T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage–defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.

This article is featured in Top Reads, p.1625

High-affinity Abs are central to immune protection elicited following vaccination. Germinal centers (GCs) are essential for the production of such Abs (1). T follicular helper (TFH) CD4 T cells provide essential help signals to Ag-specific B cells for the formation of GC B cells that culminate in the formation of long-lived Ab-secreting plasma cells and memory B cells (1). Differentiation of CD4 T cells into TFH cells is initiated upon interaction of activated CD4 T cells with Ag-presenting dendritic cells (1). Expression of the transcriptional repressor Bcl6 in CD4 T cells induces the TFH transcriptional program. Bcl6 directs TFH differentiation by suppressing alternative Th cell fates and by promoting expression of gene products associated with TFH cells and function (1–5). Other transcription factors such as LEF-1/TCF-1 (Lef1/Tcf7) (6–8), Batf (9, 10), Maf (11, 12), or E proteins (13, 14) play critical roles in supporting TFH differentiation. These transcription factors are balanced by inhibitors of TFH differentiation such as Blimp-1 (5) and Klf2 (2, 15).

Differentiation of CD4 T cells into a wide variety of helper subsets is tightly controlled by chromatin modifications (16). Transcription factors driving TFH differentiation have been thoroughly characterized, but the role of epigenetics in controlling this process remains mostly unexplored. TFH cells possess cell type–specific enhancers identified by monomethylation of histone 3 of lysine 4 (H3K4me1) and acetylation at lysine 27 of histone 3 (H3K27ac) (17, 18). Additionally, TFH cells possess unique patterns of chromatin accessibility (2, 17). All of the above suggest an epigenetic program specific for TFH cells.

Bcl6 plays a central role in establishing the TFH-specific epigenetic program. The middle domain of Bcl6, which recruits the corepressor MTA3, is necessary for full activity of Bcl6 and proper formation of TFH cells (19). Additionally, depletion of the corepressor NCoR1, or deletion of BCoR, impairs TFH formation (2, 20). TCF-1 recruits the histone methyltransferase EZH2 to activate Bcl6 expression (21), and the absence of EZH2 abrogates TFH differentiation (21, 22). The histone methyltransferase Nsd2 influences TFH differentiation by promoting expression of Bcl6 in the early stages of CD4 T cell activation (23). Bcl6, of course, is not the only TFH-associated target of chromatin regulators (CRs). The histone demethylase UTX impacts TFH differentiation by regulating Il6ra expression (24). We sought to identify novel CRs essential for TFH formation. To this end, we adapted our in vivo short hairpin RNA (shRNA)–based screening approach (25) to survey a library of microRNA-adapted shRNAs (shRNAmirs) targeting all known CRs in mice. We identified mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation and function.

SMARTA mice (transgenic expression of an I-Ab–restricted TCR specific for lymphocytic choriomeningitis virus [LCMV] glycoprotein aa 66–77) (26) and CD45.1+ mice were on a full C57BL/6 background and bred in-house. C57BL/6 and Cas9Tg mice (stock no. 028555) were purchased from The Jackson laboratory. Both male and female mice were used throughout the study, with sex- and age-matched T cell donors and recipients. All mice were maintained in specific pathogen-free facilities and used according to protocols approved by the Animal Care and Use Committees of La Jolla Institute for Immunology. Mice were infected via the i.p. route with 2 × 105, 4 × 105, or 5 × 105 PFUs of LCMV-Armstrong (LCMV-Arm) for days 6, 4, or 3 analysis, respectively. Ten micrograms of keyhole limpet hemocyanin (KLH) conjugated to gp66–77 peptide of LCMV mixed with 2.5 μg of saponin/monophosphoryl lipid A nanoparticles (SMNP) (27) in 100 μl of PBS was injected at the base of the tail. One microgram of BrdU in PBS was injected via the i.p. route 16 h before analysis.

We included a set of 42 control shRNAmir-retroviral vectors (RVs). Three hundred twenty-two CRs genes expressed in mice are targeted by three or four shRNAmirs each for a total of 1230 shRNAmir-RVs in the library. The initial screen was performed twice as previously described (25). Subsequent screens were modified as follows. Equal amounts of every shRNAmir were pooled to create 22 tubes. Plat-E cells were transfected with 2 μg of pooled shRNAmir (1 pool/well) and 1 μg of pCL-Eco plasmids. Anti-CD3/CD28–activated SMARTA CD4 T cells were transduced once with RV supernatant. Sorted shRNAmir-RV+ cells (5 × 105) were transferred into 20 recipient mice by i.v. injection. An aliquot of 1.1–1.5 × 106 sorted cells was saved for RNA isolation (input). Mice were infected with 2 × 106 PFU of LCMV-Arm 4 d after transfer.

At 3 d postinfection, spleens were isolated and pooled. CD45.1+ SMARTA cells were pre-enriched and sorted into CXCR5+SLAMlo TFH and CXCR5SLAM+ TH1 SMARTA cells. 1.7 × 105 TFH and 1.6 × 106 TH1 cells were sorted for the first screen, and 7.2 × 105 TFH and 3.2 × 106 TH1 cells were sorted for the second screen. Total RNA was isolated using a mirVana miRNA isolation kit (Invitrogen). RNA was quantified with high-sensitivity RNA ScreenTape kit and TapeStation (Agilent Technologies). Seventy nanograms of RNA was used to synthesize cDNA using random oligonucleotides in triplicate reactions that were subsequently pooled.

The amount of shRNAmir-derived cDNA template in each sample preparation was quantified using quantitative PCR and a standard curve prepared from known molar amounts of gel-purified shRNAmir amplicons. To build shRNAmir amplicon libraries capturing passenger strand sequences, an amount of cDNA template in each sample resulting in equivalent amplification to previously analyzed libraries amplified from 100 ng of total genomic DNA template from transduced T cells was amplified using primers including Illumina P5 (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTCGAGAAGGTATATTGCT-3′) and P7 (5′-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCGATCTACATCTGTGGCTTCACTA-3′) adapter sequences. The miR30 (P5) and shRNAmir-loop (P7) annealing sequences are underlined and the custom barcode is indicated (Ns). Sequencing libraries were amplified in triplicate reactions using Phusion high-fidelity DNA polymerase in 1× high-fidelity buffer for 24 PCR cycles. Replicate reactions were pooled, purified with 1.6× AMPure XP beads (Beckman Coulter), and quantified with a Qubit DNA assay and Agilent 2100 Bioanalyzer (Agilent Technologies). The individually barcoded libraries were pooled in equimolar ratios, mixed with separate base-balanced libraries to increase diversity, and sequenced on the NextSeq 500 at a 1.8 pM concentration with paired-end 75-bp reads.

A FASTA-formatted reference database comprising 97-mer sequences of all analyzed shRNAmirs in the library was built. Raw sequencing reads were merged using FLASH, and the merged reads were compared with the reference database using nucleotide BLAST. Only the top alignment for each read was retained and the number of times each reference sequence was aligned in each sample library was counted and reported. Normalized reads for all shRNAmirs targeting the same gene were averaged to create a single gene read number and gene log2 TFH/TH1 ratios were calculated.

pMIG, Lef1, Tcf7-p45, and Bcl6 pMIG were described previously (6, 7, 19). Guide RNAs (gRNAs) were selected by CHOP-CHOP (https://chopchop.cbu.uib.no) and cloned into the BbsI site of a modified LMPd-Ametrine vector. Transduction and transfer of CD4 T cells were performed as previously detailed (25). Transferred cells were allowed to rest in host mice for 3–4 d before infection or immunization. 2 × 104, 5 × 104, or 4 × 105 transduced CD4+ T cells were transferred for day 6, 4, or 3 analysis, respectively. For protein immunization, 2 × 105 transduced cells were transferred. For TH1 cultures, cells were cultured in the presence of 10 ng/ml IL-12 (PeproTech) and 10 μg/ml anti–IL-4 (R&D Systems).

Single-cell suspensions of spleen or draining inguinal lymph nodes were prepared by standard gentle mechanical disruption. Surface staining for flow cytometry was done with mAbs against CD4 (RM4-5, 1:400), CD8 (53-6.7, 1:400), CD45.1 (A20, 1:400), and B220 (RA3-6B2, 1:400) (eBioscience); PSGL-1 (2PH1, 1:400) (BD Biosciences); and SLAM (TC15-12F12.2, 1:400), CD25 (PC61, 1:400), and CD44 (IM7, 1:400) (BioLegend). CXCR5 staining was done using biotinylated anti-CXCR5 (SPRCL5, eBioscience), followed by PE-Cy7-, BV421- or allophycocyanin-labeled streptavidin (BioLegend). Intranuclear staining was performed with a mAb to Bcl6 (K112-91, BD Biosciences), TCF1 (C63D9), LEF-1 (C12A5, Cell Signaling Technology), or Blimp-1 (5E7, BioLegend) and the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen). For measurement of cytokines, cells from spleen were cultured with 10 μg/ml gp66 peptide and brefeldin A. Intracellular staining for cytokines was performed with mAbs to IFN-γ (XMG1.2), CD40L (MR1), IL-2 (JES6-5H4, Invitrogen), and recombinant mouse IL-21 receptor Fc (R&D Systems), followed by anti-human IgG (Invitrogen), using the Fixation/Permeabilization buffer kit (BD Biosciences). BrdU staining was performed with the BrdU flow kit (BD Biosciences). Stained cells were analyzed using LSR II or FACSCelesta (BD Biosciences) and FlowJo software (Tree Star). Anti-TBP (ab63766, Abcam) and anti-Mll1 (14689, Cell Signaling Technology) were used in Western blots.

CD45.1+gRNA+ Cas9Tg SMARTA cells were pre-enriched from spleens pooled from four mice per group. Five thousand to 150,000 CXCR5+SLAMlo TFH cells, CXCR5SLAM+ TH1 SMARTA cells, or naive SMARTA cells were sorted into TRIzol LS (Invitrogen). RNA sequencing (RNA-seq) was performed as previously described (2). cDNA (0.5 ng) was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA library preparation kit and index kit; Illumina). Libraries were sequenced using a HiSeq 2500 (Illumina) to generate 50-bp single-end reads, generating a median of >17 million mapped reads per sample. Three biological replicates were generated for TFH and TH1 samples. RNA-seq was analyzed as previously described (2) except for the following modifications. The reads were aligned with the mm10 reference genome using STAR (v2.6.1) and the RefSeq gene annotation downloaded from the UCSC Genome Bioinformatics site. Read counts to each genomic feature were obtained with the featureCounts (v1.6.5) using the default option along with a minimum quality cutoff (Phred > 10). We considered genes differentially expressed between two groups of samples when the DESeq2 analysis resulted in an adjusted p value of <0.05 and the difference in gene expression was 1.4-fold. Transcripts per million were calculated. Gene set enrichment analysis (GSEA) was run on gene lists with GSEA v3.0 (Broad Institute). Gene signatures from an in vitro TH2, regulatory T (Treg), Bcl6-bound gene list from human tonsil GC-TFH cells (18), Blimp-1–bound genes from activated CD8 T cells (28) or from in vitro–differentiated TH17 cells (29), and genes signatures from TFH, TH1, and Tcf7−/−Lef1−/− GC-TFH cells isolated after LCMV-Arm infection and from in vitro–differentiated Lef1+-RV TH1 cells (6) were previously published. The RNA-seq data have been deposited to Gene Expression Omnibus under accession number GSE226341 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE226341).

Statistical tests were performed using Prism 8.0 (GraphPad). Significance was determined by an unpaired Student t test with a 95% confidence interval, by one-way ANOVA with a Tukey’s multiple comparison test or by two-way ANOVA with a Sidak’s multiple comparison test. The p values are defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To discover novel CRs regulating TFH differentiation, we generated an shRNAmir-RV library targeting all known mouse CRs, 322 genes, each targeted by 3–4 shRNAmirs for a total of 1230 shRNAmir-RVs (Fig. 1A). Briefly, LCMV gp66–77-specific TCR transgenic SMARTA CD4 T cells were transduced once with shRNAmir-RV–containing supernatant. shRNAmir-RV+ SMARTA CD4 T cells were sorted based on Ametrine marker expression and adoptively transferred into recipient C57BL/6 mice (Fig. 1B). Total RNA was isolated from an aliquot of CD4 T cells for the input sample. Recipient mice were infected with LCMV-Arm and, at 3 d postinfection, SMARTA CD4 T cells were sorted into CXCR5+SLAMlo TFH and CXCR5SLAM+ TH1 populations (Fig. 1C). cDNA sequencing libraries were prepared for the SMARTA input, TFH and TH1 samples. Sequenced cDNA was aligned to a list of all shRNAmir sequences in the library to determine read counts for every shRNAmir in the library. The screen was performed twice in independent experiments.

FIGURE 1.

An in vivo RNA interference screen identifies Mll1 as a novel regulator of TFH differentiation. (A) Scheme for screening the shRNAmir library targeting CRs. (B) Transduction efficiency of SMARTA CD4 T cells infected with pooled shRNAmir-RVs from the CR library. (C) Sorting strategy of shRNAmir+ SMARTA CXCR5+SLAMlo TFH and CXCR5SLAM+ TH1 cells 3 d after LCMV infection. (D) Construct TFH/TH1 ratio Z scores for all shRNAmirs in the library in two independent experiments. (E) Gene TFH/TH1 ratio Z scores for all genes in the library in two independent experiments.

FIGURE 1.

An in vivo RNA interference screen identifies Mll1 as a novel regulator of TFH differentiation. (A) Scheme for screening the shRNAmir library targeting CRs. (B) Transduction efficiency of SMARTA CD4 T cells infected with pooled shRNAmir-RVs from the CR library. (C) Sorting strategy of shRNAmir+ SMARTA CXCR5+SLAMlo TFH and CXCR5SLAM+ TH1 cells 3 d after LCMV infection. (D) Construct TFH/TH1 ratio Z scores for all shRNAmirs in the library in two independent experiments. (E) Gene TFH/TH1 ratio Z scores for all genes in the library in two independent experiments.

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We calculated Z score values of the TFH/TH1 ratio for every shRNAmir in the library. Direct comparison of Z scores per shRNAmir construct was not informative (Fig. 1D). It is possible that differences in shRNA knockdown efficacy was the source of the variability. We averaged the read numbers for each shRNA targeting the same gene to create a single Z score for each targeted gene. We hypothesized that calculating a single Z score value for each targeted gene would reduce variability and be more informative. This analysis method showed improved reproducibility between experiments (Fig. 1E, Supplemental Table I). We assessed the gene Z score distribution of known positive and negative regulators of TFH differentiation. Bcl6 and Itch had negative Z score values in both experiments, indicating that their shRNAmir constructs were depleted from the TFH population (Fig. 1E), consistent with their known roles as positive TFH regulators (3–5, 30). Conversely, Prdm1, Tbx21, and Id2 had positive Z score values, indicative of enrichment in TFH cells (Fig. 1E), consistent with their known roles in directly inhibiting TFH differentiation or promoting TH1 cells (5, 13, 31). We then filtered the gene Z score data for novel regulators of TFH differentiation by ranking genes depleted from TFH cells. The gene coding for the histone methyltransferase Mll1 was one of the most depleted genes from TFH cells, with Z score values of −1.4 and −4.8, suggesting that Mll1 is a positive regulator of TFH differentiation (Fig. 1E). Mll1 methylates H3K4, a mark associated with active transcription, and assembles with numerous regulatory proteins such as RbBP5, Wdr5, Ash2l, Dpy30, and Menin-1 (32). Mll1 regulates the expression of TH1 and TH2 cytokines in CD4 T cells (33, 34), but a role for Mll1 in TFH was unknown. Our shRNA library included shRNA for the Ezh2 and NCoR1, two CRs known to regulate Tfh differentiation (2, 23). NCoR1 had Z score values of −0.50 and −2.46 whereas Ezh2 had Z score values of −1.29 and −0.91, suggesting that our screen can detect CRs known to regulate TFH differentiation.

In published RNA-seq data (2, 6), we observed that Mll1 is expressed at similar levels between naive CD4, TFH, and TH1 cells (Supplemental Fig. 1A). Two shRNAmirs that efficiently inhibited Mll1 expression in vitro were tested individually for their effect on TFH differentiation in vivo (Supplemental Fig. 1B). We transduced SMARTA CD4 T cells with shMll1-RV or shCd8-RV, transferred the SMARTA CD4 T cells into C57BL/6 mice, and analyzed TFH differentiation at 3 d postinfection with LCMV-Arm. shMll1+ SMARTA CD4 T cells exhibited a significantly smaller early TFH cell population (CXCR5+CD25 or CXCR5+Bcl6+) when compared with shCd8+ SMARTA cells (Fig. 2A, 2B, Supplemental Fig. 1C, 1D). shRNAmirs targeting Mll1 had a small but significant impact on the accumulation of SMARTA CD4 T cells in vivo (Fig. 2C, Supplemental Fig. 1E, 1F). Importantly, we observed reduced expression of Bcl6 in Mll1-deficient CXCR5+CD25 early TFH cells (Fig. 2D). These results confirmed the identification of Mll1 as, to our knowledge, a novel positive regulator of TFH differentiation after LCMV infection.

FIGURE 2.

Mll1 is required for proper differentiation of early TFH cells after LCMV infection. (AH) shRNA+ SMARTA (A–D) or gRNA+ Cas9Tg SMARTA (E–H) CD4 T cells were transferred into C57BL/6 mice and analyzed 3 d after infection with LCMV-Arm. (A) CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells and (B) CXCR5+Bcl6+ TFH cells in SMARTA CD4 T cells. (C) Quantitation of SMARTA CD4 T cells. (D) Bcl6 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histogram indicate geometric mean fluorescence intensity (GeoMFI) values. (E) CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells and (F) CXCR5+Bcl6+ TFH cells in CasTg SMARTA CD4 T cells. (G) Quantitation of Cas9Tg SMARTA CD4 T cells. (H) Bcl6 expression in CXCR5+CD25 Cas9Tg SMARTA CD4 T cells. Data shown are from one experiment representative of five (A–D) or three (E–H) independent experiments with three to four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

FIGURE 2.

Mll1 is required for proper differentiation of early TFH cells after LCMV infection. (AH) shRNA+ SMARTA (A–D) or gRNA+ Cas9Tg SMARTA (E–H) CD4 T cells were transferred into C57BL/6 mice and analyzed 3 d after infection with LCMV-Arm. (A) CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells and (B) CXCR5+Bcl6+ TFH cells in SMARTA CD4 T cells. (C) Quantitation of SMARTA CD4 T cells. (D) Bcl6 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histogram indicate geometric mean fluorescence intensity (GeoMFI) values. (E) CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells and (F) CXCR5+Bcl6+ TFH cells in CasTg SMARTA CD4 T cells. (G) Quantitation of Cas9Tg SMARTA CD4 T cells. (H) Bcl6 expression in CXCR5+CD25 Cas9Tg SMARTA CD4 T cells. Data shown are from one experiment representative of five (A–D) or three (E–H) independent experiments with three to four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

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We next sought to confirm the role of Mll1 using an independent experimental approach. Cas9Tg SMARTA CD4 T cells transduced with a control gCd8-RV or gMll1-RV (Supplemental Fig. 1G) were transferred into C57BL/6 mice and analyzed 3 d after LCMV-Arm infection. gMll1+ Cas9Tg SMARTA CD4 T cells had substantially reduced potential to differentiate into CXCR5+CD25 or CXCR5+Bcl6+ TFH cells (Fig. 2E, 2F, Supplemental Fig. 1H, 1I). Accumulation of gMll1+ Cas9Tg SMARTA CD4 T cells was normal postinfection (Fig. 2G, Supplemental Fig. 1J). Bcl6 expression was also significantly reduced in gMll1+ CXCR5+CD25 cells (Fig. 2H). Thus, disruption of Mll1 expression by gRNA phenocopied shRNA-mediated knockdown of Mll1, establishing Mll1 as, to our knowledge, a novel positive regulator of TFH differentiation.

To assess whether Mll1 is dispensable for later stages of TFH differentiation, we analyzed TFH differentiation of Mll1-deficient CD4 T cells at 6 d postinfection. Compared to control shCd8+ cells, shMll1+ SMARTA CD4 T cells showed impaired formation of TFH cells (CXCR5+SLAMlo, Fig. 3A). We next analyzed formation of GC-TFH cells. The percentages of CXCR5+Bcl6+ GC-TFH cells in shMll1+ SMARTA CD4 T cells were comparable to those of control shCd8+ SMARTA (Fig. 3B). Unlike what we observed at 3 d postinfection, the reduction in TFH differentiation was coupled to a significant decrease in the accumulation of shMll1+ SMARTA CD4 T cells by day 6 (Fig. 3C, Supplemental Fig. 1K).

FIGURE 3.

Mll1 is required for proper differentiation of TFH cells. (AH) shRNA+ SMARTA (A–C) or gRNA+ Cas9Tg SMARTA (D–H) CD4 T cells were transferred into C57BL/6 mice and analyzed 6 d after infection with LCMV-Arm (A–F) or 8 d after immunization with GP-KLH mixed with SMNP (G–H). (A) CXCR5+SLAMlo TFH cells or (B) CXCR5Bcl6 TH1, CXCR5+Bcl6 TFH, and CXCR5+Bcl6+ GC-TFH cells in SMARTA CD4 T cells. (C) Quantitation of SMARTA CD4 T cells. (D) CXCR5+SLAMlo TFH cells or (E) CXCR5Bcl6 TH1, CXCR5+Bcl6 TFH, and CXCR5+Bcl6+ GC-TFH cells in Cas9Tg SMARTA CD4 T cells. (F) Quantitation of Cas9Tg SMARTA CD4 T cells. (G) CXCR5PSGL-1+ TH1 and CXCR5+PSGL-1lo TFH cells in Cas9Tg SMARTA CD4 T cells. (H) Quantitation of Cas9Tg SMARTA CD4 T cells. (IK) shRNA+ SMARTA CD4 T cells were transferred into C57BL/6 mice and mice were infected with LCMV-Arm. Mice were injected with BrdU 3 d after infection and analyzed 16 h later. (I) BrdU+ cells in SMARTA CD4 T cells, (J) CXCR5+SLAMlo TFH SMARTA CD4 T cells and (K) CXCR5SLAM+ TH1 SMARTA CD4 T cells. Data shown are from one experiment representative of five (A–C), one (D–G), or two (I–K) independent experiments with four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

FIGURE 3.

Mll1 is required for proper differentiation of TFH cells. (AH) shRNA+ SMARTA (A–C) or gRNA+ Cas9Tg SMARTA (D–H) CD4 T cells were transferred into C57BL/6 mice and analyzed 6 d after infection with LCMV-Arm (A–F) or 8 d after immunization with GP-KLH mixed with SMNP (G–H). (A) CXCR5+SLAMlo TFH cells or (B) CXCR5Bcl6 TH1, CXCR5+Bcl6 TFH, and CXCR5+Bcl6+ GC-TFH cells in SMARTA CD4 T cells. (C) Quantitation of SMARTA CD4 T cells. (D) CXCR5+SLAMlo TFH cells or (E) CXCR5Bcl6 TH1, CXCR5+Bcl6 TFH, and CXCR5+Bcl6+ GC-TFH cells in Cas9Tg SMARTA CD4 T cells. (F) Quantitation of Cas9Tg SMARTA CD4 T cells. (G) CXCR5PSGL-1+ TH1 and CXCR5+PSGL-1lo TFH cells in Cas9Tg SMARTA CD4 T cells. (H) Quantitation of Cas9Tg SMARTA CD4 T cells. (IK) shRNA+ SMARTA CD4 T cells were transferred into C57BL/6 mice and mice were infected with LCMV-Arm. Mice were injected with BrdU 3 d after infection and analyzed 16 h later. (I) BrdU+ cells in SMARTA CD4 T cells, (J) CXCR5+SLAMlo TFH SMARTA CD4 T cells and (K) CXCR5SLAM+ TH1 SMARTA CD4 T cells. Data shown are from one experiment representative of five (A–C), one (D–G), or two (I–K) independent experiments with four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

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gRNA-mediated disruption of Mll1 also significantly reduced CXCR5+SLAMlo TFH differentiation 6 d after LCMV infection (Fig. 3D). gMll1+ Cas9Tg SMARTA CXCR5+Bcl6+ GC-TFH cells were reduced compared with gCd8+ (Fig. 3E). These results suggest that Mll1 mostly regulates TFH differentiation with less impact on GC-TFH differentiation because a reduction in CXCR5+Bcl6+ GC-TFH cells was observed only in gMll1+ Cas9Tg SMARTA CD4 T cells but not in shMll1+ SMARTA CD4 T cells. gMll1+ Cas9Tg SMARTA CD4 T cells also accumulated significantly less than gCd8+ Cas9Tg SMARTA CD4 T cells (Fig. 3F). These results showed that Mll1 is a positive regulator of TFH differentiation after an acute viral infection.

We next tested Mll1 disruption in the context of protein immunization. C57BL/6 mice that received gCd8+ or gMll1+ Cas9Tg SMARTA CD4 T cells were immunized s.c. with KLH conjugated to gp66–77 peptide from LCMV (GP-KLH) mixed with the SMNP adjuvant. Draining lymph nodes were harvested for analysis 8 d later. Differentiation of gMll1+ Cas9Tg SMARTA CD4 T cells into CXCR5+PSGL-1lo TFH cells was reduced following immunization (Fig. 3G) whereas accumulation of Mll1-deficient SMARTA CD4 T cells was not compromised (Fig. 3H). These experiments demonstrate a role for Mll1 in TFH cell differentiation in different contexts.

We tested whether reduced proliferation of Mll1-deficient CD4 T cells was causing their reduced accumulation after LCMV infection. At 4 d postinfection, total shMll1+ SMARTA CD4 T cells showed significantly reduced incorporation of BrdU when compared with shCd8+ SMARTA cells (Fig. 3I). The decrease in BrdU incorporation was observed for both CXCR5+SLAMlo TFH and CXCR5SLAM+ TH1 cells (Fig. 3J, 3K). These results indicate that Mll1 is required for optimal proliferation of CD4 T cells following infection and suggest that the reduced TFH differentiation of shMll1+ SMARTA CD4 T cells was not caused by a selective proliferation defect of TFH cells. These experiments demonstrated the importance for Mll1 in supporting TFH differentiation.

To test the functionality of Mll1-deficient TFH cells, we assessed cytokine production in shCd8+ and shMll1+ SMARTA CD4 T cells at 6 d postinfection. Substantial fractions of IL-21–producing cells were observed in control shCd8+ SMARTA cells, but IL-21 expression was greatly reduced in shMll1+ SMARTA cells (Fig. 4A, Supplemental Fig. 2A). Expression of CD40L or IFN-γ was not affected by loss of Mll1 (Fig. 4B, Supplemental Fig. 2B). Production of IL-2, whose gene is immediately adjacent to Il21, was mostly unaffected by Mll1 deficiency (Supplemental Fig. 2C). IL-21 expression was similarly disrupted in gMll1+ Cas9Tg SMARTA CD4 T cells (Fig. 4C). A small but significant reduction in CD40L was observed whereas IFN-γ production was not significantly impacted (Fig. 4D, Supplemental Fig. 2D). IFN-γ expression in TH1 cells in response to LCMV infection was also reduced after gRNA-mediated abrogation of Mll1 expression (Supplemental Fig. 2E, 2F), similar to what was observed in Mll1+/− CD4 T cells responding to tuberculosis Ags (34).

FIGURE 4.

Mll1 is required for production of IL-21. (AE) shRNA+ SMARTA (A, B, and E) or gRNA+ Cas9Tg SMARTA (C and D) CD4 T cells were transferred into C57BL/6 mice. At 6 d after infection with LCMV-Arm, splenocytes were restimulated with LCMV gp66–77 peptide. (A, B, and E) Expression of IL-21 (A) and CD40L (B) in SMARTA CD4 T cells or TFH and TH1 SMARTA CD4 T cells (E). (C and D) Expression of IL-21 (C) and CD40L (D) in Cas9Tg SMARTA CD4 T cells. (F) shRNA+ CD4 T cells were cultured in TH1 conditions. Expression of IL-21 in shRNA+ CD4 T cells restimulated with anti-CD3 is shown. (G) gRNA+ Cas9Tg SMARTA CD4 T cells were cultured in TH1 conditions. Expression of IL-21 in gRNA+ Cas9Tg SMARTA CD4 T cells restimulated with anti-CD3 is shown. Data shown are from one experiment representative of five (A, B, and E), three (C and D), or two (F and G) independent experiments with three to four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

FIGURE 4.

Mll1 is required for production of IL-21. (AE) shRNA+ SMARTA (A, B, and E) or gRNA+ Cas9Tg SMARTA (C and D) CD4 T cells were transferred into C57BL/6 mice. At 6 d after infection with LCMV-Arm, splenocytes were restimulated with LCMV gp66–77 peptide. (A, B, and E) Expression of IL-21 (A) and CD40L (B) in SMARTA CD4 T cells or TFH and TH1 SMARTA CD4 T cells (E). (C and D) Expression of IL-21 (C) and CD40L (D) in Cas9Tg SMARTA CD4 T cells. (F) shRNA+ CD4 T cells were cultured in TH1 conditions. Expression of IL-21 in shRNA+ CD4 T cells restimulated with anti-CD3 is shown. (G) gRNA+ Cas9Tg SMARTA CD4 T cells were cultured in TH1 conditions. Expression of IL-21 in gRNA+ Cas9Tg SMARTA CD4 T cells restimulated with anti-CD3 is shown. Data shown are from one experiment representative of five (A, B, and E), three (C and D), or two (F and G) independent experiments with three to four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (unpaired two-tailed Student t test).

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Notably, we observed reduction of IL-21 production by both TH1 and TFH cells (Fig. 4E), suggesting that Mll1 regulates IL-21 production independently of TFH differentiation. We next tested whether Mll1 is required for IL-21 production in a system independent of TFH differentiation. In vitro shMll1+ TH1 cells and gMll1+ Cas9Tg TH1 cells exhibited impaired IL-21 expression (Fig. 4F, 4G). Impacts on IFN-γ in vitro were variable with shMll1+ TH1 cells producing normal levels of IFN-γ whereas IFN-γ production was reduced in gMll1+ TH1 cells (Supplemental Fig. 2G, 2H). Altogether, these results demonstrate that IL-21 production by CD4 T cells depends on Mll1.

To gain further understanding of the function of Mll1, we performed a transcriptomics analysis of Mll1-deficient TFH and TH1 cells (Fig. 5A). Mll1-deficient TFH cells had expression changes of a selected number of genes in comparison with control TFH cells (89 upregulated and 29 downregulated genes, |fold change| > 1.4, adjusted p < 0.05, Fig. 5B, 5C). In TH1 cells, loss of Mll1 resulted in the downregulation of 91 genes and the upregulation of 72 genes (Fig. 5B, 5C). We next asked how different gene expression signatures associated with CD4 T cell subsets were affected by loss of Mll1 expression. We performed GSEA of TFH, TH1, TH17, TH2, and Treg gene signatures against control and Mll1-deficient TH1 and TFH populations. In these analyses, a negative normalized enrichment score denotes enrichment of the indicated gene signature in the gMll1+ populations whereas a positive normalized enrichment score indicates enrichment of the specified gene signature in gCd8+ cells. TFH-associated gene expression was depleted from Mll1-deficient TFH cells (Fig. 5D). Conversely, a TH1 signature was enriched in Mll1-deficient TFH cells (Fig. 5D). Enrichment of a TH1 gene signature was also observed in Mll1-deficient TH1 cells (Fig. 5D). Genetic signatures associated with TH2, TH17, and Treg cells were slightly enriched in Mll1-deficient TFH cells (Fig. 5D). In a separate analysis, we observed that wild-type TH1 cells were enriched for genes upregulated in Mll1-deficient cells (Fig. 5E). It was possible that Mll1 controls TFH differentiation by acting in concert with Bcl6 or Blimp-1. We tested the enrichment of Bcl6-bound and Blimp-1 genes in control and Mll1-deficient TH1 and TFH populations but found no strong evidence of Mll1 involvement in the regulation of Bcl6-bound genes (Fig. 5F). Expression of Blimp-1–bound genes was decreased in Mll1-deficient TH1 cells, suggesting dysregulation of Blimp-1 activity in the absence of Mll1 (Fig. 5F). Taken together, these GSEAs suggest that in the absence of Mll1, TFH cells gain expression of genes associated with the TH1 program.

FIGURE 5.

Transcriptomics analysis of Mll1-deficient TFH cells. (A) gRNA+ CasTg SMARTA CD4 T cells were transferred into C57BL/6 mice and sorted into CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells 3 d after infection with LCMV-Arm. (B) Quantitation of differentially expressed genes in gCd8+ and gMll1+ TFH and TH1 cells. The numbers of differentially expressed genes are indicated in the columns. (C) Heatmaps of differentially expressed genes in gCd8 and gMll1 TFH and TH1 cells. (D and F) Bubblegum plots of GSEAs of genes differentially expressed between gCd8+ and gMll1+ TFH or TH1 cells against the indicated gene signatures. (E) GSEA of genes downregulated or upregulated in gMll1+ TFH cells compared with genes differentially expressed in gCd8+ TFH and TH1 cells.

FIGURE 5.

Transcriptomics analysis of Mll1-deficient TFH cells. (A) gRNA+ CasTg SMARTA CD4 T cells were transferred into C57BL/6 mice and sorted into CXCR5+CD25 TFH and CXCR5CD25+ TH1 cells 3 d after infection with LCMV-Arm. (B) Quantitation of differentially expressed genes in gCd8+ and gMll1+ TFH and TH1 cells. The numbers of differentially expressed genes are indicated in the columns. (C) Heatmaps of differentially expressed genes in gCd8 and gMll1 TFH and TH1 cells. (D and F) Bubblegum plots of GSEAs of genes differentially expressed between gCd8+ and gMll1+ TFH or TH1 cells against the indicated gene signatures. (E) GSEA of genes downregulated or upregulated in gMll1+ TFH cells compared with genes differentially expressed in gCd8+ TFH and TH1 cells.

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We searched a curated list of known TFH regulators for genes with dysregulated expression in Mll1-deficient TFH cells. Mll1-deficient TFH cells expressed significantly less Lef1 and Tcf7 (Fig. 6A, 6B). We also observed that Prdm1, the gene encoding Blimp-1, had higher expression in Mll1-deficient TFH cells in comparison with control TFH cells (Fig. 6A). Id2 and Il2ra, two genes associated with suppression of TFH fate (13, 35, 36), were also upregulated in Mll1-deficient TFH cells (Fig. 6A). Interestingly, Lef1 also showed reduced expression in Mll1-deficient TH1 cells (Fig. 6A, 6B).

FIGURE 6.

Mll1 regulates expression of LEF-1 and TCF-1 in TFH cells. (A) Volcano plots of gene expression in gCd8+ and gMll1+ TFH or TH1 cells plotted against adjusted p value. Genes of interest are indicated. (B) Transcripts per million (TPM) values for Tcf7 and Lef1 in the indicated populations. (C) GSEA of genes downregulated or upregulated in Lef-RV+ TH1 cells compared with genes differentially expressed in gCd8+ TFH and gMll1+ TFH cells. (D) GSEA of genes downregulated or upregulated in Lef1−/−Tcf7−/− GC-TFH cells compared with genes differentially expressed in gCd8+ TFH and gMll1+ TFH cells. Selected genes that form the core enrichment signatures are listed. NES, normalized enrichment score. (E and F) shRNA+ SMARTA CD4 T cells were transferred into C57BL/6 mice and analyzed 3 d after infection with LCMV-Arm. (E) LEF-1 or (F) TCF-1 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histograms indicate geometric mean fluorescence intensity (GeoMFI) values. Data shown are from one experiment representative of three (E and F) independent experiments with four mice per group. (B) **p ≤ 0.01, ***p ≤ 0.001 (two-way ANOVA with Sidak’s multiple comparison test); (E and F) *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

FIGURE 6.

Mll1 regulates expression of LEF-1 and TCF-1 in TFH cells. (A) Volcano plots of gene expression in gCd8+ and gMll1+ TFH or TH1 cells plotted against adjusted p value. Genes of interest are indicated. (B) Transcripts per million (TPM) values for Tcf7 and Lef1 in the indicated populations. (C) GSEA of genes downregulated or upregulated in Lef-RV+ TH1 cells compared with genes differentially expressed in gCd8+ TFH and gMll1+ TFH cells. (D) GSEA of genes downregulated or upregulated in Lef1−/−Tcf7−/− GC-TFH cells compared with genes differentially expressed in gCd8+ TFH and gMll1+ TFH cells. Selected genes that form the core enrichment signatures are listed. NES, normalized enrichment score. (E and F) shRNA+ SMARTA CD4 T cells were transferred into C57BL/6 mice and analyzed 3 d after infection with LCMV-Arm. (E) LEF-1 or (F) TCF-1 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histograms indicate geometric mean fluorescence intensity (GeoMFI) values. Data shown are from one experiment representative of three (E and F) independent experiments with four mice per group. (B) **p ≤ 0.01, ***p ≤ 0.001 (two-way ANOVA with Sidak’s multiple comparison test); (E and F) *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

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GSEA revealed that genes upregulated following ectopic expression of Lef1 in CD4 T cells are depleted from Mll1-deficient TFH cells (Fig. 6C). Conversely, genes suppressed when Lef1 is overexpressed are overrepresented in TFH cells lacking Mll1 (Fig. 6C). In a separate GSEA, Mll1-deficient TFH cells were enriched for genes upregulated in Lef1−/−Tcf7−/− GC-TFH cells (Fig. 6D). These findings indicated that LEF-1– and TCF-1–regulated genes were dysregulated, implicating roles for LEF-1 and TCF-1 downstream of Mll1. We measured the expression of LEF-1 and TCF-1 in Mll1-deficient TFH cells by flow cytometry. Disruption of Mll1 reduced expression of both LEF-1 and TCF-1 in early TFH cells (CXCR5+CD25, Fig. 6E, 6F, Supplemental Fig. 3A–C) These experiments demonstrate that in the absence of Mll1, LEF-1 and TCF-1 expression is impaired.

We sought to confirm the increased Prdm1 and Il2ra mRNA level in Mll1-deficient TFH by analyzing Blimp-1 and CD25 expression by flow cytometry. We first validated the staining of Blimp-1 by showing that CXCR5CD25+ early TH1 cells express more Blimp-1 than do CXCR5+CD25 early TFH cells (Supplemental Fig. 3D). We observed sporadic increases in Blimp-1 expression in Mll1-deficient CXCR5+Bcl6+ TFH cells 3 d after LCMV infection (Supplemental Fig. 3E). Similarly, CD25 expression was not reproducibly increased in Mll1-deficient TFH cells (Supplemental Fig. 3F).

We hypothesized that ectopic expression of Bcl6, TCF-1, or LEF-1 in Mll1-deficient CD4 T cells may rescue TFH differentiation, as the expression of all three is decreased in the absence of Mll1. To test this hypothesis, we transduced shCd8+ and shMll1+ SMARTA CD4 T cells with RV-Lef1, RV-Tcf7-p45 (full-length isoform of TCF-1), RV-Bcl6, or control RV expressing GFP alone. RV+shRNAmir+ SMARTA CD4 T cells were transferred into C57BL/6 recipient mice and TFH differentiation was analyzed 3 d after LCMV-Arm infection. The expression of LEF-1 in shMll1+Lef1-RV+ TFH cells was equivalent to that of shCd8+GFP+ TFH cells (Fig. 7A). As expected, forced expression of LEF-1 resulted in increased TFH differentiation of shCd8+ SMARTA CD4 T cells (Fig. 7B). However, ectopic expression of wild-type levels of LEF-1 in shMll1+ SMARTA CD4 T cells did not rescue TFH differentiation (Fig. 7B). The levels of LEF-1 may not have quantitatively compensated for the reduced levels of TCF-1 (Supplemental Fig. 3G), as Tcf7 is more highly expressed than Lef1 at the mRNA level in mouse CD4 T cells (Fig. 6B), supporting a speculation that TCF1 protein is more highly expressed than LEF1.

FIGURE 7.

TCF-1 partially rescues TFH differentiation of Mll1-deficient CD4 T cells. SMARTA CD4 T cells transduced with the indicated RV were transferred into C57BL/6 mice and analyzed 3 d after LCMV-Arm infection. (A) LEF-1 or (C) TCF-1 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histogram indicate geometric mean fluorescence intensity (GeoMFI) values. (B and D) CXCR5+CD25 TFH or (E) CXCR5+Bcl6+ TFH cells in SMARTA CD4 T cells. Data shown are from one experiment representative of two to three independent experiments with four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

FIGURE 7.

TCF-1 partially rescues TFH differentiation of Mll1-deficient CD4 T cells. SMARTA CD4 T cells transduced with the indicated RV were transferred into C57BL/6 mice and analyzed 3 d after LCMV-Arm infection. (A) LEF-1 or (C) TCF-1 expression in CXCR5+CD25 SMARTA CD4 T cells. Numbers in the histogram indicate geometric mean fluorescence intensity (GeoMFI) values. (B and D) CXCR5+CD25 TFH or (E) CXCR5+Bcl6+ TFH cells in SMARTA CD4 T cells. Data shown are from one experiment representative of two to three independent experiments with four mice per group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

Close modal

Expression of Tcf7-p45 in Mll1-deficient CD4 T cells resulted in TCF-1 expression equivalent to control shCd8+GFP+ cells (Fig. 7C). TCF-1 partially rescued the differentiation of early TFH differentiation by Mll1-deficient SMARTA CD4 T cells (Fig. 7D), including minimal changes in expression of Bcl6 and LEF-1 (Supplemental Fig. 3H). Overall, the results indicate that Mll1 regulates multiple aspects of TFH differentiation, which can only be partially restored by TCF-1 expression.

If Mll1 primarily functions upstream of Bcl6 in TFH differentiation, ectopic expression of Bcl6 may bypass the loss of Mll1 and support TFH differentiation. TFH differentiation of shMll1+ SMARTA CD4 T cells was rescued by ectopic expression of Bcl6 (Fig. 7E) but did not increase LEF-1 and TCF-1 expression in TFH cells (Supplemental Fig. 3I). Overall, these data indicate that Mll1 regulates TFH differentiation by regulating expression of a network of transcription factors that includes, but is most likely not limited to, Bcl6, LEF-1, and TCF-1.

In this study, we identified Mll1 as a critical regulator of TFH cells. Our work shows that Mll1 is required for optimal TFH cell differentiation and production of IL-21. We demonstrate that absence of Mll1 results in impaired expression of Bcl6, LEF-1, and TCF-1, each of which is an important regulator of TFH differentiation. Our findings add Mll1 to the CRs that regulate TFH differentiation (19–24). The results in the present study demonstrate that Mll1 is required for TFH differentiation. Mll1 is also involved in the expression of TH1 and TH2 cytokines (33, 34). Thus, Mll1 has roles in multiple CD4 T cell subsets similar to other CRs and transcription factors. The histone methyltransferase EZH2 is required for both TFH and Treg cell differentiation (21, 22, 37, 38). Similarly, the transcription factors Maf, IRF4, and Batf are all required for both TH17 and TFH differentiation (9–12, 39, 40).

Accumulation of Mll1-deficient CD4 T cells was mildly impaired 3 d after LCMV infection but was more dramatically impaired 3 d later. Loss of Mll1 expression resulted in a decreased TFH population at both times analyzed after LCMV infection. It is possible that Mll1 plays a more prominent role in the early stages of TFH differentiation, at a time when the impact of Mll1 deficiency on CD4 T cell accumulation is marginal. Mll1 impact on CD4 T cell accumulation may become more prominent at later times as evidenced by decreased BrdU labeling observed 4 d after LCMV infection and the substantial loss in Mll1-deficient CD4 T cells 2 d later. Six days after LCMV infection, the decrease in TFH cell frequencies may be secondary to the decreased accumulation of Mll1-deficient CD4 T cells. Nevertheless, in a protein immunization system, TFH cells were decreased 8 d after immunization even when accumulation of CD4 T cells was not impaired in the absence of Mll1.

Our study provides a mechanistic explanation for the defective TFH differentiation in the absence of Mll1. First and foremost, expression of the critical TFH regulator Bcl6 is reduced in Mll1-deficient CD4 T cells. A small reduction in Bcl6 expression is sufficient to negatively impact TFH differentiation, as seen in CD4 T cells lacking a single Bcl6 allele (13). The reduced expression of Bcl6 in the absence of Mll1 most likely impairs suppression of alternative cell fates. Partial loss of Bcl6 is probably not the only cause for defective TFH differentiation in the absence of Mll1. LEF-1 and TCF-1 have largely redundant functions to each other in CD4 T cells, and reduced expression of both is likely a key contributing factor to the observed phenotype of Mll1-deficient CD4 T cells. Ectopic expression of LEF-1 did not rescue the defective TFH in Mll1-deficient CD4 T cells. It is plausible that forced expression of LEF-1 was not elevated enough to compensate for both LEF-1 and TCF-1. TCF-1 is expressed at substantially higher levels than LEF-1 and may explain why ectopic expression of TCF-1 partially compensated for Mll1 deficiency. We suggest that Mll1 controls a network of genes that includes Bcl6, LEF-1, and TCF-1 to promote TFH differentiation. We did not observe any effect of Mll1 on the expression of Bcl6- or Blimp-1–bound genes, suggesting that Mll1 does not influence their transcriptional activities. Mll1 may still act on a small subset of such genes, and this influence was not detected in our bioinformatics analysis. It is also possible that Mll1 is required for the function of other transcription factors controlling formation of TFH cells.

In addition to driving TFH differentiation, Mll1 promotes production of IL-21. Loss of Mll1 does not impact expression of Maf (data not shown), a transcription factor essential for IL-21 production (11, 12). Deletion of Mll1 could limit H3K4 methylation at the Il21 locus, reducing its transcription leading to impaired production of IL-21. Mll1 is required for normal hematopoiesis (41, 42), but this function does not require the histone methyltransferase domain of Mll1 (43). It is conceivable that the enzymatic activity of Mll1 is not involved in promoting IL-21 production and TFH differentiation.

Chromatin modification is a complex process, and absence of a single CR is often compensated by other enzymes. Reduction in TFH differentiation of Mll1-deficient CD4 T cells may be partially due to compensatory mechanisms by other Mll1 family members (Mll2, Mll3, or Mll5), all of which form complexes with Wdr5, Ash2l, and RbBp5 (32). Expression of Mll family members and genes coding for components of the Mll1 complex (Wdr5, Ash2l, Dpy30, RbBp5, Menin-1) was normal in the absence of Mll1 (data not shown). The phenotype of Mll1-deficient CD4 T cells is similar to the incomplete loss of TFH cells when other CRs are deleted (21–24). Further studies are required to obtain a detailed understanding the role of Mll1 in the control of epigenetic modifications of enhancers and promoters and how this impacts general chromatin accessibility. The roles of members of the Mll1 complex in regulating Mll1 function in the context of TFH differentiation should also be studied.

Disruption of the interaction between Mll1 and the core complex protein Wdr5 with a small-molecule drug inhibits the growth of Mll1 leukemic cells (44). Similarly, a small-molecule inhibitor blocking the interaction between Mll1 and Menin-1 reduces tumor burden in mice (45). Targeting Mll1 to inhibit TFH differentiation or function could be a promising therapeutic approach for autoimmune disorders characterized by elevated TFH activity. Alternatively, targeting the Mll1 complex to enhance its activity may be used in the design of better vaccines.

S.B. is a current employee of VIR Biotechnology and owns shares of VIR Biotechnology. The other authors have no financial conflicts of interest.

We thank members of the Crotty laboratory for critical reading of the manuscript, D. Hinz, M. Haynes, S. Sehic, S. Ellis, and C. Dillingham of the LJI Flow Cytometry core for cell sorting, as well as K. Fung and J. Greenbaum of the LJI Bioinformatics Core and G. Seumois, H. Simon, and A. Wang of the LJI Sequencing core.

This work was supported by National Institute of Allergy and Infectious Diseases Grants U19 AI109976, R01 AI72543, and S10 RR027366 (to La Jolla Institute for Immunology), as well as by internal La Jolla Institute institutional funds (to S.C.). This work was partially supported by the Achievement Rewards for College Scientists Foundation of San Diego (S.H.).

The online version of this article contains supplemental material.

The RNA-seq data presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE226341) under accession number GSE226341.

CR

chromatin regulator

GC

germinal center

gRNA

guide RNA

GSEA

gene set enrichment analysis

H3K4

histone 3 of lysine 4

KLH

keyhole limpet hemocyanin

LCMV

lymphocytic choriomeningitis virus

LCMV-Arm

LCMV-Armstrong

Mll1

mixed lineage leukemia 1

RNA-seq

RNA sequencing

RV

retroviral vector

shRNA

short hairpin RNA

shRNAmir

microRNA-adapted shRNA

SMNP

saponin/monophosphoryl lipid A nanoparticles

TFH

T follicular helper

Treg

regulatory T

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Supplementary data