Dynamic regulation of T Follicular Regulatory cell responses by interleukin 2 during influenza infection

SUMMARY Interleukin 2 (IL-2) promotes Foxp3+-regulatory T (Treg) cell responses, but inhibits T follicular helper (TFH) cell development. However, it is not clear how IL-2 affects T follicular regulatory (TFR) cells, a cell type with properties of both Treg and TFH cells. Using an influenza infection model, we demonstrated that high IL-2 concentrations at the peak of the infection prevented TFR cell development by a Blimp-1–dependent mechanism. However, once the immune response resolved, some Treg cells down-regulated CD25, up-regulated Bcl-6 and differentiated into TFR cells, which then migrated into the B cell follicles to prevent the expansion of self-reactive B cell clones. Thus, unlike its effects on conventional Treg cells, IL-2 inhibits TFR cell responses.


INTRODUCTION
Interleukin-2 (IL-2) is essential for the development and maintenance of Foxp3 + CD4 + T regulatory (T reg ) cells, which prevent autoimmune disease development 1 . The principal mechanism by which IL-2 promotes T reg cell development is by triggering STAT5 activation, which binds to the Foxp3 locus and promotes Foxp3 expression [2][3][4] . IL-2 signaling is also required to maintain the competitive fitness of T reg cells in secondary lymphoid organs 5,6 and for reinforcing their suppressive activity 7,8 . Hence, mice lacking IL-2 or IL-2Rα (CD25) fail to maintain peripheral tolerance and develop autoimmune disease 9 .
T reg cells express high amounts of CD25, the α chain of the high-affinity IL-2 receptor, allowing them to effectively compete with other cells for available IL-2 [10][11][12] . Indeed, IL-2consumption by T reg cells is one of the main mechanisms by which they prevent effector-T cell (T eff ) responses 13 . Conversely, IL-2 consumption by T reg cells facilitates CD4 + T follicular helper (T FH ) cell development 10 , since IL-2 signaling inhibits T FH cell differentiation [14][15][16] . Interestingly, some activated T reg cells down-regulate CD25, and do not require IL-2 for their homeostatic maintenance 17 . Instead, their survival is dependent on ICOS-ICOS-L interactions 17 . Similarly, antigen-experienced T reg cells in the skin 18 and in aged mice 19 express less CD25, and depend on IL-7 and IL-15 rather than IL-2 for their maintenance, thus suggesting that IL-2 might be dispensable for the homeostasis of some T reg cell subsets.
In this study, we investigated the role of IL-2 in T FR cell responses to influenza. We demonstrated that high concentrations of IL-2 at the peak of the infection promoted the expression of Blimp-1 in T reg cells, which suppressed Bcl-6 expression and thereby precluded T FR cell development. As a consequence, T FR cells failed to accumulate at the peak of the influenza infection. However, once the virus was eliminated and the IL-2 concentrations declined, some CD25 hi T reg cells down-regulated CD25, up-regulated Bcl-6 and differentiated into T FR cells, which migrated into the B cell follicles to prevent the accumulation of self-reactive B cell clones. Collectively, our data demonstrate that IL-2 signaling differentially controls conventional T reg and T FR cell responses to influenza virus, and reveal an important role for T FR cells in maintaining B-cell tolerance after influenza infection.
infection, we determined the placement of Foxp3 + cells relative to B cell follicles and GCs in sections of mLNs obtained from day 30 influenza-infected mice ( Supplementary Fig. 1a). As expected, we identified CD4 + Foxp3 + cells within the B cell follicles, the interfollicular area and inside the GCs ( Supplementary Fig. 1a,b). These results indicated that bona fide T FR cells did develop following influenza virus infection.
We next infected B6 mice with influenza and enumerated T FR cells and conventional T reg cells in the mLN at different times after infection (Fig. 1f-h). Bcl-6 hi CXCR5 hi T FR cells were barely detectable at the peak of the infection (day 7 to 15), but largely accumulated during the late phase of the primary response (day 30 to 60) (Fig. 1f,g). In contrast, conventional T reg cells rapidly expanded between days 3 and 7 (Fig. 1h). Importantly, the paucity of T FR cells at the peak of the infection was not due to a lack of GC B cells, since GC B cells were easily detected at day 10, continued to expand through day 15, and declined thereafter (Fig. 1i,j). We also observed that Bcl-6 hi CXCR5 hi T FH cells (Fig. 1k,l) peaked between days 7 and 15 after infection, and subsequently contracted between days 15 and 30. Thus, in contrast to GC cells, T FH cells and conventional T reg cells, T FR cells failed to accumulate at the peak of the infection.
To further confirm this conclusion, we evaluated the presence of Foxp3 + cells within the GCs at days 10 and 30 after infection by immunohistochemistry ( Supplementary Fig. 1c). Foxp3 + cells were easily detected in the B cell follicles, interfollicular area and GCs on day 30. By contrast, very few Foxp3 + cells were present within the GCs at day 10, despite being abundant in the T cell area. Collectively, these results indicated that the T FR cells failed to develop during the peak of the influenza infection, but accumulated in the mLN at later time points.
Previous studies have shown that T FR cells develop quickly (days 7-14) upon immunization with soluble antigens 20,21,23 . To test whether the delayed appearance of T FR cells was unique to influenza, we enumerated T FR cells in B6 mice that were either immunized with influenza hemagglutinin (HA) adsorbed to alum (Supplementary Fig.1 d,e) or infected with LCMV-Armstrong (Supplementary Fig. 1 f,g). In agreement with the published studies, we found that T FR cells were readily detected at day 9 after HA immunization and were maintained for at least 30 days (Supplementary Fig. 1 d,e). By contrast, T FR cells were virtually undetectable at day 9 following LCMV-Armstrong infection, but accumulated in large numbers at day 30 after infection (Supplementary Fig. 1 f,g). Collectively, these results suggested that, whereas T FR cell responses quickly develop following soluble protein immunization, T FR cells fail to differentiate at the peak of acute viral infections.

T FR cells exhibit low expression of CD25
T FR cells depend on Bcl-6 20-22 , whose expression is inhibited by IL-2 10,14,16 . To examine the relationship between T FR cells and IL-2 signaling, we divided the CD69 + Foxp3 + CD4 + T-cell population into CD25 hi and CD25 lo cells and analyzed the expression of T FR cell markers in these subpopulations (Fig. 2a,c). We found that CD25 lo Foxp3 + cells, but not CD25 hi Foxp3 + cells, up-regulated T FR markers following infection (Fig. 2a,c). Importantly, although CD25 lo Foxp3 + cells were present at the peak of the infection (d10), they expressed low amount of CXCR5, Bcl-6 and PD-1 relative to later time points, suggesting that lack of T FR cells at the peak of the infection was not due to a lack of CD25 lo Foxp3 + cells ( Supplementary Fig. 2a-c). Similar results were obtained in LCMV-infected mice or HA-Alum-immunized mice ( Supplementary Fig. 2d,e). Thus, unlike conventional T reg cells, which are CD25 hi , T FR cells are CD25 lo .
We next evaluated STAT5 phosphorylation in T FR and conventional T reg cells and found that pSTAT5 was reduced in T FR cells compared to conventional T reg cells (Fig. 2d), suggesting that T FR and conventional T reg cells responded differently to IL-2. To confirm this conclusion, we sorted conventional T reg cells (Foxp3 + CD69 hi PD-1 lo CXCR5 lo CD25 hi ) and T FR cells (Foxp3 + CD69 hi PD-1 hi CXCR5 hi CD25 lo ) from the mLN of day 30 infected B6.Foxp3-DTR-GFP mice and performed RNA-sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) to identify hallmark-signaling pathways differentially enriched in these populations (Fig. 2e,f). Approximately 2,000 genes were differentially expressed between conventional T reg and T FR cells (Supplementary Table 1). Importantly, the IL-2-STAT5 signaling pathway had the highest normalized enrichment score (NES=3.54; FDR < 0.001) and contained the largest number of genes up-regulated in T reg cells relative to T FR cells (Fig. 2e). Of the 200 genes in the hallmark IL-2-STAT5 signaling pathway, 72 were significantly downregulated in T FR vs T reg cells (Fig. 2f and  Supplementary Tables 2,3). Interestingly, despite their low expression of CD25 (Fig. 2g), T FR cells expressed high amounts of IL-2Rβ (CD122) (Fig. 2h), suggesting that T FR cells may have some remaining capacity to respond to IL-2, particularly when its physiological concentration is sufficiently high 27 . Altogether, our results indicated that the IL-2-STAT5 signaling pathway was downregulated in T FR cells compare to conventional T reg cells.

CD25 + T reg cells are the precursors of T FR cells
Previous studies showed that T FR cells differentiate from pre-existing Foxp3 + T reg precursors 20,21 . Given that T FR cells were CD25 lo , we used adoptive-transfer experiments to test whether T FR cells were derived from pre-existing CD25 hi Foxp3 + or CD25 lo Foxp3 + T cells. Therefore, we sorted CD25 hi Foxp3 + or CD25 lo Foxp3 + CD4 + T cells from the spleen of naïve B6.Foxp3-DTR-GFP (CD45.2 + ) mice, which express the diphtheria-toxin receptor and the eGFP genes under the control of the Foxp3 promoter, and adoptively transferred equivalent numbers of these cells into naïve Tcrb −/− Tcrd −/− mice, that lack T cells. We also transferred total CD8 + and CD4 + T cells from B6.CD45.1 mice to provide a competent Tcell environment. One day later, mice were infected with influenza and the donor-derived Foxp3 + cells were characterized on day 30. The frequencies and numbers of activated Foxp3 + cells derived from the CD45.2 + donors were increased in recipients of CD25 hi Foxp3 + cells compared to recipients of CD25 lo Foxp3 + cells (Fig. 3a,b). We also found that nearly 6% of the progeny derived from CD25 hi Foxp3 + cells up-regulated Bcl-6 and CXCR5, whereas few of the progeny derived from CD25 lo Foxp3 + cells up-regulated Bcl-6 and CXCR5 (Fig. 3c). As a result, the numbers of CD45.2 + T FR cells were significantly higher in recipients of CD25 hi Foxp3 + cells compared to recipients of CD25 lo Foxp3 + cells (Fig. 3d). Importantly, the number of CD45.1 + Foxp3 + cells, and the number of CD45.1 + T FR cells were similar in the two groups (Fig. 3e,f). These data indicated that pre-existing CD25 hi Foxp3 + cells, but not pre-existing CD25 lo Foxp3 + cells, differentiate into T FR cells following influenza infection.
Recent data suggest that T FR cells can also be derived from CD4 + Foxp3 − precursors 28 . Therefore, we performed adoptive-transfer experiments to compare the capacity of CD4 + Foxp3 − and CD25 hi Foxp3 + cells to differentiate into T FR cells following infection. Although some CD4 + Foxp3 − cells up-regulated Foxp3 after infection (data not shown), CD45.2 + T FR cells were only generated from CD25 hi Foxp3 + cell precursors (Fig. 3g,h). Collectively, these data indicated that CD25 hi Foxp3 + cells are the precursors of T FR cells after influenza infection.
We next tested whether the Foxp3 + cells derived from the CD25 hi Foxp3 + donors maintained CD25 expression (Fig. 3i). We found that, although the majority of Foxp3 + progeny derived from the CD25 hi Foxp3 + donors were CD25 hi , some cells lost expression of CD25 and only the CD25 lo cells up-regulated T FR markers (Fig. 3i). Collectively, these results demonstrated that a fraction of the activated CD25 + Foxp3 + CD4 + T cells down-regulate CD25 and upregulate Bcl-6, PD-1 and CXCR5 late after infection.

IL-2 signaling precludes T FR cell development
Given that IL-2 signaling inhibits Bcl-6 expression 10,14,16 , we next tested whether T FR cells could develop in a high-IL-2 environment. To do this, B6 mice were infected with influenza, treated them with either 30,000 units (U) of recombinant IL-2 (rIL-2) or PBS for nine consecutive days starting on day 20 and T FR cells were enumerated in the mLN on day 30. As a control, we analyzed day 10 infected mice. As expected, the frequencies and numbers of T FR cells were higher in day 30 infected mice relative to day 10 infected mice ( Fig. 4a,b). By contrast, T FR cells failed to accumulate in rIL-2-treated mice (Fig. 4a,b), suggesting that a high-IL-2 environment prevented the accumulation of T FR cells.
We next determined the kinetic of IL-2 production during influenza infection using Il21-mCherry-Il2-emGFP dual reporter transgenic mice 29 (Fig. 4c). We found that the frequency of IL-2-producing CD4 + T cells peaked at day 10 and declined thereafter (Fig. 4c). We also found that the frequency of IL-2-producing CD4 + T cells was significantly increased in day 10 influenza-infected mice relative to day 10 HA-immunized mice ( Supplementary Fig. 3). Given that IL-2 is highly produced at the peak of the infection, we hypothesized that strong IL-2 signals prevented the differentiation of T FR cells at this time. To address this possibility, we infected B6 mice with influenza, treated them daily from day 3 to day 9 with neutralizing IL-2 antibodies (JES6-1A12 + S4B6-1) or control antibody (2A3), and analyzed the Foxp3 compartment in the mLN on day 10 ( Fig. 4d,e). As expected, we detected very few T FR cells in control-treated mice. The frequency and number of T FR cells was, however, significantly increased in the anti-IL-2-treated mice (Fig. 4d,e). These results suggested that an elevated concentration of IL-2 at the peak of the infection prevented Foxp3-expressing cells from differentiating into T FR cells. We also found more T FH cells, but less conventional CD25 + T reg cells in the anti-IL-2-treated mice compared to control mice ( Fig. 4f-h), which is consistent with previous studies showing that IL-2 signaling is required for CD25 hi Foxp3 + T reg cell expansion 2,7,30-32 , but prevents T FH cell differentiation [14][15][16]26 .
To confirm that IL-2 signaling inhibited T FR cell differentiation, we sorted CD25 hi Foxp3 + cells from day 7 influenza-infected B6.Foxp3-DTR-GFP mice, activated them in vitro with anti-CD3 and anti-CD28 beads in the presence of high (200U/ml) or low (5U/ml) rIL-2 concentrations and assessed the phenotype of Foxp3 + cells three days later ( Fig. 4i-o). Foxp3 + cells activated in the presence of high-IL-2 concentrations expressed high levels of CD25, Blimp-1 and T-bet relative to Foxp3 + cells cultured in low-IL-2 conditions ( Fig. 4ik). In contrast, Bcl-6 and CXCR5 were up-regulated in Foxp3 + cells cultured in low-IL-2 concentrations (Fig. 4l,m). As a result, Bcl-6 hi CXCR5 hi cells differentiated in the low but not high-IL-2 cultures (Fig. 4n,o). Collectively, our data indicated that, while elevated IL-2 signaling at the peak of the infection promoted CD25 hi T reg cell responses, it simultaneously prevented Foxp3-expressing cells from up-regulating Bcl-6 and CXCR5 and differentiating into T FR cells.
To investigate the role of these transcription factors in T FR cell development, we made mixed-bone marrow (BM) chimeras in which irradiated Tcrb −/− Tcrd −/− recipient mice were reconstituted with a 50:50 mixture of BM obtained from CD45.1 + wild-type and CD45.2 + Tbx21 −/− (T-bet-deficient) B6 donor mice. Two months later, chimeras were infected with influenza and the frequency of T FR cells derived from each donor was determined on day 10. As expected, we failed to detect Bcl-6 hi CXCR5 hi cells within the CD25 hi Foxp3 + compartment of either donor (Fig. 5d). Although we did observe approximately 5% Bcl-6 hi CXCR5 hi cells in the CD25 lo Foxp3 + compartment, there was no difference between the wild-type and Tbx21 −/− donor cells (Fig. 5d). These results suggested that T-bet did not prevent T FR cell differentiation at the peak of the infection.
To address a potential role for Blimp-1 in preventing T FR differentiation, we generated mixed-BM chimeras using wild-type (CD45.1 + ) and Prdm1 fl/fl -Lck-cre/+ (CD45.2 + ) donors (WT-Prdm1 −/− chimeras). After reconstitution, chimeras were infected with PR8 and analyzed at day 10. As expected, very few wild-type CD25 lo Foxp3 + cells expressed T FR markers at day 10 post-infection. However, 20% of the Prdm1 −/− CD25 lo Foxp3 + cells were T FR cells (Fig. 5e). These results suggested that Blimp-1 prevented the development of T FR cells at the peak of the influenza infection.
Finally, we examined whether IL-2 prevents T FR cell differentiation by a Blimp-1-dependent mechanism. Thus, WT-Prdm1 −/− chimeras were infected with influenza, treated daily with rIL-2 or control PBS starting at day 20, and the T FR cell response was assessed within the wild-type and Prdm1 −/− compartments on day 30 after infection ( Fig. 5f,g). Similar to our prior experiment, we found more CD25 lo Foxp3 + cells with a T FR cell phenotype in the Prdm1 −/− compartment relative to the wild-type compartment in the PBS-treated mice (Fig.   5f). We also found diminished frequencies of T FR cells in the wild-type compartment of the rIL-2 treated mice relative to PBS-treated mice (Fig. 5f). However, we observed similar frequencies of Prdm1 −/− T FR cells in both PBS control and IL-2-treated mice (Fig. 5f). As a consequence, the ratio of Prdm1 −/− to wild-type T FR cells was increased in the rIL-2-treated mice compared to PBS control counterparts (Fig. 5g). These results indicated that high-IL-2 signaling directly prevented T FR cell differentiation by an intrinsic IL-2/Blimp-1-dependent mechanism.

T FR cells maintain B-cell tolerance after infection
We next used three independent approaches to test the effect of T FR cells on the B cell response to influenza. First, we crossed Bcl-6 fl/fl mice 36 to Foxp3 YFP/Cre mice 37 to generate Bcl-6 fl/fl Foxp3 YFP/Cre mice, in which the zinc finger-domains of Bcl-6 are conditionally deleted in Foxp3-expressing T cells 36 . Bcl-6 fl/fl Foxp3 YFP/Cre mice were infected with influenza and analyzed them on day 30. As expected, the number of T FR cells was reduced in Bcl-6 fl/fl Foxp3 YFP/Cre mice compared to control mice ( Fig. 6a,b). In contrast, T FH cells normally accumulated in Bcl-6 fl/fl Foxp3 YFP/Cre mice (Fig. 6c,d). Similar results were obtained when we used fluorochrome-labeled MHC Class II tetramers to identify influenza nucleoprotein (NP)-specific T FH cells ( Supplementary Fig. 4a,b). We also found GC B cells normally accumulated in control and Bcl-6 fl/fl Foxp3 YFP/Cre mice (Fig. 6e,f). Importantly, however, the frequency and number of CD138 + antibody-secreting cells (ASCs) were increased in Bcl-6 fl/fl Foxp3 YFP/Cre mice relative to control mice (Fig. 6g,h). These results suggested that lack of T FR cells did not change T FH cell or the GC B-cell responses, but promoted the accumulation of CD138 + ASCs.
To confirm this observation, we generated BM chimeras in which T FR cells were selectively depleted after diphtheria toxin (DT) administration ( Fig. 7a-f). Thus, we reconstituted irradiated Tcrb −/− Tcrd −/− recipient mice with a 50:50 mix of Cxcr5 −/− and Foxp3-DTR BM (Foxp3-Cxcr5 −/− chimeras), or wild-type BM and Foxp3-DTR BM (Foxp3-WT chimeras). Because T FR cells cannot be produced from Cxcr5 −/− precursors 20 , all the T FR cells in the influenza-infected Foxp3-Cxcr5 −/− chimeras developed from the Foxp3-DTR donors (Fig.  7a). In contrast, T FR cells in the Foxp3-WT chimeras developed equally from the WT and Foxp3-DTR donors (Fig. 7a). As a consequence, T FR cells were depleted following DT administration in the Foxp3-Cxcr5 −/− but not in the Foxp3-WT influenza-infected chimeras (Fig. 7b,c). The T reg cell response was, however, similar in the two groups (Fig. 7d). Therefore, we infected the chimeric mice with influenza, treated them with DT every four days starting on day 15 and enumerated T FH cells, GC B cells and CD138 + ASCs on day 50 in the mLN. We found similar frequencies and numbers of T FH cells and GC B cells ( Supplementary Fig. 5a-d) in the DT-treated Foxp3-Cxcr5 −/− and Foxp3-WT chimeras. However, CD138 + ASCs accumulated to a greater frequency in Foxp3-Cxcr5 −/− chimeras compared to Foxp3-WT controls (Fig. 7e,f). Importantly, no differences were detected between PBS-treated Foxp3-Cxcr5 −/− and Foxp3-WT chimeras ( Supplementary Fig. 5e). These findings indicated that lack of T FR cells promotes the expansion of CD138 + ASCs.
To confirm our observations, we treated influenza-infected B6 mice daily with 15,000U of rIL-2 starting on day 20 to deplete T FR cells, and evaluated the -cell response in the mLN on day 30. T FR cells were significantly depleted in rIL-2-treated mice (Supplementary Figure 5f). In contrast, rIL-2 treatment did not affect the accumulation of total or NPspecific T FH cells at this time and dosage ( Supplementary Fig. 5g-j). Similarly, the GC B cell response was similar in PBS and rIL-2 treated mice ( Supplementary Fig. 5k,l). In contrast, the frequencies and numbers of CD138 + ASCs were increased in rIL-2-treated mice (Fig. 7g,h). Collectively, our data indicated that absence of T FR cells late after infection promoted the expansion of CD138 + ASCs.
Lack of T FR cell promotes the outgrowth of self-reactive B cell clones following immunization with T-dependent antigens 21 . To characterize the role of T FR cells in controlling the influenza-specific B-cell response, we used fluorochrome-labeled recombinant NP-tetramers 10,16 to identify NP-specific B cells in IL-2-treated and Bcl-6 fl/fl Foxp3 YFP/Cre mice (Fig. 8a-h). The frequencies and numbers of NP-specific GC B cells were similar in control and rIL-2 treated mice (Fig. 8a,b). In contrast, while 30% of the CD138 + ASCs were NP-specific in control mice, only 8% of the CD138 + ASCs were NPspecific in the rIL-2-treated mice (Fig. 8c). As a consequence, the number of NP-specific CD138 + ASCs was similar in control and rIL-2 treated mice (Fig. 8d). Similar results were obtained when comparing Bcl-6 fl/fl Foxp3 YFP/Cre and control counterparts (Fig. 8e,f). Finally, the titers of influenza-specific serum IgG were similar in PBS and rIL-2-treated mice (Fig. 8g), or when we compared the serum from Bcl-6 fl/fl Foxp3 YFP/Cre and control mice (Fig. 8h). These results suggested that a lack of T FR cells did not significantly affect the influenza-specific B-cell response, but instead promoted the accumulation of noninfluenza-specific CD138 + ASCs.
We next hypothesized that these ASCs represented the differentiated daughter cells of selfreactive B cells that are known to be generated in the GCs during infections 38 . To test this possibility, we first enumerated histone-specific, IgG-secreting cells by ELISPOT in the mLNs of day 30-infected PBS and rIL-2-treated mice. As expected, we found very few histone-specific ASCc in the mLN of PBS-treated mice (Fig. 8i). In contrast, the frequency of anti-histone, IgG ASCs was increased in rIL-2-treated mice, suggesting that lack of T FR cells resulted in the development of anti-nuclear antibody (ANA) responses (Fig. 8i). Thus, we evaluated the serum samples for ANA reactivity. As expected, PBS-treated control mice lacked ANAs, whereas IL-2-treated mice were ANA positive (Fig. 8j). Finally, we evaluated the presence of ANAs in the serum of Bcl-6 fl/fl Foxp3 YFP/Cre mice. We found that, while the sera from B6 infected mice (Fig. 8k) or naïve Bcl-6 fl/fl Foxp3 YFP/Cre mice ( Supplementary  Fig. 6) were negative for ANA staining, the sera obtained from day 30-infected Bcl-6 fl/fl Foxp3 YFP/Cre mice were positive (Fig. 8k). Therefore, our data indicated that T FR cells prevented the expansion of self-reactive ASCs after influenza infection.

DISCUSSION
We show here that T FR cells are characterized by low expression of CD25 and that IL-2 signaling inhibits, rather than promotes, the development of T FR cells. Correspondently, T FR cells fail to accumulate at the peak of the influenza infection, a time during which IL-2 is highly produced. However, after IL-2 withdrawal, some Foxp3 + cells down-regulate CD25, up-regulate Bcl-6, express CXCR5 and differentiate into T FR cells, which migrate into the B-cell follicles to prevent self-reactive B-cell responses. Thus, unlike conventional T reg cells, high-IL-2 signaling precludes T FR cell development. Importantly, IL-2 consumption by CD25 + T reg cells is required for the initial development of virus-specific T FH cells 10 . Thus, IL-2-consumption acts as a rheostat that, while facilitating virus-specific T FH cell responses, it selectively prevents T FR cell development at the peak of the infection.
T FR cells prevented self-reactive ASCs, but had no significant effect on the influenzaspecific B-cell response, suggesting that T FR cells may be self-specific rather than influenzaspecific. This idea is consistent with data showing that the TCR repertoire of T FR cells is skewed towards self-antigens 39 , and so is the TCR repertoire of thymic-T reg cells 40,41 , which we show here are the likely precursors of T FR cells after influenza infection. However, T FR cells prevent antigen-specific B-cell responses following immunization with soluble antigens, suggesting that antigen-specific T FR cells can develop under some circumstances 20,21,23,28 . In this regard, a recent study suggests that a fraction of the T FR cells differentiate from naïve T-cells precursors following immunization and are specific for the immunizing Ag 28 . Thus, depending on the specific nature of the immune response, T FR cells can prevent foreign and self-reactive B-cell responses based on their origin and TCRspecificity. In any case, the capacity of T FR cells to prevent influenza-specific effector B responses is overcome in the context of influenza infection. Indeed, influenza-NP is normally complexed with viral-RNA 42 , which targets it to the intracellular TLR7 compartment following BCR stimulation 43

. Thus, it is possible that co-ligation of the BCR and pathogen-recognition-receptors in virus-specific B cells synergize to overcome T FRmediated suppression during influenza infection.
We found that Blimp-1, but not T-bet, suppresses T FR cell development. However, we failed to detect Bcl-6 up-regulation in CD25 hi T reg cells even in Blimp-1-deficient cells, suggesting that Bcl-6 expression in CD25 hi cells is prevented by additional Blimp-1-independent mechanisms. Given that STAT5 binds to the Bcl-6 promoter and directly represses Bcl-6 expression in response to high-IL-2 signaling 26,44-46 , it is likely that strong IL-2 signaling through CD25 prevents Bcl-6 expression by a direct STAT5-dependent mechanism.
Our data showing that IL-2 prevents T FR cell responses are in conflict with the notion that IL-2-STAT5 signaling is required for maintaining Foxp3 expression 1 . However, T FR cells express high amounts of CD122. Thus, although insufficient for inducing sustained Blimp-1 expression in low-IL-2 environments, basal IL-2-STAT5 signaling through the intermediateaffinity IL-2R may be sufficient to prevent Foxp3 down-regulation in T FR cells. Alternatively, T FR cell homeostasis may be partially independent of IL-2, but may require signals from other common-γ chain cytokines and costimulatory molecules, such as IL-7, IL-15 or ICOS which can contribute towards the maintenance of Foxp3-expressing cells in the absence of IL-2 18,19,47,48 . In any case, it is likely that conventional CD25 + T reg and T FR cells use different cellular and molecular pathways for their homeostatic maintenance.
In summary, our data demonstrate that IL-2 signaling temporarily inhibits T FR cell responses during influenza infection. However, once the immune response is resolved, T FR cells differentiate and home to B cell follicles, where are required for maintaining B cell tolerance after infection. Thus, the same mechanism that promotes conventional T reg cell responses, namely IL-2 signaling, also prevents T FR cell formation. Collectively, our data provide a new perspective into how IL-2 dynamically regulates T reg cell homeostasis and function along the course of a relevant pathogen infection. All mice were bred in the University of Alabama at Birmingham (UAB) animal facility. All experimental procedures involving animals were approved by the UAB Institutional Animal Care and Use Committee and were performed according to guidelines outlined by the National Research Council.

Infections, BM chimeras and in vivo treatments
Influenza virus infections were performed intranasally (i.n) with 6,500 VFU of A/PR8/34 (PR8) in 100 μl of PBS. Acute LCMV infections were established by intraperitoneal injection with 2 × 10 5 PFU LCMV-Armstrong. In some experiments mice were immunized with 50 μg of recombinant hemagglutinin (HA) adsorbed to Alum (Imject® Alum, Thermo Scientific™). BM chimeric mice were generated by irradiating the indicated recipient mice with 950 Rads from an X-ray source delivered in 2 equal doses administered 4-5 h apart. Following irradiation, mice were intravenously injected with 5 × 106 total BM cells and were allowed to reconstitute for 8-10 weeks before influenza infection. In indicated experiments, experimental animals received an intraperitoneal injection of 50 μg/kg of diphtheria toxin (DT -Sigma) at the indicated time points. In some experiments, mice were intraperitoneally administered recombinant IL-2 (National Cancer Institute) at the indicated time points. Control mice received injections of PBS. In some experiments mice were treated with 500 μg of a mix of anti-IL-2 neutralizing antibodies (JES6-1A12 and S4B6) or 500 μg of Isotype control (2A3), all obtained from BioXcell.

96-well plates (Corning™ Clear
Polystyrene 96-Well Microplates) were coated overnight with purified PR8 proteins 50 at 1 μg/ml in 0.05 M Na 2 CO 3 pH 9.6. Coated plates were then blocked for 1 h with 1% BSA in PBS. Serum from PR8-infected mice was collected and serially diluted (3-fold) in PBS with 10 mg/ml BSA and 0.1% Tween 20 before incubation on coated plates. After washing, bound antibody was detected with HRP-conjugated goat Anti-Mouse, IgG (g heavy chain specific) Ab (Southern Biotech) and quantified by spectrophotometry at 405 nm (OD).

ELISPOT
Multiscreen cellulose filter plates (Millipore) were coated overnight with 10 μg of histone from calf thymus (Sigma-Aldrich). Cells from the mLN were collected and plated starting at 3 × 10 6 /well and in 2-fold serial dilutions in complete RPMI 1640 containing 10% FBS. After 5 h, the wells were washed with PBS containing 0.5% BSA and 0.05% Tween 20, and IgG was detected using alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson immunoresearch). Spots were counted using a dissecting microscope. Images were taken using a CTL-immunoSpot S5 analyzer.

ANA analysis
For ANA analysis, serum was diluted 1:5 in PBS/0.2% BSA and incubated on Kallestad® HEp-2 Slides (Bio-rad). Slides were washed and stained with anti-mouse IgG FITC (SouthernBiotech). Slides were mounted with Aqua PolyMount and analyzed on a fluorescent microscope (Axio Observer.Z1). Images were obtained using a Zeiss Axioplan 2 microscope with a Zeiss Axiocam digital camera (Zeiss).

RNA-sequencing (RNA-seq)
Conventional T reg cells (CD19 − CD4 + Foxp3 + CD69 hi PD-1 lo CXCR5 lo CD25 hi ) and T cells T FR cells (CD19 − CD4 + Foxp3 + CD69 hi PD-1 hi CXCR5 hi CD25 lo ) were sorted from the mLN of Foxp3-DTR/GFP mice at day 30 after influenza infection using a FACSAria (BD Biosciences) after positive selection with anti-CD4 MACS beads (Miltenyi Biotec). RNA was isolated from the sorted cells using the Single cell RNA purification kit (Norgen Biotek Corp). Three replicates from three independent experiments for each condition were analyzed with RNA-seq. Library preparation and RNA sequencing was conducted through Genewiz. Libraries were sequenced using a 1×50bp single end rapid run on the HiSeq2500 platform. Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality (error rate < 0.05) at the end. After trimming, sequence reads shorter than 30 nucleotides were discarded. Remaining sequence reads were mapped to the Mus musculus mm10 reference genome using CLC genomics workbench v. 9.0.1 Differential gene expression was determined using DESeq2. Genes with an adjusted P-value <0.05 and an absolute Log 2 -fold change > 1 were considered significantly differentially expressed genes between conventional T reg cells and T FR cells.

GSEA, hierarchical clustering and visualization of RNA sequencing results
GSEA was performed using the Molecular Signatures Database on the publically available MIT BROAD Institute server. We ranked the 2002 genes obtained from RNA sequencing (Genewiz) according to a logarithmic transformation of each gene's P-value multiplied by the sign of the corresponding logarithmic fold change, and subsequently utilized these rank lists to perform a gene set enrichment analysis (Broad Institute's GSEA Java app, version 2.2.4). Given gene sets can include both activated and repressed genes, we additionally performed an extended assessment of gene enrichment of the two complementary gene sets against N ranked genes 51, 52 , to confirm the original GSEA results. Separately, we performed hierarchical clustering analysis 53, 54 of IL-2-induced genes that are differentially expressed in T reg cells vs T FR cells, using Matlab (version R2016b). Differential clusters are presented in the form of an annotated heatmap based on standardized expression values, along with the resulting hierarchical clustering dendogram. We additionally plotted in Matlab (version R2016b) the number and proportion of genes expressed within hallmark sets (GSEA), and their corresponding p-values.

Statistical analysis
GraphPad Prism software (Version 5.0a) was used for data analysis. The statistical significance of differences in mean values was determined using a two-tailed Student's ttest. P values of less than 0.05 were considered statistically significant.

Data availability
The data that support the findings of this study are available from the corresponding author upon request. RNA-seq data are available from GEO under accession code GSE101016.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student's t-test. . P values were determined using a two-tailed Student's t-test. (D) B6 mice were infected with PR8 and STAT5 phosphorylation in CD4 + B220 − CD25 lo Bcl-6 hi CXCR5 hi Foxp3 + T FR cells, CD4 + B220 − CD25 hi Bcl-6 lo CXCR5 lo Foxp3 + conventional T reg cells and The frequency (A) and number (B) of CD45.2 + FoxP3 + cells in recipients receiving CD25 hi FoxP3 + and CD25 lo FoxP3 + cells are shown. Plots were gated on FoxP3 + CD69 hi CD4 + T cells. The frequency (C) and number (D) of CD45.2 + CD69 + FoxP3 + CD4 + T cells with a Bcl-6 hi CXCR5 hi phenotype in recipients receiving CD25 hi FoxP3 + and CD25 lo FoxP3 + cells are shown. (E) The number of donor-derived CD45.1 + CD69 + FoxP3 + CD4 + T cells in recipients receiving CD25 hi FoxP3 + and CD25 lo FoxP3 + cells are shown. (F) The number of donor-derived CD45.1 + CD69 + FoxP3 + T cells with a Bcl-6 hi CXCR5 hi phenotype in recipients receiving CD25 hi FoxP3 + and CD25 lo FoxP3 + cells are shown. Data were pooled from two independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed  the expression of T-bet in CD25 hi Blimp hi and CD25 lo Blimp lo FoxP3 + CD69 hi CD4 + T cells was analyzed at days 10 and 30 by intracellular staining. Data are representative of two independent experiments (mean ± S.D of 3-5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student's t-test. (D) Tcrb −/− Tcrd −/− mice were irradiated and reconstituted with a 50:50 mix of BM from CD45.1 + WT and CD45.2 + Tbx21 −/− donors. Eight weeks later, reconstituted mice were infected with PR8 and cells from the mLN were analyzed on day 10. The frequency of CD25 lo FoxP3 + and CD25 hi FoxP3 + cells with a Bcl-6 hi CXCR5 hi phenotype was determined in the CD45.1 + and CD45.2 + compartments. Representative plots are shown. Data in the graph are shown as the mean ± SD (n=4 mice). Data are representative of two independent experiments. (E) Tcrb −/− Tcrd −/− mice were irradiated and reconstituted with a 50:50 mix of BM from CD45.1 + WT and CD45.2 + Prdm1 fl/fx -Lck cre/+ donors. Eight weeks later, reconstituted mice were infected with PR8 and cells from the mLN were analyzed on day 10. The frequency of CD25 lo FoxP3 + and CD25 hi FoxP3 + cells with a Bcl-6 hi CXCR5 hi phenotype were calculated in the CD45.1 + and CD45.2 + compartments. Representative plots are shown. Data in the graph are shown as the mean ± SD (n=5 mice). Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a twotailed Student's t-test. (F-G) WT-Prdm1 fl/fl -Lck cre/+ chimeric mice were infected with PR8 and treated daily with 30,000 U of rIL-2 or PBS (control) starting 20 days after infection. Cells from the mLNs were analyzed by flow cytometry on day 30. (F) Frequency of CD45.1 + and CD45.2 + CD25 lo FoxP3 + CD4 + T cells with a Bcl-6 hi CXCR5 hi T FR -cell phenotype. Data in the graph are shown as the mean ± SD (n=5-7 mice/group). Representative plots are shown. (G) Ratio of Prdm1 −/− to WT T FR cell was calculated in control and rIL-2-treated mice. Data are shown as the mean ± SD (n=5-7 mice/group). Data are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student's t-test.  Data are shown as the mean ± SD (n=4-5 mice/group). Data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a two-tailed Student's t-test. (G-H) B6 mice were infected with PR8 and treated daily with 15,000 U of rIL-2 or PBS starting on day 20. Cells from the mLNs were analyzed by flow cytometry on day 30. Frequency (G) and number (H) of CD138 + ASCs. Data are shown as the mean ± SD (n=4-5 mice/group). Data are representative of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using a twotailed Student's t-test. Frequency (A) and number (B) of CD19+CD138-GL-7 + CD38 lo CD95 hi GC B cells that were NP-specific. Frequency (C) and number (D) of CD138 + ASCs that were influenza-NP specific. Data are shown as the mean ± SD (n=4-5 mice/group). Data are representative of four independent experiments. (E-F) B6 and Bcl-6 fl/fl Foxp3 YFP/Cre mice were infected with PR8 and CD138 + ASCs from the mLN were analyzed by flow cytometry on day 30. Frequency (E) and number (F) of CD138 + ASCs that were NP-specific. Data are shown as the mean ± SD (n=3-4 mice/group). Data are representative of three independent experiments. (G) B6 mice were infected with PR8 and treated daily with 15,000 U of rIL-2 or PBS starting on day 20. Serum was obtained on day 30 and PR8-specific IgG Abs were measured by ELISA. Data are representative of three independent experiments (mean ± SD of 5 mice per group). (H) B6 and Bcl-6 fl/fl Foxp3 YFP/Cre mice were infected with PR8. Serum from was obtained on day 30 and PR8-specific IgG Abs were measured by ELISA. (I) B6 mice were infected with PR8 and treated daily with 15,000 U of rIL-2 or PBS starting 20 days after infection. Histone-specific IgG-secreting cells in the mLN were enumerated by ELISPOT on day 30. Data are representative of two independent experiments (mean ± SD of 4-5 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. All P values were determined using a two-tailed Student's t-test. (J) The presence of ANAs in the serum from day 30 influenza-infected control and IL-2-treated mice was determined by fluorescence microscopy using HEp-2 slides. Images are representative of two independent experiments