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 β-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 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 infection.
In response to a viral infection, naive T cells that recognize their cognate Ags become activated, expand prolifically, and differentiate into effector T cells equipped with diverse functions. Effector CD8+ T cells acquire cytotoxic functions and eliminate virus-infected cells (1, 2). In contrast, activated CD4+ T cells predominantly differentiate into two types of effectors: TH1 cells that secrete IFN-γ and enhance the cytotoxicity of effector CD8+ T cells, and T follicular helper (TFH) cells that secrete IL-4 and IL-21 and provide essential help to Ab-producing B cells (3–5). Effector T cells are heterogeneous and contain subsets that have different kinetics of contraction following the peak responses and, hence, different potential to give rise to memory T cells. Although memory CD8+ T cells are more durable than memory CD4+ T cells, both populations contribute to enhanced responses upon rechallenge with the same Ag.
Differentiation of effector T cells and their transition to memory T cells are coordinated by transcriptional regulators (5, 6). In activated CD8+ T cells, T-bet and Blimp1 transcription factors, as well as the Id2 cofactor, are potently induced and critically regulate CD8+ effector cell differentiation and acquisition of cytotoxic functions (7–9). In contrast, Eomes, Bcl6, and Id3 promote the transition and survival of memory CD8+ T cells (9–11). In CD4+ T cells, T-bet and Bcl6 are the lineage-specifying master regulators for TH1 and TFH cells, respectively (3, 4), and induction of Blimp1 and Id2 favors TH1 differentiation at the expense of the TFH lineage (12, 13). In contrast to advances in elucidating the transcriptional networks in CD4+ lineage differentiation at the effector phase, little is known about transcriptional regulation involved in memory CD4+ T cell formation and functions.
T cell factor 1 (Tcf1) has been known as a transcription factor acting downstream of the Wnt pathway and can interact with β-catenin coactivator. β-catenin is posttranslationally regulated and stabilized by Wnt- or PG-derived signals. In addition to its essential role for T cell development (14, 15), recent studies have revealed that Tcf1 critically regulates mature T cell responses. Although loss of Tcf1 modestly diminished production of effector CD8+ T cells, Tcf1 is essential for maturation, longevity, and secondary expansion of memory CD8+ T cells (16, 17). In activated CD4+ T cells, Tcf1 appears to restrain TH1 differentiation in vitro (18) but is essential for activating the TFH program by acting upstream of Bcl6 (12, 19, 20). As a result of differential promoter usage and alternative splicing, multiple Tcf1 isoforms can be detected in T cells (21). All isoforms contain a C-terminal HMG DNA-binding domain, which can also interact with Groucho/transducin-like enhancer of split corepressor proteins, and a newly discovered HDAC domain (22). The Tcf1 long isoforms (p45 and p42) contain an N-terminal β-catenin–binding domain, whereas the Tcf1 short isoforms (p33 and p30) lack this domain and, hence, cannot interact with β-catenin. Most of the previous loss-of-function studies ablated all Tcf1 isoforms. The specific requirements for the Tcf1 long isoforms in effector and memory T cell responses have not been elucidated.
In this study, we specifically ablated Tcf1 long isoforms in mouse and coupled this model with MHC class I– and MHC class II–restricted TCR transgenes to dissect the roles of Tcf1 isoforms in regulating mature CD8+ and CD4+ T cell responses. Our data showed that Tcf1 long isoforms were dispensable for generation of cytotoxic CD8+ effector T cells and TH1 cells in response to viral infection, whereas Tcf1 short isoforms were sufficient for maintaining memory CD8+ T cell pool size. In contrast, Tcf1 long isoforms remained critical for TFH differentiation at the effector phase and for generation of memory TH1 and memory TFH cells. This study reveals a functional complementation among Tcf1 isoforms in programming mature T cell responses and further suggests that an inter-isoform coordination is necessary to constitute a fully functional gene product.
Materials and Methods
p45+/GFP (i.e., p45+/−) mice were generated as previously described (23) and cross-bred with P14 or SMARTA TCR transgenes to generate P14 p45−/− or SMARTA p45−/− mice, respectively. Our recent analysis of thymocyte development in p45-targeted mice showed no detectable differences between p45+/+ and p45+/− animals (23), suggesting that heterozygosity of the p45-targeted allele does not significantly impact T cell biology. As such, we used T cells from p45+/− mice as controls in this study, and the Tcf1-EGFP reporter embedded in the p45+/− allele was used to mark Tcf1 expression in CD4+ and CD8+ T cells at different stages of 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 genders were used without randomization or blinding. All mouse experiments were performed under protocols approved by the Institutional Animal Use and Care Committees of the University of Iowa.
Single-cell suspensions were prepared from the spleen, lymph nodes (LNs), or PBLs and surface or intracellularly stained as described (19). The following fluorochrome-conjugated Abs were used: anti-CD8 (53-6.7), anti-CD4 (RM4-5), anti-CD44 (IM7), anti-CD62L (MEL-14), anti–PD-1 (J43), anti-CD45.1 (A20), anti-CD45.2 (104), anti-ICOS (C398.4A), anti–IFN-γ (XMG1.2), anti–TNF-α (MP6-XT22), anti-Eomes (Dan11mag), anti–T-bet (eBio4B10), anti–IL-2 (JES6-5H4), anti–IL-7Rα (eBio17B7), and anti-KLRG1 (2F1) (all from eBioscience); anti-Bcl6 (K112-91; BD Biosciences); anti-human granzyme B (FGB12) and corresponding isotype control (both from Invitrogen/Life Technologies); and anti-SLAM (TC15-12F12.2; BioLegend). For detection of CXCR5, a three-step staining protocol was used with unconjugated anti-CXCR5 (2G8; BD Biosciences) (19). For detection of Bcl6, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), followed by incubation with a fluorochrome-conjugated Ab or isotype control. Peptide-stimulated cytokine production and detection by intracellular staining were as described (24). Data were collected on a FACSVerse (BD Biosciences) and were analyzed with FlowJo software (TreeStar).
Adoptive transfer and viral infection
Naive CD45.2+ P14 CD8+ T cells or SMARTA CD4+ T cells were isolated from the LNs of P14- or SMARTA-transgenic p45+/− or p45−/− mice. For characterization of CD8+ T cell responses, 2 × 104 Vα2+ P14 CD8+ T cells were injected i.v. into CD45.1+ B6.SJL recipient mice and infected i.p. with 2 × 105 PFU lymphocytic choriomeningitis virus (LCMV) Armstrong strain (LCMV-Arm). For characterization of CD4+ T cell responses, 2 × 105 Vα2+ SMARTA CD4+ T cells were injected i.v. into CD45.1+ hosts and infected similarly. For examining CD4+ T cell responses under a competitive condition, 5000 p45+/− or p45−/− SMARTA CD4+ T cells were mixed with the same number of wild-type (WT) CD45.1+CD45.2+ SMARTA CD4+ T cells and adoptively transferred. At ≥40 d postinfection (dpi), the immune mice were infected i.v. with 2 × 106 PFU LCMV clone 13 (LCMV-Cl13) to elicit secondary CD8+ or CD4+ T cell responses.
CD44loCD62L+ naive P14 CD8+ T cells or SMARTA CD4+ T cells were sort purified from the spleens of P14- or SMARTA-transgenic p45+/− or p45−/− mice. Cell lysates were prepared from the sorted cells, resolved on SDS-PAGE, and immunoblotted with an anti-Tcf1 Ab (C46C7; Cell Signaling Technology), as previously described (23).
RNA sequencing and quantitative RT-PCR
p45+/− or p45−/− SMARTA CD4+ T cells were adoptively transferred, and recipients were infected as described above. On 8 dpi, PD-1lo CXCR5+ cells were sort purified from CD45.2+CD4+ splenocytes. The total RNA was extracted and subjected to RNA sequencing (RNA-Seq) analysis, as previously described (19). The RNA-Seq data were deposited at the Gene Expression Omnibus under accession number GSE98347 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse98347). For validation of key gene expression changes, the RNA was reverse transcribed and analyzed by quantitative PCR, as previously described (19). The primers used for Id2 were 5′-CATCAGCATCCTGTCCTTGC-3′ and 5′-GTGTTCTCCTGGTGAAATGG-3′, and those for Id3 were 5′-ATCTCCCGATCCAGACAGC-3′ and 5′-GAGAGAGGGTCCCAGAGTCC-3′.
WT SMARTA CD4+ T cells were adoptively transferred, and the recipients were infected as above. On 8 dpi, CXCR5−SLAMhi TH1 cells and CXCR5+SLAMlo TFH cells were sort purified from CD45.2+CD4+ splenocytes and cross-linked, and the resulting chromatin fragments were immunoprecipitated with an anti-Tcf1 Ab (C46C7) or normal rabbit IgG, as previously described (19). Enriched Tcf1 binding at the Id3 transcription start site was determined by quantitative PCR. The primers are 5′-GTAAGCTTTCTCCTGGCGC-3′ and 5′-CCGACTGAACCCTAAGCCTT-3′.
Data from multiple experiments were analyzed using a Student t test with a two-tailed distribution, assuming equal sample variance when p > 0.05 with an F test. When p ≤ 0.05 with an F test comparing the variances, the Welch correction was applied.
Tcf1 long isoforms are dispensable for CD8+ effector T cell differentiation
Tcf1 proteins are encoded by the Tcf7 gene, with long isoforms produced from transcripts initiated at exon 1 and short isoforms produced from transcripts initiated at exon 3. Previously, we have generated a Tcf1-EGFP reporter mouse strain, in which we inserted an IRES-EGFP cassette into the first intron of Tcf7 gene (25). In front of the cassette, we placed an En2 splice acceptor that forced the splicing of exon 1 to this cassette instead of exon 2. Thus, mice homozygous for the Tcf1-EGFP reporter alleles failed to produce Tcf1 long isoforms (p45 and p42); for simplicity, this strain is called p45−/− in this article. To assess the importance of Tcf1 long isoforms (p45 and p42) in CD8+ T cell responses to viral infection, we crossed p45−/− mice with the P14 TCR transgene, which encodes a TCR specific for the gp33 epitope of LCMV. As expected, p45−/− P14 CD8+ T cells were deficient for Tcf1 long isoforms but retained the expression of Tcf1 short isoforms, albeit at a reduced level (Fig. 1A). Loss of Tcf1 long isoforms moderately reduced the frequency of P14 CD8+ T cells in the LNs, but it did not result in aberrant activation of peripheral CD8+ T cells, as determined by the CD62Lhi phenotype (Fig. 1B).
To facilitate functional characterization on a per-cell basis, we adoptively transferred the same numbers (2 × 104) of p45+/− and p45−/− P14 CD8+ T cells into congenic recipients, followed by infection with LCMV-Arm. As tracked in PBLs, p45+/− and p45−/− CD45.2+ CD8+ effector T cells showed a similar frequency among Ag-experienced CD11ahiCD8int T cells (Fig. 1C). In the spleens, similar numbers of p45+/− and p45−/− CD45.2+CD8+ effector T cells were generated at the peak response (i.e., 8 dpi) (Fig. 1D). In comparison, loss of all Tcf1 isoforms diminishes CD8+ effector T cells by ∼50% in PBLs and spleens (16). CD8+ effector T cells are heterogeneous, with KLRG1hiCD127− cells as terminally differentiated effector CD8+ T cells, and the KLRG1loCD127+ subset showing increased potential to give rise to memory CD8+ T cells (6). As observed in p45+/−CD8+ T cells, Tcf1-EGFP reporter expression is potently diminished in KLRG1hiCD127− cells, but it is partially retained in the KLRG1loCD127+ subset (Fig. 1E). This is consistent with our previous observations that Tcf1 and Lef1 are required for generation of the KLRG1loCD127+ subset (26). However, the KLRG1loCD127+ subset in p45−/−CD8+ effectors was only modestly reduced in frequency and numbers (Fig. 1F). p45+/− and p45−/− CD8+ effectors exhibited a similar capacity to produce IFN-γ, TNF-α, and granzyme B (Fig. 1G, 1H); in comparison, these functional aspects are not affected by the loss of all Tcf1 isoforms, as shown in our previous study (16). Collectively, these data indicate that Tcf1 long isoforms are dispensable for clonal expansion and cytotoxic functions of CD8+ effector T cells.
Tcf1 long isoforms contribute to maturation of central memory CD8+ T cells
Following the peak response to LCMV infection, p45+/− and p45−/− CD45.2+CD8+ effector T cells contracted similarly (Fig. 1C), and, at ≥60 dpi, similar numbers of p45+/− and p45−/− memory CD8+ T cells were found in the recipient spleens (Fig. 2A). Memory CD8+ T cells in the secondary lymphoid organs are heterogeneous, with the CD62L− subset as effector memory T (TEM) cells that reside in nonlymphoid tissues and the CD62L+ subset as central memory T (TCM) cells that are capable of self-renewal and conferring long-term protection (27). Compared with CD8+ effector T cells, most of the memory CD8+ T cells upregulated Tcf1, and the CD62L+ subset expressed higher levels of Tcf1, as determined by Tcf1-EGFP reporter activity (Fig. 2B versus Fig. 1E). Our previous studies showed that deletion of all Tcf1 isoforms almost completely abrogated generation of CD62L+ TCM cells and caused a substantial decrease in Eomes expression (16). In the p45−/− memory CD8+ T cell pool, CD62L+ cells were generated, but at diminished frequency and numbers (Fig. 2C). In addition, p45−/− memory CD8+ T cells showed a modest reduction in Eomes expression, accompanied by a modest increase in T-bet expression (Fig. 2D). With regard to the functional aspects, fewer p45−/− memory CD8+ T cells were capable of producing IL-2 compared with p45+/− cells (Fig. 2E), whereas they showed similar production of IFN-γ and TNF-α (data not shown). Additionally, p45−/− memory CD8+ T cells showed a modest increase in granzyme B production compared with p45+/− cells (Fig. 2F). These observations indicate that loss of Tcf1 long isoforms does not affect the pool size of memory CD8+ T cells, but predisposes them to a TEM cell phenotype.
To assess the recall response by memory CD8+ T cells, we rechallenged the immune recipient mice with LCMV-Cl13. As detected on day 5 postinfection, p45+/− and p45−/− memory CD8+ T cells underwent substantial secondary expansion, although the numbers of p45−/− secondary CD8+ effector T cells in the spleens were modestly decreased (Fig. 2G), which is in contrast to the severely impaired secondary expansion in the absence of all Tcf1 isoforms (16). Similar to CD8+ effector T cells in the primary response, the secondary CD8+ effector T cells were heterogeneous, with the KLRG1loCD127+ subset retaining substantially higher Tcf1-EGFP reporter activity compared with the KLRG1hiCD127− subset (Fig. 2H). Loss of Tcf1 long isoforms did not noticeably perturb the subset composition or IFN-γ and TNF-α production in the secondary effectors (Fig. 2H, data not shown). Collectively, these data suggest that Tcf1 long isoforms contribute to optimal maturation of TCM cells and optimal secondary expansion in a recall response. It is also noteworthy that reduced expression of Tcf1 short isoforms in p45−/− CD8+ T cells may be a contributing factor as well.
Tcf1 long isoforms are required for TFH cell differentiation at the effector phase of CD4+ T cell responses
We (19) and other investigators (12, 20) have recently demonstrated that deletion of all Tcf1 isoforms impairs TFH cell differentiation in response to viral infections. To determine the requirements for Tcf1 long isoforms during CD4+ T cell responses, we crossed the p45−/− strain with the SMARTA TCR transgene, which encodes a TCR specific for the gp61 epitope of LCMV. By immunoblotting, we validated that p45−/− SMARTA CD4+ T cells were deficient for Tcf1 long isoforms but retained expression of the Tcf1 short isoforms, also at reduced levels (Fig. 3A). Although the frequency of SMARTA CD4+ T cells in the LNs was diminished in p45−/− mice, the majority of p45−/− SMARTA CD4+ T cells exhibited a CD44loCD62Lhi phenotype, suggesting that loss of Tcf1 long isoforms did not result in aberrant activation of peripheral CD4+ T cells (Fig. 3B).
To investigate the regulatory roles of Tcf1 long isoforms on a per-cell basis, we adoptively transferred the same numbers (2 × 105) of p45+/− and p45−/− SMARTA CD4+ T cells from LNs into congenic recipients, followed by infection with LCMV-Arm. At the peak of effector CD4+ T cell responses (i.e., 8 dpi), p45+/− and p45−/− SMARTA CD4+ T cells expanded to similar levels, showing similar frequency and numbers of effector CD4+ T cells in the spleen of recipient mice (Fig. 3C). Viral infection elicits two major forms of effector CD4+ T cells, TH1 cells and TFH cells, which exhibit CXCR5−SLAMhi and CXCR5+SLAMlo phenotypes, respectively. p45+/− effector CD4+ T cells contained approximately equal amounts of TH1 cells and TFH cells; in contrast, p45−/− effector CD4+ T cells were strongly skewed toward the TH1 phenotype (Fig. 3D). As a result, p45−/− SMARTA CD4+ T cells generated markedly fewer TFH cells compared with their p45+/− counterparts (Fig. 3D), similar to the TFH cell differentiation defects observed in the absence of all Tcf1 isoforms (12, 19, 20). In contrast, p45−/− and p45+/− SMARTA CD4+ T cells gave rise to similar amounts of effector TH1 cells (Fig. 3D). These data suggest that Tcf1 long isoforms are important in directing effective TFH cell differentiation but are dispensable for TH1 cell responses.
In the experiments above, we transferred a relatively large number of SMARTA CD4+ T cells so as to obtain sufficient cells for RNA-Seq and memory-phase analysis (see below). To exclude a secondary impact derived from a higher precursor frequency and demonstrate an intrinsic requirement for Tcf1 long isoforms, we transferred a small number of p45+/− or p45−/− SMARTA CD4+ T cells in a competitive setting. Specifically, we mixed CD45.2+ p45+/− or p45−/− SMARTA CD4+ T cells (as test cells) with the same number of CD45.1+CD45.2+ WT SMARTA CD4+ T cells (as competitors) (Fig. 4A). We then adoptively transferred a total of 10,000 SMARTA CD4+ T cells (5000 each of test and competitor cells) into CD45.1+ recipients, followed by LCMV-Arm infection. On 8 dpi, within the TH1 compartment, the p45+/−/WT ratio was largely maintained at 1:1, and the p45−/−/WT ratio was modestly decreased (Fig. 4B, 4C). However, within the TFH cells, the p45−/−/WT ratio was reduced by >5-fold compared with the p45+/−/WT ratio (Fig. 4B, 4C). Thus, analysis under the competitive setting at a lower precursor frequency further corroborated a specific, intrinsic requirement for Tcf1 long isoforms in TFH cell differentiation.
Tcf1 long isoforms regulate a unique subset of genes in the TFH program
We previously showed that Tcf1 critically regulates activation of the TFH program in primed CD4+ T cells, and deletion of all Tcf1 isoforms diminishes the expression of Bcl6 and ICOS (19). However, p45+/− and p45−/− TFH cells showed similar Bcl6 and ICOS expression (Fig. 5A, 5B), suggesting that Tcf1 short isoforms, although reduced in protein expression, are sufficient for supporting Bcl6 and ICOS induction. TFH cell differentiation is known as a step-wise process (4), with TFH lineage–committed cells undergoing further maturation and becoming germinal center (GC) TFH cells, as marked by high PD-1 expression. Although there was an overall reduction in p45−/− TFH cells, PD-1hiCXCR5+ GC TFH cells were detected at a similar frequency and number in p45+/− and p45−/− effector SMARTA CD4+ T cells (Fig. 5C). Thus, loss of Tcf1 long isoforms showed a stronger impact on non-GC TFH cells, causing subset composition changes within the TFH population. In comparison, non-GC TFH cells and GC TFH cells were diminished in the absence of all T cell isoforms (19). Therefore, Tcf1 short isoforms appear to be adequate for promoting GC TFH cell maturation.
To search for downstream genes that account for defective p45−/− TFH cell differentiation, we specifically sorted p45+/− and p45−/− PD-1loCXCR5+ non-GC TFH cells at 8 dpi and performed RNA-Seq analysis. Using ≥2-fold expression changes and a false discovery rate q value < 0.05, we found 89 downregulated genes and 119 upregulated genes in p45−/− TFH cells compared with their p45+/− counterparts (Supplemental Table I). Transcriptomic changes have been profiled in TFH cells lacking all Tcf1 isoforms or both Tcf1 and Lef1 proteins (19, 20). Although differences in the specific cell subsets analyzed and the profiling platforms used among these studies prevented a meaningful and fair comparison of the specific genes affected, the number of differentially expressed genes was substantially lower in p45−/− TFH cells than in Tcf1/Lef1-deficient TFH cells, suggesting a relatively limited impact of the deficiency in Tcf1 long isoforms on the TFH transcriptome. Functional annotation using the DAVID Bioinformatics Resources revealed a partial activation of the TH1 program in p45−/− TFH cells, including upregulation of Prdm1, Il2ra, and Ifng (encoding Blimp1 transcription factor, CD25, and IFN-γ, respectively) (Fig. 5D). Increased expression of Prdm1 was validated by quantitative RT-PCR (Fig. 5G). Blimp1 is known to promote activated CD4+ T cells to a TH1 cell fate at the expense of TFH cell differentiation (28), and Tcf1 and Bcl6 co-occupy an intron 3 regulatory region in the Prdm1 gene locus (19, 29). Our data suggest that Tcf1 long isoforms are critical in repressing the TH1 potential in TFH cells.
The upregulated genes included Maf, Fos, and Fosb (Fig. 5E). Maf is necessary for promoting TFH cell differentiation and inducing IL-21 (30, 31). AP-1 factors, including Fos and FosB, may contribute to recruiting Bcl6 to its target genes in TFH cells (29). Among the downregulated genes, a prominent subset was involved in transcriptional regulation, including Irf6, Nfe2, Sox4, and Id3 (Fig. 5F). Importantly, the first three genes were detected at low abundance in TFH cells (<5 FPKMs); in contrast, Id3 was highly abundant in TFH cells (132 FPKMs on average). As validated in Fig. 5G, Id3 expression was diminished in p45−/− TFH cells, whereas Id2 was not noticeably altered. Using chromatin immunoprecipitation, we further found that Tcf1 exhibited enriched binding to the transcription start site of the Id3 gene in TFH cells but not in TH1 cells (Fig. 5H), establishing Id3 as a direct target gene of Tcf1 in TFH cells. It has been recently shown that Id3 deficiency results in the marked accumulation of GC TFH cells (13). These data collectively suggest that another important function of Tcf1 long isoforms appears to be restraining GC TFH cell differentiation by positive regulation of Id3, possibly aided by suppression of Maf and AP-1 factors.
Tcf1 long isoforms are essential for formation of memory TH1 and TFH cells
We continued to track SMARTA CD4+ T cell responses during the memory phase (i.e., >40 dpi) and found significantly fewer p45−/− memory CD4+ T cells compared with their p45+/− counterparts (Fig. 6A). Compared with CD4+ effector T cells, the number of p45+/− memory CD4+ T cells was a result of an ∼20-fold contraction; in contrast, the number of p45−/− memory CD4+ T cells represented an almost 200-fold contraction of corresponding CD4+ effector T cells (Fig. 6B). Phenotypic analysis using SLAM and CXCR5 expression further revealed that p45−/− memory CD4+ T cells were strongly skewed toward TH1 cells, with very few SLAMloCXCR5+ memory TFH cells detected (Fig. 6C). Despite the lineage bias in the absence of Tcf1 long isoforms, p45−/− memory TH1 and memory TFH cells both showed more potent contraction than their p45+/− counterparts (Fig. 6C). As detected by the Tcf1-GFP reporter in p45+/− CD4+ T cells, although Tcf1 was downregulated in TH1 effectors, it was upregulated in memory TH1 cells, whereas the Tcf1-GFP reporter was detected at high levels in effector and memory TFH cells (Fig. 6D). We next assessed the recall response by memory CD4+ T cells by rechallenging the immune recipient mice with LCMV-Cl13. As detected on day 5 postinfection, p45−/− secondary CD4+ effectors were greatly diminished in numbers and remained strongly skewed toward TH1 cells, containing very few TFH cells (Fig. 6E). These data suggest that Tcf1 long isoforms are essential for generation of memory TH1 cells, as well as memory TFH cells, and the ensuing secondary responses. It should be noted that reduced expression of Tcf1 short isoforms in p45−/− CD4+ T cells may also have contributed to defects in memory CD4+ T cells.
Tcf1 is known for its versatile functions in regulating T cell development and mature CD8+ and CD4+ T cell responses to infections (15, 32). Most of the previous studies involved deletion of all Tcf1 isoforms. Until recently, Tcf1 short isoforms have been generally considered to function as dominant negatives for the full-length molecule, because of their inability to interact with the coactivator β-catenin. Using a new mouse model in which generation of transcripts for Tcf1 long isoforms was abrogated, we achieved specific retention of only Tcf1 short isoforms. We have recently showed that Tcf1 short isoforms are sufficient to support developing thymocytes in traversing through maturation stages without causing obvious developmental blocks, whereas Tcf1 long isoforms remain necessary for optimal thymic survival (23). Coupled with MHC class I– or MHC class II–restricted TCR transgenes, in this study we used Tcf1 long isoform–targeted mice to reveal isoform-specific requirements for CD8+ and CD4+ T cell differentiation in response to viral infection.
At the peak of the CD8+ T cell response to LCMV, the terminally differentiated KLRG1hiCD127− CD8+ effector T cells exhibit strong downregulation of all Tcf1 isoforms. Therefore, it is not surprising that loss of Tcf1 long isoforms did not result in a noticeable impact on the numbers or functions of CD8+ effector T cells. Recent studies examined early events of CD8+ T cell activation in vivo and found that Tcf1 expression is retained at high levels in all daughter cells during the first three or four divisions. In the ensuing divisions, as a result of asymmetrical signaling via the PI3K pathway, most daughter cells obliterate Tcf1 expression and become differentiated CD8+ effector T cells. Meanwhile, some daughter cells maintain higher Tcf1 expression, acquire “self-renewal” capacity, and give rise to memory CD8+ T cells (33, 34). In this scenario, retention of Tcf1 short isoforms alone appears to be adequate to maintain the self-renewing Tcf1hi population, despite the diminished amount of protein expression, because the memory CD8+ T cell pool was similar between the p45+/− and p45−/− genotypes. It is currently unknown whether the Tcf1hi population at the effector phase is responsible for generation of both TEM and TCM cells. Loss of all Tcf1 isoforms almost completely abrogated TCM maturation (16); in contrast, p45−/− TCM cells were indeed generated, albeit at a modestly lower frequency. These data suggest that Tcf1 long isoforms remain necessary to promote optimal TCM maturation.
Similar to the terminally differentiated CD8+ effector T cells, CXCR5−SLAMhi TH1 cells at the effector phase of CD4+ T cell responses potently downregulated Tcf1 expression, and loss of Tcf1 long isoforms was rather inconsequential for generation of TH1 effectors. Unlike memory CD8+ T cells, whose numbers were unaffected by the loss of Tcf1 long isoforms, p45−/− memory TH1 cells were greatly diminished. It has also been shown that after four or five divisions during early CD4+ T cell activation, Tcf1 expression is specifically retained in a subset of activated CD4+ T cells as a result of asymmetrical signaling (35). The Tcf1hi CD4+ cells seem to possess “self-renewal” capacity as well, although whether they are direct precursors of memory CD4+ T cells remains to be conclusively demonstrated. Nonetheless, formation of memory TH1 cells is strongly dependent on Tcf1 long isoforms.
Another major CD4+ T cell response to viral infection is differentiation to the TFH lineage, and deletion of all Tcf1 isoforms severely impairs generation of TFH cells and GC TFH cells as a result of insufficient induction of Bcl6 and ICOS (12, 19, 20). This study shows that loss of Tcf1 long isoforms greatly diminished the total number of TFH cells but only modestly affected GC TFH cells. Transcriptomic analysis, coupled with phenotypic characterization, revealed that ablating Tcf1 long isoforms had a rather limited impact on the gene expression profile of TFH cells compared with deleting all Tcf1 isoforms, similar to what we have observed in early thymocytes (23). Apparently, Tcf1 short isoforms, even at a reduced amount of protein, appeared to be sufficient to support induction of Bcl6 and ICOS in TFH cells. Differentiation of non GC TFH cells to GC TFH cells is a controlled process, and a recent study identified Id3 as a restraining factor (13). Our data demonstrated that Tcf1 directly bound to the Id3 gene locus, and Tcf1 short isoforms alone were not sufficient to support optimal Id3 expression in TFH cells. Paradoxically, p45−/− TFH cells upregulated Maf, another key TFH cell regulator, and AP-1 factors that potentially cooperate with Bcl6. These gene expression changes may all indirectly contribute to promoting GC TFH cell differentiation, in addition to direct activation of Bcl6 transcription by Tcf1 itself. p45−/− TFH cells also exhibited inappropriate activation of TH1 cell–associated genes, including Prdm1, Il2ra, and Ifng, representing another unique requirement for Tcf1 long isoforms in protecting TFH lineage integrity (36).
Recent studies have provided strong evidence that TH1 and TFH cells are largely committed to their respective lineages at the effector and memory phases (37, 38). Loss of Tcf1 long isoforms impaired TFH cell differentiation at the effector phase of CD4+ T cell responses and also caused a stronger contraction, resulting in even fewer memory TFH cells. It has been suggested that stronger TCR stimulation during CD4+ effector differentiation is associated with a predisposition to form memory CD4+ T cells (39). Increased TCR signaling, resulting from extended dwell time of TCR with peptide–MHC class II complex, is considered to favor TFH cell differentiation (40). Because memory CD4+ T cell formation and TFH cell differentiation were affected by the loss of Tcf1 long isoforms, an interesting possibility is that Tcf1 contributes to regulation of TCR signaling outcome, without affecting TCR avidity or dwell time per se. In light of our recent finding that Tcf1 has intrinsic HDAC activity (22), Tcf1 may have global impact on the epigenome of T cells and, hence, affect their response to external stimuli. This scenario merits further investigation.
Collectively, our study identified differential requirements for Tcf1 long and short isoforms in mature CD8+ and CD4+ T cell responses. It is of interest that the expression levels of Tcf1 short isoforms in p45−/− cells were modestly reduced compared with those in control cells. Despite this, the Tcf1 short isoforms were adequate in supporting the memory CD8+ T cell pool and induction of Bcl6 and ICOS in TFH cells, indicating a positive regulatory role rather than being merely dominant negatives. In contrast, Tcf1 long isoforms, plus an appropriate amount of short isoforms, remained important for supporting TFH cell differentiation at the effector phase and memory TH1 and TFH cell formation. It remains to be determined whether the defects associated with p45−/− cells are solely ascribed to lack of interaction with β-catenin. Nevertheless, our study supports the notion that generation of multiple isoforms from one gene may represent an evolutionarily conserved fail-safe mechanism to ensure preservation of key biological functions.
We thank the University of Iowa Flow Cytometry Core facility (J. Fishbaugh, H. Vignes, M. Shey, and G. Rasmussen) for cell sorting and Igor Antoshechkin (California Institute of Technology) for RNA-Seq.
This work was supported by the National Institute of Health (Grants AI112579 and 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., and Grant AI113806 to W.P.) and the U.S. Department of Veterans Affairs (Grant I01 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 (AI007485), and the Ballard and Seashore Dissertation Fellowship. The flow cytometry core facility at the University of Iowa is supported by the Carver College of Medicine, the Holden Comprehensive Cancer Center, and the Iowa City Veteran’s Administration Medical Center, as well as by grants from the National Cancer Institute (P30CA086862) and the National Center for Research Resources, National Institutes of Health (S10 OD016199).
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 GSE98347.
The online version of this article contains supplemental material.
Abbreviations used in this article:
lymphocytic choriomeningitis virus
LCMV Armstrong strain
LCMV clone 13
T cell factor 1
central memory T
effector memory T
T follicular helper
The authors have no financial conflicts of interest.