Heterogeneous Tfh cell populations that develop during enteric helminth infection predict the quality of type 2 protective response

T follicular helper (Tfh) cells are an important component of the germinal centre (GC)-mediated humoral immunity. Yet, how regulation of Tfh- GC responses impacts on effective responses to helminth infection are poorly understood. Here we show that chronic helminth Trichuris muris infection fails to induce Tfh-GC B cell responses, with Tfh cells expressing T-bet and IFN-γ. In contrast, Tfh cells that express GATA-3 and IL-4 dominate responses to an acute, resolving infection. Accordingly, heightened expression and increased chromatin accessibility of Th1- and Th2 cell-associated genes is observed in chronic and acute induced Tfh cells, respectively. However, both acute and chronic Tfh cell populations retained the capacity to produce IL-21 in spite of the Th-biased response. Blockade of Tfh-GC interactions impaired type 2 immunity, highlighting the protective role of GC-dependent Th2-like Tfh cell responses against helminths. Collectively, these results provide new insights into the protective roles of Tfh-GC responses and identify distinct transcriptional and epigenetic features of Tfh cells that emerge during resolving or chronic helminth infections. Author summary About a quarter of the world population is afflicted with parasitic worm infection. Although deworming drugs can reduce the levels of the infection, they fail to prevent reinfections. Therefore, the most sustainable goal is to develop vaccines against human helminth parasites, which has been extremely challenging due to the lack of understanding of host-parasite interactions. While the protective roles of T helper 2 (Th2) cells are well established, the regulation of T follicular helper (Tfh) cells and their roles during helminth infection remain poorly defined. In this study, we describe the differential regulation of Tfh cell responses during chronic non-protective vs acute protective responses during helminth infection. We show that Tfh cells during chronic infection are rare and have strikingly different characteristics to acute-induced Tfh cells, which appear to be more like Th2 cells. Specifically, we show that blockade of Th2-like Tfh cell response during acute infection results in the host failing to expel the worms. Our study identifies that Tfh cell populations that emerge during chronic and acute infection are strikingly heterogeneous and critically important in mediating protective immune responses against helminths.


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The germinal center (GC) reaction is a highly dynamic process through which highaffinity and long-lived antibodies and memory B cells develop in response to infections, and is 60 often used as the basis of vaccinations, primarily against viral pathogens. However, there are currently no viable human vaccines against helminth parasites that afflict billions of people 62 world-wide [1]. The lack of helminth vaccines may reflect the complexity and diversity of helminths, the biology of the infectious cycle, as well as the lack of knowledge on the precise 64 requirements for immunity to infection. Although Th2 cells are the key to protective responses against helminths, the role of GCs in promoting effective responses to acute helminth infection, 66 or whether their dysregulation contributes to ineffective clearance during chronic infection, remains poorly defined [2]. 68 CXCR5-expressing T follicular helper (Tfh) cells aid B cell responses through the production of 70 cytokines and direct contact-mediated interactions such as CD40-CD40L [3]. Tfh cell differentiation is dependent upon Bcl6, the Tfh cell lineage-defining transcription factor (TF). 72 Mechanistically, Bcl6 represses non-Tfh cell lineage genes, including Th1 cell-defining T-bet and Th2 cell-associated Gata3 [4][5][6], as well as through the repression-of-repressors mechanism 74 [7, 8]. Despite Bcl6-dependent repression of T-bet and Gata3, distinct subpopulations of Tfh cells that share Th1 and Th2 phenotypes develop during different types of infection [9,10]. In 76 response to viral infection, Tfh cells initially co-express Bcl6 and T-bet and the interplay between these TFs plays an important role in the establishment of Tfh versus Th1 cell lineage 78 [11,12] in a context-dependent manner [13][14][15]. Type 2 immune stimuli such as allergens and helminths promote Tfh cells that mainly produce IL-4, a cytokine that is required for 80 coordinating the overall type 2 immune response, as well as IgG1 and IgE class switching [16][17][18], as well as for coordinating the overall type 2 immune response. The diverse Tfh cell 82 phenotypes suggest that tailoring of Tfh cells is central to the development of protective CD4 + T cell responses across pathogens [9,10]. However, how the type of the immune response (type 1 84 vs type 2) against one class of pathogen regulates the flexibility and functionality of Tfh cell responses remains unclear. 86 In the current study, we characterized Tfh cell and GC B cell responses in the draining  (Fig 1A). Consistent with previous studies [21,22], worm burden in HD-infected mice progressively decreased from 7 to 14 days pi, with almost all worms cleared by day 21 pi. (Fig   118   1B). In contrast to HD infection, LD-infected mice failed to clear the parasite, maintaining a consistent and chronic worm burden to day 35 pi. (Fig 1B). These results indicate that C57BL/6J 120 mice were capable of effectively clearing worms post-HD infection, whereas LD infection results in susceptibility to the parasite. 122 We next characterised the Tfh/GC response during non-protective and protective responses using 124 LD-and HD-infection models. We used the canonical Tfh cell (CD4 + BCL6 + CXCR5 + PD-1 + ) and GC B cell (B220 + IgD -CD95 hi CD38 + ) markers to identify Tfh and GC B cells in the mLNs 126 at day 7, 14, 21 and 35 following LD or HD infection. Flow cytometric analysis revealed the frequencies and numbers of HD-induced Tfh cells peaked at d21 pi (Fig 1C-1E). In contrast, Tfh 128 cell frequencies and numbers were lower during LD infection at all timepoints (Fig 1C-1E). The GC B cell response paralleled the Tfh cell response during LD and HD infection (Fig 1F-1H). 130 We found significantly fewer GCs in the mLNs during LD infection (Figs 2A-2C and S1A).
Further, the size of the GCs as determined by the GL7 + /IgD + area was also significantly smaller 132 in the LD-infected mLNs (Fig 2D), suggesting that LD infection fails to induce GC B cell expansion. Consistent with the development of a protective Th2 cell response in resistant mice 134 [23], we found high levels of serum parasite-specific IgG1 and low levels of IgG2c (Fig 2E and   2F). Surprisingly, despite a poor Tfh-GC response, LD-infected mice were capable of producing 136 class-switched IgG2c (Fig 2E and 2F). Taken together, our results show that potent Tfh-GC responses correlate with type 2-mediated protection following HD T. muris infection. 140 As we had observed robust GC responses that developed progressively throughout HD but not LD T. muris infection, we next generated IL-4-AmCyan/IL-13-DsRed/IL-21-GFP (IL-4-142 13-21) triple reporter mice [24][25][26] to dissect the temporal expression of these cytokines in Tfh cells. First, we failed to detect any IL-13-DsRed expression in both CD4 + CD44 hi CXCR5 -PD-1 -144 non-Tfh cells and Tfh cells at d7-21 post-LD-and -HD T. muris infection (S1B Fig). This finding complement previous studies showing that IL-13 expression is typically expressed in 146 CD4 + T cells in the non-lymphoid tissue such as lungs but not draining lymph nodes (LNs) [27,28]. In addition, the absence of IL-13-producing Tfh cells in response to T. muris infection is 148 consistent with results from infections with the helminth Nippostrongylus brasiliensis (N.

Kinetics of IL-4 and IL-21 expression during Th1-and Th2-skewed helminth infection
brasiliensis) [29]. The expression of IL-4 single-producing Tfh cells in both LD and HD 150 infections peaked at d14, with the higher levels of single IL-4-expressing Tfh cells observed in HD than that of LD infection (Fig 3A and 3B). Consistent with a population of Tfh cells that co-152 produce both IL-4 and IL-21 in response to N. brasiliensis infection [18], we also observed Tfh cells that co-expressed IL-4 and IL-21 in both LD and HD T. muris infections (Fig 3A-3C). 154 Similar to single IL-4-producing Tfh cells, both LD and HD infection drove comparable levels of IL-4 and IL-21 double-positive Tfh cell frequencies prior to the peak of Tfh-GC responses (i.e., 156 d7 and d14 pi) (Fig 3A-3C). Unlike single IL-4 expression that peaked at d14, the expansion of single IL-21 expression in both LD-and HD-induced Tfh cells dropped at this time point and 158 both infection doses consistently drove a comparable proportion of IL-21 expression in Tfh cells throughout the course of infection (Fig 3A and 3D). Collectively, these results indicate that HD-160 but not LD-induced Tfh cells are potent producers of IL-4, yet LD-induced cells remained able to produce similar levels of IL-21 during HD infection.
Multiple reports demonstrate that Tfh cells are major producers of IL-4 in draining LNs in response to type 2 inflammatory stimuli [16,17,24]. Given HD T. muris infection potently drives 164 IL-4-mediated Th2 cell responses [30], we sought to further clarify the producers of IL-4 during T. muris infection. At d14 and d21 pi, we observed that LD-and HD-induced Tfh cells were 166 indeed the major producers of IL-4 with a substantial proportion that co-expressed IL-21 when compared to non-Tfh cell populations (Fig 3E-3J). Interestingly, non-Tfh cell populations also 168 expressed IL-21 at d14 and the levels of IL-21 expression in these cells were more prominent at d21 pi (Fig 3E and 3H). Thus, IL-21 production was not expressed exclusively by Tfh cells. 170 Taken together, these data suggest that Tfh cells are major producers of IL-4, a proportion of which is co-expressed with IL-21.  To begin to identify the mechanisms controlling the differential Tfh cell/GC responses following LD and HD T. muris infection, we examined the transcriptional profile of CD4 + 204 CD44 hi FOXP3 -Ly6C -CD162 -PD-1 + CXCR5 + Tfh cells isolated from the mLN of HD-and LDinfected mice at day 21 pi. This gating strategy discriminates CD4 + CD44 hi FOXP3 + PD-1 + 206 CXCR5 + T follicular regulatory (Tfr), CD4 + CD44 hi FOXP3 -Ly6C + CD162 + Th1, and CD4 + CD44 hi FOXP3 -Ly6C -CD162 -PD-1 -CXCR5 -Th2 cells in our genome-wide expression analysis by RNA-sequencing (RNA-seq). Additionally, we included naive CD4 + FOXP3 -CD44 lo CD62L hi T cells in RNA-seq analysis as a control. Multidimensional scaling (MDS) analysis 210 revealed that the overall transcriptomic profile of naive CD4 + T cells, LD-and HD-Tfh cells is highly distinct from each other and the two biological replicates from each group are clustered 212 closely, suggesting high similarities between replicates (Fig 4A). In comparison to downregulated genes, the numbers of up-regulated genes such as Bcl6 and Cd44 (S3A- S3B Fig) were 214 higher in both LD-and HD-Tfh cells over naive CD4 + T cells (Fig 4B), indicating the Tfh cellassociated activated state. Analysis of differentially expressed genes between LD-and HD-Tfh 216 cells revealed 748 genes with higher expression in HD-induced Tfh cells, and 360 genes whose expression was higher in LD-induced Tfh cells (Fig 4C). Although there was no observable 218 difference in gene expression of conventional Th-associated master transcriptional regulator and cytokine transcripts such as Bcl6/Il21, Gata3/Il4 and Tbx21/Ifng, HD-induced Tfh cells had 220 increased expression of genes that have been previously shown to be associated with Th2 cells such as Il4ra [32] and Bhlhe41 (encodes for Dec2) [33], as well as Tfh-associated genes such 222 Pou2af1 (encodes for Bop1) [34] and Ascl2 [35] (Fig 4C). Conversely, the Th1-associated genes such as Il2ra [36], Il12rb2 [37] and Il18r1 [21] were highly expressed in LD-induced Tfh cells 224 (Fig 4C), suggesting the resemblance of Th1-associated transcriptomic profile in LD-Tfh cells. 226 To gain further insights into how similar or different LD/HD-Tfh cells are to bona fide Th1 and Th2 cells, respectively, we first generated LD-and HD-Tfh signature gene sets by listing 228 differentially expressed genes (i.e. ≥ 2-fold) that were highly expressed in LD-or HD-Tfh cells.
Using gene set enrichment analysis (GSEA) [38], we examined whether these LD/HD-Tfh gene 230 signatures were enriched in in vitro polarised IFN-γ + Th1 and IL-4/13 + Th2 cells [39]. As expected, in vitro Th2 cells exhibited enriched HD-Tfh but reduced LD-Tfh gene expression 232 signatures (Fig 4D-4E), whereas in vitro Th1 cells were enriched in LD-Tfh signatures (Fig 4E). Th1-like Tfh cells are more transcriptionally similar to Th1 or   234 Tfh cells, we also performed GSEA analysis on acute LCMV-induced CXCR5 + SLAM lo Tfh and LD-and HD-Tfh cells is distinct from each other (Fig 5A). We identified over 80,000 accessible 248 genomic regions, where differentially accessible regions in HD-Tfh cells over naive CD4 + T cells are higher (i.e., 19,927) when compared to that of LD-Tfh cells (i.e., 16,485) (Fig 5B). In 250 contrast, we observed a smaller difference in the numbers of less accessible genomic regions between LD-and HD-Tfh (Fig 5B). In addition, we found 7,946 differentially accessible regions 252 between the two groups, in which the majority of these regions (i.e., 5,537) were observed in HD-Tfh cells, while the remaining 2,409 regions were more accessible in LD-Tfh cells (Fig 5C). However, these differential accessible regions did not include Tfh-associated locus such as Bcl6, Il6st and Il21r (Figs 5C and S4A). Further, the GSEA analysis showed that LD-and HD-Tfh Given the earlier transcriptomic evidence that suggests Th1-or Th2-like Tfh cells, we next focused on genes related to Th1 and Th2 cells. Despite the higher numbers of accessible regions 278 in HD-Tfh cells (Fig 5B to 5C), we found that the chromatin accessibility in Th1-associated genes such as Tbx21, Ifng and Cxcr3 locus was reduced in HD-Tfh cells compared to Tfh cells 280 from LD-infected mice (Fig 5C to 5F). In contrast, the accessibility at Il4 promoter and enhancer (Intron 2) was increased in HD-Tfh cells (Fig 5C to 5F), which is consistent with lower levels of 282 IL-4 expression compared to HD-Tfh cells at d21 pi (Fig 3B to 3C). Using TF binding motif HOMER de novo analysis [42], we identified top 10 motifs for LD and HD-Tfh cells that include 284 Th1 and Th2 master TF such as T-bet ( Fig 5G) and Gata3 (Fig 5H), respectively. This TF binding prediction analysis suggests the presence of active T-bet binding activity in LD-Tfh cells 286 that likely binds to accessible Ifng locus, leading to IFN-γ expression [43]. In addition, we also found a TF motif for BATF enrichment in LD-Tfh cells (Fig 5G). Although BATF is critical for the analysis also showed the enrichment of TFs such as SpiB, Pou2f3 and PU.1 (Fig 5H), with the latter previously implicated in Th2 cell heterogeneity [47] and Tfh cell development [48]. 298 Interestingly, it has been previously shown that Pou2f3 inhibits Gata3 binding activity in Th2 cells [49], and thus pointing to the potential regulation by Pouf3-PU.1 in Tfh cells to regulate 300 Gata3-mediated IL-4 expression. Collectively, these results support the idea that the epigenetic landscape of Th1-and Th2-like Tfh cells are influenced by Th1-and Th2-associated TF binding   302 activity, respectively.

To better understand whether LD
Th2 cytokines maintain the stability of ex vivo generated Th2-like Tfh cells 304 As transcriptomic and chromatin landscape analysis suggests that HD-Tfh cells share certain attributes of Th2 cells, this prompted us to assess the stability of HD-induced IL-21 + Tfh 306 cells under in vitro Th1 and Th2 polarizing conditions. HD-induced IL-21 + Tfh cells were sorted at d21 pi, and cultured ex vivo for 3 days in either Th1 or Th2 polarizing conditions. We found a 308 higher proportion of cultured cells maintaining the expression of PD-1 and CXCR5 under Th2 than Th1 conditions (Fig 6A to 6C (Fig 6A, 6B and 6D), and most of these IL-4 + cells co-expressed IL-21 (Fig 6A,   314 6B and 6E). Further, single IL-21 + cells were mainly found as CXCR5 + PD-1 + cells under both Th1 and Th2 conditions, which suggests that IL-21 expression by Tfh cells is not extrinsically 316 influenced by Th1 or Th2 polarising cytokines (Fig 6A, 6B and 6F). Unexpectedly, the expanded CXCR5 -PD-1non-Tfh cell population also expressed comparable levels of IL-21-318 GFP under both Th1 and Th2 conditions (Fig 6A, 6B and 6F), suggesting that non-Tfh cells upon Tfh cell development in response to Th1-biased LD T. muris infection. Indeed, Tbx21ΔT 342 mice were resistant to LD infection (Fig 7D). Importantly, we observed that the loss of T-bet rescued the lack of Tfh cells (Fig 7E and 7F) and GC B cell expansion (Fig 7G and 7H), which is associated with increased Il13 (Fig 7I) but a concomitant decrease in a non-protective Th1 cytokine Ifng (Fig 7J) in the proximal colon tissue. As expected, LD-infected Tbx21ΔT mice had 346 increased parasite-specific IgG1 but reduced levels IgG2c (Fig 7K and 7L), which was also consistent with the prior observation in intact HD-infected C57BL/6J mice (Fig 2E and 2F). 348 These results demonstrate that T-bet not only inhibits Th2 cell development [52,53], but also the Tfh cell lineage pathway during type 1-induced chronic helminth infection. Th2-like Tfh-GC B cell interaction is required for type 2 protective response. We used anti-CD40L (aCD40L) Ab to disrupt the interaction between Tfh and GC B cells [55] (Fig 8A). 360 Worm burden analysis revealed that aCD40L Ab-treated mice cleared most of the parasites; however, there remained low levels of worm burden that had not been fully resolved by d21 pi 362 (Fig 8B). We further checked worm burden at d35 pi, a time point by which parasites developmentally moult into an adult stage, and thus is typically used to evaluate complete worm 364 clearance. Similar to LD-infected mice, the parasites remained present in the caecum of aCD40L Ab-treated but not control mice (Fig 8C), suggesting that aCD40L Ab treatment compromises 366 protection to the parasite. Immune response analysis in the mLNs showed that the disruption of Tfh-GC responses via aCD40L Ab was successful, as evidenced by impaired Tfh cell 368 development (Fig 8D-8E). In addition, we showed that blockade of Tfh-GC interactions resulted in fewer Gata3 expressing Tfh cells (Fig 8F-8G), suggesting that cognate B cell-mediated CD40-370 CD40L signals are indispensable for Gata3 expression in Th2-like Tfh cells. Consistent with reduced Tfh cell numbers, activated B cell (Fig 8H to 8I), and GC B cell development (Fig 8J- induced disrupted Tfh-GC responses (Fig 8L), the numbers of IgG1-switched GC B cells were reduced (Fig 8M) due to the decreased numbers of GC B cells (Fig 8K). Further, we showed that 382 levels of parasite-specific IgG1 (Fig 8N), and surprisingly, IgG2c were reduced upon aCD40L treatment (Fig 8O), despite no difference in plasma cell numbers (S5G and S5H Fig). In support 384 of the idea that Tfh-GC responses regulate the type 2 protective response, we found that defective GCs also led to decreased Il13 (Fig 8P) but increased Ifng expression (Fig 8Q) in the 386 proximal colon tissue, implying that Tfh-GC responses in mLNs indirectly regulate the quality of protective responses at the effector site. Thus, these results demonstrate that Tfh-GC responses 388 are a necessary component of the type 2 protective-dependent immunity to helminth infection.

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[26] with 4C13R mice [24]. Tbx21ΔT mice [13] were bred and maintained with Cd4-Cre + mice. Leica ImageScope processing software was used to add 100 μM scale bars. 538 Immunofluorescence mLNs were extracted and were fixed in 4% paraformaldehyde for 6 h and were subsequently 540 immersed in 30% sucrose overnight prior to being embedded in OCT compound. 12-20 μm sections were stained with CD4, GL7 and IgD antibodies as described previously [68]. Images