Differential Requirements for Tcf1 Long Isoforms in CD8+ and CD4+ T Cell Responses to Acute Viral Infection

Differential Requirements for Tcf1 Long Isoforms in CD8 + and CD4+ T Cell Responses to Acute Viral Infection

This information is current as Jodi A. Gullicksrud, Fengyin Li, Shaojun Xing, Zhouhao of September 26, 2021. Zeng, Weiqun Peng, Vladimir P. Badovinac, John T. Harty and Hai-Hui Xue J Immunol 2017; 199:911-919; Prepublished online 26 June 2017;

doi: 10.4049/jimmunol.1700595 Downloaded from http://www.jimmunol.org/content/199/3/911

Supplementary http://www.jimmunol.org/content/suppl/2017/06/24/jimmunol.170059 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 40 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/199/3/911.full#ref-list-1

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Differential Requirements for Tcf1 Long Isoforms in CD8+ and CD4+ T Cell Responses to Acute Viral Infection

Jodi A. Gullicksrud,*,† Fengyin Li,* Shaojun Xing,* Zhouhao Zeng,‡ Weiqun Peng,‡ Vladimir P. Badovinac,*,†,x John T. Harty,*,† and Hai-Hui Xue*,†

In response to acute viral infection, activated naive T cells give rise to effector T cells that clear the pathogen and memory T cells that persist long-term and provide heightened protection. T cell factor 1 (Tcf1) is essential for several of these differentiation processes. Tcf1 is expressed in multiple isoforms, with all isoforms sharing the same HDAC and DNA-binding domains and the long isoforms containing a unique N-terminal b-catenin–interacting domain. In this study, we speciﬁcally ablated Tcf1 long isoforms in mice, while retaining expression of Tcf1 short isoforms. During CD8+ T cell responses, Tcf1 long isoforms were dispensable for generating cytotoxic CD8+ effector T cells and maintaining memory CD8+ T cell pool size, but they contributed to optimal maturation of central memory CD8+ T cells and their optimal secondary expansion in a recall response. In contrast, Tcf1 long Downloaded from isoforms were required for differentiation of T follicular helper (TFH) cells, but not TH1 effectors, elicited by viral infection. Although Tcf1 short isoforms adequately supported Bcl6 and ICOS expression in TFH cells, Tcf1 long isoforms remained important for suppressing the expression of Blimp1 and TH1-associated genes and for positively regulating Id3 to restrain germinal center TFH cell differentiation. Furthermore, formation of memory TH1 and memory TFH cells strongly depended on Tcf1 long isoforms. These data reveal that Tcf1 long and short isoforms have distinct, yet complementary, functions and may represent an evolutionarily conserved means to ensure proper programming of CD8+ and CD4+ T cell responses to viral http://www.jimmunol.org/ infection. The Journal of Immunology, 2017, 199: 911–919.

n response to a viral infection, naive T cells that recognize T cells predominantly differentiate into two types of effectors: their cognate Ags become activated, expand proliﬁcally, and TH1 cells that secrete IFN-g and enhance the cytotoxicity of ef- + differentiate into effector T cells equipped with diverse fector CD8 T cells, and T follicular helper (TFH) cells that secrete I + functions. Effector CD8 T cells acquire cytotoxic functions and IL-4 and IL-21 and provide essential help to Ab-producing B cells eliminate virus-infected cells (1, 2). In contrast, activated CD4+ (3–5). Effector T cells are heterogeneous and contain subsets that have different kinetics of contraction following the peak responses by guest on September 26, 2021 and, hence, different potential to give rise to memory T cells. *Department of Microbiology, University of Iowa, Iowa City, IA 52242; Although memory CD8+ T cells are more durable than memory † Interdisciplinary Immunology Graduate Program, University of Iowa, Iowa City, CD4+ T cells, both populations contribute to enhanced responses IA 52242; ‡Department of Physics, The George Washington University, Washington, DC 20052; and xDepartment of Pathology, Carver College of Medicine, University of upon rechallenge with the same Ag. Iowa, Iowa City, IA 52242 Differentiation of effector T cells and their transition to memory ORCIDs: 0000-0003-0777-2196 (S.X.); 0000-0003-3180-2439 (V.P.B.); 0000-0001- T cells are coordinated by transcriptional regulators (5, 6). In 7266-2802 (J.T.H.); 0000-0002-9163-7669 (H.-H.X.). activated CD8+ T cells, T-bet and Blimp1 transcription factors, as Received for publication January 6, 2017. Accepted for publication June 7, 2017. well as the Id2 cofactor, are potently induced and critically reg- This work was supported by the National Institute of Health (Grants AI112579 and ulate CD8+ effector cell differentiation and acquisition of cyto- AI115149 to H.-H.X., Grant AI042767 to J.T.H., Grant AI119160 to H.-H.X. and V.P.B., Grants AI114543 and GM113961 to V.P.B., Grant AI121080 to H.-H.X. and W.P., toxic functions (7–9). In contrast, Eomes, Bcl6, and Id3 promote and Grant AI113806 to W.P.) and the U.S. Department of Veterans Affairs (Grant I01 the transition and survival of memory CD8+ T cells (9–11). In BX002903 to H.-H.X.). J.A.G. is the recipient of a University of Iowa Presidential + Graduate Research Fellowship, a T32 Predoctoral Training Grant in Immunology CD4 T cells, T-bet and Bcl6 are the lineage-specifying master (AI007485), and the Ballard and Seashore Dissertation Fellowship. The ﬂow cytom- regulators for TH1 and TFH cells, respectively (3, 4), and induction etry core facility at the University of Iowa is supported by the Carver College of of Blimp1 and Id2 favors T 1 differentiation at the expense of the Medicine, the Holden Comprehensive Cancer Center, and the Iowa City Veteran’s H Administration Medical Center, as well as by grants from the National Cancer Insti- TFH lineage (12, 13). In contrast to advances in elucidating the tute (P30CA086862) and the National Center for Research Resources, National transcriptional networks in CD4+ lineage differentiation at the ef- Institutes of Health (S10 OD016199). fector phase, little is known about transcriptional regulation in- The RNA sequencing data presented in this article have been submitted to the Gene + Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number volved in memory CD4 T cell formation and functions. GSE98347. T cell factor 1 (Tcf1) has been known as a transcription factor Address correspondence and reprint requests to Dr. Hai-Hui Xue, University of Iowa, acting downstream of the Wnt pathway and can interact with 51 Newton Road, BSB 3-772, Iowa City, IA 52242. E-mail address: hai-hui-xue@uiowa. b-catenin coactivator. b-catenin is posttranslationally regulated edu and stabilized by Wnt- or PG-derived signals. In addition to its The online version of this article contains supplemental material. essential role for T cell development (14, 15), recent studies have Abbreviations used in this article: dpi, day postinfection; GC, germinal center; revealed that Tcf1 critically regulates mature T cell responses. LCMV, lymphocytic choriomeningitis virus; LCMV-Arm, LCMV Armstrong strain; LCMV-Cl13, LCMV clone 13; LN, lymph node; RNA-Seq, RNA sequencing; Tcf1, Although loss of Tcf1 modestly diminished production of effector + T cell factor 1; TCM, central memory T; TEM, effector memory T; TFH, T follicular CD8 T cells, Tcf1 is essential for maturation, longevity, and helper; WT, wild-type. secondary expansion of memory CD8+ T cells (16, 17). In acti- + Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 vated CD4 T cells, Tcf1 appears to restrain TH1 differentiation www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700595 912 Tcf1 LONG ISOFORMS IN MATURE T CELL RESPONSES

+ 3 4 + + in vitro (18) but is essential for activating the TFH program by characterization of CD8 T cell responses, 2 10 Va2 P14 CD8 + acting upstream of Bcl6 (12, 19, 20). As a result of differential T cells were injected i.v. into CD45.1 B6.SJL recipient mice and infected 3 5 promoter usage and alternative splicing, multiple Tcf1 isoforms can i.p. with 2 10 PFU lymphocytic choriomeningitis virus (LCMV) Armstrong strain (LCMV-Arm). For characterization of CD4+ T cell re- be detected in T cells (21). All isoforms contain a C-terminal HMG sponses, 2 3 105 Va2+ SMARTA CD4+ T cells were injected i.v. into DNA-binding domain, which can also interact with Groucho/ CD45.1+ hosts and infected similarly. For examining CD4+ T cell re- 2 2 2 transducin-like enhancer of split corepressor proteins, and a sponses under a competitive condition, 5000 p45+/ or p45 / SMARTA + newly discovered HDAC domain (22). The Tcf1 long isoforms (p45 CD4 T cells were mixed with the same number of wild-type (WT) CD45.1+CD45.2+ SMARTA CD4+ T cells and adoptively transferred. and p42) contain an N-terminal b-catenin–binding domain, whereas At $40 d postinfection (dpi), the immune mice were infected i.v. with 2 3 the Tcf1 short isoforms (p33 and p30) lack this domain and, hence, 106 PFU LCMV clone 13 (LCMV-Cl13) to elicit secondary CD8+ or CD4+ cannot interact with b-catenin. Most of the previous loss-of- T cell responses. function studies ablated all Tcf1 isoforms. The specific require- Immunoblotting ments for the Tcf1 long isoforms in effector and memory T cell lo + + + responses have not been elucidated. CD44 CD62L naive P14 CD8 T cells or SMARTA CD4 T cells were sort purified from the spleens of P14- or SMARTA-transgenic p45+/2 or In this study, we specifically ablated Tcf1 long isoforms in 2/2 p45 mice. Cell lysates were prepared from the sorted cells, resolved on mouse and coupled this model with MHC class I– and MHC class SDS-PAGE, and immunoblotted with an anti-Tcf1 Ab (C46C7; Cell Sig- II–restricted TCR transgenes to dissect the roles of Tcf1 isoforms naling Technology), as previously described (23). in regulating mature CD8+ and CD4+ T cell responses. Our data RNA sequencing and quantitative RT-PCR showed that Tcf1 long isoforms were dispensable for generation +/2 2/2 + + p45 or p45 SMARTA CD4 T cells were adoptively transferred, and Downloaded from of cytotoxic CD8 effector T cells and TH1 cells in response to recipients were infected as described above. On 8 dpi, PD-1lo CXCR5+ viral infection, whereas Tcf1 short isoforms were sufficient for + + + cells were sort purified from CD45.2 CD4 splenocytes. The total RNA maintaining memory CD8 T cell pool size. In contrast, Tcf1 long was extracted and subjected to RNA sequencing (RNA-Seq) analysis, as isoforms remained critical for TFH differentiation at the effector previously described (19). The RNA-Seq data were deposited at the Gene Expression Omnibus under accession number GSE98347 (https://www. phase and for generation of memory TH1 and memory TFH cells. This study reveals a functional complementation among Tcf1 ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse98347). For validation of key gene expression changes, the RNA was reverse transcribed and ana- isoforms in programming mature T cell responses and further lyzed by quantitative PCR, as previously described (19). The pri- http://www.jimmunol.org/ suggests that an inter-isoform coordination is necessary to con- mers used for Id2 were 59-CATCAGCATCCTGTCCTTGC-39 and stitute a fully functional gene product. 59-GTGTTCTCCTGGTGAAATGG-39, and those for Id3 were 59- ATCTCCCGATCCAGACAGC-39 and 59-GAGAGAGGGTCCCAG- AGTCC-39. Materials and Methods Mice Chromatin immunoprecipitation + p45+/GFP (i.e., p45+/2) mice were generated as previously described (23) WT SMARTA CD4 T cells were adoptively transferred, and the recipients 2 hi and cross-bred with P14 or SMARTA TCR transgenes to generate P14 were infected as above. On 8 dpi, CXCR5 SLAM TH1 cells and 2/2 2/2 + lo + + p45 or SMARTA p45 mice, respectively. Our recent analysis of CXCR5 SLAM TFH cells were sort purified from CD45.2 CD4 thymocyte development in p45-targeted mice showed no detectable dif- splenocytes and cross-linked, and the resulting chromatin fragments were by guest on September 26, 2021 ferences between p45+/+ and p45+/2 animals (23), suggesting that het- immunoprecipitated with an anti-Tcf1 Ab (C46C7) or normal rabbit IgG, erozygosity of the p45-targeted allele does not significantly impact T cell as previously described (19). Enriched Tcf1 binding at the Id3 transcrip- biology. As such, we used T cells from p45+/2 mice as controls in this tion start site was determined by quantitative PCR. The primers are 59- study, and the Tcf1-EGFP reporter embedded in the p45+/2 allele was used GTAAGCTTTCTCCTGGCGC-39 and 59-CCGACTGAACCCTAAGCCTT-39. + + to mark Tcf1 expression in CD4 and CD8 T cells at different stages of Statistical analyses responses to viral infection. C57BL/6J and B6.SJL mice were from The Jackson Laboratory. All mice analyzed were 6–12 wk of age, and both Data from multiple experiments were analyzed using a Student t test with a genders were used without randomization or blinding. All mouse experi- two-tailed distribution, assuming equal sample variance when p . 0.05 ments were performed under protocols approved by the Institutional An- with an F test. When p # 0.05 with an F test comparing the variances, the imal Use and Care Committees of the University of Iowa. Welch correction was applied. Flow cytometry Results Single-cell suspensions were prepared from the spleen, lymph nodes (LNs), Tcf1 long isoforms are dispensable for CD8+ effector or PBLs and surface or intracellularly stained as described (19). The following fluorochrome-conjugated Abs were used: anti-CD8 (53-6.7), anti- T cell differentiation CD4 (RM4-5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti–PD-1 (J43), Tcf1 proteins are encoded by the Tcf7 gene, with long isoforms anti-CD45.1 (A20), anti-CD45.2 (104), anti-ICOS (C398.4A), anti–IFN-g produced from transcripts initiated at exon 1 and short isoforms (XMG1.2), anti–TNF-a (MP6-XT22), anti-Eomes (Dan11mag), anti–T-bet (eBio4B10), anti–IL-2 (JES6-5H4), anti–IL-7Ra (eBio17B7), and anti- produced from transcripts initiated at exon 3. Previously, we have KLRG1 (2F1) (all from eBioscience); anti-Bcl6 (K112-91; BD Biosci- generated a Tcf1-EGFP reporter mouse strain, in which we ences); anti-human granzyme B (FGB12) and corresponding isotype inserted an IRES-EGFP cassette into the first intron of Tcf7 gene control (both from Invitrogen/Life Technologies); and anti-SLAM (TC15- (25). In front of the cassette, we placed an En2 splice acceptor that 12F12.2; BioLegend). For detection of CXCR5, a three-step staining forced the splicing of exon 1 to this cassette instead of exon 2. protocol was used with unconjugated anti-CXCR5 (2G8; BD Biosciences) (19). For detection of Bcl6, surface-stained cells were fixed and per- Thus, mice homozygous for the Tcf1-EGFP reporter alleles failed meabilized with the Foxp3/Transcription Factor Staining Buffer Set to produce Tcf1 long isoforms (p45 and p42); for simplicity, this 2 2 (eBioscience), followed by incubation with a fluorochrome-conjugated Ab strain is called p45 / in this article. To assess the importance of or isotype control. Peptide-stimulated cytokine production and detection Tcf1 long isoforms (p45 and p42) in CD8+ T cell responses to by intracellular staining were as described (24). Data were collected on a viral infection, we crossed p452/2 mice with the P14 TCR FACSVerse (BD Biosciences) and were analyzed with FlowJo software (TreeStar). transgene, which encodes a TCR specific for the gp33 epitope of LCMV. As expected, p452/2 P14 CD8+ T cells were deficient for Adoptive transfer and viral infection Tcf1 long isoforms but retained the expression of Tcf1 short Naive CD45.2+ P14 CD8+ T cells or SMARTA CD4+ T cells were isolated isoforms, albeit at a reduced level (Fig. 1A). Loss of Tcf1 long from the LNs of P14- or SMARTA-transgenic p45+/2 or p452/2 mice. For isoforms moderately reduced the frequency of P14 CD8+ T cells The Journal of Immunology 913 in the LNs, but it did not result in aberrant activation of peripheral cells, but it is partially retained in the KLRG1loCD127+ subset CD8+ T cells, as determined by the CD62Lhi phenotype (Fig. 1B). (Fig. 1E). This is consistent with our previous observations that To facilitate functional characterization on a per-cell basis, we Tcf1 and Lef1 are required for generation of the KLRG1loCD127+ adoptively transferred the same numbers (2 3 104) of p45+/2 and subset (26). However, the KLRG1loCD127+ subset in p452/2 p452/2 P14 CD8+ T cells into congenic recipients, followed by CD8+ effectors was only modestly reduced in frequency and infection with LCMV-Arm. As tracked in PBLs, p45+/2 and numbers (Fig. 1F). p45+/2 and p452/2 CD8+ effectors exhibited p452/2 CD45.2+ CD8+ effector T cells showed a similar fre- a similar capacity to produce IFN-g, TNF-a, and granzyme B quency among Ag-experienced CD11ahiCD8int T cells (Fig. 1C). (Fig. 1G, 1H); in comparison, these functional aspects are not In the spleens, similar numbers of p45+/2 and p452/2 CD45.2+ affected by the loss of all Tcf1 isoforms, as shown in our previous CD8+ effector T cells were generated at the peak response (i.e., 8 dpi) study (16). Collectively, these data indicate that Tcf1 long iso- (Fig. 1D). In comparison, loss of all Tcf1 isoforms diminishes forms are dispensable for clonal expansion and cytotoxic func- CD8+ effector T cells by ∼50% in PBLs and spleens (16). CD8+ tions of CD8+ effector T cells. effector T cells are heterogeneous, with KLRG1hiCD1272 cells as + lo Tcf1 long isoforms contribute to maturation of central memory terminally differentiated effector CD8 T cells, and the KLRG1 + CD127+ subset showing increased potential to give rise to memory CD8 T cells CD8+ T cells (6). As observed in p45+/2CD8+ T cells, Tcf1-EGFP Following the peak response to LCMV infection, p45+/2 and p452/2 reporter expression is potently diminished in KLRG1hiCD1272 CD45.2+CD8+ effector T cells contracted similarly (Fig. 1C), and, Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Tcf1 long isoforms are dispensable for effector CD8+ T cell responses. (A) Detection of Tcf1 isoforms by immunoblotting in naive CD8+ T cells. CD62LhiCD44loVa2+CD8+ cells were sorted from the LNs of p45+/2 or p452/2 P14 transgenic mice and immunoblotted with an anti-Tcf1 or b-actin Ab. (B) Characterization of naive P14 CD8+ T cells. LNs from uninfected p45+/2 or p452/2 P14 transgenic mice were surface stained to determine the frequency (left panel) and immunophenotype (right panel) of Va2+CD8+ T cells (n = 4 from four experiments). (C) Kinetics of CD8+ T cell responses in PBLs. CD45.2+ p45+/2 or p452/2 P14 CD8+ T cells (2 3 104 each) were adoptively transferred into CD45.1+ recipients, followed by infection with LCMV- Arm. The frequency of CD45.2+ CD8+ effector T cells in CD11ahiCD8dim Ag-experienced CD8+ T cells were monitored in the PBLs on the indicated days postinfection (n = 10–20 from three experiments). (D) Numbers of P14 CD8+ effector T cells detected in the spleen on 8 dpi (n = 8 from three experiments). (E) Detection of the Tcf1-EGFP reporter in p45+/2 P14 CD8+ effector subsets; the geometric mean ﬂuorescent intensity (gMFI) of EGFP is shown. The dashed line marks EGFP expression in naive CD8+ T cells for a direct comparison. (F) Detection of P14 CD8+ effector subsets. CD45.2+CD8+ effector T cells in the spleen (8 dpi) were analyzed for KLRG1 and CD127 expression, and the percentages of KLRG1hiCD1272 and KLRG1loCD127+ subsets are shown in representative contour plots (left panel). Cumulative data on the frequency and numbers of the KLRG1loCD127+ subset are summarized in the bar graphs (n = 10 from three experiments) (middle and right panels). (G) Cytokine production by P14 CD8+ effector T cells. Splenocytes (8 dpi) were incubated with gp33 peptide for 5 h, and CD45.2+CD8+ T cells were intracellularly stained to detect IFN-g and TNF-a. The percentage of each population is shown in representative contour plots from three experiments. (H) Granzyme B expression in P14 CD8+ effector T cells detected by intracellular staining. gMFI of Granzyme B (GzmB) is shown in the representative line graph. The dashed line represents isotype control staining. Data in bar graphs are mean 6 SEM. *p , 0.05, Student t test. 914 Tcf1 LONG ISOFORMS IN MATURE T CELL RESPONSES at $60 dpi, similar numbers of p45+/2 and p452/2 memory T cells showed a modest increase in granzyme B production CD8+ T cells were found in the recipient spleens (Fig. 2A). compared with p45+/2 cells (Fig. 2F). These observations indi- Memory CD8+ T cells in the secondary lymphoid organs are cate that loss of Tcf1 long isoforms does not affect the pool size 2 + heterogeneous, with the CD62L subset as effector memory T of memory CD8 T cells, but predisposes them to a TEM cell + (TEM) cells that reside in nonlymphoid tissues and the CD62L phenotype. + subset as central memory T (TCM) cells that are capable of self- To assess the recall response by memory CD8 T cells, we renewal and conferring long-term protection (27). Compared rechallenged the immune recipient mice with LCMV-Cl13. As with CD8+ effector T cells, most of the memory CD8+ T cells detected on day 5 postinfection, p45+/2 and p452/2 memory upregulated Tcf1, and the CD62L+ subset expressed higher CD8+ T cells underwent substantial secondary expansion, although levels of Tcf1, as determined by Tcf1-EGFP reporter activity the numbers of p452/2 secondary CD8+ effector T cells in the (Fig. 2B versus Fig. 1E). Our previous studies showed that de- spleens were modestly decreased (Fig. 2G), which is in contrast letion of all Tcf1 isoforms almost completely abrogated gener- to the severely impaired secondary expansion in the absence + + ation of CD62L TCM cells and caused a substantial decrease in of all Tcf1 isoforms (16). Similar to CD8 effector T cells in the Eomes expression (16). In the p452/2 memory CD8+ T cell pool, primary response, the secondary CD8+ effector T cells were het- CD62L+ cells were generated, but at diminished frequency and erogeneous, with the KLRG1loCD127+ subset retaining substan- numbers (Fig. 2C). In addition, p452/2 memory CD8+ T cells tially higher Tcf1-EGFP reporter activity compared with the showed a modest reduction in Eomes expression, accompanied KLRG1hiCD1272 subset (Fig. 2H). Loss of Tcf1 long isoforms by a modest increase in T-bet expression (Fig. 2D). With regard did not noticeably perturb the subset composition or IFN-g and 2 2 to the functional aspects, fewer p45 / memory CD8+ T cells TNF-a production in the secondary effectors (Fig. 2H, data not Downloaded from were capable of producing IL-2 compared with p45+/2 cells shown). Collectively, these data suggest that Tcf1 long isoforms (Fig. 2E), whereas they showed similar production of IFN-g and contribute to optimal maturation of TCM cells and optimal sec- TNF-a (data not shown). Additionally, p452/2 memory CD8+ ondary expansion in a recall response. It is also noteworthy that http://www.jimmunol.org/ by guest on September 26, 2021

+ FIGURE 2. Tcf1 long isoforms contribute to optimal TCM maturation and secondary expansion. (A) Numbers of memory P14 CD8 T cells detected in the spleen at $60 dpi (n = 5–6 from two experiments). (B) Detection of Tcf1-EGFP reporter in CD62L2 and CD62L+ memory CD8+ T cell subsets. CD45.2+ memory CD8+ T cells from p45+/2 mice were analyzed for EGFP expression, and values in the line graphs denote EGFP geometric mean + + + fluorescent intensity (gMFI). (C) Detection of P14 CD8 TCM cells. CD45.2 memory CD8 T cells in the spleen were analyzed for CD62L expression, and + + the percentages of CD62L TCM cells are shown in the representative line graph (left panel). Cumulative data on the frequency and numbers of CD62L + TCM cells are summarized in bar graphs (n = 5–6 from two experiments) (middle and right panels). (D) T-bet and Eomes expression in memory P14 CD8 T cells detected by intranuclear staining. gMFI of T-bet or Eomes is shown in representative line graphs (left panels), and cumulative data are shown in bar graphs (right panels) (for T-bet, n = 2–3 from one experiment; for Eomes, n = 5–6 from two experiments). Dashed lines represent isotype control staining. (E) Cytokine production by memory P14 CD8+ T cells. Splenocytes were harvested at $60 dpi and incubated with gp33 peptide, followed by intracellular detection of IFN-g and IL-2 in CD45.2+CD8+ T cells. The percentage of each population is shown in representative contour plots (n = 8 from three experiments; left panel) and that of IFN-g and IL-2 double producers is summarized in the bar graph (right panel). (F) Granzyme B expression in memory P14 CD8+ T cells detected by intracellular staining. gMFI of granzyme B is shown in a representative line graph (left panel), and cumulative data are shown in the bar graph (right panel) (n = 8 from three experiments). The dashed line represents isotype control staining. (G) Numbers of secondary CD8+ effector T cells. The immune CD45.1+ recipients were infected with LCMV-Cl13, and the numbers of CD45.2+CD8+ secondary effector T cells were determined in the spleen at 5 dpi (n = 5 from two experiments). (H) Characterization of P14 CD8+ secondary effector T cells. CD45.2+CD8+ secondary effector T cells in the spleen were surface stained; the percentages of KLRG1hiCD1272 and KLRG1loCD127+ subsets are shown in representative contour plots (left panel). Both subsets were analyzed for Tcf1-EGFP reporter expression in p45+/2 recipients, with EGFP gMFI shown (right panel) (n = 5–6 from two experiments). Data in bar graphs are mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, Student t test. The Journal of Immunology 915 reduced expression of Tcf1 short isoforms in p452/2 CD8+ T cells ation defects observed in the absence of all Tcf1 isoforms (12, 19, may be a contributing factor as well. 20). In contrast, p452/2 and p45+/2 SMARTA CD4+ T cells gave rise to similar amounts of effector T 1 cells (Fig. 3D). These data Tcf1 long isoforms are required for T cell differentiation at H FH suggest that Tcf1 long isoforms are important in directing ef- the effector phase of CD4+ T cell responses fective TFH cell differentiation but are dispensable for TH1 cell We (19) and other investigators (12, 20) have recently demon- responses. strated that deletion of all Tcf1 isoforms impairs TFH cell differ- In the experiments above, we transferred a relatively large entiation in response to viral infections. To determine the number of SMARTA CD4+ T cells so as to obtain sufficient cells + requirements for Tcf1 long isoforms during CD4 T cell re- for RNA-Seq and memory-phase analysis (see below). To exclude 2/2 sponses, we crossed the p45 strain with the SMARTA TCR a secondary impact derived from a higher precursor frequency and transgene, which encodes a TCR specific for the gp61 epitope of demonstrate an intrinsic requirement for Tcf1 long isoforms, we 2/2 LCMV. By immunoblotting, we validated that p45 SMARTA transferred a small number of p45+/2 or p452/2 SMARTA CD4+ + CD4 T cells were deficient for Tcf1 long isoforms but retained T cells in a competitive setting. Specifically, we mixed CD45.2+ expression of the Tcf1 short isoforms, also at reduced levels p45+/2 or p452/2 SMARTA CD4+ T cells (as test cells) with the + (Fig. 3A). Although the frequency of SMARTA CD4 T cells in same number of CD45.1+CD45.2+ WT SMARTA CD4+ T cells (as 2/2 2/2 the LNs was diminished in p45 mice, the majority of p45 competitors) (Fig. 4A). We then adoptively transferred a total of + lo hi SMARTA CD4 T cells exhibited a CD44 CD62L phenotype, 10,000 SMARTA CD4+ T cells (5000 each of test and competitor suggesting that loss of Tcf1 long isoforms did not result in aber- cells) into CD45.1+ recipients, followed by LCMV-Arm infection. + 2

+/ Downloaded from rant activation of peripheral CD4 T cells (Fig. 3B). On 8 dpi, within the TH1 compartment, the p45 /WT ratio was To investigate the regulatory roles of Tcf1 long isoforms on a largely maintained at 1:1, and the p452/2/WT ratio was modestly 3 2/2 per-cell basis, we adoptively transferred the same numbers (2 decreased (Fig. 4B, 4C). However, within the TFH cells, the p45 5 +/2 2/2 + 10 ) of p45 and p45 SMARTA CD4 T cells from LNs into /WT ratio was reduced by .5-fold compared with the p45+/2/WT congenic recipients, followed by infection with LCMV-Arm. At ratio (Fig. 4B, 4C). Thus, analysis under the competitive setting at a + +/2 the peak of effector CD4 T cell responses (i.e., 8 dpi), p45 and lower precursor frequency further corroborated a speciﬁc, intrinsic 2/2 + p45 SMARTA CD4 T cells expanded to similar levels, requirement for Tcf1 long isoforms in TFH cell differentiation. http://www.jimmunol.org/ showing similar frequency and numbers of effector CD4+ T cells in the spleen of recipient mice (Fig. 3C). Viral infection elicits two Tcf1 long isoforms regulate a unique subset of genes in the + TFH program major forms of effector CD4 T cells, TH1 cells and TFH cells, which exhibit CXCR52SLAMhi and CXCR5+SLAMlo pheno- We previously showed that Tcf1 critically regulates activation of +/2 + + types, respectively. p45 effector CD4 T cells contained ap- the TFH program in primed CD4 T cells, and deletion of all Tcf1 proximately equal amounts of TH1 cells and TFH cells; in contrast, isoforms diminishes the expression of Bcl6 and ICOS (19). 2/2 + +/2 2/2 p45 effector CD4 T cells were strongly skewed toward the However, p45 and p45 TFH cells showed similar Bcl6 and 2/2 + TH1 phenotype (Fig. 3D). As a result, p45 SMARTA CD4 ICOS expression (Fig. 5A, 5B), suggesting that Tcf1 short iso-

T cells generated markedly fewer TFH cells compared with their forms, although reduced in protein expression, are sufﬁcient for by guest on September 26, 2021 +/2 p45 counterparts (Fig. 3D), similar to the TFH cell differenti- supporting Bcl6 and ICOS induction. TFH cell differentiation is

+ hi lo FIGURE 3. Tcf1 long isoforms regulate optimal TFH differentiation. (A) Detection of Tcf1 isoforms by immunoblotting in CD4 T cells. CD62L CD44 Va2+CD4+ cells were sorted from the LNs of p45+/2 or p452/2 SMARTA-transgenic mice and immunoblotted as in Fig. 1A. (B) Characterization of naive SMARTA CD4+ T cells. LNs were isolated from p45+/2 or p452/2 SMARTA-transgenic mice and surface stained to detect the frequency (left panel) and immunophenotype (right panel) of Va2+CD4+ T cells (n = 8 from eight experiments). (C) Detection of effector SMARTA CD4+ T cells at the peak response. CD45.2+ p45+/2 or p452/2 SMARTA CD4+ T cells from LNs (2 3 105 each) were adoptively transferred into CD45.1+ recipients, followed by infection with LCMV-Arm. On 8 dpi, CD45.2+CD4+ T cells were detected and enumerated in the spleens of recipient mice (n = 8 from three experiments). + + + (D) Detection of TH1 and TFH effector CD4 T cells. CD45.2 effector CD4 T cells (as in C) were analyzed for CXCR5 and SLAM expression. 2 hi + lo The percentages of CXCR5 SLAM TH1 cells and CXCR5 SLAM TFH cells are shown in representative contour plots (left panel). Cumulative data on the frequency and numbers of each subset are summarized in bar graphs (n = 5–8 from two or three experiments). Data are mean 6 SEM. **p , 0.01, ***p , 0.001, Student t test. 916 Tcf1 LONG ISOFORMS IN MATURE T CELL RESPONSES

+ + +/2 FIGURE 4. CD4 T cells lacking Tcf1 long isoforms are less competitive in generating TFH cells. (A) Relative abundance of CD45.2 test cells (p45 or p452/2) and CD45.1+CD45.2+ competitors (WT CD4+ T cells) in the cell mixture used in competitive transfer experiments. (B) Relative contribution of test + 2 hi + lo and competitor SMARTA CD4 T cells to TH1 and TFH responses. CXCR5 SLAM TH1 cells and CXCR5 SLAM TFH cells were detected in the recipient spleens on 8 dpi (left panel). Within each subset, the percentages of test and competitor cells were determined and are shown in representative contour plots (right panel). (C) Cumulative data for test/competitor ratio within TH1orTFH subsets at the effector phase (8 dpi). Data are from two experiments (n = 5–6). Data are mean 6 SEM. *p , 0.05, ***p , 0.001, Student t test. Downloaded from known as a step-wise process (4), with TFH lineage–committed abundance in TFH cells (,5 FPKMs); in contrast, Id3 was highly cells undergoing further maturation and becoming germinal center abundant in TFH cells (132 FPKMs on average). As validated in 2/2 (GC) TFH cells, as marked by high PD-1 expression. Although Fig. 5G, Id3 expression was diminished in p45 TFH cells, 2/2 hi there was an overall reduction in p45 TFH cells, PD-1 whereas Id2 was not noticeably altered. Using chromatin immu- + CXCR5 GC TFH cells were detected at a similar frequency and noprecipitation, we further found that Tcf1 exhibited enriched 2 2 2 +/ / + http://www.jimmunol.org/ number in p45 and p45 effector SMARTA CD4 T cells binding to the transcription start site of the Id3 gene in TFH cells (Fig. 5C). Thus, loss of Tcf1 long isoforms showed a stronger but not in TH1 cells (Fig. 5H), establishing Id3 as a direct target impact on non-GC TFH cells, causing subset composition changes gene of Tcf1 in TFH cells. It has been recently shown that Id3 within the TFH population. In comparison, non-GC TFH cells and deficiency results in the marked accumulation of GC TFH cells GC TFH cells were diminished in the absence of all T cell isoforms (13). These data collectively suggest that another important (19). Therefore, Tcf1 short isoforms appear to be adequate for function of Tcf1 long isoforms appears to be restraining GC TFH promoting GC TFH cell maturation. cell differentiation by positive regulation of Id3, possibly aided by 2 2 To search for downstream genes that account for defective p45 / suppression of Maf and AP-1 factors. T cell differentiation, we specifically sorted p45+/2 and p452/2 FH Tcf1 long isoforms are essential for formation of memory T 1 by guest on September 26, 2021 PD-1loCXCR5+ non-GC T cells at 8 dpi and performed RNA-Seq H FH and T cells analysis. Using $2-fold expression changes and a false discovery FH + rate q value , 0.05, we found 89 downregulated genes and 119 We continued to track SMARTA CD4 T cell responses during the 2 2 2/2 +/2 . / upregulated genes in p45 TFH cells compared with their p45 memory phase (i.e., 40 dpi) and found significantly fewer p45 + +/2 counterparts (Supplemental Table I). Transcriptomic changes have memory CD4 T cells compared with their p45 counterparts + been profiled in TFH cells lacking all Tcf1 isoforms or both Tcf1 (Fig. 6A). Compared with CD4 effector T cells, the number of +/2 + and Lef1 proteins (19, 20). Although differences in the specific cell p45 memory CD4 T cells was a result of an ∼20-fold con- 2/2 + subsets analyzed and the profiling platforms used among these traction; in contrast, the number of p45 memory CD4 Tcells + studies prevented a meaningful and fair comparison of the specific represented an almost 200-fold contraction of corresponding CD4 genes affected, the number of differentially expressed genes was effector T cells (Fig. 6B). Phenotypic analysis using SLAM and 2/2 2/2 + substantially lower in p45 TFH cells than in Tcf1/Lef1-deficient CXCR5 expression further revealed that p45 memory CD4 TFH cells, suggesting a relatively limited impact of the deficiency in T cells were strongly skewed toward TH1 cells, with very few lo + Tcf1 long isoforms on the TFH transcriptome. Functional annotation SLAM CXCR5 memory TFH cells detected (Fig. 6C). Despite the 2/2 using the DAVID Bioinformatics Resources revealed a partial ac- lineage bias in the absence of Tcf1 long isoforms, p45 memory 2/2 tivation of the TH1 program in p45 TFH cells, including upreg- TH1 and memory TFH cells both showed more potent contraction 2 ulation of Prdm1, Il2ra,andIfng (encoding Blimp1 transcription than their p45+/ counterparts (Fig. 6C). As detected by the Tcf1- +/2 + factor, CD25, and IFN-g, respectively) (Fig. 5D). Increased ex- GFP reporter in p45 CD4 T cells, although Tcf1 was down- pression of Prdm1 was validated by quantitative RT-PCR (Fig. 5G). regulated in TH1 effectors, it was upregulated in memory TH1cells, + Blimp1 is known to promote activated CD4 TcellstoaTH1cell whereas the Tcf1-GFP reporter was detected at high levels in ef- fate at the expense of TFH cell differentiation (28), and Tcf1 and fector and memory TFH cells (Fig. 6D). We next assessed the recall Bcl6 co-occupy an intron 3 regulatory region in the Prdm1 gene response by memory CD4+ T cells by rechallenging the immune locus (19, 29). Our data suggest that Tcf1 long isoforms are critical recipient mice with LCMV-Cl13. As detected on day 5 postinfec- 2/2 + in repressing the TH1 potential in TFH cells. tion, p45 secondary CD4 effectors were greatly diminished in The upregulated genes included Maf, Fos, and Fosb (Fig. 5E). numbers and remained strongly skewed toward TH1cells,con- Maf is necessary for promoting TFH cell differentiation and in- taining very few TFH cells (Fig. 6E). These data suggest that Tcf1 ducing IL-21 (30, 31). AP-1 factors, including Fos and FosB, may long isoforms are essential for generation of memory TH1cells,as contribute to recruiting Bcl6 to its target genes in TFH cells (29). well as memory TFH cells, and the ensuing secondary responses. It Among the downregulated genes, a prominent subset was involved should be noted that reduced expression of Tcf1 short isoforms in 2 2 in transcriptional regulation, including Irf6, Nfe2, Sox4, and Id3 p45 / CD4+ T cells may also have contributed to defects in (Fig. 5F). Importantly, the first three genes were detected at low memory CD4+ Tcells. The Journal of Immunology 917 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

+/2 2/2 FIGURE 5. Tcf1 long isoforms are essential for regulating a subset of TFH transcriptional program. Bcl6 (A) and ICOS (B) in p45 and p45 + lo CXCR5 SLAM TFH cells (8 dpi). Geometric mean ﬂuorescent intensity (gMFI) of each protein is shown in representative line graphs (left panels), and +/2 2/2 cumulative data on the relative expression between p45 and p45 TFH cells are shown in bar graphs (right panels) (n = 11 from four experiments). (C) hi + + + Detection of PD-1 CXCR5 GC TFH cells (8 dpi). The percentages of GC TFH cells among CD45.2 SMARTA CD4 T cells are shown in representative contour plots (left panel), and cumulative data in the bar graphs (middle and right panels) are from two experiments (n = 6). (D–F) Heat maps of select +/2 2/2 lo + + + differentially expressed genes. p45 and p45 PD-1 CXCR5 non-GC TFH cells were sorted from splenic CD45.2 SMARTA CD4 T cells in the recipients on 8 dpi. Two biological replicates were collected for each genotype and analyzed with RNA-Seq. (G) Detection of select gene expression in non- +/2 GC TFH cells by quantitative RT-PCR. The cells were sorted using the same strategy as above. For each gene of interest, its expression in p45 cells was set as 1, and its relative expression in p452/2 cells was calculated accordingly. Data are from two independent experiments (n = 3–4). (H) Detection of enriched Tcf1 binding. WT TH1 and TFH cells were sorted and subjected to chromatin immunoprecipitation analysis. The relative enrichment at the control Hprt locus and Id3 transcription start site by an anti-Tcf1 Ab was determined by quantitative PCR. Data are from two independent experiments with each sample measured in duplicates or triplicates. Data in bar graphs are mean 6 SEM. *p , 0.05, **p , 0.01, Student t test.

Discussion to reveal isoform-specific requirements for CD8+ and CD4+ T cell Tcf1 is known for its versatile functions in regulating T cell de- differentiation in response to viral infection. + velopment and mature CD8+ and CD4+ T cell responses to in- At the peak of the CD8 T cell response to LCMV, the termi- hi 2 + fections (15, 32). Most of the previous studies involved deletion of nally differentiated KLRG1 CD127 CD8 effector T cells all Tcf1 isoforms. Until recently, Tcf1 short isoforms have been exhibit strong downregulation of all Tcf1 isoforms. Therefore, it generally considered to function as dominant negatives for the is not surprising that loss of Tcf1 long isoforms did not result in a full-length molecule, because of their inability to interact with the noticeable impact on the numbers or functions of CD8+ effector coactivator b-catenin. Using a new mouse model in which gen- T cells. Recent studies examined early events of CD8+ T cell eration of transcripts for Tcf1 long isoforms was abrogated, we activation in vivo and found that Tcf1 expression is retained at achieved specific retention of only Tcf1 short isoforms. We have high levels in all daughter cells during the first three or four di- recently showed that Tcf1 short isoforms are sufficient to support visions. In the ensuing divisions, as a result of asymmetrical developing thymocytes in traversing through maturation stages signaling via the PI3K pathway, most daughter cells obliterate without causing obvious developmental blocks, whereas Tcf1 long Tcf1 expression and become differentiated CD8+ effector T cells. isoforms remain necessary for optimal thymic survival (23). Meanwhile, some daughter cells maintain higher Tcf1 expression, Coupled with MHC class I– or MHC class II–restricted TCR acquire “self-renewal” capacity, and give rise to memory CD8+ transgenes, in this study we used Tcf1 long isoform–targeted mice T cells (33, 34). In this scenario, retention of Tcf1 short isoforms 918 Tcf1 LONG ISOFORMS IN MATURE T CELL RESPONSES

+ FIGURE 6. Tcf1 long isoforms are critical for formation of memory TH1 and memory TFH cells. (A) Detection of memory SMARTA CD4 T cells. Adoptive transfer and infection were performed as in Fig. 3. On .40 dpi, CD45.2+CD4+ T cells were detected and enumerated in the spleens of recipient Downloaded from mice (n = 7–8 from three experiments). (B) Population fold changes due to contraction. p45+/2 and p452/2 SMARTA CD4+ T cells were enumerated at the peak (8 dpi) and memory (.40 dpi) phases in response to viral infection, and fold reduction between the two phases was calculated (n = 8 from three + + experiments). (C) Detection of memory TH1 and TFH T cells. CD45.2 CD4 T cells were analyzed for CXCR5 and SLAM expression. The percentages of 2 hi + lo CXCR5 SLAM memory TH1 and CXCR5 SLAM memory TFH cells are shown in representative contour plots (left panel). Cumulative data on the numbers of TH1 and TFH cells at effector and memory phases are summarized in line graphs (middle and right panels), and the fold reduction during 2 hi contraction of each cell type was calculated (n = 8 from three experiments). (D) Detection of the Tcf1-EGFP reporter in CXCR5 SLAM TH1 cells and + lo +/2 http://www.jimmunol.org/ CXCR5 SLAM TFH cells. p45 TH1 and TFH cells at the effector (8 dpi, left) and memory (.40 dpi, right) phases were analyzed for EGFP expression, and values in the line graphs denote EGFP geometric mean ﬂuorescent intensity. (E) Detection of secondary TH1 and TFH effector responses. The immune + + CD45.1 recipients were infected with LCMV-Cl13, and 5 d later, CD45.2 secondary TH1 and TFH effector T cells were detected in the spleen. The frequency of each subset is shown in representative contour plots (left panel), and the cumulative data on cell numbers are summarized in bar graphs (middle and right panels) (n = 6–8 from three experiments). Data are mean 6 SEM. **p , 0.01, ***p , 0.001, Student t test.

hi alone appears to be adequate to maintain the self-renewing Tcf1 impact on the gene expression profile of TFH cells compared with population, despite the diminished amount of protein expression, deleting all Tcf1 isoforms, similar to what we have observed in because the memory CD8+ T cell pool was similar between the early thymocytes (23). Apparently, Tcf1 short isoforms, even at a by guest on September 26, 2021 p45+/2 and p452/2 genotypes. It is currently unknown whether the reduced amount of protein, appeared to be sufficient to support hi Tcf1 population at the effector phase is responsible for genera- induction of Bcl6 and ICOS in TFH cells. Differentiation of non tion of both TEM and TCM cells. Loss of all Tcf1 isoforms almost GC TFH cells to GC TFH cells is a controlled process, and a recent 2/2 completely abrogated TCM maturation (16); in contrast, p45 study identified Id3 as a restraining factor (13). Our data dem- TCM cells were indeed generated, albeit at a modestly lower fre- onstrated that Tcf1 directly bound to the Id3 gene locus, and Tcf1 quency. These data suggest that Tcf1 long isoforms remain nec- short isoforms alone were not sufficient to support optimal Id3 2/2 essary to promote optimal TCM maturation. expression in TFH cells. Paradoxically, p45 TFH cells upregu- + Similar to the terminally differentiated CD8 effector T cells, lated Maf, another key TFH cell regulator, and AP-1 factors that 2 hi + CXCR5 SLAM TH1 cells at the effector phase of CD4 T cell potentially cooperate with Bcl6. These gene expression changes responses potently downregulated Tcf1 expression, and loss of may all indirectly contribute to promoting GC TFH cell differen- Tcf1 long isoforms was rather inconsequential for generation of tiation, in addition to direct activation of Bcl6 transcription by + 2/2 TH1 effectors. Unlike memory CD8 T cells, whose numbers were Tcf1 itself. p45 TFH cells also exhibited inappropriate activa- 2/2 unaffected by the loss of Tcf1 long isoforms, p45 memory TH1 tion of TH1 cell–associated genes, including Prdm1, Il2ra, and cells were greatly diminished. It has also been shown that after Ifng, representing another unique requirement for Tcf1 long iso- + four or five divisions during early CD4 T cell activation, Tcf1 forms in protecting TFH lineage integrity (36). + expression is specifically retained in a subset of activated CD4 Recent studies have provided strong evidence that TH1 and TFH T cells as a result of asymmetrical signaling (35). The Tcf1hi cells are largely committed to their respective lineages at the ef- CD4+ cells seem to possess “self-renewal” capacity as well, fector and memory phases (37, 38). Loss of Tcf1 long isoforms + + although whether they are direct precursors of memory CD4 impaired TFH cell differentiation at the effector phase of CD4 T cells remains to be conclusively demonstrated. Nonetheless, T cell responses and also caused a stronger contraction, resulting formation of memory TH1 cells is strongly dependent on Tcf1 in even fewer memory TFH cells. It has been suggested that long isoforms. stronger TCR stimulation during CD4+ effector differentiation is Another major CD4+ T cell response to viral infection is dif- associated with a predisposition to form memory CD4+ T cells ferentiation to the TFH lineage, and deletion of all Tcf1 isoforms (39). Increased TCR signaling, resulting from extended dwell time severely impairs generation of TFH cells and GC TFH cells as a of TCR with peptide–MHC class II complex, is considered to + result of insufficient induction of Bcl6 and ICOS (12, 19, 20). This favor TFH cell differentiation (40). Because memory CD4 T cell study shows that loss of Tcf1 long isoforms greatly diminished the formation and TFH cell differentiation were affected by the loss of total number of TFH cells but only modestly affected GC TFH cells. Tcf1 long isoforms, an interesting possibility is that Tcf1 Transcriptomic analysis, coupled with phenotypic characteriza- contributes to regulation of TCR signaling outcome, without tion, revealed that ablating Tcf1 long isoforms had a rather limited affecting TCR avidity or dwell time per se. In light of our recent The Journal of Immunology 919

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