where Th1 cells now became the dominant subset (**Figures 6A,B**). Skewed differentiation was even more pronounced in *Rag2*−/− hosts, where

Friend virus (FV) infected recipients (*n* = 10). (B) Characterization of clonal expansion and expression of Vα2, PD-1, and LAG3 in env-reactive donor CD4+ T cells, 7 days post-transfer in WT recipient mice infected with ~104 infectious units of F-MLV-B, F-MLV-NB envL128I, or F-MLV-N. (C) Absolute number (*left*) and mean frequency (±SEM) (*right*) of Th subsets, defined by PSGL1 and CXCR5 expression, in env-reactive donor CD4+ T cells from the same donor cells as in (B). One representative of two experiments with *n* = 4, *n* = 3, and *n* = 3 mice for F-MLV-B, F-MLV-NB envL128I, and F-MLV-N infection, respectively, is shown.

env-reactive EF4.1 CD4+ T cells developed almost exclusively (~75%) into Th1 cells (**Figures 6A,B**). This shift in favor of Th1 differentiation in *Tcra*−/− and *Rag2*−/− hosts was driven by approximately 60-fold higher expansion of Th1 cells in such T cell-lymphopenic hosts, in comparison with T cell-replete hosts.

Together, these results highlighted the multitude of intrinsic and extrinsic factors that influence the balance of Tfh and Th1 differentiation in response to F-MLV env, but also indicated that each of these factors may contribute to a different degree, with T cell lymphopenia exerting the strongest influence.

### Reduced IL-2 Availability During FV Infection Facilitates Tfh Development

In addition to increased availability of antigenic peptide-MHC complexes, a well-described effect of T cell lymphopenia is reduced T cell competition for other growth signals, such as cytokines. Consumption of effector CD4+ T cell-produced IL-2 by Treg cells or dendritic cells has been shown to promote Tfh differentiation, at the expense of Th1 differentiation in a variety of experimental systems (20, 24–26). It was, therefore, possible that the strong bias toward Tfh differentiation in FV infection was due to defective IL-2 signaling, either due to lack of production

or to increased consumption by cells other than effector CD4+ T cells.

Consistent with this hypothesis, expression levels of CD25 were lower when env-specific EF4.1 CD4+ T cells were primed in WT than in or *Rag2*−/− hosts (**Figure 7A**), although most env-reactive donor CD4+ T cells did not exhibit detectable CD25 expression in either type of host. As expression of CD25 may also be induced by IL-2 signaling, it was possible that, as well as reduced responsiveness of EF4.1 CD4+ T cells to IL-2, production or availability of IL-2 was reduced in WT hosts. Indeed, serum IL-2 was minimally detected during FV infection of lymphocyte-replete hosts, even when B6 mice with increased genetic susceptibility, provided by the *Fv2*s allele (36), were used (**Figure 7B**). In contrast, transfer of EF4.1 CD4+ T cells into FV-infected lymphocyte-deficient *Rag1*−/− hosts led to readily detectable serum IL-2 (**Figure 7C**).

IL-2 transcription was previously found to be reduced in envspecific EF4.1 CD4+ T cells responding to FV infection than to Ad5.pIX-gp70 immunization (37). However, relative reduction of IL-2 transcription in FV infection could simply reflect the strong skewing toward Tfh cells, which may not produce IL-2. To further investigate the nature of IL-2-producing CD4+ T cells, we searched for correlates of IL-2 production in single-cell RNAsequencing data obtained with env-specific EF4.1 CD4+ T cells primed by FV or Ad5.pIX-gp70 (39). This analysis revealed that most EF4.1 CD4+ T cells transcribing *Il2* in response to FV infection, also transcribed *Il21*, *Il10* or *Ifng* (**Figure 7D**). In contrast, transcription of distinct cytokine genes was largely restricted to different EF4.1 CD4+ T cells responding to Ad5.pIX-gp70 immunization, with most *Il2*-positive cells lacking transcripts for other cytokines, with the exception of *Gzmb* (**Figure 7D**). These data suggest that env-specific EF4.1 CD4+ T cells producing IL-2 following FV infection or Ad5.pIX-gp70 immunization display disparate functional properties. Together, these results highlighted the important contribution of reduced IL-2 production and availability to the dominant Tfh skewing in the CD4+ T cell response to FV infection.

spleens of WT recipients infected with FV or immunized with Ad5.pIX-gp70. Each column represents an individual cell.

### Distinct Correlates of Tfh and Th1 Differentiation in Response to FV Infection

To investigate possible correlates of Tfh bias in FV infection, we compared a number of variables relating to the strength of TCR signaling. This was assessed independently by the degree of clonal expansion and resulting clonotypic composition, the degree of surface TCR downregulation, the activity of a Nur77- GFP reporter (**Figure 8A**) (31), and the degree of PD-1 and

FIGURE 8 | Correlates of follicular helper cells (Tfh) and Th1 CD4+ T cell response to F-MLV env. (A) Representative flow cytometric profile of GFP expression in Nur77-GFP EF4.1 doubly transgenic env-reactive donor CD4+ T cells, 7 days post transfer into Friend virus (FV)-infected WT recipients. (B) Mean fluorescence intensity (MFI) (±SEM) of Nur77-GFP expression (*left*) and levels of TCRβ expression (*right*) in env-reactive donor CD4+ T cells, 7 days post transfer into WT (*n* = 4), *Tcra*−*/*− (*n* = 3) or *Rag2*−*/*− (*n* = 4) recipients, infected with an intermediate dose of FV. One representative of three experiments is shown. (C) Flow cytometric profile (*left*) and MFI of Nur77-GFP expression (*right*) and (D) flow cytometric profile (*left*) and levels of TCRβ expression (*right*) in T helper (Th) subsets, defined by PSGL1 and CXCR5 expression, in env-reactive donor CD4+ T cells, 7 days after transfer into FV-infected WT recipients (*n* = 19). Lines connect values from individual recipients. (E) Matrix of correlation coefficients between the indicated variables and Th subset differentiation.

LAG3 expression. Although all these parameters can be directly affected by the strength of TCR signaling, their precise relationship or indeed their effect on Tfh differentiation is not necessarily linear.

For example, EF4.1 CD4+ T cells primed in WT hosts exhibited the strongest Nur77-GFP signals and highest degree of TCR downregulation, whereas those primed in *Rag2*−/− hosts displayed the opposite phenotype (**Figure 8B**), indicating a direct correlation between Nur77-GFP intensity and TCR downregulation. However, EF4.1 CD4+ T cells primed in *Tcra*−/− hosts downregulated their TCRs to the same degree as in WT hosts, but without the accompanying increase in Nur77-GFP reporter activity (**Figure 8B**). Also, partly discordant were the degree of TCR downregulation and Nur77-GFP reporter activity in Th functional subsets primed in the same host (**Figures 8C,D**). Th1 EF4.1 CD4+ T cells retained significant amounts of surface TCR, compared with Tfh or Th0 cells primed in WT hosts (**Figures 8C,D**), likely due to infrequent interaction of the former subset with B cells. However, Th0 cells exhibited significantly higher Nur77-GFP reporter activity, than either Th1 or Tfh cells in these hosts, despite comparable TCR downregulation with Tfh cells (**Figures 7C,D**). These results suggested that, although modulation of TCR signal strength is evidently different in EF4.1 CD4+ T cells primed in T cell-replete or T cell-deficient hosts, the effect of T cell lymphopenia on Th differentiation operated through additional mechanisms.

Given the complex patterns of correlation between independently measured variables and the degree of Tfh and Th1 differentiation, we next calculated a correlation matrix (**Figure 8E**). To this end, we assessed the relative contribution and possible interaction of 13 variables controlling Tfh differentiation measured in the 11 separate combinations of host and immunization or infection regimen described here. This analysis indicated three distinguishable clusters across all conditions, corresponding to each of the three major Th subsets observed in FV infection. Development of Tfh cells correlated most strongly with the activity of the Nur77-GFP reporter, taken to indicate the strength of signaling EF4.1 CD4+ T cells received, and also with the intensity of PD-1 expression (**Figure 8E**). In contrast, Th1 development exhibited strong anti-correlation with Nur77-GFP reporter activity and PD-1 expression levels and instead correlated with T cell lymphopenia and the availability of IL-2, together likely driving T cell clonal expansion (**Figure 8E**). Finally, uncommitted Th0 cells, although sharing many attributes with Tfh cells, appear to cluster separately, correlating strongly with LAG-3 expression, the degree of TCR downregulation and, the use of high-affinity TCRs (**Figure 8E**). Thus, our data suggest that Tfh differentiation is promoted by an optimal degree of TCR signaling, as well as by T cell competition.

### DISCUSSION

It is now well recognized that both T cell-intrinsic and T cellextrinsic factors shape the balance of CD4+ T cell differentiation into distinguishable functional subsets (1–8). However, the relative contribution of each of these factors in isolation or their potential intersection with each other in the context of diverse immunological challenges is still not fully understood. Here, we provided evidence to suggest that the CD4+ T cell response to a model retroviral antigen, presented during natural infection, is heavily skewed toward Tfh differentiation. This allowed us to identify the variables that best correlate with the degree of Tfh differentiation in this model and to accurately quantify their contribution.

The variable that exhibited the closest positive correlation with Tfh differentiation of env-reactive CD4+ T cells in all the conditions studied was the strength of TCR signaling, translating to increased transcription of the Nur77-GFP reporter. Also, directly correlating with both the degree of Tfh differentiation and Nur77-GFP reporter activity was the intensity of PD-1 expression. These findings are consistent with an instructive model, whereby stronger TCR signaling in Th cell precursors favors Tfh development, likely through stronger induction of the Tfh-promoting cytokine IL-21 (42, 48, 49).

Although stronger TCR signaling in Tfh cells was indicated by the increase in Nur77-GFP reporter activity and the intensity of PD-1 expression, it should be noted that not all correlates of TCR signaling followed a similar pattern. For example, the degree of surface TCR downregulation, generally proportional to TCR signal strength (50), did not correlate with the degree of Tfh differentiation. Moreover, expression of LAG3, which is also transiently induced by strong TCR signals (51, 52), showed no positive correlation with Tfh differentiation. Instead, the envreactive CD4+ T cells with the highest TCR signal, assessed by both the activity of the Nur77-GFP reporter and the degree of surface TCR downregulation, appeared inhibited in their commitment to either the Th1 or Tfh subsets. These cells, which we refer to as uncommitted Th0 cells (44), also strongly correlated with co-expression of the inhibitory receptors PD-1 and LAG3.

Seemingly uncommitted Th0 cells are not typically observed in acute viral infections (44), but a similar population lacking either Th1 or Tfh characteristics has been described in CD4+ T cells primed during the chronic phase of LCMV Cl13 infection (18). In that model, the emergence of Th0 cells was linked to an initial defect in effector differentiation, indirectly caused by chronic IFN type I production (18). Eventually, CD4+ T cells primed during chronic LCMV-specific developed almost exclusively into Tfh cells (18).

The env-reactive CD4+ T cells with Th0 characteristics observed during FV infection are also closely related to Tfh cells, highlighting parallels between acute FV infection and chronic LCMV infection. However, as IFN type I production is not a prominent feature of acute FV infection (40), defective effector differentiation must have alternative explanations. Our results with the FV model suggest that TCR signaling above an optimal strength restrains differentiation of env-reactive CD4+ T cells, which would otherwise be committed to the Tfh subset. Indeed, in addition to displaying the highest Nur77-GFP reporter activity and preferentially expressed genes involved in active metabolism, Th0 cells were most noticeable in conditions associated with the highest antigenic load or potency. Furthermore, the frequency of Th0 in env-reactive CD4+ T cells was positively correlated with the percentage of PD-1 and LAG3 coexpressing cells across the various conditions studied and was reduced by anti-PD-1 and anti-LAG3 treatment during FV infection, which promoted their differentiation into Tfh cells. Notably, Tfh differentiation was also reported to be enhanced by anti-PD-1 and anti-LAG3 treatment during *Plasmodium yoelii* infection of mice (53), indicating that this pathway is restricting Tfh differentiation in persistent parasitic, as well as viral infection. Thus, our results suggest that Tfh differentiation is most efficiently induced by an optimal range of TCR signal strength.

Although evidently influenced by TCR signal strength, Tfh differentiation of the CD4+ T cell response to F-MLV env was independent of TCR clonotypic affinity, as previously suggested (37, 41, 42). Env-reactive CD4+ T cells bearing identical highaffinity clonotypic TCRs (identified by the use of endogenous Vα2 chains) were found both in the Tfh and Th1 subsets at comparable frequencies. Notably, despite comparable clonotypic TCR usage and Nur77-GFP reporter activity, Th1 env-reactive CD4+ T cells retained nearly the full amount of surface TCR, when compared with their Tfh counterparts in the same host, which had nearly lost their surface TCR expression. Therefore, the differential strength of TCR signals received by Th1 and Tfh cells cannot be solely attributed to differences in clonotypic TCR affinity. Instead, at least part of the differential TCR signaling between Th1 and Tfh cells is secondary to their differentiation and likely the result of their anatomic localization and interaction with distinct APC types. Supporting this notion, downregulation of surface TCR expression in CD4+ T cells has been previously shown, in FV infection (42), as well as in other model systems (54, 55), to be dependent on interaction with antigen-presenting B cells. Retention of surface TCR preferentially in Th1 cells would, therefore, suggest reduced B cell interaction. A central role for the APC type in determining or consolidating Th1 or Tfh effector differentiation may also underlie the propensity of different vaccine vectors or immunization regimens to induce distinct ratios of Th1 and Tfh response to a given antigen.

In addition to interaction with distinct APC types, our findings also support the concept that interaction and/or competition between T cells are also critical in determining effector differentiation, particularly, of Th1 cells. Although expectedly B cell deficiency did promote Th1 responses at the expense of Tfh responses, the strongest positive effect on Th1 differentiation was T cell lymphopenia. Indeed, the proportion of Th1 cells was approximately threefold higher in T cell deficiency than in B cell deficiency. Conditions conducive for Th1 differentiation in T cell lymphopenia are likely to involve deficiency in Treg cells, which can reduce availability of IL-2, thus promoting Tfh differentiation (24). However, an additional role for primary IL-2 production by effector CD4+ T cells was also indicated by

### REFERENCES

single-cell RNA-sequencing analysis. Although Th1-promoting *Il2* transcription was still detected in single CD4+ T cells despite the heavy skewing toward a Tfh response following FV infection, this was always accompanied by transcription of Th1-suppressing cytokines, such as *Il21* and *Il10*. In contrast, *Il2* transcription in single CD4+ T cells primed by Ad5.pIX-gp70 vaccination partly overlapped only with *Gzmb* transcription.

Together, our results highlight the potent contribution of T cell-extrinsic variables to determine the relative balance of Th1 and Tfh responses. Manipulating these variables in vaccination regimens in order to achieve a balance of CD4+ T cell effector differentiation appropriate for the respective context (e.g., viral infection or cancer) will be the next important challenge.

### ETHICS STATEMENT

All animal experiments were approved by the ethical committee of the Francis Crick Institute, and conducted according to local guidelines and UK Home Office regulations under the Animals Scientific Procedures Act 1986 (ASPA).

### AUTHOR CONTRIBUTIONS

LD and TD performed the experiments and analyzed the data. LD and GK wrote the manuscript. GK supervised the study and contributed to data analysis.

### ACKNOWLEDGMENTS

We wish to thank Dr. Kristin A. Hogquist for the Nur77-GFP mice, Dr. Kim Hasenkrug for FV and FBL-3 stocks, and Dr. Ulf Dittmer for the Ad5.pIX-gp70 stocks. We are grateful for assistance from the Flow Cytometry and Biological Resource Facilities at the Francis Crick Institute.

### FUNDING

This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001099), the UK Medical Research Council (FC001099), and the Wellcome Trust (FC001099).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01260/ full#supplementary-material.

function: separable roles in delivery of ICOS ligand and antigen. *J Immunol* (2014) 192(7):3166–79. doi:10.4049/jimmunol.1302617

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Danelli, Donnarumma and Kassiotis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Unexpected Help: Follicular Regulatory T Cells in the Germinal Center

### *Markus M. Xie and Alexander L. Dent\**

*Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, United States*

Follicular helper T (Tfh) cells are necessary for germinal center (GC) formation and within the GC, provide key signals to B cells for their differentiation into plasmablasts and plasma cells that secrete high-affinity and isotype-switched antibody (Ab). A specialized subset of Foxp3+ T cells termed T follicular regulatory (Tfr) cells, also regulate the differentiation of Ab-secreting cells from the GC. Tfr-cell function in the GC is not well understood, however, the dominant paradigm currently is that Tfr cells repress excessive Tfh and GC B cell proliferation and help promote stringent selection of high-affinity B cells. A mouse model where the Bcl6 gene is specifically deleted in Foxp3+ T cells (Bcl6FC mice) allows the study of Tfr cell function with more precision than other approaches. Studies with this model have shown that Tfr cells play a key role in maintaining GC B cell proliferation and Ab levels. Part of the mechanism for this positive "helper" effect of Tfr cells on the GC is Tfr cell-derived IL-10, which can promote B cell growth and entry into the dark zone of the GC. Recent studies on Tfr cells support a new paradigm for Tfr cell function in the GC reaction. Here, we review studies on Tfr cell functions and discuss the evidence that Tfr cells can have a major helper role in the GC-dependent Ab response.

#### *Edited by:*

*Georgia Fousteri, San Raffaele Hospital (IRCCS), Italy*

### *Reviewed by:*

*Luis Graca, Universidade de Lisboa, Portugal Talal A. Chatila, Harvard University, United States*

> *\*Correspondence: Alexander L. Dent<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 20 April 2018 Accepted: 21 June 2018 Published: 02 July 2018*

#### *Citation:*

*Xie MM and Dent AL (2018) Unexpected Help: Follicular Regulatory T Cells in the Germinal Center. Front. Immunol. 9:1536. doi: 10.3389/fimmu.2018.01536*

Keywords: T follicular regulatory cells, germinal center, follicular helper T cell, regulatory T cells, T cell differentiation

### INTRODUCTION

A major function of the adaptive immune response is to produce highly specific antibodies (Abs) that bind to antigen (Ag) with high affinity and help to eliminate pathogens and foreign substances. A specialized subset of differentiated CD4 T cells, follicular helper T (Tfh) cells, are required in the germinal center (GC) reaction to help B cells generate high-affinity Abs to Ag (1, 2). Tfh cells control the initiation as well as the outcome of the GC B cell response (3–6). Tfh cells are critical for the proper production of protective Abs during an infection, however, the over-production of Tfh cells can also lead to autoimmunity since Tfh cells can help B cells to produce self-reactive Abs (6–8). Thus, the proper regulation of Tfh cell differentiation is essential both for normal immune function and for preventing autoimmune disease.

Germinal center B cell responses are also regulated by T follicular regulatory (Tfr) cells, which develop from regulatory T cells (Tregs) and localize to the GC (9–16) (**Figure 1**). Tfr cells are generally thought to limit the function of Tfh cells in the GC (9–13, 16).  The prevailing model for Tfr cell function currently is that Tfr cells repress excessive Tfh and GC B cell proliferation and promote the selection of high-affinity B cells (9–13, 16), however, the complete range of Tfr cell functions are poorly understood. A Tfr-deficient mouse model where the *Bcl6* gene is specifically deleted in Foxp3+ T cells (*Bcl6* fl/fl *F*oxp3-*C*re or Bcl6FC mice) has been used by us and others to study Tfr cell function

Figure 1 | Follicular helper T (Tfh) and T follicular regulatory (Tfr) cells both act in the germinal center (GC) to regulate the generation of antigen (Ag)-specific antibody-secreting cells. Tfh cells differentiate from conventional CD4 T cells after activation with Ag and dendritic cell (DC) presentation. Tfr cells differentiate from conventional regulatory T cells (Tregs) and migrate into the GC.

in immune and autoimmune responses (14, 17–19). With this model, we and others have unexpectedly found that the major Tfr cell function may not be inhibiting the GC response but instead helping promote the Ab response and even the magnitude of the GC response. Here, we discuss the current understanding of the differentiation and physiological functions of Tfr cells. We also discuss how Tfr cells balance suppressive and helper functions, the potential mechanisms underlying Tfr cell functions, and directions for future investigations.

### DIFFERENTIATION AND REGULATION OF Tfr CELLS

Several studies have shown that Tfr cells primarily differentiate from Foxp3+ Treg precursor cells (10, 11, 20–22) (**Figure 2**), however, like Tfh cells, Tfr cells can also develop from naïve CD4 T cells (23). Tregs are generated either during T cell differentiation in the thymus (tTregs) or from mature CD4 T cells in the periphery (pTregs) (24, 25), but whether Tfr cells preferentially develop from tTregs or pTregs is not known. Tregs in the intestinal mucosa are predominantly pTregs that develop to Ags derived from microbiota and diet as a tolerance mechanism (24, 25). Tfr cells that develop in the gut lymphoid tissues such as Peyer's patches may therefore differentiate from pTregs, and so ultimately may have a naïve CD4 T cell origin. Interestingly, Peyer's patch Tfr cells have a markedly different transcriptome than peripheral lymph node Tfr cells, possibly suggesting a different origin (26).

T follicular regulatory cells express Tfh cell surface markers such as PD-1, CXCR5, and ICOS, Treg surface markers such as CTLA-4 and GITR, and the master regulatory transcription Xie and Dent Follicular Tregs as B Cell Helpers

factors for both Tregs (Foxp3) and Tfh cells (Bcl6) (9–11, 14, 27). Thus, Tfr cells display a hybrid or mixed Tfh/Treg phenotype. Most studies have analyzed Tfr cells in the mouse, but phenotypically similar Tfr cells have also been described for humans (28, 29) and macaques (30). Tfr cells also express significant levels of Blimp1, a transcriptional repressor protein that suppresses Bcl6 expression (10, 31). Notably, Tfr cells express lower CD25 compared with non-Tfr Tregs (14, 29). Together with Bcl6, Nfat2 upregulates CXCR5 expression on Tregs and enables them to migrate to GC, take on the follicular phenotype and become Tfr cells (9–11, 14, 32). Recent work has revealed that the mTor pathway is a key regulator of Tfr cells. The mTorc1 complex is essential in regulating the conversion of Tregs to Tfr cells and this is potentially through a Stat3–Tcf-1–Bcl6 pathway (33, 34). Our lab has also specifically found that in contrast to Tfh cells which can develop in the absence of Stat3, Stat3 is essential for Tfr cell development (15). Deletion of *Pten* in Tregs leads to upregulated mTorc2 activity and heightened Tfr cell development (35). Thus, the Akt–mTor2 kinase pathway promotes Tfr cell development and the Pten phosphatase helps restrain excessive Tfr cell development (35).

Antigen exposure triggers the differentiation of Tfr cells and this process is dendritic cell (DC)-dependent (10, 11, 23, 27). Sage et al. used mice that express diphtheria toxin receptor specifically on DCs to test this (12). DC-depletion led to substantially decreased Tfr cells, however, it is unknown which specific DC subsets directly contribute to Tfr cell differentiation. At the same time, PD-1-ligand expressed on DCs has an inhibitory role on Tfr cell development (36). Tregs can repress the function of Ag presenting cells (APCs) including DCs (37), but whether Tfr cells can affect DCs or other APCs and how this might affect the GC response is unknown. Precisely what Ags and signals that Tregs respond to in order to become Tfr cells is not well understood. Tfr cells respond more strongly to self-Ags than foreign Ags, which fits with the self-reactive nature of tTregs (23, 38). While Tfr cells can be found that have specificity for the immunizing Ag (23), a recent study on the TCR specificity of Tfh and Tfr cells indicated that in contrast to Tfh cells, Tfr cells do not respond well to the cognate Ag after immunization (22). Furthermore, an analysis of TCR gene sequences in Tfh and Tfr cells indicated that Tfh cells are a sub-population of cells related to naïve CD4 T cells, whereas Tfr cells showed a TCR profile very similar to the total Treg population (22). These findings are consistent with the model that Tfh cells are Ag-specific T cells that proliferated after Ag stimulation, while Tfr cells develop in a polyclonal and Ag-independent manner from Tregs. Therefore, Tfr cells either develop from Tregs in a polyclonal TCR-dependent response involving recognition of self-Ag, or Tfr cells expand and differentiate by an Ag-independent and TCR independent pathway [e.g., Jagged1 plus Ox40 stimulation (39)]. Note that the Maceiras et al. study (22) of Tfr cell TCR sequences analyzed Tfr cells from peripheral LNs, and the TCR specificity of Peyer's patch Tfr cells may be more similar to naïve CD4 T cells that are responsive to gut Ags.

T cell co-stimulation is required for Tfr cell differentiation as either CD28 or ICOS deficiency leads to reduction of Tfr cells (10, 27, 40). Mice with CD28 deficiency specifically in Tregs (using Foxp3-cre) had a large reduction in Tfr cells in the draining lymph node after NP-OVA immunization (40). This is largely due to the roles of CD28 in inducing Foxp3 expression as well as Tfr cell proliferation (10, 41–44). Similarly, Tfr cell development is abrogated in ICOS-deficient mice (27). ICOS signaling modulates the expression of Bcl6 and c-Maf in Tfh cells and might play a similar role in Tfr cells (45–47). Bcl6 is an essential transcription factor for Tfr cells, and recent studies suggest that c-Maf is also pivotal for Tfr cell differentiation (10, 11, 14, 48, 49). Bcl6 and Blimp1 reciprocally repress expression of the other factor in both Tfh and Tfr cells (31, 50). The regulation of Tfh cell differentiation by Blimp1 is Bcl6-dependent while Blimp1 controls Tfr cell differentiation independent of Bcl6 (31). One mechanism for Bcl6-independent Blimp1 activity may relate to regulation of Nfat2, which has been shown to be important for upregulation of CXCR5 on Tfr cells as well as for expression of PD-1 (32, 51). Blimp1 has been shown to repress Nfat2 expression (51), and thus Blimp1 could have a suppressive role for CXCR5 and PD-1, both of which are key genes increased in Tfr cells. Increased expression of Nfat2 in Blimp1-deficient Tregs could then lead to Bcl6-independent expression of CXCR5 and PD-1, and appearance of Tfr-like cells (31). Tfr cells were repressed by high IL-2 levels at the peak of influenza infection and this was through a Blimp1-dependent mechanism (19). IL-2 is also a negative signal for Bcl6 expression, and decreased IL-2 promotes induction of Tfr cells. After the peak anti-flu virus immune response, CD25 expression is downregulated in some Tregs while Bcl6 is increased, leading to Tfr programming (19). Thus, IL-2 is a key factor regulating Tfr differentiation, promoting Blimp1 expression while repressing Bcl6 in Tregs to preclude Tfr cell development.

PD-1, which is expressed by both Tfh and Tfr cells, inhibits Tfr differentiation and their suppressive function (10, 11, 16, 27). Sage et al. showed that Tfr cells in *Pdcd1-*deficient mice had greater suppressive function and resulted in decreased Ab production both *in vitro* and *in vivo* (27). The exact mechanism for the increased inhibitory function of *Pdcd1-*deficient Tfr cells remains unclear. At the same time, PD-1 ligand is required for Tfr cell generation, however, it is not clear if this is a direct or indirect effect on Tfr cells (23). Similarly, CTLA-4, the inhibitory receptor which binds to CD80 and CD86, limits the differentiation of Tfr cells (13, 52, 53). However, restricted CTLA-4 deficiency in Tregs contributes not only to enhanced Tfr cells but also enhanced Tfh, GCB cells, and Ab responses (53). One explanation is that in the absence of CTLA-4 function in Tregs, there is uncontrolled inflammation that drives higher Tfh cell and GCB responses. However, since it is not clear what drives the enhanced Tfh, GCB, and IgE responses, a "helper" role of Tfr cells cannot be completely excluded (53). Deletion of CTLA-4 results in increased IL-10 production by Tregs (54). Since IL-10 can promote GC responses (17, 55), it is possible that increased IL-10 production by Tfr cells contributes to the increased GC and Ab response in CTLA-4 KO mice.

The majority of research on Tfr cells has been conducted in the mouse system but a few recent studies have elucidated Tfr cell populations in human GCs that are basically similar to Tfr cells in mice (29, 56, 57). CXCR5+ Tfh-like cells in blood, also known as circulating Tfh (cTfh) cells, are typically used as a proxy marker for the GC Tfh cell response in humans. By assessing cTfh cell frequency in patients with monogenic mutations leading to immunodeficiency, a large number of genes controlling human Tfh cell development have been categorized (58). Circulating Tfr (cTfr) cells in blood are also used as a correlate of the Tfr cell response (28, 59–62), however, in contrast to Tfh cells (58), relatively few genes that control Tfr cell development and function in humans have been characterized to date (e.g., *LRBA* and *CTLA4*) (62–64). Thus, much work remains in fully understanding specific genes and pathways that regulate human Tfr cells.

## SUPPRESSIVE FUNCTIONS OF Tfr CELLS

T follicular regulatory cells have been described in the literature mainly as suppressors of the GC reaction and the Ab response, repressing the proliferation of Tfh cells and GC B cells, and limiting the generation of Ab-secreting cells and overall Ab responses. However, the experimental approaches taken in many studies can give rise to alternative interpretations. *In vitro*, Tfr cells can suppress the proliferation and cytokine production of Tfh cells as well as the proliferation and Ig secretion of B cells, similar to the *in vitro* suppressive function seen with non-Tfr Tregs (13, 27–29, 38, 65). *In vivo* studies have demonstrated that Tfr cells, analyzed initially by depletion of total Tregs, can suppress the numbers of GC B cells and Tfh cells (9–11, 13, 27, 53). However, these studies may not represent specific effects of Tfr cell depletion or physiological Tfr cell function. Total Treg deletion (10, 11, 13, 66) provokes severe inflammation and causes a very broad effect on T cell responses, thus obscuring the specific functions of Tfr cells. Studies using adoptive transfer of Tfr cells along with other T cells into T cell-deficient mice or Tfhcell-deficient mice might have non-physiological effects due to the abnormal immune environment of the recipient mice (10, 11, 27, 33). Studies where Tfr cell numbers are greatly enhanced due to deletion of Roquin (34), or where Tregs are forced to migrate to the B cell follicle by ectopic CXCR5 expression (32) might also lead to non-physiological suppression and/or non-specific suppression of GC responses. Mice with the Nfat2 gene deleted in Tregs with Foxp3-cre showed a partial loss of Tfr cells and augmented numbers of GC B cells, Tfh cells, and Ag-specific Abs after immunization (32). However, a more general loss of Treg function by loss of Nfat2 affecting Tfh cell expansion cannot be discriminated from the specific effects from loss of Tfr cells. *In vitro* studies of Tfr cells cannot mimic the complex *in vivo* environment of the GC reaction and cannot analyze affinity selection of GC B cells. Together, a re-interpretation of the Tfr cell literature helps to explain why the function of Tfr cells assessed using Bcl6FC mice (14, 17, 19), is strikingly different from many other studies on Tfr cell function.

Nonetheless, it is clear that under some conditions, Tfr cells can negatively regulate the GC reaction, and the precise mechanisms that Tfr cells use to negatively regulate the GC is one of the unsolved mysteries in the Tfr cell field. Tregs can suppress immune responses by multiple known mechanisms: IL-2 consumption, secretion of inhibitory factors (IL-10, TGF-β, IL-35, granzyme B, CD39, CD73, and TRAIL), and CTLA-4-mediated inhibition of Tfh cell co-stimulation (67–69). Of these known suppressor factors, we can narrow down mechanisms for Tfr cells based on previous data. Tfh cell differentiation is inhibited by IL-2 (70–72), and IL-2 consumption by Tfr cells could be predicted to help stabilize Tfh cell responses. However, Tfr cells have low levels of the high-affinity IL-2 receptor CD25 (14), which indicates a lessened capacity to compete for available IL-2. IL-10 is unlikely to be the key suppressor factor, since IL-10 is a stimulatory or growth factor for GC B cells (17), and furthermore, IL-10 expression by Tfh cells is increased in the absence of Tfr cells in Bcl6 mice (14). IL-35 is unlikely to be a Tfr suppressor factor as it primarily affects T cell proliferation (73), and data with Bcl6FC mice does not indicate an effect of Tfr cells on the number of Tfh cells (14). Granzyme B is unlikely as a major mechanism as it is decreased in Tfr cells compared to Tregs (10). Metabolic suppressor pathways such as CD39 and CD73 have not been extensively characterized and are possible effectors of suppression by Tfr cells as they could potentially affect cell proliferation in the GC. In mice, TGF-β is known to stabilize Tfh cell responses (74), and prevent excess Tfh cell responses (75). In humans, TGF-β is required for Tfh cell differentiation (76). A lack of TGF-β signaling from loss of Tfr cells does not clearly explain the normal Tfh cell numbers in the presence of increased Tfh cytokine expression in Bcl6FC mice (14). TRAIL is cytotoxic to follicular B cell lymphomas, which have a GC phenotype (77), but otherwise there is no data about TRAIL activity in GCs, particularly regarding Tfr cells. CTLA-4 expression by Tfr cells may inhibit the ability of Tfh cells to receive key co-stimulation signals from GC B cells, thus limiting Tfh cell and thus Tfh cell-driven GC B cell expansion. Unfortunately, studies on the role of CTLA-4 function in Tfr cells are difficult to interpret, as noted above (13, 52, 53).

A recently described mechanism for Tfr cells to inhibit Tfh cells and the GC is secretion of a decoy IL-1 receptor that inhibits Tfh cell differentiation (38). This pathway appears to be most critical during early Tfh cell activation and differentiation rather than during the GC reaction itself. Furthermore, data pointing to this decoy IL-1 receptor pathway being used specifically by Tfr cells to control Tfh cells *in vivo* is lacking. Another potential pathway used by Tfr cells to control Tfh cells and GC B cells is the inhibitory receptor TIGIT, that is important for Treg suppressive function (14, 78). Intriguingly, the two major suppressive pathways utilized by TIGIThigh Tregs are IL-10 and Fibrinogen-like protein 2 (Fgl2) (78). Fgl2 is a secreted protein that binds the inhibitory IgG receptor FcγRIIB (79). As noted above, it is unlikely that loss of IL-10 from Tfr cells contributes to the deregulated cytokine expression of Tfh cells. Thus, Fgl2 may be a key factor used by Tfr cells to regulate GC B cells. Interestingly, FcγRIIB KO mice are known to develop lupus (80), and Fgl2 KO mice develop glomerulonephritis, a pathologic manifestation of auto-Abs in severe lupus disease also seen in IgA nephropathy (81, 82). Fgl2 KO mice have Treg defects, but the GC response in these mice has not been characterized (81). TIGIThigh Tregs can also affect cell activation by inducing tolerogenic DCs *via* CD155 (83). But currently, data showing Tfr cells controlling the GC *via* TIGIT is lacking.

In analyzing mice with deletions of Bcl6 or Stat3 specifically in the Treg lineage (Bcl6FC and Stat3FC mice, respectively), we found noteworthy differences between how Tfr cells are regulated by Stat3 and Bcl6 (14, 15). While Tfr cells are strongly depleted in both Bcl6FC and Stat3FC mice (14, 15), there are significant differences in the phenotype. First, in Bcl6FC but not Stat3FC mice, Tfh cells produce higher levels of cytokines compared to control mice. Second, Ag-specific IgA is increased in Bcl6FC mice whereas Ag-specific IgG is increased in Stat3FC mice (14, 15). At the same time, Tfh cell and GC B cell numbers are not altered in either Bcl6FC or Stat3FC mice compared to control mice (14, 15). The function of Stat3 in Tfr cells is not understood. Analogous to Tfh cells (84, 85), Stat3 may be important for Tfr cell development by inducing Bcl6 expression in Tregs in response to cytokines such as IL-6 and IL-21. Stat3 expression is also activated in Tfr cells by the mTorc1 pathway (33). Bcl6 is required for the development of the CXCR5+PD-1+ follicular T cell phenotype, and the induction of Bcl6 by STAT factors may be essential for both Tfr cell as well as Tfh cell development. If this is the case though, why does deletion of Stat3 in Foxp3-expressing cells produce a different phenotype than deletion of Bcl6 in Foxp3-expressing cells? Why are cytokines upregulated from Tfh cells in Bcl6FC mice but not in Stat3FC mice? The answer to this question is currently unknown but is essential for fully understanding Tfr cell development and function. One possible explanation for the difference is that there is a larger population of residual Tfr cells in Stat3FC mice compared to Bcl6FC mice and these residual Tfr cells in Stat3FC mice are enough to negatively regulate Tfh cells. Thus, there is a greater deletion of Tfr cells in Bcl6FC mice, leading to a more complete loss of repression by Tfr cells, and thus increased Tfh cell activity. The increased Tfh cell cytokines in Bcl6FC mice might promote the elevated IgA response that is not seen in Stat3FC mice. In summary, in the Bcl6FC model, Tfr cells repress Tfh cell activity but not proliferation. Why Ag-specific IgG is increased in Stat3FC mice is unclear, but possibly the residual Tfr cells in Stat3FC mice have augmented GC helper activity.

T follicular regulatory cells have been studied extensively in immune responses induced by model protein Ags and adjuvants. Studies on Tfr cell function in regulating the gut microbiota (86) or in viral infection (17–19) have also been performed. More than the type of immune challenge, the model system used to assess Tfr cell function (e.g., Treg depletion in mice versus Bcl6FC mice) determines whether suppression by Tfr cells is observed.

### ROLE OF Tfr CELLS IN AUTOIMMUNE DISEASE

An important area where Tfr cells have a clear suppressive effect on the GC and Ab response, even in Bcl6FC mice, is in suppression of auto-Abs that drive autoimmune disease (14, 18, 19, 32, 40). This role of Tfr cells in suppressing auto-Ab production was elucidated most thoroughly by Fu et al. who showed that Bcl6FC mice developed late-onset Sjogren's-like autoimmune disease and autoimmunity could be induced in young mice by immunizing mice with salivary gland extracts (18). The precise mechanisms for how Tfr cells can suppress auto-Abs while at the same time promote the Ab response to foreign Ags remains unexplored. One possible explanation is that since Tfr cells, like Tregs, have a bias toward self-Ag recognition (22, 23, 38), they are able to inhibit self-reactive Tfh cells that might develop in the GC by competing with them for recognition of self-Ags on GC B cells and binding and blocking B7 co-stimulatory receptors *via* CTLA-4. Little is known about the role of Tfr cells in human autoimmune disease, but increased levels of cTfr cells are observed in patients with Sjogren's disease (28, 59) and systemic lupus erythematosus (60). Interestingly, an increased ratio of cTfr to cTfh cells is strongly associated with more severe disease in the case of Sjogren's syndrome (59). Whether high levels of cTfr cells simply represent the presence of active GC responses or whether cTfr cells are especially elevated in autoimmune disease is not clear. The data with Sjogren's cTfr cells is particularly hard to interpret since the cells have an immature CD25+ Tfr phenotype and their relationship to GC-localized Tfr cells is unclear (59).

### "HELPER" FUNCTIONS OF Tfr CELLS

Although Tregs themselves are overwhelmingly described as suppressor cells, there are several reports that Tregs can promote immune responses in certain circumstances. Under inflammatory conditions or in mice with mutations in genes that affect Foxp3 expression, a fraction of Tregs can become "ex-Tregs" and differentiate into proinflammatory cells (87, 88). Surprisingly, ex-Tregs can also convert into functional Tfh cells in Peyer's patches (89) and in atherosclerosis (90).

The first published characterization of Tfr cells in 2009 by Linterman et al. showed that Tfr cells had a key "helper" role in terms of helping Tfh cells select high-affinity Ag-specific B cell clones (7). In their proposed model, Tfr cells restrict the outgrowth of non-Ag-specific B cell clones in the GC, presumably allowing for more efficient interaction of Tfh cells with selection of specific high-affinity Abs (7). At the same time, the Linterman et al. data can also be interpreted as showing evidence for a major helper function for Tfr cells in the GC. For instance, a significant decrease in Ag-specific GC B cells is observed after total Treg depletion at the same time that total GC B cells increased (7). This can be interpreted as two distinct processes: (1) loss of Tfr cells leads to a loss of Tfr cell helper activity and thus reduced Ag-specific GC B cells and (2) because total Tregs are depleted, there is a massive increase in GC responses to commensals and self-Ags—responses that are normally inhibited by Tregs. Even though these latter commensal-specific and self-Ag-specific GCs may be weakened by loss of Tfr cell helper activity, the large number of these responses leads to a total increase in GC B cells.

A different Treg–Tfh helper pathway was shown by Leon et al., who found that Tregs are required for the normal anti-influenza Tfh cell response (66). In this study, ex-Tregs were not converting into Tfh cells, and Leon et al. proposed a mechanism where CD25+ Tregs take up IL-2 and limit the overall availability of IL-2, thereby promoting Tfh cell differentiation (66). Importantly, however, Leon et al. did not investigate loss of Tfr cells (which would occur with Treg depletion) as a mechanism for the Treg helper effect, and their data does not eliminate a helper role for Tfr cells in the Tfh/GC response in the virus infection system.

Because of the problems associated with deleting total Tregs and the lack of specific and robust models to deplete Tfr cells *in vivo*, we developed Bcl6FC mice (14). In these mice, Tfr cell development is specifically blocked without a loss in total Tregs or Treg function (14). We determined that loss of Tfr cells led to a significantly decreased IgG response and that Tfr cells were required to produce the highest affinity Ag-specific Abs (14). These results are consistent with a critical helper role for Tfr cells in the GC. In our published results, we did not observe a loss of GC B cells or Tfh cells in Bcl6FC mice despite the decreased IgG response (14). This could be due to the time-point where we analyzed the GC or the type of Ag used to induce the GC.

In 2017, Laidlaw et al. presented the clearest evidence to date that Tfr cells can act as essential helper cells in the GC (17). In this study, mice were infected with lymphocytic choriomeninigitis virus (LCMV) and the GC and Ab response analyzed (17). Importantly, Laidlaw et al. used Bcl6FC mice and Treg-specific IL-10 cKO mice to demonstrate that Tfr cells are a critical source of IL-10 in the GC and that IL-10 drives the growth of GCs by promoting entry of GC B cells into the dark zone (17). In the absence of IL-10-producing Tfr cells, GC B cell numbers and the LCMV-specific Ab response were decreased (17). A recent study with malaria infection in mice also showed that IL-10 was critical for the maintenance of the GC and GC-derived Ab response (55). Overall, these recent findings strongly support the idea that IL-10-producing Tfr cells have a major role in maintaining the GC reaction and thus act as "helper cells." In our lab, we have been using Bcl6FC mice and analyzing the role of Tfr cells in a food allergy model with peanut Ag. In this model, we find that Tfr cells help maintain the peanut-specific GC response and IgE response (Markus M. Xie and Alexander L. Dent, manuscript in preparation). Tfr cells thus appear to have a key role in allergic immune responses, and represent a novel target for allergyspecific immunotherapy.

### OUTSTANDING QUESTIONS AND FUTURE DIRECTIONS

As of this writing, Tfr cells have only been analyzed in a very small fraction of infectious disease models and immunological diseases such as allergy and autoimmunity. Testing Tfr cell function in various disease states will be an important area for future research on Tfr cells. Also unknown is if Tfr cells affect diseases that are not driven by Ab-mediated pathology. Whether Tfr cells play a regulatory role in cancer, diabetes, heart disease, atherosclerosis, or other types of inflammatory diseases, is ripe for exploration. The mechanism of Tfr cell help in the GC is not completely understood and an important topic is why some types of GC responses seem to rely on Tfr cells for help whereas other GC responses are only mildly affected or not affected at all by loss of Tfr cells. A major issue for future studies is whether Tfr cells switch between help and suppression in the GC for foreign Ag, or primarily

act as helpers for foreign Ag and suppressors of autoimmune responses. If Tfr cells act as suppressor cells of non-autoimmune responses in the GC, what mechanism of suppression do they use, and what controls whether Tfr cells act as suppressors versus helpers? Do human and mouse Tfr cells have similar helper and repressor functions? Tfr-like cells have been found circulating in both mouse and human; what is the relationship of these cells to Tfr cells in the GC? Also unclear is how Tfr cells regulate Ab affinity maturation and Tfh responses. A major question is whether Tfr cells regulate the generation or differentiation or survival of memory B cells. Finally, almost nothing is known about what signals drive Tfr cell responses to the GC and what Ags do they recognize? Thus, there are huge numbers of vital questions about Tfr cells that need to be answered through more research.

### CONCLUSION

Even though Tregs act overwhelmingly as major suppressors of the immune response, Tfr cells provide a striking and clear example of Tregs acting as "helper" cells for the immune response. At least part of this Tfr cell helper function is producing IL-10 that promotes GC B cell growth and the GC-dependent high-affinity

### REFERENCES

Ab response. Thus, in the context of the GC response, Tfr cells appear to maintain a key balance between help (GC maintenance, Ab response, and Ab affinity) and suppression (Tfh cell numbers, GC B cell numbers, Tfh cell cytokines, and auto-Abs) (**Figure 3**). One interesting idea is that the autoreactivity and suppressive capability of Tregs is used to help control autoimmunity in the GC but has been co-opted to also promote the overall GC response. Much work remains to fully understand the role of Tfr cells in the overall humoral immune response, and in the larger scope of the immune system.

### AUTHOR CONTRIBUTIONS

AD and MX both wrote and made equal contributions to the manuscript.

### ACKNOWLEDGMENTS

This work was supported by National Institutes of Health R01 AI132771-01 to AD. MX was supported by a Careers in Immunology Fellowship from American Association of Immunologists. We thank Dr. Mark Kaplan for critically reviewing the manuscript.

independently of interleukin 2. *Nat Immunol* (2005) 6:152–62. doi:10.1038/ ni1160

Th1 and Th17 cell responses. *Immunity* (2014) 40:569–81. doi:10.1016/j. immuni.2014.02.012

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Xie and Dent. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# T Follicular Helper Cells in Autoimmune Disorders

*Noémie Gensous, Manon Charrier, Dorothée Duluc, Cécile Contin-Bordes, Marie-Elise Truchetet, Estibaliz Lazaro, Pierre Duffau, Patrick Blanco and Christophe Richez\**

*ImmunoConcept, UMR-CNRS 5164, Université de Bordeaux, Bordeaux, France*

T follicular helper (Tfh) cells are a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They are required for the generation of germinal center reactions, where selection of high affinity antibody producing B cells and development of memory B cells occur. Owing to the fundamental role of Tfh cells in adaptive immunity, the stringent control of their production and function is critically important, both for the induction of an optimal humoral response against thymusdependent antigens but also for the prevention of self-reactivity. Indeed, deregulation of Tfh activities can contribute to a pathogenic autoantibody production and can play an important role in the promotion of autoimmune diseases. In the present review, we briefly introduce the molecular factors involved in Tfh cell formation in the context of a normal immune response, as well as markers associated with their identification (transcription factor, surface marker expression, and cytokine production). We then consider in detail the role of Tfh cells in the pathogenesis of a broad range of autoimmune diseases, with a special focus on systemic lupus erythematosus and rheumatoid arthritis, as well as on the other autoimmune/inflammatory disorders. We summarize the observed alterations in Tfh numbers, activation state, and circulating subset distribution during autoimmune and some other inflammatory disorders. In addition, central role of interleukin-21, major cytokine produced by Tfh cells, is discussed, as well as the involvement of follicular regulatory T cells, which share characteristics with both Tfh and regulatory T cells.

#### *Edited by:*

*Georgia Fousteri, San Raffaele Hospital (IRCCS), Italy*

#### *Reviewed by:*

*Tri Giang Phan, Garvan Institute of Medical Research, Australia Laurence Morel, University of Florida, United States*

#### *\*Correspondence:*

*Christophe Richez<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 05 May 2018 Accepted: 03 July 2018 Published: 17 July 2018*

#### *Citation:*

*Gensous N, Charrier M, Duluc D, Contin-Bordes C, Truchetet M-E, Lazaro E, Duffau P, Blanco P and Richez C (2018) T Follicular Helper Cells in Autoimmune Disorders. Front. Immunol. 9:1637. doi: 10.3389/fimmu.2018.01637*

Keywords: auto-immunity, auto-immune diseases, T cells, T helper follicular cells, systemic lupus erythematosus

### INTRODUCTION

T follicular helper (Tfh) cells represent a distinct CD4+ helper T cell subset, specialized for provision of help to B cells (1–4). They develop within secondary lymphoid organs (SLO) and can be identified based on their unique surface phenotype, cytokine secretion profile, and signature transcription factor. They support B cells to produce high-affinity antibodies toward antigens (Ag), in order to develop a robust humoral immune response and they are crucial for the generation of B cell memory. Whereas they are essential for infectious disease control and optimal antibody responses after vaccination, an excessive response can lead to a breakdown of tolerance. In this review, after introducing the biology of Tfh cells, we will consider in detail their role in the pathogenesis of autoimmune diseases and inflammatory disorders.

### Tfh CELLS: OVERVIEW ON CHARACTERIZATION, GENERATION, AND FUNCTIONS

T follicular helper cells were initially described in 2000 and 2001 in humans (1–4). They have been identified as a distinct T helper cell subset, based on their unique combination of surface

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markers [abundant expression of chemokine (C-X-C motif) receptor 5 (CXCR5) (1, 4), downregulation of C-C chemokine receptor type 7 (CCR7), and expression of the co-stimulatory molecules inducible co-stimulator (ICOS) (1) and programmed cell death protein-1 (PD-1)], cytokine production [expression of high levels of interleukin 21 (IL-21)], and specific transcription factor [expression of the nuclear transcriptional repressor B cell lymphoma 6 (Bcl-6)]. All are necessary for the development, the maintenance, and the functions of these cells. Tfh cells are crucial for the generation of germinal centers (GC) (5, 6), unique structures formed in SLO where antigen-primed B cells undergo proliferation, immunoglobulin (Ig) class switching, somatic hypermutation, and differentiation (7). Strong expression of CXCR5 by Tfh cells is pivotal for their migration into B cell follicles, in which they are attracted in response to the gradient expression of chemokine ligand 13 (CXCL13) (8–10). Within GC, Tfh cells are important regulators of B cell maturation and help signals rely on both cell-to-cell interactions and on the production of cytokines. Inside GC, Tfh cells are equipped with a specific combination of surface molecules, such as ICOS, a co-stimulatory molecule that belongs to the CD28 superfamily, PD-1, a potent inhibitory receptor, or CD40 ligand (CD40L), a member of the tumor necrosis factor family. Engagement of these molecules on their receptors on B cells delivers signals of survival, differentiation, or isotype switching (11–22). Tfh cells are also characterized by the secretion of a diversity of cytokines (23–26). Production of high levels of IL-21 is one hallmark of Tfh cells (23). This cytokine promotes B cells differentiation into plasma cells, somatic hypermutation, and Ig isotype switching (23, 27–30). Moreover, IL-21 promotes Tfh cells differentiation in a positive autocrine feedback loop (31–35).

Differentiation and development of Tfh cells is an extremely complex process, occurring in a multi-stage way. The essential transcription factor of Tfh cells, Bcl-6, controls their differentiation, their full maturation, and the expression of Tfh cells signature molecules (36–38). Bcl-6 expression, associated with the downregulation of its antagonist Blimp-1, leads to the inhibition of the other transcription factors specific to other T helper cell lineages (T-bet, GATA3, and RORγt specially). In addition to Bcl-6, other transcription factors, such as c-MAF, achaete–scute complex homolog 2, basic leucine zipper transcription factor (39), or IFN regulatory factor 4 are also crucial for Tfh cells differentiation (39–41). While Bcl-6 expression is essential for Tfh cells, it should be noted that it is not perfectly Tfh cell-specific, as it can be transiently up-regulated in dividing CD4+ T cells after activation by dendritic cells (42, 43). Micro-environmental factors are necessary for T cells to acquire a Tfh profile. First, Ag stimulation and constant initial interaction with Ag-presenting cells (APCs) and activated B cells play a crucial role in their differentiation from naive CD4+ T cells (2, 44–46). Second, a specific cytokinic milieu is necessary as IL-12, and to a lesser extent TGFβ, seem to be of particular importance for Tfh cells differentiation in humans (47–50). In addition, although originally recognized as an essential T-cell growth factor, IL-2 is a potent inhibitor of Tfh cells differentiation as the ligation of its receptor leads to STAT5 activation, promoting the formation of non-Tfh effector cells (51–53).

One layer of complexity in the study of Tfh cell populations is their substantial heterogeneity. Tfh cells are not a monolithic population with fixed phenotype and functions. They undergo changes over time in the expression of surface molecules and in the secretion of cytokines, in order to shape more efficiently the help delivered to B cells (45, 54, 55).

The biology of Tfh cells has been extensively studied in SLO in mouse models, but due to the difficulties of sampling in humans, different research groups have focused on identifying the circulating counterparts of bona fide Tfh cells in peripheral blood. Description of a CXCR5+ subset of memory CD4+ T cells has emerged few years ago (56–58) and has been subsequently referred to as circulating Tfh (cTfh) cells (59). Although these cells do not express Bcl6, they share phenotypic and functional properties with tissue Tfh cells (15, 56, 58–60). However, several questions are still under investigation regarding this population: their exact biology is still poorly defined, and how they relate to bona fide lymphoid Tfh cells remains unclear. Literature data, arising specially from SAP-deficient mice, suggest that development of cTfh cells does not require GC formation. These cells are committed to the Tfh lineage and are primarily generated before participating in GC reaction (54, 58). There is actually no consensus regarding phenotypic markers that should be used for the identification of this population. It is clear that other activated CD4+ T cell subsets, with other effector fates, can express transiently CXCR5, ICOS, or PD-1, but we can consider that sustained and/or high levels of expression of these molecules are characteristic of Tfh cells (59). The heterogeneous population of cTfh cells contains three major subsets, defined by their expression of the chemokine receptors CXCR3 and CCR6. They present similar properties with Th1, Th2, and Th17 cells: CXCR3+CCR6− represent cTfh1 cells, CXCR3−CCR6− cTfh2 cells and CXCR3−CCR6+ cTfh17 cells. cTfh2 and cTfh17 are more efficient than cTfh1 cells in helping B cells to proliferate and differentiate into plasma cells (56). Finally, within each of these subsets (cTfh1, cTfh2, or cTfh17), it is possible to subdivide other distinct subpopulations, according to the differential expression of the markers ICOS and PD-1 which can be used as indicators of active state of Tfh cells (58, 61, 62).

Regulatory pathways need to be engaged to control the development and maturation of Tfh cells, as well as their interactions with B cells. Within the diverse regulating mechanisms that we will not develop here, it is of particular importance to mention the role of another specific subset of CD4+Foxp3+ regulatory T cells, called follicular regulatory T (Tfr) cells, that has been more recently described. These cells share phenotypic characteristics with Tfh cells, with the expression of Bcl-6, CXCR5, PD-1, and ICOS. They are found within the GC, where they act to limit the size of the response, regulating the frequencies of both Tfh and GC B cells (63–66). Lack of Tfr cells is associated with greater GC reactions *in vivo*.

### Tfh CELLS AND AUTO-IMMUNITY: GENERALITIES

Autoimmune diseases are caused by a breakdown of immune tolerance. Proliferation of self-reactive B cells with generation of high-affinity autoantibodies participate to the pathophysiology of these disorders and, in this way, have led to consider Tfh cells as possible actors in their pathogenesis. Moreover, in autoimmune diseases, inflammatory sites develop frequently lymphoid cell aggregations which include B and T helper cells, leading to the formation of ectopic lymphoid structures (67). These tissuelocalized T–B-cell interactions which can promote the maturation of B cells and can potentiate the production of pathogenic autoantibodies (68), even if the exact nature of helper T cells in inflamed tissues is not completely understood currently.

Studies of the contribution of Tfh cells in autoimmune disorders have been initially limited to animal models, essentially because of difficulties in investigating Tfh cells from human SLO. The ulterior identification of circulating Tfh-like cells, which are more accessible than Tfh cells in tissues, has provided an opportunity to analyze these cells in the context of human autoimmune disorders. Since then, alterations in Tfh cells have been described in a broad range of autoimmune diseases, including systemic diseases, or organ- and cell-specific disorders. Increased numbers of cTfh cells, often correlating with the frequency of circulating plasmablasts or plasma cells, have been observed in several auto-immune diseases and alterations have also been reported regarding their polarization, their function, and the quality of help they provide. All the data collected so far in auto-immunity and in inflammatory diseases should not mask our lack of knowledge regarding the exact biology of cTfh cells and that analysis could be confounded by our incomplete understanding. Moreover, it is to mention that combination of markers used to define cTfh cell populations in the studies that will be mentioned in the next sections often differ among the laboratories. Some studies have defined cTfh cells as total CXCR5+CD4+ T cells, while other have investigated a subset of CXCR5+CD4+ T cells (CXCR5+ICOS+, CXCR5+PD1+, CXCR5+ICOS+PD1+, or CXCR5+IL-21+).

### SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)

Systemic lupus erythematosus is characterized by the presence of high-affinity pathogenic autoantibodies directed against nuclear components (69). Autoreactive B cells in SLE are derived from GC reactions (70); therefore, investigations of Tfh cell roles have been intensive since many years, both in murine models and in human SLE. In different mouse models sharing immunopathologic features with human SLE, it has been demonstrated that Tfh cells contribute to the activation of the immune system and to the pathogenesis of the disease. Animals in these different models have an aberrant expansion of Tfh cells, associated with dysregulation of the GC responses. BXD2 mice, which develop spontaneously autoantibodies and glomerulonephritis, are characterized by an increased frequency of PD1+ Tfh cells as compared to wild-type animals, correlating positively with the frequency of GC B cells and with levels of anti-dsDNA antibodies (71). Sanroque mice have a particular single recessive mutation in *roquin*, a gene encoding for a RING-type ubiquitin ligase family member that disrupts a repressor of ICOS expression. Animals are characterized by a SLE-like phenotype with lymphadenopathy and splenomegaly, high levels of anti-nuclear antibodies (AAN), autoimmune cytopenia, and immune complex deposits (72). Even without antigenic stimulation, a spontaneous formation of GC is observed, with an increase in the number of Tfh cells and an increase in their activity, particularly marked regarding the excessive production of IL-21 (72–75). The other murine model that has provided evidence of the implication of Tfh cells in the development of systemic autoimmunity is represented by BXSB mice, in which the duplication of the *Tlr7* gene promotes excessive signaling by self-autoantigens, leading to a severe inflammatory disease, with high levels of autoantibodies and proliferative glomerulonephritis (76). These animals have expanded numbers of B cells and Tfh cells, especially in the spleen (77). Besides, IL-21 is elevated in the serum and over-expressed in splenocytes (78). In this mouse model, IL-21R deficiency induces a decrease in the serum levels of AAN and prevents the apparition of renal disease (77). Moreover, therapeutic blockade of IL-21, by the administration of an IL-21R-Fc fusion protein, seems to have a biphasic response characterized by an aggravation of the disease when the treatment is given during early life and an anti-inflammatory effect (decrease in IgG1 serum levels, in proteinuria levels, and in histological glomerulonephritis) when it is administrated later in the disease course (79, 80). Implication of IL-21 in the pathogenesis of SLE is also supported by data obtained in two other murine models of the disease. In MRL-Fas(lpr) mice, accumulation of activated B cells, activated T cells, plasma cells, and spontaneous GC formation is dependent on IL-21R signaling. Administration of IL-21R-Fc fusion protein reduces disease severity (81, 82). Finally, in the NZB/NZW mouse model, blockade of IL-21R inhibits the progression of the pre-established disease (83). Murine models also point out the role of interactions between ICOS and its ligand in the development of systemic autoimmunity and suggest that this pathway could possibly be an interesting novel therapeutic target. In the NZB/NZW mice, blockade of ICOS pathway, by the use of a monoclonal antibody directed against ICOS-L, leads to the inhibition of the development of Tfh cells, to a decrease in anti-dsDNA antibody titers and to an improvement in kidney function (84, 85). Reduction in titers of anti-dsDNA antibodies is also observed in the mouse model MRL-Fas(lpr) when animals have an additional genetic deletion in ICOS (86). Finally, in B6.Sle1 mice, elevated expression of ICOS contributes to the expansion of Tfh cells and to the breakdown in peripheral tolerance (87).

Several lines of evidence also support a pathogenic role of Tfh cells and IL-21 in human SLE (**Table 1**). Data are available mostly for cTfh cells, which have been showed to be increased and have an active phenotype in SLE patients as compared to controls (88–98). This activated phenotype correlates with the number of circulating plasmablasts and with the levels of pathogenic autoantibodies (58, 88, 90, 91, 93–95, 98), but the correlation with disease activity observed in some studies (58, 90, 92, 97) has been considered inconsistent by other research teams (88, 94). Of particular interest are the observations made on alterations in the composition of cTfh cells subsets in SLE, associated with disease activity (99). Ratio of cTfh2 and cTfh17 (which are both considered as efficient B helpers) over cTfh1 are increased in SLE patients as compared to controls and disease activity correlates with the frequency of cTfh2 cells (99). Higher plasma levels of

#### Table 1 | T follicular helper (Tfh) cells in human SLE.

*cTfh cells, circulating Tfh cells; GC, germinal center; SLE, systemic lupus erythematosus.*

IL-21, as well as an increase in the frequency of IL-21-expressing CD4+ T cells, are found in SLE patients as compared to controls, correlating with the number of switched memory B cells and with several markers of disease severity (89, 91, 95, 100–104). Moreover, Tfh cells have been also investigated at tissue level in SLE: Tfh-like cells are present in inflamed kidney of patients with nephritis (105, 106), where they cluster with B cells building structures similar to ectopic GCs (106).

The role of Tfr in SLE remains unclear and literature data are inconsistent. Some authors observed a reduction in the frequency of circulating Tfr cells in SLE patients (107, 108) with an increase in the ratio Tfh/Tfr, associated with a negative correlation with disease activity and titers of anti-dsDNA antibodies (107). On the contrary, another study revealed a significant increase of Tfr cells in peripheral blood with an increase in the Tfr/Tfh ratio (109). Discrepancies could be related to the strategy used to define Tfr cells by flow cytometry (CD4+CD25+CD127low-intermediateCXCR5+

(107) or CD4+CXCR5+FoxP3+ (109)), as well as the different recruitment of subjects within each study.

T follicular helper cell expansion and aberrant GC responses play clearly a crucial role in the pathogenesis of lupus in mice and data obtained so far in human SLE seem to go in the same direction. Tfh cells could, therefore, represent an interesting therapeutic target. Conventional treatments used in SLE (steroids, immunosuppressive drugs) result in a decrease in the number of activated cTfh cells (90, 91), associated with a clinical improvement (90). A treatment with low-dose recombinant human IL-2 in SLE patients leads also to a decrease in the relative number of cTfh cells (110). However, the specific targeting of these cells requires a better understanding of the mechanisms involved in their aberrant activation during SLE. In this way, OX40 ligand (OX40-L)–OX40 axis has been identified as a key player in the increased Tfh responses (111): OX40-L, highly expressed by myeloid APCs in active SLE patients in response to TLR7 activation by RNP-anti-RNP immune complexes, promotes the differentiation of CD4+ T cells in functional Tfh cells (111). In this way, in human SLE, the OX40-L–OX40 axis provides an amplification loop in the generation of autoantibodies (112) and could represent an attractive pathway to target. However, although anti-OX40 and anti-OX40L antibodies exist, there is for instance no ongoing trial of these molecules in human SLE.

### RHEUMATOID ARTHRITIS (RA)

Rheumatoid arthritis is a systemic auto-immune disease, associated with acute and chronic synovial inflammation, cartilage lesions, and bone erosion (113). Various autoantibodies associated with RA and which play important roles in the pathological progression of the disease have been identified, including rheumatoid factor and antibodies against cyclic citrullinated peptides (anti-CCP). Exact pathogenic processes of RA are not totally understood and involve many types of immunocompetent cells. Nevertheless, interactions between T and B cells are required for antibody production and, similarly to what are observed during SLE, many of the RA-related autoantibodies are high-affinity IgG antibodies, indicative of the involvement of GC reactions and, therefore, suggesting a critical role of Tfh cells in the disease pathogenesis.

Data from murine models of arthritis seem to indicate that some essential Tfh cells associated molecules are required for the disease development. For example, in the model of collageninduced arthritis (CIA), CXCR5 was identified as an essential factor for the induction of the inflammatory autoimmune arthritis, as CXCR5-deficient mice and mice selectively lacking CXCR5 on T cells are resistant to CIA (114). In another model (transfer of self-reactive CD4+ cells from KRN-TCR transgenic mice into recipient animals), Chevalier et al. observed that deficiency in CD4+ cells of signaling of lymphocytic activation molecule-associated protein (SAP) (a protein known to promote Tfh cell differentiation during GC formation) protects mice from the induction of arthritis, indicating that long-lasting interactions between T and B cells and GC formation are required for the development of the disease (115).

In human RA, it is worth noting that literature data are conflicting regarding Tfh cells frequencies (**Table 2**). In five studies, it was observed increased frequencies of cTfh cells in patients as compared to controls (116–120), especially in those with newonset disease. For some authors, this increase is constitutive and is not associated with the activity of arthritis, as both patients with active or inactive disease have higher frequencies of activated cTfh cells and of subsets with B cell helper phenotype (Tfh2 and Tfh17) (116). Still these findings contrast with other reports where percentages of cTfh cells have been associated with the disease activity [mostly assessed by 28-joint count disease activity score (DAS28)] (117–119, 121) and with the levels of autoantibodies (119–121). An enrichment of Tfh cells was observed in synovium tissues of patients with RA whereas they were absent in both osteoarthritis and normal synovium tissues (122, 123). By contrast of the five studies mentioned above, three other ones dismiss the presence of increased frequencies of Tfh cells during

*Ab, antibody; Anti-CCP, antibodies against cyclic citrullinated peptides; cTfh cells, circulating Tfh cells; CXCR5, chemokine (C-X-C motif) receptor 5; DAS, disease activity score; ICOS, inducible co-stimulator; RA, rheumatoid arthritis.*

RA. No differences were found in the percentages of cTfh cells or within Tfh1, Tfh2, and Tfh17 subsets in RA patients, as compared to healthy controls or patients with undifferentiated arthritis, without correlation with DAS28 (124, 125) and Penatti et al. even observed lower frequencies of ICOS+ cTfh cells in patients with RA (122).

Investigations on the implication of Tfh cells in the pathogenesis of the disease have also led to the evaluation of the impact of their signature cytokine, IL-21. RA patients have higher serum levels of IL-21 as compared to controls (117, 120, 121), correlating with DAS28 (117, 121), serum anti-CCP antibodies, and frequencies of cTfh cells (121). IL-21R is highly expressed in inflamed synovial tissues of RA patients (126, 127), by macrophages but also by fibroblasts with activated phenotype (126). IL-21 has an impact on local T-cell activation and proliferation, but also promotes the aggressive migration, invasion, and metalloproteinase secretion by fibroblast-like synoviocytes (128). In RA synovial cell cultures, neutralization of IL-21 and IL-15 inhibits their pro-inflammatory cytokine production (129). Finally, in the K/BxN mouse model of RA, IL-21R deficiency is sufficient to protect from arthritis (130).

Potential use of Tfh cells as biomarkers of RA has conducted to the evaluation of the impact of treatments on these populations. In 2013, Wang et al. observed a significant reduction in the percentages of ICOS+ Tfh cells following 1 month of disease-modifying antirheumatic drugs (DMARDs) and *T. wilfordii* (Chinese herb) in the drug-responding patients (119). On the contrary, no decrease in the frequency of Tfh cells in response to 24 weeks of DMARD therapy (mostly methotrexate monotherapy) was observed, whereas IL-21 concentrations were remarkably reduced (117). In patients with RA treated by abatacept, which modulates CD28 mediated T cells co-stimulation, the treatment is associated with a marked decrease in the proportion of activated cTfh (118). Data from murine model of RA associated with a breach of self-tolerance brought some mechanistic explanations on this observation made in RA patients: abatacept is associated with a failure of T cells to acquire a follicular helper cell phenotype (CXCR5+ICOS+), to proliferate, to enter B cell follicles, leading to a reduction in antibodies responses (131).

It is known that environmental factors are implicated in the development of arthritis and two recent studies suggest that generation of RA-related Tfh cells possibly relies on gut microbiota. Using the K/BxN autoimmune arthritis model, Teng et al. evaluated the role of a specific type of commensal gut bacteria, named segmented filamentous bacteria (SFB), in the regulation of Tfh cell responses (132). SFB are known to drive autoimmune arthritis in this mouse model (133) and it was shown that SFB induce an increase in the systemic Tfh cell populations by driving differentiation and egress of Tfh cells from intestinal Peyer's patches into systemic lymphoid sites, leading to an increase in auto-antibody responses and to the development of arthritis (132). Using the same murine models, Block et al. confirmed the requirement of gut microbiota in the differentiation of Tfh cells, the formation of GCs, the autoantibody production, and the development of the experimental arthritis. Depletion of gut microbiota in the animals, by the use of antibiotics, reduced the number of Tfh cells and the levels of antibodies (134).

Finally, we would like to highlight the results published recently by Rao et al. in Nature, who identified a novel subset of T cells in the synovium of patients with RA (135). Using multidimensional cytometry, transcriptomics, and functional assays to analyze activated T cells in joint tissues, authors observed, in seropositive RA patients, abundant populations of a specific subset of T cells, defined as PD-1hiCXCR5−CD4+ and which they named "peripheral helper" cell population. This population, not expanded in patients with seronegative RA and of whom frequencies are highly correlated with disease activity in response to therapy, shares B-cell-helper functions with Tfh cells but seems to differ from them by a specific migratory program targeting inflamed tissues rather than lymphoid tissues (135). These results need to be confirmed and some questions need to be addressed, but this study raises the possibility of the existence in RA of a subset with high pathologic abilities that could be of great interest in a therapeutic perspective.

T follicular helper cells have also been investigated in patients with ankylosing spondylitis (AS), another rheumatologic disease, with contradictory results. Higher frequencies of cTfh cells have been observed in AS patients as compared to controls, associated with elevated concentrations of serum IL-21 (136, 137), whereas Bautista-Caro et al. reported decreased frequencies of cTfh cells, plasmablasts and underrepresentation of cTfh subsets with a B helper phenotype in patients with AS naïve from TNF blockers (138). Frequencies of Tfr cells were also reported to be significantly higher in AS patients than in healthy controls, with higher ratio of Tfr/Tfh cells (137).

### SJOGREN SYNDROME

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease, presented as a disabling sicca syndrome associated with asthenia and pain (139). It is characterized by lymphocytic infiltration and destruction of exocrine glands, primarily the lacrimal and salivary ones. Crucial role of B cells in pSS pathogenesis is illustrated by the emergence of circulating immune complexes, autoantibodies, ectopic GCs in the affected tissues, and enhanced risk of developing B cell lymphoma (140) underlying a possible important role played by Tfh cells in the pathogenesis of this disease. Increased percentages of cTfh cells (especially cTfh17 subset) have been observed in peripheral blood of patients with pSS (141, 142). This increase seems to be limited to pSS patients with severe manifestations of the disease. Indeed, in patients with pSS who present extraglandular manifestations (EGMs), Szabo et al. observed an increase in activated cTfh cells (ICOS+ or PD1+ cTfh) as compared to pSS patients without EGMs and to healthy controls, whereas there was no difference in cTfh percentages between patients without EGMs and controls (143). Elevated Tfh percentages were also observed in the anti-SSA/ SSB positive patients (143). Similarly, a significant elevation in the proportion of IL-21-producing PD1+ cTfh cells was described in pSS patients with EGMs and in patients with autoantibodies against anti-SSA/Ro (95). More interestingly, correlation between cTfh cells percentages and importance of glandular involvement assessed by the focus scores of labial salivary gland biopsies has been estimated with a coefficient R equal to 0.6984 (143). Otherwise, increased serum levels of IL-21 are observed in pSS patients with a significant association with the systemic DAS28 (ESSDAI) (144, 145). Regarding salivary glands, Jin et al. reported increased numbers of Tfh cells in pSS patients as compared to controls (141). When analyzing the expression of cytokines and transcription factors associated with the different Th subsets (Tfh, but also Th1, Th2, and Th17) in labial salivary glands, Maehara et al. observed an increase in the expression of all Th subset-related molecules in pSS patients as compared to controls. However, expression of Tfh-related molecules was associated with a strong lymphocytic infiltration and especially in the presence of GCs (146). Differentiation of naïve CD4+ T cells seems to be particular during pSS and implies the interactions with salivary gland epithelial cells such as IL-6 and ICOS-L expressed by these cells contribute to the direct induction of Tfh cells differentiation (145).

Frequency of cTfh cells in pSS patients is almost reduced by 50% after B cell depletion therapy and reach levels comparable to controls, in association with a significant lowering of serum levels of IL-21, with a decrease in anti-SSA/Ro and antiSSB/ La antibodies and with the improvement of the disease activity measured by ESSDAI (147). During B cell repopulation, frequencies of cTfh cells return to baseline levels. Like described in RA, specific effects of abatacept on Tfh cells are observed during pSS treatment (148). Abatacept results in the predominant reduction of percentages and numbers of cTfh cells (other effector subsets are not affected, except Treg cells), of serum levels of IL-21 and of autoantibodies. A decrease in ICOS expression on CD4+ T cells is observed in both peripheral blood and parotid gland tissue and this phenomenon correlates significantly with a lowering in the DAS28 (148).

### NEUROLOGICAL DISEASES

### Multiple Sclerosis (MS)

Multiple sclerosis is a chronic, inflammatory, and autoimmune disease affecting the central nervous system (CNS), leading to the destruction of myelin and axons (149). In pathological studies, important meningeal inflammation with ectopic lymphoid follicles, B and plasma cells is observed (150, 151). This suggests a possible implication of T helper cells and especially Tfh cells in the pathogenesis of the disease. In peripheral blood from patients with relapsing–remitting or secondary progressive forms of the disease, an increase in the frequencies of ICOS+ cTfh cells is observed, associated with an increased expression of Tfh and plasmablast markers by cerebrospinal fluid (CSF) cells (152). Activated memory cTfh cells (CCR7+ICOS+) were also found increased in another study in patients with relapsing MS. Moreover, they observed increased levels of plasma and CSF IL-21 correlating positively with the score of severity of the disease (EDSS score) and with levels of auto-antibodies directed against myelin basic protein or myelin oligodendrocyte glycoprotein (153). Treatment with systemic steroids (methylprednisolone) leads to a decrease in the numbers of activated cTfh cells and in the plasmatic levels of IL-21 (153).

### Neuromyelitis Optica Spectrum Disorders (NMOSDs)

Neuromyelitis optica spectrum disorders are also inflammatory demyelinating diseases affecting CNS, characterized clinically by attacks of myelitis and optic neuritis and biologically by the presence of highly specific and pathogenic autoantibodies directed against the extracellular domain of the water channel protein aquaporin-4 expressed in astrocytes (NMO-IgG) (154). Activated Tfh cell frequencies are higher in NMOSD subjects, as compared to healthy controls and even as compared to MS patients and are associated with disease activity and with increased levels of plasma and CSF IL-21 (155, 156). Treatment with methylprednisolone is associated with a significant decrease in the proportion of cTfh cells in NMOSD patients (155).

### Myasthenia Gravis (MG)

Myasthenia gravis is characterized by the production of anti-acetylcholine receptor (AchR) antibodies, leading to a dysfunction of the neuromuscular junction and *in fine* to muscle weakness (157). Ocular and generalized MG are the two major clinical forms of the disease. Expansion of cTfh cells, concomitant with expansion of circulating plasmablasts, is observed in patients with MG as compared to healthy control subjects, and is associated with the clinical severity and the form of the disease (158–161). Strong correlations between cTfh cells and titers of anti-AChR autoantibodies are frequently described (159–161). On the contrary, proportions of Tfr cells are decreased in MG patients as compared to controls, leading to an imbalance in the Tfr/Tfh ratio (159, 162). One important specificity of MG is the frequent association with thymoma and the use of thymectomy as a treatment, leading to the possible pathological analysis of thymus obtained from MG patients. Clinical severity of the disease (subjects with generalized MG versus subjects with ocular MG and controls) is correlated with higher mRNA expression of four markers by thymoma cells (CXCR5, Bcl-6, ICOS, and IL-21) (163). Higher percentages of thymic Tfh cells are present in MG patients, as compared to patients with thymoma without MG or patients with healthy thymus (control thymic biopsies from cardiac surgery cases) (164).

## IgG4-RELATED DISEASE (IgG4-RD)

IgG4-related disease consists in lymphoplasmacytic infiltrates of CD4+ T cells and IgG4+ plasma cells associated with fibrosis. It affects mainly males over 50 years and may involve all tissues (pancreatitis, retroperitoneal fibrosis…) (165). IgG4-RD patients are characterized by higher proportions of cTfh cells, as well as increased proportions of plasmablasts as compared to controls (166). The two populations correlate positively within each other and cTfh cell rates are positively correlated with EGMs of IgG4-RD (166). Proportions of cTfh cells that express a high level of PD-1 positively correlate with serum levels of IgG4, IgG4/IgG ratio, and number of involved organs (167). When cTfh subsets are analyzed, a significant specific expansion of Tfh2 cells is observed, associated with serum levels of IgG4, IgG4/IgG ratio, number of plasmablasts, disease activity, and number of affected organs (168–170). Tfh2 cells appear to be responsible, in the pathogenesis of the disease, for the induction of the differentiation of naïve B cells into plasmablasts and of the increase in the production of IgG4 in patients with active disease (169). Contradictory results have been published regarding the population of Tfh1 cells: a Japanese team observed an increase in the number of activated Tfh1 cells in IgG4-RD, correlating with disease activity but not with serum IgG4 levels, whereas another team reported a decrease in the proportion of cTfh1 cells in patients as compared with controls (170).

One particularity of IgG4-RD is the existence of numerous data with histopathological examination. In fact, in IgG4-RD, it is necessary to get a biopsy-proven diagnosis, allowing the analysis of Tfh cells directly in the involved tissues. Histological data reveal marked Tfh cell infiltration or overexpression of Tfh-related molecules in the different affected organs studied (166, 171–173), colocalizing with B cells and plasma cells (171). Tfh cells infiltrating the submandibular glands (SMGs) of patients suffering from IgG4 dacryoadenitis and sialadenitis are characterized by high expression levels of Bcl6, PD-1, and ICOS, as compared with SMGs from control patients with head and neck cancers, and functional analysis reveal that they have a higher capacity than tonsillar Tfh cells to help B cells to produce IgG4 (167). Expression of IL-21 in labial salivary glands from patients with IgG4-RD correlates with the number of GCs and with the IgG4/IgG ratio (172). All teams working on Tfh cells and IgG4-RD showed a decrease in Tfh percentages in patients treated by steroids or rituximab, correlating to the improvement of symptoms (166, 167, 169, 170).

## VASCULITIS

### Large Vessels Vasculitis

To our knowledge, role of Tfh cells in the physiopathology of large vessels vasculitis has been rarely investigated. One team studied Tfh cells in granulomatosis with polyangiitis (GPA) (174), disease characterized, in the majority of patients, by the development of ANCA autoantibodies with specificity for PR3 (ANCA/anti-PR3). Because GPA has benefited recently from B-cell depletion therapy, it raises the question of the underlying mechanism of its effectiveness. Authors found, in GPA patients on conventional therapies, an increased frequency of cTfh cells. This increase is not observed in GPA patients treated with rituximab who are clinically improved and in whom cTfh cells frequencies are statistically not different from those seen in healthy controls (174).

### Small Vessels Vasculitis

Ig A vasculitis, also called Henoch Schönlein Purpura (HSP), is the most common small vessel vasculitis which affects particularly children. Vascular deposits of IgA-related immune complexes characterize this systemic inflammatory disease. Circulating Tfh cells are increased in children with acute HSP as compared to healthy controls and are associated with higher levels of IL-21 (175, 176). Levels of cTfh and IL-21 lowered significantly following disease remission (175). Adult patients with HSP nephritis (HSPN) display same features, with increased levels of cTfh cells and IL-21 as compared to healthy controls and a significant reduction after treatment (177). When further investigating Tfh subpopulations, an increase in cTfh2 and cTfh17 is present in HSP patients compared to healthy controls and their levels positively correlated with serum IgA ones (178). The increases in cTfh2 and cTfh17 cells counts are abrogated by treatment.

### IDIOPATHIC THROMBOCYTOPENIC PURPURA (ITP)

Idiopathic thrombocytopenic purpura is characterized by a low platelet count, which is the result of both insufficient platelet production and increased platelet destruction by auto-antibodies directed against platelet glycoproteins. Main antibody isotype in ITP is represented by IgG, which underlies a class-switch recombination, a mechanism supported by Tfh cell help to B cells. In adult ITP patients, there is an increase in the proportion of cTfh cells with high expression of ICOS or PD-1 (179). This is particularly marked in the sub-group of patients with positivity of anti-platelet antibodies as compared to platelet-antibody-negative patients. Plasma levels of IL-21 are also significantly increased in patients with active ITP as compared to controls (179, 180). In the pediatric population, which is frequently impacted by ITP, similar results are observed: frequencies of cTfh cells are markedly increased during ITP, with a strong negative correlation between the proportion of cTfh cells and platelet count, as well as with increased serum levels of IL-21 (181). It should be highlighted that the frequencies of cTfh cells return to normal levels after therapy (intravenous Ig, corticosteroids, or both) in patients with newly diagnosed ITP, whereas children who fall in chronic ITP (cITP) have a persistent increase in both cTfh cell frequencies and IL-21 levels. Moreover, in cITP subjects, overexpression of ICOS-L by CD19+ B cells remains after treatment, while it markedly decreases in the other group (181).

Spleen is the site of platelet destruction by splenic macrophages phagocytosis and as it is the primary site for the activation of B cells and for the autoimmune response and, therefore, an organ of interest in ITP. Splenectomy is one possible therapeutic option for resistant ITP and pathological analysis of the resected organs gives precious information. Audia et al. characterized Tfh cells in the spleen of 13 ITP patients compared to 8 controls (splenectomy because of spleen traumatism) (182). Splenic Tfh cells frequencies (CXCR5+ICOS+PD-1hi) are increased during ITP, concomitant with an expansion of GCs and with an increase in splenic CD38+ B cell subsets (pre-GC B cells, GC B cells, and plasma cells). Furthermore, IL-21 expression in splenic CD4+ T cells correlates with Tfh cell abundance. Finally, *in vitro* stimulation of B cells with IL-21, in the presence of CD40 engagement, induces the differentiation of B cells in antiplatelet antibodies secreting plasmablasts in ITP patients (182). The expansion of splenic Tfh cells is dramatically decreased after B cell depletion induced by rituximab, associated with a decrease in the absolute count of cTfh cells, even if the therapy is not clinically effective (183).

### CHRONIC INFLAMMATORY SKIN DISEASES

### Psoriasis