NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection

Cutting Edge: NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection

This information is current as Gustavo J. Martinez, Joyce K. Hu, Renata M. Pereira, Jordan of September 28, 2021. S. Crampton, Susan Togher, Nicholas Bild, Shane Crotty and Anjana Rao J Immunol published online 5 February 2016 http://www.jimmunol.org/content/early/2016/02/04/jimmun

ol.1501841 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2016/02/04/jimmunol.150184 Material 1.DCSupplemental http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 5, 2016, doi:10.4049/jimmunol.1501841 Th eJournal of Cutting Edge Immunology

Cutting Edge: NFAT Transcription Factors Promote the Generation of Follicular Helper T Cells in Response to Acute Viral Infection † ,1 † Gustavo J. Martinez,* Joyce K. Hu, Renata M.x Pereira,* Jordan{ S.‖ Crampton, Susan Togher,* Nicholas Bild,‡ Shane Crotty,†, and Anjana Rao*, , ,# Follicular CD4+ Th (Tfh) cells provide B cell help in motif) receptor 5 (CXCR5) and the lineage-defining tran- germinal center reactions that support class switching, scription factor B-cell CLL/lymphoma 6 (Bcl6) (8–10). Bcl6 somatic hypermutation, and the generation of high- does not act alone, and other transcription factors have also affinity Abs. In this article, we show that deficiency been identified as key regulators of Tfh differentiation, in- + in NFAT1 and NFAT2 in CD4 T cells leads to im- cluding STATs, Maf, BATF, IRF4, ASCL2 (reviewed in Refs. Downloaded from paired germinal center reactions upon viral infection 11, 12), and LEF-1 and TCF-1 (13, 14). NFAT2 is highly because of reduced Tfh cell differentiation and defec- expressed in Tfh cells (15); however, the roles played by tive expression of proteins involved in T/B interactions NFAT family members in Tfh cells are not well understood. and B cell help, including ICOS, PD-1, and SLAM Tfh cells have enhanced calcium signaling with NFAT nu- family receptors. Genome-wide chromatin immuno- clear translocation compared with Th1 cells, suggesting that precipitation data suggest that NFAT proteins likely NFATs may have preferential responsibilities in Tfh cells http://www.jimmunol.org/ compared with Th1 cells (16). In this article, we investigated directly participate in regulation of genes important the role of NFAT1 and NFAT2 in the generation of Tfh cells for Tfh cell differentiation and function. NFAT pro- and GC responses to acute viral infection. teins are important TCR and Ca2+-dependent regulators of T cell biology, and in this article we demonstrate Materials and Methods a major positive role of NFAT family members in Mice and infections Tfh differentiation. The Journal of Immunology, SMARTA [lymphocytic choriomeningitis virus (LCMV) gp66-77-IAb–spe- 2 2 2016, 196: 000–000. cific] CD45.1+ mice (17) were crossed with Nfat1 / , Nfat2fl/fl CD4Cre,or 2 2 Nfat1 / Nfat2fl/fl CD4Cre mice (2). All mice were maintained in specific by guest on September 28, 2021 pathogen-free barrier facilities and used according to protocols approved by uclear factor of activated T cells (NFAT) transcription the La Jolla Institute for Allergy and Immunology animal care and use 3 5 factors are key regulators of T cell activation (1) and committees. Mice were infected i.p. with 2 10 PFU LCMV Armstrong 5 strain. The adoptive transfer experiments using SMARTA cells were per- N exhaustion (2). NFAT1, NFAT2, and NFAT4 are formed as previously described (17), unless otherwise stated. Postinfection, expressed in cells of the immune system and have important splenocytes were harvested and cells were stained with Abs against cell-surface roles in T cell development and function (3). Although NFAT markers as previously described (17). LCMV-specific serum IgG was quan- tified by ELISA as previously described (18). ELISA data were analyzed by family members make similar contacts with DNA (4), they area under the curve (AUC). AUC analysis better accounts for both the show distinct expression patterns and functions, as judged by quantity and the quality of the IgG, because it accounts for the shape of the the nonoverlapping phenotypes of mice deficient in individual curve. AUC total peak area above baseline calculations (GraphPad Prism 6.0) NFAT family members (5, 6, and reviewed in Ref. 7). was done for each individual sample, log transformed. + Follicular CD4 Th (Tfh) cells are essential for mediating Isolation and culture of T cells B cell help and inducing germinal center (GC) responses re- Spleens and lymph nodes were harvested from 6- to 8-wk-old mice. Naive quired for most high-affinity Ab responses. Tfh cells have CD4+ cells were purified using EasySep kit (Stem Cell) and activated with been characterized by their expression of chemokine (C-X-C anti-CD3 and anti-CD28 as previously described (2).

*Department of Signaling and Gene Expression, La Jolla Institute for Allergy and postdoctoral fellowship from the Jane Cofﬁn Childs Memorial Fund (to G.J.M.); and a Immunology, La Jolla, CA 92037; †Department of Vaccine Discovery, La Jolla Institute postdoctoral fellowship from the Pew Latin American Fellows Program in the Biomed- for Allergy and Immunology, La Jolla, CA, 92037; ‡Genomics Core, The Scripps ical Sciences (to R.M.P.). x Research Institute, Jupiter, FL, 33458; Division of Infectious Diseases, Department { Address correspondence and reprint requests to Dr. Gustavo J. Martinez at the current of Medicine, University of California, San Diego, La Jolla, CA 92037; Department ‖ address: Department of Microbiology and Immunology, Chicago Medical School, Ro- of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Moores salind Franklin University of Medicine and Science, 3333 Green Bay Road, North Cancer Center, University of California, San Diego, La Jolla, CA 92093; and #Sanford Chicago, IL 60088. E-mail address: [email protected] Consortium for Regenerative Medicine, La Jolla, CA 92037 The online version of this article contains supplemental material. 1Current address: Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Abbreviations used in this article: AUC, area under the curve; Bcl6, B-cell CLL/lymphoma 6; ChIP-seq, chromatin immunoprecipitation followed by next generation sequencing; ORCIDs: 0000-0003-0178-3329 (G.J.M.); 0000-0002-6484-6262 (S.C.); 0000-0002- GC, germinal center; LCMV, lymphocytic choriomeningitis virus; RNA-seq, RNA 1870-1775 (A.R.) sequencing; Tfh, follicular CD4+ Th; WT, wild type. Received for publication August 19, 2015. Accepted for publication January 5, 2016. Ó This work was supported by National Institutes of Health Grants R01AI 109842 (to Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 A.R.), AI40127 (to A.R.), R01AI072543 (to S.C.), and U19 AI109976 (to S.C.); a

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501841 2 CUTTING EDGE: NFAT PROTEINS DRIVE TFH CELL GENERATION AND MAINTENANCE

RNA sequencing and chromatin immunoprecipitation followed by next develop in response to acute viral infections, and KLRG1 generation sequencing data analyses expression is associated with highly polarized Th1 cells in Previously published human RNA sequencing (RNA-seq) data from Weinstein some contexts (20, 21). The increased KLRG1 expression on et al. (19) were used. The mouse homologs of genes differentially expressed NFAT-deficient cells suggests that, in the absence of NFATs, in Tfh versus non-Tfh cells were identified. NFAT1 chromatin immuno- the cells become even more polarized toward Th1 (Fig. 1F, precipitation followed by next generation sequencing (ChIP-seq) data for memory-like CD8+ T cells were from our previous report (2). 1G). In summary, our results suggest that NFAT family members have an important role in the generation or main- Statistics tenance of Tfh cells in vivo upon viral infection, without Statistical analyses were done using Prism 6.0 (GraphPad). The p values were which GC and antiviral Ab responses are defective. calculated using two-tailed unpaired Student t tests with a 95% confidence interval. T cell–intrinsic defect in Tfh cell generation in NFAT1,2-deficient Results and Discussion mice Reduced Tfh cells and GCs in NFAT1,2-deficient mice To assess CD4 T cell–intrinsic activities of NFAT1 and 2 2 We investigated the role of two NFAT family members, NFAT2, we generated Nfat1 / , Nfat2fl/fl Cd4-Cre CD45.1+ NFAT1 and NFAT2, in the generation of Tfh cells using SMARTA TCR transgenic mice. Naive SMARTA CD45.1+ 2 2 single- and double-deficient Nfat1 / , Nfat2fl/fl Cd4-Cre mice CD4+ T cells were transferred into congenic mice that were (2). The frequency of total B cells was not affected in these then infected with LCMV. Expansion of the adoptively mice (data not shown). The mice were infected with LCMV transferred cells and expression of the activation marker Downloaded from Armstrong strain, which causes an acute viral infection, and CD44 was similar between WT and NFAT1,2-deficient the frequency of GC B cells and Tfh cells were determined in SMARTA cells 8 d postinfection (Supplemental Fig. 1B–D, spleens 8 d postinfection. Single NFAT1 or NFAT2 defi- 1J), indicating the capacity of NFAT-deficient CD4 T cells to ciency did not significantly affect the generation of GL7+ Fas+ mount an Ag-specific response. Mice receiving NFAT1,2- GC B cells compared with wild type (WT) control mice. deficient SMARTA CD4 T cells developed fewer GC

However, deficiency of both NFAT1 and NFAT2 led to an B cells compared with WT controls (Fig. 2A, 2B), correlating http://www.jimmunol.org/ almost complete absence of GC B cells (Fig. 1A, 1B). LCMV- with a decrease in the generation of GC Tfh (CXCR5+ Bcl6+) specific IgG responses were severely decreased in NFAT1,2- (Fig. 2C, 2D, Supplemental Fig. 1H), as well as all CXCR5- deficient mice, commensurate with the failure to develop GCs expressing SMARTA cells (Supplemental Fig. 1E–G). Ex- (Fig. 1C, Supplemental Fig. 1A). pression of the PD-1 and ICOS receptors associated with Tfh We then sought to determine whether lack of GC responses cell functions was also affected, in this case predominantly was due to impaired Tfh cell generation. Similar frequencies of by NFAT2 (Fig. 2E, 2F). More severe GC defects had been total CD4 T cells were observed between WT and NFAT- observed in germline knockout mice (Fig. 1), suggesting a

deficient animals at day 8 after acute viral infection, and possibility for non-CD4 T cell–intrinsic effects. However, the by guest on September 28, 2021 CD44 expression was comparable (data not shown). In con- SMARTA transfer experiment results were also consistent with trast, a substantial decrease in the frequency of CXCR5+ Bcl6+ the presence of endogenous WT T cell responses capable of GC Tfh cells was observed in NFAT1,2-deficient compared providing T cell help to B cells. In summary, our results dem- with WT mice (Fig. 1D, 1E). Moreover, expression of key onstrate an essential role of NFAT family members in the gen- receptors associated with Tfh cell function, such as ICOS, eration or maintenance of Tfh cells in vivo upon viral infection. Ly108, PD-1, and CXCR5, were all reduced in NFAT1,2- Among the non-Tfh SMARTA cells, a dramatic increase deficient CD4+ T cells (Fig. 1F, 1G). Both Th1 and Tfh cells in KLRG1+ cells was observed for NFAT2- and NFAT1,2-

FIGURE 1. Decreased GC reaction and Tfh cells in NFAT1,2-deficient mice. WT, NFAT1-, NFAT2-, and NFAT1,2-deficient mice were infected with LCMV Armstrong (2 3 105 PFU). (A, B, and E–H) On day 8, spleens were harvested, and expression of cell-surface markers was determined by flow cytometry. (A and 2 2 B) Frequency of GL7+ Fas+ GC B cells (gated on B220+ CD19+ CD8 CD4 cells). (C) Serum LCMV-specific from infected mice were determined by ELISA 2 2 (n = 5–6 mice/group). (D and E) Frequency of CXCR5+ Bcl6+ GC Tfh cells (gated on CD4+ CD44+ CD8 B220 cells). (F and G) Expression of cell-surface 2 2 receptors on CD4+ CD44+ CD8 B220 cells. Contour plots (A and D) or histogram (F) are from a representative mouse in each group; the combined results from a total of five mice per group are shown (B, E, and G). A representative experiment out of two is shown. Statistical analysis was performed using t test. *p , 0.05, **p , 0.01, ***p , 0.005, ****p , 0.0001. The Journal of Immunology 3

FIGURE 2. T cell–intrinsic defect in Tfh generation/ maintenance in NFAT1,2-deficient mice. SMARTA CD45.1+ WT, NFAT1-, NFAT2-, or NFAT1,2-deficient cells were adoptively transferred into congenic CD45.2+ C57BL/6 congenic mice. One day later, mice were infected with LCMV Armstrong (2 3 105 PFU). On day 8, spleens were harvested, and expression of cell-surface markers was determined by flow cytometry. (A and B) Frequency of GL7+ Fas+ GC B cells 2 2 (gated on B220+ CD19+ CD8 CD4 cells). (C–H) Expres- sion of CXCR5 and Bcl6 (C and D), ICOS or PD-1 (E and F), CXCR5 and KLRG1 (G and H), KLRG1 and Tbet (I–K)on + + A C G I

CD4 CD45.1 cells. Contour plots ( , , , and ) or his- Downloaded from tograms (E and J) are from a representative mouse in each group; the combined results from a total of ﬁve mice per group are shown (B, D, F, H, and K). A representative experiment out of two is shown. Statistical analysis was performed using t test. *p , 0.05, **p , 0.01, ***p , 0.005, ****p , 0.0001. http://www.jimmunol.org/

deficient cells (Fig. 2G–I, Supplemental Fig. 1L). This ob- to B cells (23–25). Thus, our results indicate that NFAT by guest on September 28, 2021 servation is consistent with these cells being an unusual form family members have a major role in the induction of re- of highly polarized Th1 effector cells previously identified in ceptors involved in Tfh cell biology. mice with Mycobacterium tuberculosis infection (20). More- Given the early defects in upregulation of ICOS on NFAT1,2- over, we observed no significant differences in Tbet expression deficient CD4 T cells, we asked whether early stages of Tfh between WT and NFAT1,2-deficient cells (Fig. 2I, 2J). Thus, differentiation in vivo depend on NFAT transcription factors. our results indicate that NFAT expression prevents the gen- Pilot experiments revealed NFAT1,2-deficient SMARTA CD4 eration of KLRG1+ effector CD4 T cells in response to in- T cells exhibited a delay in accumulation compared with WT fection and specifically regulates Tfh cell generation. SMARTA CD4 T cells in vivo by day 3 in response to an acute viral infection (Supplemental Fig. 1J). Thus, in subsequent Defective induction of key receptors in NFAT1,2-deficient mice experiments, 4-fold more NFAT1,2-deficient SMARTA cells accounts for the defect in GC formation were transferred to partially compensate for the delayed ex- To examine potential mechanisms by which NFATs control pansion (Supplemental Fig. 1J). Nascent Tfh cells can be Tfhdifferentiationandfunction,weactivatedWTand identified as CXCR5+ Bcl6+ CD4 T cells at day 3 postinfection NFAT1,2-deficient CD4 T cells with anti-CD3 and anti- (Fig. 3B, 3C). NFAT1,2-deficient cells had severely diminished CD28 in vitro and characterized expression of genes of in- frequencies of CXCR5+ Bcl6+ (or CXCR5+PD-1+) nascent Tfh terest. Rapid induction of CD69 and CD25 by NFAT1,2- cells in comparison with WT CD4 T cells (Fig. 3B, 3C, deficient CD4 T cells was comparable with that of WT Supplemental Fig. 1K). The Tfh deficiency was not simply a controls at early times (3, 6 h), although expression of CD69 reflection of a generalized T cell activation defect, because the 2 and CD25 was not maximally maintained in the absence of frequency of early Th1 cells (CD25hi Bcl6 ) was elevated (Fig. NFAT1 and NFAT2 (Supplemental Fig. 2A). In contrast, 3D, 3E) and mean fluorescence intensities of the activation induction of ICOS protein expression was severely impaired marker CD25 were high (Fig. 3D and data not shown). Thus, on NFAT1,2-deficient naive CD4+ T cells compared with our results demonstrate that NFAT family members are selec- their WT counterparts at all time points examined (Fig. 3A). tively important for early Tfh differentiation, most likely via ICOS expression is critical for Tfh differentiation (17, 22), regulation of Icos and other Tfh-associated differentiation genes. and therefore loss of ICOS expression is likely one major To identify direct roles of NFAT in the regulation of Tfh cause of Tfh loss in the absence of NFATs. Expression of differentiation and function, we examined binding of NFAT1 CD40L, PD-1, and SLAM was also significantly impaired to genes that influence Tfh differentiation or are significantly on in vitro–activated NFAT1,2-deficient CD4 T cells expressed on Tfh cells (Fig. 4A). ChIP-Seq analysis from (Supplemental Fig. 2); each receptor has roles in Tfh help memory-like CD8+ T cells stimulated in vitro with PMA and 4 CUTTING EDGE: NFAT PROTEINS DRIVE TFH CELL GENERATION AND MAINTENANCE

FIGURE 3. Early defect in Tfh cell generation in NFAT1,2-deficient mice in vivo. (A) WT or NFAT1,2-deficient naive T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time points. Expression of ICOS was determined by flow cytometry. A representative example of two independent experiments with two biological replicates each is shown. (B–E) SMARTA+ CD45.1+ WT (5 3 105) or NFAT1,2-deficient (2 3 106) cells were adoptively transferred into CD45.2+ C57BL/6 congenic mice. One day later, mice were infected with LCMV Armstrong (2 3 105 PFU). On day 3, spleens were harvested, and expression of cell-surface markers was determined by flow cytometry. Expression of CXCR5 and Bcl6 (B and C) and CD25 and Bcl6 (D and E) on CD4+ CD45.1+ adoptively transferred cells is shown. Contour plots (B and D) are from a representative mouse in each group; combined results from a total of four to five mice per group from two independent experiments are shown (C and E). Statistical analysis was performed using t test. **p , 0.01, ****p , 0.0001. ionomycin (2) shows NFAT1 binding sites in the proximal junction with Tfh gene expression profiles. It was recently promoters and/or within the transcribed regions of the Icos, reported that Ca2+ signaling was also necessary for Tfh IL-21 Il2ra,andIL6ra genes, which encode receptors that have expression (16), suggesting that NFATs are required for many

prominent roles in early Tfh differentiation (17, 26, 27). functions of Tfh cells during an antiviral response. Downloaded from These ChIP-seq associations, in conjunction with the ICOS A previous study reported that NFAT2-deficient mice have expression defects observed in NFAT1,2-deficient CD4 T enhanced GCs in the context of an immunization model; this cells and the early Tfh differentiation defects of NFAT1,2- was associated with a loss of follicular regulatory T cells deficient CD4 T cells in vivo (Fig. 3), suggest that NFAT through regulation of CXCR5 expression, but no direct im- transcription factors might regulate Tfh differentiation up- pact on Tfh cells was ascribed to NFAT2 (15). In contrast, in

stream of Bcl6, as also recently observed for TCF-1 and LEF- the experiments reported in this article, NFAT2 positively http://www.jimmunol.org/ 1 transcription factors (13, 14). NFAT1 binding sites are also regulates Tfh differentiation. More strikingly, acute infection in the vicinity of the Cxcr5 and Slamf1 genes. NFAT1 did not of NFAT1,2-deficient mice demonstrated that NFAT1 and bind to the Bcl6 locus in CD8 T cells (data not shown), but NFAT2 cooperate for the generation of Tfh cells during whether NFAT proteins bind to Bcl6 in CD4 T cells is un- antiviral responses. One explanation for the discrepancies known. Whether NFAT transcription factors cooperate with could be that immunization and viral infection elicit different LEF-1, TCF-1, or other transcription factors to regulate Bcl6 in vivo responses. In fact, we have not observed follicular expression and Tfh differentiation remains to be determined. regulatory T cell differentiation by SMARTA CD4 T cells in When comparing global gene expression patterns of genes LCMV infections (data not shown). differentially upregulated in human Tfh versus non-Tfh cells Consistent with the findings presented in this report, mice by guest on September 28, 2021 (19) (Fig. 4B), 40% of Tfh-associated upregulated genes deficient in both Stim1 and Stim2, endoplasmic reticulum possessed at least one NFAT1 binding site within 10 kb of calcium sensors that control the gating of the store-operated their transcription start sites (Fig. 4B, dark blue dots). Fur- calcium released–activated calcium channel ORAI1 up- thermore, 47% of genes downregulated in Tfh compared with stream of NFAT activation (28), displayed decreased Ab re- non-Tfh cells also possessed an NFAT1 binding site within sponses and decreased CD40L expression in CD4+ T cells 10 kb of their transcription start sites (Fig. 4B, dark red dots). upon LCMV infection (29). Moreover, GC Tfh cells ex- Thus, these bioinformatics analyses suggest that NFAT family hibit extensive calcium signaling in GCs (30, 31) and were members extensively regulate Tfh-associated genes. also observed to have enhanced NFAT nuclear localization Overall, our results highlight important roles for NFAT (16), in accord with our work. It has been previously observed family members in Tfh biology. NFAT expression was re- that Tfh differentiation is associated with TCRs capable of quired for appropriate expression of ICOS and CD40L, which greater MHC class II:peptide complex binding capacity (TCR are essential for Tfh differentiation, and NFATs likely regulate dwell time) compared with other Th subsets (32, 33). Given additional essential components of Tfh differentiation based that NFAT activity is TCR activation dependent, we infer on bioinformatics analysis of NFAT ChIP-seq data in con- from the Tfh defects associated with NFAT1,2 deficiency that

FIGURE 4. NFAT directly regulates expression of Tfh-associated genes. (A) Genome browser views of indicated gene loci showing NFAT1 binding. (B) Changes in gene expression in human Tfh versus non-Tfh cells plotted against overall gene expression. Genes significantly upregulated in Tfh compared with non- Tfh are shown in blue, and genes significantly upregulated in non-Tfh compared with Tfh cells are shown in red. Genes with NFAT1 binding within 10 kb of their transcription start site are shown with dark colors (dark blue and dark red for genes that are significantly upregulated or downregulated, respectively). The Journal of Immunology 5

NFAT1 and NFAT2 synergy might occur optimally in vivo in humoral autoimmunity via NFAT2-regulated CXCR5 expression. J. Exp. Med. 211: 545–561. the context of extended TCR dwell times. Overall, our results 16. Ray, J. P., M. M. Staron, J. A. Shyer, P. C. Ho, H. D. Marshall, S. M. Gray, have significant implications for therapeutic approaches to B. J. Laidlaw, K. Araki, R. Ahmed, S. M. Kaech, and J. Craft. 2015. The Interleukin-2-mTORc1 Kinase Axis Defines the Signaling, Differentiation, and modulate B cell function by modulating the activity of NFAT Metabolism of T Helper 1 and Follicular B Helper T Cells. Immunity 43: 690–702. transcription factors. 17. Choi, Y. S., R. Kageyama, D. Eto, T. C. Escobar, R. J. Johnston, L. Monticelli, C. Lao, and S. Crotty. 2011. ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6. Im- Acknowledgments munity 34: 932–946. 18. Kageyama, R., J. L. Cannons, F. Zhao, I. Yusuf, C. Lao, M. Locci, We thank Ryan Hastie for help in maintaining the mouse colony, and C. Kim, P. L. Schwartzberg, and S. Crotty. 2012. The receptor Ly108 functions as a SAP K. Gunst, and L. Nosworthy at the La Jolla Institute Flow Cytometry Facility adaptor-dependent on-off switch for T cell help to B cells and NKT cell develop- for help with cell sorting experiments. ment. Immunity 36: 986–1002. 19. Weinstein, J. S., K. Lezon-Geyda, Y. Maksimova, S. Craft, Y. Zhang, M. Su, V. P. Schulz, J. Craft, and P. G. Gallagher. 2014. Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes. Disclosures Blood 124: 3719–3729. The authors have no financial conflicts of interest. 20. Reiley, W. W., S. Shafiani, S. T. Wittmer, G. Tucker-Heard, J. J. Moon, M. K. Jenkins, K. B. Urdahl, G. M. Winslow, and D. L. Woodland. 2010. Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis References infection. Proc. Natl. Acad. Sci. USA 107: 19408–19413. 1. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT family: 21. Sakai, S., K. D. Kauffman, J. M. Schenkel, C. C. McBerry, K. D. Mayer-Barber, regulation and function. Annu. Rev. Immunol. 15: 707–747. D. Masopust, and D. L. Barber. 2014. Cutting edge: control of Mycobacterium 2. Martinez, G. J., R. M. Pereira, T. Aïjo¨, E. Y. Kim, F. Marangoni, M. E. Pipkin, tuberculosis infection by a subset of lung parenchyma-homing CD4 T cells. Downloaded from S. Togher, V. Heissmeyer, Y. C. Zhang, S. Crotty, et al. 2015. The transcription J. Immunol. 192: 2965–2969. factor NFAT promotes exhaustion of activated CD8⁺ T cells. Immunity 42: 265– 22. Akiba, H., K. Takeda, Y. Kojima, Y. Usui, N. Harada, T. Yamazaki, J. Ma, 278. K. Tezuka, H. Yagita, and K. Okumura. 2005. The role of ICOS in the CXCR5+ 3. Muller,€ M. R., and A. Rao. 2010. NFAT, immunity and cancer: a transcription follicular B helper T cell maintenance in vivo. J. Immunol. 175: 2340–2348. factor comes of age. Nat. Rev. Immunol. 10: 645–656. 23. Cubas, R. A., J. C. Mudd, A. L. Savoye, M. Perreau, J. van Grevenynghe, 4. Chen, L., J. N. Glover, P. G. Hogan, A. Rao, and S. C. Harrison. 1998. Structure of T. Metcalf, E. Connick, A. Meditz, G. J. Freeman, G. Abesada-Terk, Jr., et al. the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. 2013. Inadequate T follicular cell help impairs B cell immunity during HIV in- Nature 392: 42–48. fection. Nat. Med. 19: 494–499.

5. Dietz, L., F. Frommer, A. L. Vogel, M. Vaeth, E. Serfling, A. Waisman, 24. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, and G. Kelsoe. 1995. http://www.jimmunol.org/ M. Buttmann, and F. Berberich-Siebelt. 2015. NFAT1 deficit and NFAT2 deficit Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in attenuate EAE via different mechanisms. Eur. J. Immunol. 45: 1377–1389. established germinal centers. J. Immunol. 155: 556–567. 6. Reppert, S., E. Zinser, C. Holzinger, L. Sandrock, S. Koch, and S. Finotto. 2015. 25. Renshaw, B. R., W. C. Fanslow, III, R. J. Armitage, K. A. Campbell, D. Liggitt, NFATc1 deficiency in T cells protects mice from experimental autoimmune en- B. Wright, B. L. Davison, and C. R. Maliszewski. 1994. Humoral immune re- cephalomyelitis. Eur. J. Immunol. 45: 1426–1440. sponses in CD40 ligand-deficient mice. J. Exp. Med. 180: 1889–1900. 7. Macian, F. 2005. NFAT proteins: key regulators of T-cell development and func- 26. Choi, Y. S., D. Eto, J. A. Yang, C. Lao, and S. Crotty. 2013. Cutting edge: STAT1 tion. Nat. Rev. Immunol. 5: 472–484. is required for IL-6-mediated Bcl6 induction for early follicular helper cell differ- 8. Johnston, R. J., A. C. Poholek, D. DiToro, I. Yusuf, D. Eto, B. Barnett, A. L. Dent, entiation. J. Immunol. 190: 3049–3053. J. Craft, and S. Crotty. 2009. Bcl6 and Blimp-1 are reciprocal and antagonistic 27. Johnston, R. J., Y. S. Choi, J. A. Diamond, J. A. Yang, and S. Crotty. 2012. STAT5 regulators of T follicular helper cell differentiation. Science 325: 1006–1010. is a potent negative regulator of TFH cell differentiation. J. Exp. Med. 209: 243– 9. Nurieva, R. I., Y. Chung, G. J. Martinez, X. O. Yang, S. Tanaka, 250.

T. D. Matskevitch, Y. H. Wang, and C. Dong. 2009. Bcl6 mediates the develop- 28. Hogan, P. G., R. S. Lewis, and A. Rao. 2010. Molecular basis of calcium signaling by guest on September 28, 2021 ment of T follicular helper cells. Science 325: 1001–1005. in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28: 491–533. 10. Yu, D., S. Rao, L. M. Tsai, S. K. Lee, Y. He, E. L. Sutcliffe, M. Srivastava, 29. Shaw, P. J., C. Weidinger, M. Vaeth, K. Luethy, S. M. Kaech, and S. Feske. 2014. M. Linterman, L. Zheng, N. Simpson, et al. 2009. The transcriptional repressor CD4⁺ and CD8⁺ T cell-dependent antiviral immunity requires STIM1 and STIM2. Bcl-6 directs T follicular helper cell lineage commitment. Immunity 31: 457–468. J. Clin. Invest. 124: 4549–4563. 11. Crotty, S. 2014. T follicular helper cell differentiation, function, and roles in dis- 30. Liu, D., H. Xu, C. Shih, Z. Wan, X. Ma, W. Ma, D. Luo, and H. Qi. 2015. T-B- ease. Immunity 41: 529–542. cell entanglement and ICOSL-driven feed-forward regulation of germinal centre 12. Liu, X., R. I. Nurieva, and C. Dong. 2013. Transcriptional regulation of follicular reaction. Nature 517: 214–218. T-helper (Tfh) cells. Immunol. Rev. 252: 139–145. 31. Shulman, Z., A. D. Gitlin, J. S. Weinstein, B. Lainez, E. Esplugues, R. A. Flavell, 13. Choi, Y. S., J. A. Gullicksrud, S. Xing, Z. Zeng, Q. Shan, F. Li, P. E. Love, J. E. Craft, and M. C. Nussenzweig. 2014. Dynamic signaling by T follicular helper W. Peng, H. H. Xue, and S. Crotty. 2015. LEF-1 and TCF-1 orchestrate TFH cells during germinal center B cell selection. Science 345: 1058–1062. differentiation by regulating differentiation circuits upstream of the transcriptional 32. Tubo, N. J., A. J. Paga´n, J. J. Taylor, R. W. Nelson, J. L. Linehan, J. M. Ertelt, repressor Bcl6. Nat. Immunol. 16: 980–990. E. S. Huseby, S. S. Way, and M. K. Jenkins. 2013. Single naive CD4+ T cells from 14. Xu, L., Y. Cao, Z. Xie, Q. Huang, Q. Bai, X. Yang, R. He, Y. Hao, H. Wang, a diverse repertoire produce different effector cell types during infection. Cell 153: T. Zhao, et al. 2015. The transcription factor TCF-1 initiates the differentiation of 785–796. T(FH) cells during acute viral infection. Nat. Immunol. 16: 991–999. 33. Fazilleau, N., L. J. McHeyzer-Williams, H. Rosen, and M. G. McHeyzer-Williams. 15. Vaeth, M., G. Muller,€ D. Stauss, L. Dietz, S. Klein-Hessling, E. Serﬂing, M. Lipp, 2009. The function of follicular helper T cells is regulated by the strength of T cell I. Berberich, and F. Berberich-Siebelt. 2014. Follicular regulatory T cells control antigen receptor binding. Nat. Immunol. 10: 375–384. NFAT transcription factors promote the generation of follicular helper T cells in response to acute viral infection

Gustavo J. Martinez, Joyce K. Hu, Renata M. Pereira, Jordan S. Crampton, Susan Togher, Nicholas Bild, Shane Crotty, and Anjana Rao

SUPPLEMENTAL DATA A B NFAT1 NFAT2 NFAT1,2 C WT WT -deficient -deficient -deficient 2 NFAT1 KO ** ** NFAT2 KO 28.3 22.4 33.7 26.1 40

NFAT DKO + (%) 1 1 20 CD45 . (O.D. 450 nm) 0 CD45.1 LCMV-specific IgG 2 3 4 0 10 10 10 CD4 serum dilution

D E NFAT1 NFAT2 NFAT1,2 F WT -deficient -deficient -deficient ** * 80 *** **** *

45.7 42.3 68.7 28 **** **** 8 50.9 53.4 21.3 22.9 M- (%) CR5 + 40 A X L

4 C

S SLAM

CD45.1+ (10^6) 0 0 CXCR5 G H I ** *** 3 ** * **** ** *

*** *** * 0.8 2 2 (10^6) CR5 + CR5 + X M- (10^6) X 0.4 1 C A

C 1

L Bcl6+ S KLRG1+ (10^6) 0 0 0

J K L WT NFAT1,2-deficient NFAT1 NFAT2 NFAT1,2 5x10^5 2x10^6 WT -deficient -deficient -deficient 10 20.9 2.04 1 + (%) 1 0.1

CD45 . 0.01 CD44 0.001 CXCR5 PD-1 CD4 5

5x10^

WT DKO 5x10^5 DKO 2x10^6 Figure S1. NFAT1, 2-deficient mice show defective Tfh and GC responses in vivo. SMARTA CD45.1+ WT or NFAT-deficient cells were adoptively transferred into congenic CD45.2+ C57BL/6 congenic mice. One day later, mice were infected with LCMV Armstrong (2x10^5 PFU). (A) On day 8, serum was collected and LCMV-specific IgG determined by ELISA. (B-L) On day 8 (B-J) or day 3 (K-L), spleens were harvested, and expression of cell surface markers was determined by flow cytometry. (B-C) Frequency of CD45.1+ adoptively transferred cells gated on CD4+ cells. (D) Total CD4+ CD45.1+ cells in spleen. (E-F) Frequency of CXCR5- and SLAM-expressing cells gated on adoptively transferred cells. (G-I) Total number of cells per spleen were determined. (J) Dot plots show CD44 and CD4 markers on adoptively transferred CD45.1+ cells. (K) Frequency of CD45.1+ adoptively transferred cells (gated on CD4+ cells). (L) Expression of CXCR5 and PD-1 on CD4+ CD45.1+ adoptively transferred cells is shown. Contour plots show a representative mouse in each group. Statistical analysis was performed using t-test. *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.0001. A representative experiment out of two is shown. A Anti-CD3+ anti-CD28 3h 6h 16h WT NFAT1,2-deficient WT Naive Events CD25 Events CD69 Events CD40L Events PD-1 Events SLAM B 15 0.4 10 mRN A mRN A

d 0.2 40 l

c 5 d d C P 0 0.0 WT NFAT1,2 -deficient

0.8

0.4 Slam mRN A 0.0 WT NFAT1,2 -deficient Figure S2. T cell-intrinsic defect in cell surface receptor expression in NFAT1, 2-de cient mice in vitro. WT or NFAT1, 2-de cient naïve T cells were stimulated with anti-CD3 and anti-CD28 for the indicated time points. (A) Expression of CD25, CD69, CD40L, PD-1 and SLAM was determined by ow cytometry. A representative example of two independent experiments with two biological replicates each is shown. (B) qPCR was performed after 24 hours of stimulation to determine mRNA levels of the indicated receptors. Data was noramlized to L32 ribosomal protein levels and expression in naive CD4+ T cells was set as 1. Two biological replicates were performed.