Abstract
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract T follicular helper cells (Tfh) are crucial for the initiation and maintenance of germinal center (GC) reactions and high affinity, isotype-switched antibody responses. In this study, we demonstrate that direct TGF-β signaling to CD4 T cells is important for the formation of influenza-specific Tfh cells, GC reactions, and development of isotype-switched, flu-specific antibody responses. Early during infection, TGF-β signaling suppressed the expression of the high affinity IL-2 receptor α chain (CD25) on virus-specific CD4 T cells, which tempered IL-2 signaling and STAT5 and mammalian target of rapamycin (mTOR) activation in Tfh precursor CD4 T cells. Inhibition of mTOR allowed for the differentiation of Tfh cells in the absence of TGF-βR signaling, suggesting that TGF-β insulates Tfh progenitor cells from IL-2-delivered mTOR signals, thereby promoting Tfh differentiation during acute viral infection. These findings identify a new pathway critical for the generation of Tfh cells and humoral responses during respiratory viral infections. https://doi.org/10.7554/eLife.04851.001 eLife digest The influenza virus is thought to cause illness in up to 10% of adults and 30% of children each year worldwide. Most of these cases resolve on their own and don’t require treatment, but three to five million people are hospitalized and up to half a million people die each year. Unfortunately, the vaccines currently available to protect against influenza only target particular varieties or “strains” of the virus. The strains that circulate vary from year-to-year so it is necessary to develop new influenza vaccines every year. However, it is difficult to correctly predict which strains will circulate, so a more effective solution would be to develop a new vaccine that can help the body defend itself against many, or ideally any influenza strain. During a viral infection, a type of immune cell in the host can specialize into two different types of cells to help fight the virus: T helper 1 cells and CD4 T follicular helper cells. T helper 1 cells help to kill host cells that have become infected. CD4 T follicular helper cells promote the production of proteins called antibodies, which identify and neutralize the virus. Here, Marshall et al. studied how T helper 1 cells and CD4 T follicular helper cells form in mice suffering from a lung infection similar to influenza. It was already known that a protein called transforming growth factor beta (TGF-β) helps the immune response to mount an effective defense against an infection without causing too much harm to the host. Marshall et al. show that TGF-β increases the number of CD4 T follicular helper cells in the mice by suppressing the production of another protein—called IL-2—on the surface of CD4 T cells. Treating mice lacking the ability to detect TGF-β with a drug that blocks a protein controlled by IL-2 also allows more CD4 T follicular helper cells to be produced. Marshall et al.’s findings reveal that TGF-β is involved in controlling the balance of T helper 1 cells and CD4 T follicular helper cells produced during viral infections of the respiratory tract. Since TGF-β also has other roles in immune responses against viruses, it is now an attractive target for the development of a vaccine that may protect us against all strains of the influenza virus. https://doi.org/10.7554/eLife.04851.002 Introduction During acute viral infections, CD4 T cells differentiate into primarily T helper 1 (Th1) and T follicular helper (Tfh) effector cells (Marshall et al., 2011; Johnston et al., 2012; Hale et al., 2013). Similar to CD8 T cells, Th1 cells express the transcription factors (TF) T-bet and Blimp1, the effector molecules IFN-γ, TNFα, (and in many cases granzyme B [GrzB] and perforin) and migrate to sites of viral replication to eliminate infected cells. In contrast, Tfh cells primarily remain in secondary lymphoid tissues where they communicate with B cells in germinal centers (GC) to facilitate antibody affinity maturation and isotype switching. Tfh cells express substantially lower levels of T-bet, and instead of Blimp1 they express the TF Bcl6. Some pro-inflammatory cytokines induced during infection, such as IL-12, IFN-γ, IFN-αβ, and IL-2, promote Th1 differentiation; however, the signals required for Tfh differentiation during viral infection have not been as well characterized. Tfh cells must first encounter their cognate peptide-MHC with proper costimulation from professional antigen presenting cells such as dendritic cells. Following activation, Tfh precursor cells start to express the TF Bcl6 and the chemokine receptor CXCR5, as they downregulate CCR7 and P-selectin glycoprotein ligand 1 (PSGL1) (Johnston et al., 2009; Poholek et al., 2010; Choi et al., 2011; Pepper et al., 2011). These events allow for the migration of activated Tfh precursor cells toward the interfollicular zone and T-B border where they again meet peptide-MHC as well as other costimulatory ligands such as Inducible T cell Costimulator ligand (ICOSL) from B cells (Breitfeld et al., 2000; Schaerli et al., 2000; Hardtke et al., 2005; Kerfoot et al., 2011). These interactions result in the further upregulation of Bcl6, migration into the GC, and ability to assist B cells in affinity maturation and proper isotype switching (Poholek et al., 2010; Baumjohann et al., 2011; Choi et al., 2011). In addition to these cell surface ligand-receptor pairings, cytokines play critical roles in the full differentiation of effector Tfh cells during infection. Cytokines utilizing STAT3 signaling pathways including IL-6, IL-21, and IL-27 have been implicated in driving Tfh differentiation, but may have overlapping or compensatory effects depending on the immunizing agent and inflammatory environment (Ma et al., 2012; Ray et al., 2014). For example, IL-6 appears to act on early anti-viral Tfh precursors (Choi et al., 2013a), and while it is not absolutely required for fully differentiated Tfh effector cells during acute lymphocytic choriomeningitis (LCMV) infection (Poholek et al., 2010; Eto et al., 2011), it does promote the sustained activation of Tfh cells during chronic LCMV infection (Harker et al., 2011). Further, IL-27 is required for Tfh differentiation during protein immunization (Batten et al., 2010), while IL-21 is sometimes also involved (Nurieva et al., 2008; Vogelzang et al., 2008; Eto et al., 2011; Karnowski et al., 2012). In addition to STAT3, STAT4 signaling via IL-12 may also promote early Tfh progenitor cells during infection (Nakayamada et al., 2011) and appears to be critical for the differentiation of human Tfh cells (Schmitt et al., 2009, 2013). However, STAT4 signals are absolutely required for the differentiation of Th1 cells, suggesting that additional signals are needed to repress the expression of the Th1 TFs T-bet and Blimp1 in Tfh progenitor cells. Th1 and Tfh identities can be discerned within the first few days of viral infection indicating that early cytokine signals are involved in the initial stages of the Tfh/Th1 cell fate decision. Increased expression of the high affinity IL-2Rα chain CD25 on early effector CD4 T cells correlates with enhanced expression of the Th1 TFs T-bet and Blimp1 and lower levels of the Tfh TF Bcl6 and this is driven largely by IL-2-STAT5 signaling (Choi et al., 2011; Pepper et al., 2011; Choi et al., 2013b). In contrast, CD25lo early effectors have greater potential to generate Tfh cells (Ballesteros-Tato et al., 2012; Johnston et al., 2012; Nurieva et al., 2012; Choi et al., 2013b). Intriguingly, IL-2 signals are also important for the homeostasis of regulatory T cells (Treg). Therefore, understanding how effector and regulatory CD4 T cells listen to IL-2 will unveil pathways and targets to modulate CD4 T cell responses during infection, autoimmunity, and cancer. Another important signal at the interface of balancing effector and regulatory CD4 T cells is the cytokine TGF-β. As an immune-suppressive factor, TGF-β promotes the differentiation of peripherally derived regulatory T cells (pTreg) and inhibits the development of autoreactive T cell responses. In contrast, TGF-β can also serve a pro-inflammatory role by inducing the differentiation of effector Th17 cells. T cell-specific ablation of TGF-β signaling, either via TGF-βRII deletion or expression of a dominant negative receptor, has demonstrated that direct TGF-β signals are important for both Treg homeostasis and suppression of effector T cell activation and proliferation (Li et al., 2006; Marie et al., 2006; Sanjabi et al., 2009). The aberrant activation of effector cells in the absence of TGF-β signals cannot be rescued by addition of Treg (Li et al., 2006), indicating that direct TGF-β signaling on effector CD4 T cells is required to maintain their homeostasis. Furthermore, TGF-β suppresses T-bet expression (Gorelik et al., 2002; Park et al., 2005) and the exuberant proliferation of T cells display Th1 attributes (Ishigame et al., 2013), demonstrating that TGF-β has the capacity to suppress Th1 differentiation. In this study, we have identified a new role for TGF-β in balancing the development of Th1 and Tfh cells during acute viral infection. Specifically, we found that CD4 T cell-directed TGF-β was a critical signal for anti-viral Tfh differentiation, GC B cell reactions, and isotype-switched antibody response during influenza infection. TGF-β suppressed the expression of the high affinity IL-2Rα chain CD25, which restricted IL-2 signaling via STAT5 and mTOR in Tfh progenitor cells early during infection in vivo. Finally, we show that blockade of the mTOR signaling pathway can rescue Tfh differentiation of anti-viral CD4 T cells generated in the absence of TGF-β. Thus, we have identified that T cell-directed TGF-β insulates Tfh precursor cells from IL-2 signals and plays a critical role in the generation of effector Tfh cells and high affinity, class-switched antibodies—an essential source of protective immunity to this global health burden. Results TGF-β-associated gene expression signature in Tfh cells To better understand the specification of diverse CD4 T cell subtypes during viral infection, we compared the gene expression profiles of Tfh and Th1 effector CD4 T cell subsets that formed during acute LCMV infection (Marshall et al., 2011). This analysis revealed a number of TGF-β-associated genes commonly found in Treg cells, including Nt5e (CD73), Folr4 (folate receptor 4), Foxp3, and Ikzf2 (Helios) (Hill et al., 2007), to be more highly expressed in PSLG1lo Ly6Clo T-betlo CXCR5hi Tfh cells relative to the PSGL1hi Ly6Chi T-bethi CXCR5lo Th1 cells (Marshall et al., 2011; Hale et al., 2013) (Figure 1A). We first sought to determine if these results indicated that T follicular regulatory (Tfr) cells, a recently described immune-suppressive Tfh population (Chung et al., 2011; Linterman et al., 2011; Wollenberg et al., 2011), formed during acute LCMV infection. To assess this, we infected B6 or TCR transgenic Smarta (Stg) chimeras with acute LCMV Armstrong and monitored Tfh and Treg properties in either GP66–77 tetramer+ or Stg CD4 T cells by flow cytometry. Although we detected enhanced FoxP3 mRNA in the Tfh cells from our microarray analysis, we did not identify any LCMV-specific CD4 T cells that expressed FoxP3 protein or other Treg-associated markers such as GITR, to the level of canonical CD25+ FoxP3+ Tregs (Figure 1B and Figure 1—figure supplement 1). This suggested that LCMV-specific CD4 T cells do not differentiate into Tfr cells (Marshall et al., 2011; Srivastava et al., 2014). However, in agreement with the differential mRNA expression, we did find enhanced expression of several of the TGF-β- or Treg-associated proteins including CD73, folate receptor 4, and Helios on Tfh cells relative to the Th1 cells (Figure 1C) (Hill et al., 2007; Iyer et al., 2013). These observations suggested that conventional Tfh cells bear some similarities in their gene expression profiles with Treg cells, despite having little-to-no FoxP3 expression. Figure 1 with 1 supplement see all Download asset Open asset TGF-β-associated gene expression signature in Tfh cells. (A) Bar graph shows a selected set of genes upregulated in d8 LCMV-specific Stg PSGL1lo Ly6Clo Tfh cells relative to PSGL1hi Ly6Chi Th1 cells isolated and sorted from the spleen as measured using Illumina DNA microarrays (Marshall et al., 2011) that have been described to be induced by TGF-β or associated with Treg cells (Hill et al., 2007). (B) Representative histogram plot (top) shows amount of intracellular FoxP3 in total host splenic CD4 T cells (shaded gray) and LCMV-specific Th1 (hatched line) and Tfh (black line) Stg CD4 T cells from the spleen at day 8 p.i. Region gated identifies FoxP3+ nTregs. Bar graphs (bottom) depict the cumulative frequency (left) of FoxP3+ CD4 T cells or gMFI averages (right) of the indicated CD4 T cell populations. (C) Expression of the indicated Treg-associated proteins in (A) was compared between LCMV-specific Th1 (hatched line) and Tfh cells (black line), and FoxP3+ Treg cells gated on total host CD4 T cells (shaded gray) from the spleen at day 8 p.i. Histogram plots (top) are representative examples of individual mice and bar graphs (bottom) depict the gMFI averages of each protein in the indicated CD4 T cell populations. Graphs in B and C are representative of one of five independent experiments (n = 4–5 mice/group/experiment). *p < 0.05, ***p < 0.0005. https://doi.org/10.7554/eLife.04851.003 Direct TGF-β is a critical signal for Tfh differentiation during acute influenza virus infection We hypothesized that the expression of these Treg-associated gene products may be an indication of TGF-β signaling in the virus-specific Tfh cells. In order to assess the contribution of direct TGF-β signals on the formation of anti-viral CD4 T cell subsets, we crossed TGF-βRIIf/f CD4-cre mice to the Stg TCR transgenic mice. Fixing the TCR delays the onset of autoimmunity in the TGF-βRIIf/f CD4-cre mice (Sanjabi and Flavell, 2010); however, activated CD44hi CD4 T cells do emerge over time (data not shown). Therefore, when making chimeras, we adoptively transferred naïve CD44lo TGF-βRII+/+ CD4-cre+ Stg cells (herein referred to as WT) or naïve CD44lo TGF-βRIIf/f CD4-cre+ Stg cells (KO) into congenic C57BL/6 recipients and 1 day later infected the mice with the acute Armstrong strain of LCMV. Intriguingly, we found that direct TGF-β promoted the differentiation of Tfh precursor cells at day 3 post infection (p.i.), such that there were about 1/3 fewer CD25lo CXCR5+ Tfh precursor cells in the absence of direct TGF-β signals (WT = 60.25% ± 4, KO = 42% ± 3.3) (Figure 2—figure supplement 1A). Additionally, the TGF-βRII KO early effector CD4 T cells expressed more Th1 proteins Ly6C and T-bet and slightly lower Tfh TF Bcl6 (Figure 2—figure supplement 1B). However, by day 8 there was no phenotypic difference between TGF-βRII WT and KO CD4 T cells in the spleen (Figure 2—figure supplement 1C–D). These data indicated that TGF-β played a role in the early specification of splenic Tfh progenitor cells, but that other signals compensated for TGF-β signaling over the course of a systemic LCMV infection. Because TGF-β is a dominant regulator of T cells in mucosal tissues, we speculated that it may play a larger role in controlling anti-viral effector T cell responses during infection at mucosal sites, such as the lung. Moreover, respiratory influenza infection induces transcription of TGF-β and the influenza neuraminidase enzyme promotes the cleavage of latent TGF-β complex into its bioactive form in the lung mucosa (Schultz-Cherry and Hinshaw, 1996; Carlson et al., 2010; Roberson et al., 2012). To assess the contribution of TGF-β on the anti-viral CD4 T cell response during a respiratory infection, we infected TGF-βRII WT and KO Stg chimeras i.n. with a recombinant influenza virus expressing the LCMV GP66–77 epitope recognized by the Stg TCR (WSN-GP33/66) (Marsolais et al., 2008). First, we confirmed that the phenotypic properties of influenza-specific CD4 T cells closely mirrored that of LCMV-specific CD4 T cell populations, and importantly, verified that the influenza-specific Stg cells were neither FoxP3+ Treg nor Tfr cells (Figure 2—figure supplement 2). Moreover, we found that the proportion and total number of PSGL1lo Ly6Clo and PD-1hi CXCR5hi Tfh cells in the lung-draining mediastinal lymph node (MLN) were markedly reduced in the absence of direct TGF-β signals (Figure 2A). Furthermore, there was an increase in number of PSGL1hi Ly6Chi Th1 cells in all tissues examined (Figure 2A–C). Concomitant with the cell surface phenotypes, we found increased expression of the Th1 TF T-bet and reduced expression of the Tfh TF Bcl6 as well as increased production of IFN-γ and IL-2 in the TGF-βRII KO influenza-specific CD4 T cells compared to their WT counterparts (Figure 2D). Finally, and potentially most importantly given their function to help B cells in the GC, we also detected fewer TGF-βRII KO Stg cells localized in PNA+ GC in the MLN (Figure 2E), suggesting that TGF-β is important for optimal trafficking of Tfh cells to GCs. Together, these data suggest that direct TGF-β signaling was important for the generation of Tfh cells, while it suppressed Th1 differentiation during respiratory influenza virus infection. Figure 2 with 2 supplements see all Download asset Open asset Direct TGF-β is required for influenza-specific Tfh differentiation. 2 × 105 CD44lo TGF-βRII+/+ CD4-cre+ (WT) or TGF-βRIIf/f CD4-cre+ (KO) Stg cells were adoptively transferred into C57BL/6 congenic recipients infected with WSN-GP33/66 the following day. (A–D) On day 8 p.i, Stg cells in the MLN, spleen, and lung were stained with antibodies against the indicated proteins to distinguish Th1 and Tfh cells. Cells were also stimulated with GP66-peptide for 6 hr to assess IFN-γ and IL-2 production by intracellular cytokine staining and flow cytometry. (E) Stg cells (blue, highlighted by white arrows) located within MLN PNA+ GCs (green) were assessed using immunofluorescent microscopy and their numbers were enumerated using Imaris software. Graphs in A–D are representative of one of five independent experiments (n = 4–5 mice/group/experiment). Panel E shows representative microscopy images and the cumulative data from two independent experiments with 8 total mice/group were graphed. *p < 0.05, **p < 0.005, and colored asterisks correspond to the color in the stacked graphs. https://doi.org/10.7554/eLife.04851.005 T cell-directed TGF-β signals are required for GC B cell and switched antibody responses Due to the reduced Tfh differentiation in the absence of direct TGF-β signals, we sought to investigate this to in B cell help and formation of Stg CD4 T cell as described was not to GC B cell and antibody responses the CD4 T cells B cell help in these mice. Therefore, we set up a cell using TCR transgenic mice as recipients they have a CD4 T cell that cannot help to GC B cells during the infection. We to adoptively CD4 T cells in this to a of influenza-specific naïve precursors to B cell It also be that we switched to the deletion which is to the CD4-cre strain of a number of that differentiate into during development and autoimmunity in these mice and 2012). we set up the experiments to described by adoptively naïve CD44lo TGF-βRIIf/f (WT) or TGF-βRIIf/f (KO) CD4 T cells into congenic mice and 1 day later infected mice with influenza First, we confirmed that the CD4 T cells naïve the influenza infection (Figure In with our findings for the TGF-βRII KO Stg cells, the activated CD4 T cells lacking TGF-βRII also Tfh cell Specifically, in the MLN and spleen, there was a in Ly6Clo CXCR5+ cells and a in PSGL1lo Ly6Clo Tfh cells in the TGF-βRII KO cells compared to the WT (Figure there was a increase in Ly6Chi Th1 cells that TGF-βRII relative to the WT Figure 3 Download asset Open asset Direct TGF-β signaling is required for influenza-specific Tfh differentiation. × CD44lo CD4 T cells from TGF-βRIIf/f (WT) or TGF-βRIIf/f (KO) mice were adoptively transferred into congenic TCR transgenic mice and infected with WSN-GP33/66 the following day. days host and CD4 T cells in the MLN (A) and spleen (B) were assessed for expression of distinguish activated T cells, see histogram plots and CXCR5, and Ly6C to distinguish Tfh and Th1 plots are from representative mice and bar graphs are representative of one of independent experiments (n = 4–5 *p < and colored asterisks correspond to the color in the stacked graphs. we found that the of WT CD4 T cells largely B cell help in the mice such that there was an enhanced number GC B cells and B cells in the MLN days p.i. However, the TGF-βRII KO CD4 T cells were to rescue the formation of GC B cells in these mice (Figure Further, WT CD4 T cells rescued GC B cell switching to the while the TGF-βRII KO CD4 T cells did so in the spleen (Figure In addition to the of GC B cells by flow we also fewer and GCs from the of mice TGF-βRII KO CD4 T cells by microscopy (Figure Finally, T cell-directed TGF-β was also important for influenza-specific and in the of infected mice (Figure these data demonstrate a of TGF-β signaling in T cells for Tfh cell function as B cell and GC reactions and isotype-switched antibody responses during respiratory influenza virus infection. Figure Download asset Open asset T cell-directed TGF-β is required for GC B cell and isotype-switched antibody responses during influenza infection. × CD44lo CD4 T cells from TGF-βRIIf/f (WT) or TGF-βRIIf/f (KO) mice were adoptively transferred into congenic TCR transgenic mice and infected with WSN-GP33/66 the following day. days GC B cells (A) and GC B cells (B) in the MLN and spleen were assessed by flow and enumerated in bar graphs (C) PNA+ GCs highlighted by were assessed by immunofluorescent microscopy of and the numbers of was using Imaris software. and were measured from by at day p.i. or at the indicated time in are representative of independent experiments (n = mice/group/experiment). The bar graphs in show cumulative data from two independent experiments (n = the images in C are from representative mice of these *p < 0.05, **p < 0.005, ***p < 0.0005. Direct TGF-β suppresses the formation of Th1 precursors within days of viral infection Because CD4 T helper subsets to within the first few days of viral infection (Choi et al., and we found fewer Tfh and more Th1 precursor cells in the absence of TGF-β signals during LCMV infection (Figure 2—figure supplement we when TGF-β was required for Tfh differentiation during influenza virus infection. In order to assess this, we generated chimeras with 2 × CD44lo TGF-βRII WT or KO Stg CD4 T cells, infected the mice 1 day later with WSN-GP33/66 and assessed the of the early effector CD4 T cells in the lung-draining MLN at days 4–5 p.i. Although a difference in Bcl6 expression in the TGF-βRII KO CD4 T cells was not at this time the TGF-βRII KO CD4 T cells enhanced Th1 attributes including enhanced expression of CD25, T-bet, IFN-γ, and IL-2 (Figure These findings suggested that direct TGF-β suppressed Th1 precursor formation within the first few days of influenza virus infection. Figure Download asset Open asset Direct TGF-β suppresses early influenza-specific Th1 precursor formation in the lung-draining 2 × TGF-βRII+/+ CD4-cre+ (WT) or TGF-βRIIf/f CD4-cre+ (KO) Stg cells were adoptively transferred into C57BL/6 congenic recipients that were infected with WSN-GP33/66 the following day. On day 4–5 CD44hi Stg cells in the MLN were assessed for the indicated proteins T-bet, IFN-γ, IL-2, by flow cytometry. Cells were stimulated with and for hr to assess IFN-γ and Bar graphs are representative of three independent experiments (n = mice/group/experiment). *p < 0.05, **p < 0.005, ***p < 0.0005. Direct TGF-β IL-2 and insulates early Tfh progenitor cells from mTOR signaling Due to the enhanced expression of CD25 in the absence of TGF-βRII signaling, we TGF-β may modulate the IL-2 of early effector CD4 T cells. of recombinant TGF-β to Stg CD4 T cell stimulated with in did not T cell activation the upregulation of CD25 and the proliferation of the CD4 T cells were between or lacking TGF-β (Figure However, TGF-β the ability of the activated CD4 T cells to CD25 expression and IL-2 at day 2 post activation, the amount of surface CD25 and intracellular IL-2 was the or not TGF-β was but 1 day the T cells to TGF-β CD25 and lower IL-2 compared with that were not (Figure we assessed TGF-β CD25 expression and IL-2 signaling in virus-specific CD4 T cells in by CD25, and levels in TGF-βRII WT and KO Stg CD4 T cells isolated from day 3 post LCMV infection, a in which Th1 progenitor cells are more to facilitate analysis (Figure 2—figure supplement 1A). We a enhanced CD25+ population from TGF-βRII KO early effector cells relative to the WT cells (Figure of IL-2 signaling in the TGF-βRII KO CD4 T cells in during infection. Further, we also found enhanced CD25+ early effector cells (Figure indicating that both STAT5 and signaling are in the absence of TGF-β Together, these data demonstrate that TGF-β suppresses the expression of CD25 and of STAT5 and in early effector CD4 T cells in and promotes Tfh cell differentiation by IL-2 signaling in Tfh precursor cells. Figure 6 Download asset Open asset TGF-β IL-2 and insulates early Tfh progenitor cells from mTOR (A) Stg CD4 T cells were with and in with ± TGF-β and stained for surface expression of CD25 (top) or with IL-2 to assess are representative of independent (B) Stg mice × CD44lo TGF-βRII+/+ CD4-cre+ (WT) or TGF-βRIIf/f CD4-cre+ were infected with LCMV Armstrong and 3 days were in and stained with antibodies to surface expression of CD25, and intracellular of STAT5 and vivo. are representative of three independent experiments including total 2 × 105 CD44lo TGF-βRII+/+ CD4-cre+ (WT) or TGF-βRIIf/f CD4-cre+ (KO) Stg cells were adoptively transferred into congenic C57BL/6 recipients infected with WSN-GP33/66 the following day. were with or rapamycin On day 8 p.i, Stg cells in the MLN were assessed for expression of and Ly6C (C) or T-bet or are representative of three independent experiments a total of *p < 0.05, **p < and colored asterisks correspond to the color in the stacked graphs. Since we found enhanced we we rescue Tfh differentiation in the absence of TGF-βRII signaling by mTOR To do this, we WT and TGF-βRII KO Stg mice with the