TCR Affinity Biases Th Cell Differentiation by Regulating CD25, Eef1e1, and Gbp2 Dmitri I. Kotov, Jason S. Mitchell, Thomas Pengo, Christiane Ruedl, Sing Sing Way, Ryan A. Langlois, Brian This information is current as T. Fife and Marc K. Jenkins of October 1, 2021. J Immunol published online 11 March 2019 http://www.jimmunol.org/content/early/2019/03/08/jimmun ol.1801609 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 11, 2019, doi:10.4049/jimmunol.1801609 The Journal of Immunology

TCR Affinity Biases Th Cell Differentiation by Regulating CD25, Eef1e1, and Gbp2

Dmitri I. Kotov,*,† Jason S. Mitchell,†,‡,x Thomas Pengo,{ Christiane Ruedl,‖ Sing Sing Way,#,** Ryan A. Langlois,*,† Brian T. Fife,†,x and Marc K. Jenkins*,†

Naive CD4+ T lymphocytes differentiate into various Th cell subsets following TCR binding to microbial peptide:MHC class II (p:MHCII) complexes on dendritic cells (DCs). The affinity of the TCR interaction with p:MHCII plays a role in Th differen- tiation by mechanisms that are not completely understood. We found that low-affinity TCRs biased mouse naive T cells to become T follicular helper (Tfh) cells, whereas higher-affinity TCRs promoted the formation of Th1 or Th17 cells. We explored the basis for this phenomenon by focusing on IL-2R signaling, which is known to promote Th1 and suppress Tfh cell differentiation. SIRP⍺+ DCs produce abundant p:MHCII complexes and consume IL-2, whereas XCR1+ DCs weakly produce p:MHCII but do not consume IL-2. We found no evidence, however, of preferential interactions between Th1 cell–prone, high-affinity T cells and Downloaded from XCR1+ DCs or Tfh cell–prone, low-affinity T cells and SIRP⍺+ DCs postinfection with bacteria expressing the peptide of interest. Rather, high-affinity T cells sustained IL-2R expression longer and expressed two novel Th cell differentiation regulators, Eef1e1 and Gbp2, to a higher level than low-affinity T cells. These results suggest that TCR affinity does not influence Th cell differen- tiation by biasing T cell interactions with IL-2–consuming DCs, but instead, directly regulates genes in naive T cells that control the differentiation process. The Journal of Immunology, 2019, 202: 000–000.

D4+ T lymphocytes are critical for controlling infections B cell–helping T follicular helper (Tfh) cells (4, 5, 7). Al- http://www.jimmunol.org/ through their ability to provide help to B cells, cytotoxic though Th1 cell differentiation fostered by high-affinity TCR C T cells, or myeloid cells (1). CD4+ T cells provide these interactions is related to strong induction of the IRF4 tran- diverse functions by differentiating into specialized subsets fol- scription factor (8), additional aspects of the mechanism by lowing TCR recognition of peptide:MHC class II (p:MHCII) which TCR affinity affects T cell differentiation have yet to be complexes on the surface of dendritic cells (DCs) and in the determined. context of cytokines from the innate immune system (1–3). IL-2R signaling promotes Th1 cells and suppresses Tfh cell Work by our group and others has shown that TCR dwell time differentiation by driving STAT5 activation and induction of on p:MHCII, which strongly correlates with TCR affinity, also Blimp1, a repressor for the Tfh cell–promoting transcription factor by guest on October 1, 2021 influences Th cell differentiation (4–6). In our experiments, Bcl-6 (9–15). Potentially, TCR affinity regulates Th cell differ- increases in TCR affinity corresponding to p:MHCII dwell entiation, in part, by influencing IL-2 signaling. We therefore times of 0.9–2.3 s correlated with increased differentiation of tested two TCR affinity–regulated, IL-2 signaling–based mecha- macrophage-helping Th1 cells and decreased formation of nisms, one rooted in DC Ag presentation and another focused on IL-2R a-chain (CD25) expression. The two major classical DCs in the spleen differ in p:MHCII presentation and IL-2 consumption *Department of Microbiology and Immunology, University of Minnesota, Minneapolis, potential (16–18). XCR1+ DCs are potent producers of the Th1 MN 55455; †Center for Immunology, University of Minnesota, Minneapolis, MN 55455; ‡University Imaging Centers, University of Minnesota, Minneapolis, MN 55455; cell–inducing cytokine IL-12 (18) but are relatively poor pro- x { + Department of Medicine, University of Minnesota, Minneapolis, MN 55455; University ducers of p:MHCII complexes, whereas SIRP⍺ DCs consume of Minnesota Informatics Institute, University of Minnesota, Minneapolis, MN 55455; ‖ IL-2 and are weak IL-12 producers but are strong producers of School of Biological Sciences, Nanyang Technological University, Singapore 637551; #Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Univer- p:MHCII complexes (17). Thus, it is possible that high TCR af- sity of Cincinnati College of Medicine, Cincinnati, OH 45229; and **Perinatal Institute, finity could bias toward Th1 cell differentiation because only Th Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Med- cells with high-affinity TCRs cells could access the small number icine, Cincinnati, OH 45229 of p:MHCII complexes displayed on XCR1+ DCs, whereas low- ORCIDs: 0000-0001-7843-1503 (D.I.K.); 0000-0002-9632-918X (T.P.); 0000-0002- 5599-6541 (C.R.); 0000-0001-9826-5637 (B.T.F.); 0000-0001-8009-7655 (M.K.J.). affinity cells could access the abundant p:MHCII complexes on ⍺+ Received for publication December 10, 2018. Accepted for publication February 20, SIRP DCs favoring Tfh cell differentiation (19). Alternatively, 2019. because IL-2R expression is proportional to the strength of TCR This work was supported by National Institutes of Health Grants R01 AI039614 and signaling and drives Th1 cell–promoting STAT5 activation (9–15), P01 AI35296 (to M.K.J.), T32 AI083196 and T32 AI007313 (to D.I.K.), and R01 Th cells with high-affinity TCRs may be intrinsically more likely AI106791 and P01 AI35296 (to B.T.F.). to become Th1 cells than Th cells with low-affinity TCRs. Address correspondence and reprint requests to Prof. Marc K. Jenkins, University of We tested these models by examining the influence of TCR Minnesota Medical School, Campus Code 2641, 2101 6th Street SE, Minneapolis, MN 55455. E-mail address: [email protected] affinity on differentiation and T cell/DC interactions using two The online version of this article contains supplemental material. TCR transgenic (Tg) strains that contain T cells with differing TCR Abbreviations used in this article: B6, C57BL/6; DC, dendritic cell; Eef1e1, eukaryotic affinities for the same p:MHCII ligand (20). We found that Th cells translation elongation factor 1 ε 1; Gbp2, guanylate binding protein 2; gRNA, guide with low-, medium-, or high-affinity TCR T cells tended to adopt RNA; p:MHCII, peptide:MHC class II; qPCR, quantitative PCR; Tfh, T follicular uncommitted, Tfh, or non-Tfh cell fates, respectively, after ex- helper; Tg, transgenic; tRNA, transfer RNA; WT, wild-type. posure to bacteria or viruses expressing the relevant peptide. In all + Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 cases, Th cells interacted more frequently with SIRP⍺ DCs than

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801609 2 TCR AFFINITY–BASED CONTROL OF Th CELL FATE with XCR1+ DCs, indicating that differential interactions with CD90.1 (HIS51; Thermo Fisher Scientific). All samples were also stained DCs did not account for TCR affinity–based differences in Th1/Tfh with a fixable viability dye (Ghost Dye Red 780; Tonbo Biosciences). For cell formation. Rather, TCR affinity influenced Th differentia- transcription factor staining, samples were fixed with the eBioscience Foxp3/Transcription Factor Staining Kit (Thermo Fisher Scientific) and tion by controlling the expression of the IL-2R and eukaryotic then stained with BV421-labeled RORɣt (Q31-378; BD Biosciences), translation elongation factor 1 ε 1 (Eef1e1), which promoted Th1 BV605-labeled or BV421-labeled T-bet (4B10; BioLegend), and AF488- cells, and guanylate binding protein 2 (Gbp2), which promoted labeled Bcl-6 (K112-91; BD Biosciences) Abs. To calculate cell numbers, Tfh cells. Our results suggest that TCR affinity–based effects on fluorescent AccuCheck Counting Beads (Invitrogen) were added to each sample after the final wash step. Cells were then analyzed on an LSR II Th differentiation are related to the capacity of the TCR to induce or Fortessa (BD Biosciences) flow cytometer. Data were analyzed with different genetic programs at different affinity levels. FlowJo (Tree Star). Cell sorting and coculture Materials and Methods Mice Spleens were harvested from mice 48 h after i.v. infection with L. mono- cytogenes/P5R bacteria. The spleens were chopped into small pieces and Six- to eight-week-old C57BL/6 (B6) mice were purchased from The digested with Collagenase P (MilliporeSigma) for 20 min at 37˚C prior to Jackson Laboratory or the National Cancer Institute Mouse Repository hand-mashing in a petri dish to generate a single-cell suspension. The (Frederick, MD). Il2ra2/2 mice were purchased from The Jackson single-cell suspension was enriched for CD11c-expressing cells using a Laboratory. Rag12/2 B3K506 TCR Tg, Rag12/2 B3K508 TCR Tg (20), CD11c-positive selection kit (Miltenyi Biotec). The enriched samples were and Rag12/2 UbcGFP TEa TCR Tg mice were bred and housed in specific stained for 30 min at room temperature with BV421-labeled XCR1 (ZET; pathogen-free conditions in accordance with guidelines of the University BioLegend), PE-labeled CD64 (X54-5/7.1; BioLegend), allophycocyanin- of Minnesota Institutional Animal Care and Use Committee and National labeled SIRP⍺ (P84; BioLegend), allophycocyanin/Cy7-labeled Ly-6G DTR Institutes of Health. Clec4a4 mice (21) were backcrossed onto the B6 (1A8; BioLegend), allophycocyanin/Cy7-labeled Siglec F (E50-2440; Downloaded from background and used as bone marrow donors. The Institutional Animal BD Biosciences), allophycocyanin-eF780–labeled NKp46 (29A1.4; Care and Use Committee of the University of Minnesota approved all Thermo Fisher Scientific), allophycocyanin-eF780–labeled CD90.2 animal experiments. (53-2.1; Thermo Fisher Scientific), PE/Cy7-labeled Ly-6C (HK1.4; Thermo Fisher Scientific), FITC-labeled CD11c (N418; Thermo Fisher Sci- Infections and immunization entific) Abs, and a fixable viability dye (Ghost Dye Red 780; Tonbo Biosci- 7 ences). The cells were sorted with a FACS Aria II (BD Biosciences) to isolate For intranasal and i.v. infections, mice were injected with 1 3 10 CFUs 2 2 2 2 2 2 live XCR1+ (CD64 NKp46 Ly-6C CD90.2 Ly-6G SIRP⍺ CD11c+XCR1+) of ActA-deficient Listeria monocytogenes expressing the 3K peptide, 2 2 2 2 2 2 http://www.jimmunol.org/ and SIRP⍺+ (CD64 NKp46 Ly-6C CD90.2 Ly-6G XCR1 CD11c+SIRP⍺+) L. monocytogenes/P5R, L. monocytogenes/P2A, or L. monocytogenes/Flic DCs. bacteria, as described previously (5, 22, 23). Mice injected i.v. with 2 2 For coculture, naive B3K508 CD4+ T cells were isolated from Rag1 / L. monocytogenes/P5R or L. monocytogenes/P2A bacteria contained B3K508 TCR Tg mice as described in the cell transfer section. B3K508 similar numbers of organisms in their livers 24 h later (D.I. Kotov, un- cells were cultured with the sort-purified XCR1+ or SIRP⍺+ DCs at 1:1, published observations). L. monocytogenes/3K, L. monocytogenes/P5R, and L. monocytogenes/P2A strains were generated by inserting se- 1:3, and 1:10 ratios of DCs to T cells in complete IMDM for 24 h at 37˚C. quences encoding each peptide in frame with the Listeriolysin O signal Some T cells were also cultured without DCs to serve as negative staining sequence and promoter that drives maximal production under in vivo controls for markers of activation. After culture, T cells were stained with infection conditions, as described previously (22). The P5R epitope Ab and analyzed on a flow cytometry as described in the cell enrichment was inserted into the PR8 influenza A virus genome as previously de- and flow cytometry section. by guest on October 1, 2021 scribed (24, 25). Mice were infected intranasally with 40 PFU of PR8 Confocal microscopy influenza A virus–expressing P5R. Confocal microscopy was performed using a Leica SP5 confocal microscope Cell transfer with two hybrid detectors and two photomultiplier detectors as well as 405, 458, Lymph nodes were collected from Rag12/2 B3K506 and Rag12/2 B3K508 488, 514, 543, 594, and 633 lasers. Twenty-micrometer paraformaldehyde- (20) TCR Tg mice and hand-mashed into a single-cell suspension. Aliquots fixed splenic sections from naive or L. monocytogenes/P5R-infected mice 3 were stained with allophycocyanin-labeled CD4 (RM4–5; Tonbo Biosci- were imaged with a 63 oil immersion objective lens with a numerical ences) Ab and analyzed with fluorescent AccuCheck Counting Beads aperture of 1.4 NA. The splenic sections were stained with F4/80 BV421- (Invitrogen) to assess CD4+ T cell numbers and purity using an LSR II labeled (BM8; BioLegend), Pacific Blue–labeled B220 (RA3-6B2; Bio- 6 ⍺ (BD Biosciences) flow cytometer. For imaging experiments, 1 3 10 Legend), CF405L-labeled CD8 (53-6.7; BioLegend), AF488-labeled TCR Tg cells were transferred into B6 mice 24 h before infection. A total phospho-S6 kinase (p-S6) (2F9; Cell Signaling Technologies), CF555- of 1 3 105 TCR Tg cells were transferred into B6 mice for flow cytometry labeled CD86 (GL-1; BioLegend), AF647-labeled CD45.2 (104; Bio- experiments examining the initial 3 d following L. monocytogenes infection, Legend), AF700-labeled MHCII (M5/114.15.2; BioLegend), CF514-labeled whereas 3 3 103 TCR Tg cells were transferred for experiments examining CD11c (N418; BioLegend), BV480-labeled CD3 (17A2; BD Biosciences), the response at 7 d postinfection with L. monocytogenes or influenza. and AF594-labeled SIRP⍺ (P84; BioLegend) Abs. CF-labeled Abs were generated by conjugating purified Abs from BioLegend to CF405L, Cell enrichment and flow cytometry CF514, or CF555 with Biotium Mix-n-Stain labeling kits (Biotium). The mark and find feature in the Leica Application Suite was used to image 12 Single-cell suspensions were generated by dissociating spleens with the T cell zones in each spleen, with each image consisting of a 20-mm z-stack gentleMACS Dissociator (Miltenyi Biotec) or by hand in a petri dish. acquired at a 0.5-mm step size. Additionally, the Leica SP5 microscope Single-cell suspensions were stained for 1 h at room temperature with was used to image single color–stained Ultracomp eBeads (Thermo Fisher 0.1 mg of FITC-labeled CD90.1 Ab (HIS51; Thermo Fisher Scientific) and Scientific) for generating a compensation matrix. 2 mg of BV650-labeled (L138D7; BioLegend) or BUV395-labeled (2G8; BD Biosciences) CXCR5 Ab. Samples were then enriched for CD90.1 Ab– Image processing and histo-cytometry analysis bound cells using magnetic bead–based enrichment, as described previ- ously (26), with the minor modification that EasySep Mouse APC or FITC Image analysis was performed using Chrysalis software (27). In brief, a Positive Selection Kits (Stemcell Technologies) and EasySep Magnets compensation matrix was created by automatic image-based spectral (Stemcell Technologies) were used. measurements on calibration samples in ImageJ by using Generate Com- For identification of surface markers, the sample was stained on ice with pensation Matrix (27). The compensation matrix was used to perform various combinations of the following Abs: allophycocyanin-ef780–labeled linear unmixing on three-dimensional images and movies with Chrysalis. B220 (RA3–6B2; Thermo Fisher Scientific), allophycocyanin-ef780– Chrysalis was also used for further automated image processing, including labeled CD11b (M1-70; Thermo Fisher Scientific), allophycocyanin-ef780– merging images from the same tissue into a single stack, generating pre- labeled CD11c (N418; Thermo Fisher Scientific), BV786-labeled CD4 view time-lapse sequences to identify informative fields and generating (GK1.5; BD Biosciences), AF700-labeled CD44 (IM7; Thermo Fisher Sci- new channels based on mathematical and morphological operations on entific), FITC-labeled CD45.1 (A20; BioLegend), BUV395-labeled CD45.2 existing channels. Imaris 8.3, 8.4, 9.0, and 9.1 (Bitplane) were used for (104; BD Biosciences), BUV395-labeled CD25 (PC61; BD Biosciences), surface creation to identify cells in images and to run the Sortomato, PE-labeled CD69 (H1.2F3; Thermo Fisher Scientific), and FITC-labeled XTCreateSurfaces, and XTChrysalis Xtensions (27). These Xtensions The Journal of Immunology 3 were used for creating surfaces based on protein expression, quantifying activation. B3K508 T cells were transduced with retrovirus-encoding Cas9 cell/cell interactions, and exporting cell surface statistics. These exported and retrovirus-encoding tRNA/gRNA arrays, whereas the Cas9+ B3K508 statistics were imported into FlowJo for quantitative image analysis. T cells were only transduced with tRNA/gRNA array–encoding retro- virus. For transductions, plates were spun at 1500 rpm for 2 h at 37˚C, Quantitative PCR and then the media was exchanged for complete IMDM-containing IL-2 A total of 1 3 106 B3K506 or B3K508 TCR Tg cells were transferred into (PeproTech). After 3 d of culture, the cells were moved to plates that were not coated with CD3 or CD28 Abs, and 2 d later, the media was B6 mice 24 h before i.v. infection with L. monocytogenes/P5R bacteria. switched to complete IMDM-containing IL-7 (Tonbo Biosciences). After The spleen and lymph nodes were harvested 48 h postinfection and 24 h, cells were either directly transferred into B6 mice or stained with stainedwithFITC-labeledCD90.1(HIS51; Thermo Fisher Scientific), BV786-labeled CD4 (RM4-5; BD Biosciences) Ab and a fixable via- allophycocyanin-ef780–labeled B220 (RA3–6B2; Thermo Fisher Sci- bility dye (Ghost Dye Red 780; Tonbo Biosciences). The stained cells entific), allophycocyanin-ef780–labeled CD11b (M1-70; Thermo Fisher were sorted with a FACS Aria II (BD Biosciences) to isolate live Scientific), allophycocyanin-ef780–labeled CD11c (N418; Thermo Fisher Cas9+gRNA+CD4+ T cells, which were then transferred into B6 mice. Scientific), BV786-labeled CD4 (GK1.5; BD Biosciences), AF700-labeled Each B6 mouse received 2500 T cells. Four days after transfer, mice CD44 (IM7; Thermo Fisher Scientific), and a fixable viability dye (Ghost were infected with L. monocytogenes/3K bacteria, and the spleens were Dye Red 780; Tonbo Biosciences). The TCR Tg cells were then enriched harvested 7 d postinfection for flow cytometric analysis. with the EasySep Mouse FITC Positive Selection Kit (Stemcell Technol- + ogies) and EasySep Magnets (Stemcell Technologies). Live TCR Tg CD4 Statistical analysis T cells were sorted from the enriched sample into TRIzol (Thermo Fisher Scientific) using a FACS Aria II (BD Biosciences). RNA was extracted Statistical significance was determined using Prism (GraphPad) software for using chloroform (Thermo Fisher Scientific) and purified with the RNeasy unpaired two-tailed Student t test and one-way and two-way ANOVA tests. Micro Kit (Qiagen). RNA quantities were measured with a Qubit 2.0 Prism (GraphPad) was also used to calculate linear correlations and R2. (Thermo Fisher Scientific) and 41 ng RNA of each sample was converted Code availability to cDNA with SuperScript IV Reverse Transcriptase (Thermo Fisher Sci- Downloaded from entific). Primers (Supplemental Table I) were designed with the National All of the custom code generated for image processing or analysis can be Center for Biotechnology Information Primer-Blast tool (28). Quantitative downloaded at https://histo-cytometry.github.io/Chrysalis/. PCR (qPCR) was performed with FastStart SYBR Green (MilliporeSigma) in a thermocycler (Eppendorf). The resulting data were analyzed by nor- malizing to the housekeeping gene Gapdh and then calculating fold change Results in gene expression for B3K506 cells relative to B3K508 cells with the TCR affinity biases Th cell differentiation comparative CT method (29). We used B3K506 and B3K508 TCR Tg strains (20) to examine the http://www.jimmunol.org/ CRISPR/Cas9 screen relationship between TCR affinity and T cell differentiation. A CRISPR/Cas9 system using two MSCV-based g retroviral vectors was B3K506 and B3K508 mice contain monoclonal populations of used to target genes of interest. One virus encoded Cas9 and the fluorescent CD4+ T cells with TCRs that bind to a peptide called P5R com- protein mNeongreen (30) and the other encoded guide RNAs (gRNAs) and b the fluorescent protein mAmetrine (31). These vectors were created by plexed with the I-A MHCII molecule of B6 mice. The B3K506 modifying the LMP-Amt vector (32), which was a gift from S. Crotty (La TCR, however, binds more strongly to this ligand than B3K508 Jolla Institute). The vector encoding Cas9 and mNeongreen was generated TCR (KDs of 11 and 93 mM), whereas the B3K508 TCR binds to by replacing the short hairpin RNA–encoding segment, PGK promoter, and a related ligand P2A:I-Ab more strongly than the B3K506 TCR mAmetrine gene with Cas9-P2A and mNeongreen (Allele Biotechnology) (K s of 175 and 278 mM). The K s of 11 and 93 mM will be through In-Fusion Cloning (Takara Bio). The Cas9-P2A fragment was D D by guest on October 1, 2021 cloned from the lentiCRISPRv2 puro plasmid, which was a gift from considered high and medium affinities, respectively, whereas KDs B. Stringer (plasmid no. 98290; Addgene). of 175 and 278 mM will be considered to be low affinities. The To generate the gRNA vector, the LMP-Amt vector (32) was modified by effect of these affinity differences on differentiation was ex- removing the SapI site and replacing the short hairpin RNA–encoding amined by adoptive transfer of CD90.1+ B3K506 or B3K508 segment with the bacterial toxin gene CCDB flanked by AarI sites using T cells into CD90.2+ mice (39) that were then infected i.v. In-Fusion Cloning (Takara Bio). This CCDB-containing fragment was cloned from the pMOD_B2303 plasmid, which was a gift from D. Voytas with L. monocytogenes bacteria engineered to express P5R (University of Minnesota). This modified LMP-Amt plasmid served as the (L. monocytogenes/P5R) or P2A (L. monocytogenes/P2A) peptide. recipient for Golden Gate cloning reactions that replaced the CCDB gene As expected from the work of Huseby and colleagues (20), the with a transfer RNA (tRNA)/gRNA array (33–35) using AarI (Thermo expansion of B3K506 and B3K508 T cells in response to Fisher Scientific), SapI (New England BioLabs), and T4 (New England BioLabs) enzymes. The gRNAs used in the tRNA/gRNA arrays were L. monocytogenes/P5R and L. monocytogenes/P2A infection cor- designed in Benchling to target functional motifs identified in InterPro related positively with TCR affinity (Fig. 1A). Previous work (9) (36) with two gRNA designed for each target gene. Primers encoding indicated that L. monocytogenes infection drives the differentia- the gRNAs were then generated using the Voytas Lab Plant Genome tion of Th1 cells, which express the T-bet transcription factor (40) Engineering Toolkit (37), and the cloning of the tRNA/gRNA array was and Tfh cells, which express the CXCR5 chemokine receptor performed as described previously (37). Retrovirus was prepared as described previously (38), with minor al- (41–43). B3K506 and B3K508 cells also adopted these fates terations. In brief, Platinum-E cells (Cell Biolabs) were grown in complete (Fig. 1B), although high TCR affinity (lower KD) biased differ- DMEM (Life Technologies) prior to transfection with Polyethylenimine, entiation to the Th1 cell fate and medium affinity toward the Tfh Linear, MW 25,000 (Polysciences), pCL-Eco, and retroviral plasmids cell fate as we reported previously (5), whereas lower-affinity encoding Cas9 or tRNA/gRNA arrays. After transfection, the media was supplemented with ViralBoost (ALSTEM Cell Advancements) and 30 mM T cells predominantly remained uncommitted, as defined by a water soluble cholesterol (MilliporeSigma). The virus-containing su- lack of T-bet or CXCR5 (Fig. 1C). Despite having a lower per- pernatant was collected 24 and 48 h after transfection and filtered with a centage of Tfh cells than the medium TCR affinity population, the 0.45-mm Nylon, 25-mm Syringe Filter (Thermo Fisher Scientific). high TCR affinity T cell population had a greater absolute number 2 Aliquots were stored at 80˚C for up to 3 mo. of Tfh cells because of greater proliferation (Fig. 1C). The TCR Retroviral transduction was performed as described previously (38), with minor alterations. Specifically, naive B3K508 CD4+ T cells or naive Cas9+ affinity–mediated differentiation bias was recapitulated in mice B3K508 CD4+ T cells were isolated from Rag12/2 B3K508 TCR Tg or infected with influenza expressing P5R (PR8/P5R). After PR8/ 2 2 Rag1 / Cas9+ B3K508 TCR Tg mice, respectively, as described in the P5R infection, T cells with a higher TCR affinity were again cell transfer section. These B3K508 T cells were grown in a 96-well plate biased toward the Th1 cell fate, whereas lower TCR affinity with complete IMDM (MilliporeSigma) containing IL-7 (Tonbo Biosci- ences). Cells were activated in plates coated with anti-CD3 (2C11; Bio X biased cells toward the Tfh cell fate, demonstrating that the in- Cell) and anti-CD28 (37.51; Bio X Cell). The cells were transduced with fluence of TCR affinity on T cell differentiation is not restricted retroviral supernatant and polybrene (MilliporeSigma) 24 and 40 h after to L. monocytogenes infection (Fig. 1D). 4 TCR AFFINITY–BASED CONTROL OF Th CELL FATE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. TCR affinity differences between B3K506 and B3K508 cells influence T cell expansion and bias T cell differentiation. (A) Fold expansion of B3K506 or B3K508 cells 7 d after L. monocytogenes/P5R (n =7)orL. monocytogenes/P2A (B3K506, n = 7; B3K508, n = 8) infection plotted in relation to the affinity of the relevant TCR/p:MHCII interaction. (B) Representative flow cytometric identification of TCR Tg cells (red gates in the left and middle panels) in naive or L. monocytogenes/P5R-infected mice. Th1 (T-bet+), Tfh (CXCR5+), or uncommitted (T-bet2CXCR52) cells were identified in the right panel in the red, green, and purple gates, respectively. (C) Differentiation (Th1 cells in red, Tfh cells in green, and uncommitted cells in blue) of B3K506 or B3K508 cells 7 d after L. monocytogenes/P5R (n =7)orL. monocytogenes/P2A (B3K506, n = 7; B3K50,8 n = 8) infection plotted in relation to the affinity of the relevant TCR/p:MHCII interaction. (D) Frequency of Th1, Tfh, or uncommitted cells among B3K506 (filled circle, n = 8) or B3K508 (empty circle, n = 7) effector cell populations 7 d postinfection with influenza PR8/P5R. (E) Frequency of Th17, Th1, Tfh, or uncommitted cells among B3K506 (filled circle, n = 7) or B3K508 (empty circle, n = 7) cells 7 d after intranasal infection with L. monocytogenes/P5R. The bars in (D) and (E) represent the mean. Pooled data from two or three independent experiments are shown. Spleens were analyzed for the depicted experiments. Multiple t tests were used to determine significance for (D) and (E). A linear regression was used to calculate significance for (A), and a nonlinear fit was used to calculate the R2. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

The effect of TCR affinity on T cell differentiation was also observed after this infection was likely a consequence of low Ag tested after intranasal L. monocytogenes/P5R infection, which presentation due to poor adaptation of this enteric bacterium to the induces Th17 cells along with Th1 and Tfh cells (23). Th17 cell intranasal route. differentiation was identified by expression of the lineage-defining + ⍺+ transcription factor RORɣt (44). B3K506 cells generated larger XCR1 and SIRP DCs differ in Ag presentation and effect fractions of Th17 and Th1 cells, an equal fraction of Tfh cells, on T cell differentiation and a lower fraction of uncommitted cells than B3K508 cells Before testing the differential Ag presentation hypothesis, we first (Fig. 1E), suggesting that high TCR affinity also promotes the examined the kinetics of Th cell differentiation to identify the time Th17 cell fate. The large percentage of uncommitted Th cells when critical T cell/DC interactions occur. B3K506 and B3K508 cell The Journal of Immunology 5 differentiation was examined over the first 3 d after L. monocytogenes by administering diphtheria toxin to radiation chimeras generated infection because Th1/Tfh cell bifurcation occurred in this time with Clec4a4DTR bone marrow (21). The effect of XCR1+ DCs frame in another system (11, 38). A larger fraction of the B3K508 was studied with Batf32/2 mice, which lack these cells (45). A effector T cell population consisted of CXCR5+ cells than the 60% reduction in SIRP⍺+ DCs led to an increase in Th1 cells and B3K506 population on days 1, 2, and 3 postinfection (Fig. 2A) a decrease in Tfh cell differentiation by B3K506 and B3K508 and did not change after day 3 (Figs. 1C, 2A). These results cells following L. monocytogenes/P5R infection, irrespective of suggest that the Th1/Tfh cell bifurcation occurs within the first 3 d TCR affinity (Fig. 2C). Ablation of XCR1+ DCs had the opposite following L. monocytogenes infection. effect: increased Tfh and decreased Th1 cell formation for high- We also confirmed that SIRP⍺+ DCs produce more p:MHCII and medium-affinity TCR T cells (Fig. 2D). These results indicate complexes than XCR1+ DCs after L. monocytogenes infection, as that XCR1+ DCs are critical for optimal Th1 cells and SIRP⍺+ reported in another system (17). The amount of p:MHCII presenta- DCs for optimal Tfh cell differentiation after L. monocytogenes tion was determined by measuring the capacity of purified splenic infection for high- and low-affinity T cells. Thus, this approach DCs from day 2 i.v. L. monocytogenes/P5R-infected mice to stimu- indicated that differential Ag presentation by XCR1+ and SIRP⍺+ late CD69 and CD25 expression by cognate Th cells in vitro (10, 17) DCs cannot explain the tendency of high-affinity cells to produce (Fig. 2B). SIRP⍺+ DCs were more potent activators of B3K508 cells Th1 cells and low-affinity cells to produce Tfh cells. than XCR1+ DCs in this assay (Fig. 2B), indicating that SIRP⍺+ DCs display more P5R:I-Ab complexes than XCR1+ DCs, as expected. TCR affinity does not influence T cell/DC interactions We then tested how the different DC types affected Th cell following Listeria infection differentiation using a cell ablation strategy. The majority of Because cell ablation can result in compensatory effects (46), SIRP⍺+ DCs, which express Clec4a4 (DCIR2), were ablated we also tested the DC preference model by direct observation Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Effect of genetic ablation of XCR1+ or SIRP⍺+ DCs on Th cell differentiation. (A) Tfh cell differentiation by B3K506 (filled circle, n =6) and B3K508 (empty circle, day 1, day 2, n = 6; day 3, n = 8) cells in the initial 3 d following L. monocytogenes/P5R infection. (B) Experimental model and resulting mean frequencies 6 SD of CD69+ CD25+ B3K508 cells after 1 d of coculture with SIRP⍺+ (square, n = 3) or XCR1+ (circle, n = 3) DCs from L. monocytogenes/P5R-infected mice 2 d postinfection. (C) Frequency of Tfh, Th1, or uncommitted cells among B3K506 or B3K508 TCR Tg cells in diphtheria toxin (DT)–treated WT (filled circle) or Clec4a4DTR (empty circle) mice 3 d after L. monocytogenes/P5R (B3K506 WT, n =8;Clec4a4DTR, n = 8; B3K508 WT, n =6;Clec4a4DTR, n =8)orL. monocytogenes/P2A (B3K508 WT, n =4;Clec4a4DTR, n = 6) infection. (D) Frequency of Tfh, Th1, or uncommitted cells among B3K506 or B3K508 cells in WT (filled circle) or Batf32/2 (empty circle) mice 3 d after L. monocytogenes/P5R (B3K506 WT, n = 11; Batf32/2, n = 7; B3K508 WT, n =8;Batf32/2, n =7)orL. monocytogenes/P2A (B3K508 WT, n =5;Batf32/2, n = 7) infection. The bars in (A), (C), and (D) represent the mean. Pooled data from two or three independent experiments are shown. Spleens were analyzed for the depicted experiments. Two- way ANOVAwas used to determine significance for (B), and one-way ANOVAwith Sidak multiple comparison test was used to determine significance for (A), (C), and (D). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 6 TCR AFFINITY–BASED CONTROL OF Th CELL FATE of T cell/DC interactions in mice with a normal complement of contained voxels with strong MHCII and CD11c, but weak B220, DCs. Histo-cytometry was used to assess B3K506 and B3K508 CD3, F4/80, and CD45.2 signal (Fig. 4A). XCR1+ DCs and T cell interactions with XCR1+ and SIRP⍺+ DC because two- SIRP⍺+ DCs were identified based on CD8⍺ and SIRP⍺ ex- photon microscopy cannot simultaneously resolve all the pression, respectively (16, 18, 48, 49) (Fig. 4B). Identification T cell and DC populations of interest. The analysis focused on of XCR1+ and SIRP⍺+ DCs was validated by comparing the T cell zones of splenic sections from L. monocytogenes/P5R- DC gating strategy in wild-type (WT) and Batf32/2 mice, which infected and naive CD45.1+ mice that received CD45.2+ lack XCR1+ DCs,1dafterL. monocytogenes/P5R infection B3K506 or B3K508 cells because the T cells were primarily (Fig. 4B). DCs in the WT mice consisted of∼30% XCR1+ DCs located in this location (39). TCR Tg cells were identified and 70% SIRP⍺+ DCs, whereas over 90% of the DCs were based on the congenic marker, CD45.2, which was used to XCR1+ DCs in Batf32/2 mice (Fig. 4B). Productive T cell/DC generate digital surfaces corresponding to these cells (Fig. 3A, interactions were identified as T cells undergoing T cell acti- 3B). TCR Tg Th cells receiving acute TCR signals, and thus vation (p-S6+) and touching a DC(s) (Fig. 4C). B3K506 and likely engaged in productive interactions with DCs, were B3K508 cells interacted equally with DCs 1 and 2 d after identified by staining p-S6, a T cell activation–induced phos- L. monocytogenes/P5R infection (Fig. 4D). The ratio of T cell phorylation event associated with the mTORC1 pathway (47) contacts with SIRP⍺+ or XCR1+ DCs was analyzed to determine (Fig. 3B, 3C). whetherlow-orhigh-affinityTcells interacted preferentially Although B3K506 and B3K508 cells had equal p-S6 levels 1 d with one DC subset or the other. The analysis revealed that postinfection, B3K508 cells had significantly lower values 1 d later high-affinity B3K506 and medium-affinity B3K508 cells inter- (Fig. 3D). This result suggests that higher TCR–affinity B3K506 acted 1.5 times more frequently with SIRP⍺+ than with XCR1+ cells had more prolonged TCR signaling than lower TCR–affinity DCs 1 and 2 d postinfection (Fig. 4E). Thus, TCR affinity– Downloaded from B3K508 cells. mediated bias in T cell differentiation was not associated with Th cell interactions with the DC subsets were then assessed. preferential access of low- or high-affinity T cells to a particular DCs were identified by generating surfaces on a DC channel that DC subset. http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Histo-cytometry analysis of the effect of TCR affinity on T cell activation. (A) Representative images of TCR Tg cells identified with CD45.2 (red) and p-S6 (green) Abs in a spleen 1 d after L. monocytogenes/P5R infection. (B) Digital surfaces of the cells were created and analyzed with histo- cytometry. (C) TCR Tg cell expression of p-S6 was then quantified in FlowJo. (D) Histo-cyotmetric quantification of B3K506 (filled circle) and B3K508 (empty circle) cell numbers and their p-S6 expression in naive mice and mice during the initial 3 d following L. monocytogenes/P5R infection (naive, day 1, day 2, n = 6; day 3, n = 4). The bars in (B) represent the mean. Scale bar, 20 mm. Pooled data from three independent experiments are shown. Spleens were analyzed for the depicted experiments. One-way ANOVA was used to determine significance for (D). **p , 0.01. The Journal of Immunology 7

CD25, Eef1e1, and Gbp2 are TCR-regulated proteins that bias CD25+ Tfh cells (Fig. 5B), adding support to the hypothesis that CD25 helper T cell differentiation signaling represses Tfh cell differentiation. These results suggest that The lack of impact on T cell/DC interactions suggested that TCR high-affinity TCR/p:MHCII interactions promote Th1 cell differenti- affinity regulates differentiation in a T cell intrinsic manner. We, ation by maintaining CD25, which represses the Tfh cell fate. therefore, tested a model in which increasing TCR affinity for TCR signaling induces many proteins in addition to CD25, some p:MHCII drives greater IL-2R expression, STAT5 signaling, and of which could be mediators of TCR affinity–based effects on Th Th1 cell differentiation. As described previously (4), B3K506 and cell differentiation. We focused on nine candidates from a published B3K508 cells expressed CD25 (IL-2Ra) in a TCR affinity– list of genes induced in naive TCR Tg T cells in vitro to a greater dependent manner 2 d postinfection with L. monocytogenes/P5R, extent by a large amount of agonist peptide than by a lower amount with higher TCR affinity correlating with higher CD25 expression (10). The candidate genes were identified by excluding genes with (Fig. 5A, 5B). This difference in CD25 expression inversely ,10 reads per kilobase of transcript, per million mapped for the no- correlated with Tfh cell differentiation for high and medium peptide condition, less than a 4-fold increase in expression in the TCR–affinity T cells, in line with IL-2R signal transduction sup- high- versus no-peptide conditions, and less than a 2-fold increase pressing Tfh cell differentiation (12) (Fig. 5A). T cells with a low- in expression in the high- versus low-peptide conditions. Of the 12 affinity TCR had the lowest CD25 expression, but less Tfh cell most abundantly expressed genes, Nr4a1 and Irf4 were excluded differentiation than T cells with a medium affinity TCR, which from further analysis because of their well-established roles in could be attributed to a larger proportion of uncommitted cells T cell differentiation (8, 50–55), whereas CD25 was used as a (Fig. 5A). Moreover, CD25+ Th1 cells expressed more CD25 than positive control. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Histo-cytometry analysis of DCs and T cell/DC interactions. (A) Representative images of MHCII (gray), CD11c (yellow), and B220 (purple) staining from a spleen 1 d after L. monocytogenes/P5R infection. Histo-cytometry was used to generate surfaces for DCs (CD11c+MHCII+B2202CD32CD45.22F4/802). XCR1+ or SIRP⍺+ surfaces were identified among the DC surfaces based on the indicated gates. (B) Histo- cytometry identification of XCR1+ and SIRP⍺+ DCs in WT and Batf32/2 mice infected with L. monocytogenes/P5R for 1 d. (C) Representative histo- cytometry analysis displaying the percentage of p-S6+ TCR Tg cells interacting with XCR1+ or SIRP⍺+ DCs in mice infected with L. monocytogenes/P5R for 1 or 2 d. Interactions are defined as TCR Tg cells that are within 0 mm of either DC subset with 1 3 1021 mm added to each cell for logarithmic visualization. (D) Histo-cytometry–identified interactions between p-S6+ B3K506 (filled circle, n = 6) or B3K508 (empty circle, n = 6) T cells with XCR1+ or SIRP⍺+ DCs in mice infected with L. monocytogenes/P5R for 1 or 2 d. (E) Ratio of T cell interactions with SIRP⍺+ DC to XCR1+ DC interactions by p-S6+ B3K506 (filled circle, n = 6) or B3K508 (empty circle, n = 6) T cells. The bars in (D) and (E) represent means. Scale bar, 20 mm. Pooled data from three independent experiments are shown. Spleen was analyzed for the depicted experiments. One-way ANOVAwas used to determine significance for (D) and (E). No significant differences were detected. 8 TCR AFFINITY–BASED CONTROL OF Th CELL FATE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. CD25, Eef1e1, and CD25 are upregulated by TCR signaling and bias helper T cell differentiation. (A) Frequency of CD25+ and CXCR5+ B3K506 and B3K508 T cells 2 d postinfection with L. monocytogenes/P5R (CD25%, n = 11; CXCR5%, n =7)orL. monocytogenes/P2A (n = 7). (B) CD25 geometric mean fluorescence intensity (gMFI) for B3K506 and B3K508 T cells 2 d postinfection with L. monocytogenes/P5R (n =7)orL. monocytogenes/ P2A (n = 7) and for CD25+ Th1 (T-bet+) and Tfh (CXCR5+) B3K508 T cells 2 d postinfection with L. monocytogenes/P5R (n = 9). (C) Fold change in gene expression relative to B3K508 T cells based on qPCR analysis for B3K506 (filled circle, n = 6) and B3K508 (empty circle, n = 8) T cells 2 d postinfection with L. monocytogenes/P5R. (D) Representative flow cytometric identification of gRNA+ Cas9+ B3K508 T cells (red squares) 7 d after L. monocytogenes/ 3K infection and (E) their differentiation into Th1 (T-bet+), Tfh (CXCR5+), or uncommitted (T-bet2CXCR52) cell lineages when targeting LacZ, CD25, Eef1e1, or Gbp2. (F) Quantification of gRNA+ Cas9+ B3K508 T cells and (G) their fold change in Th1% cells relative to the LacZ group 7 d after L. monocytogenes/3K infection. The bars in (A), (B), (C), (F), and (G) represent means. Pooled data from two or three independent experiments are shown. Spleen was analyzed for the depicted experiments, except (C), which represents data from spleen and lymph nodes. Multiple t tests were used to determine discoveries based on a false discovery rate of 5% for (C) and significance for (A), (B), (F), and (G). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. #, discovery.

Expression of the candidate genes was validated by qPCR analysis targeting. Cas9-expressing B3K508 cells were transduced with a of B3K506 and B3K508 T cells 2 d after L. monocytogenes/P5R retrovirus encoding the fluorescent protein mAmetrine and infection. Of the nine genes analyzed, seven were induced to a gRNAs targeting the candidate genes or the bacterial gene LacZ greater extent in B3K506 than B3K508 T cells, indicating a as a control. The transduced T cells were then transferred into B6 dependence on TCR signaling (Fig. 5C). The role of these seven mice before i.v. infection of the mice with L. monocytogenes genes in Th cell differentiation was determined by disrupting bacteria expressing 3K peptide (L. monocytogenes/3K). The each gene in B3K508 cells using CRISPR/Cas9-mediated gene transferred gRNA+ T cells were identified based on CD90.1 and The Journal of Immunology 9 mAmetrine expression and evaluated for adoption of the Th1, Our results indicate that the Eef1e1 is another TCR signal Tfh, and uncommitted cell lineages (Fig. 5D, 5E). The expan- strength–responsive driver of the Th1 cell fate. Eef1e1 binds to sion of gRNA+ B3K508 cells in the recipient mice was unaf- Lamin A (61), and overexpression studies performed in cell lines fected in the majority of cases, with the exceptions of Eef1e1 demonstrated that Eef1e1 facilitates Lamin A ubiquitination and and Srm, loss of which resulted in reduced or enhanced ex- degradation via the ubiquitin ligase, Siah1 (62). Interestingly, pansion, respectively (Fig. 5F). As expected, targeting the Il2ra Lamin A–deficient T cells have reduced in vivo Th1 cell differ- gene–encoding CD25 reduced Th1 cell differentiation relative entiation (63), whereas Siah1 promotes Th17 cell differentiation to the LacZ control population (Fig. 5G). Of the candidate in vitro (64). Thus, strong TCR signaling may favor the differ- genes, only Eef1e1 (encoding Eef1e1, also known as AIMP3 entiation of non-Tfh cells by engaging a pathway involving and p18) and Gbp2 (encoding Gbp2) had significant effects on Eef1e1, Lamin A, and Siah1. The Th1 cell–promoting effects of T cell differentiation. Ablation of Eef1e1 reduced Th1 cell Eef1e1 may also be related to the role that we identified for this differentiation (Fig. 5G), whereas targeting Gbp2 led to en- molecule in proliferation. Craft and colleagues (65) showed hanced Th1 cell differentiation. These results demonstrate that that IL-2–mediated activation pathways associated with cellu- TCR-drivenEef1e1promotesTh1cell differentiation, whereas lar proliferation promote Th1 over Tfh cell differentiation. Gbp2 inhibits this process. Eef1e1, which interacts with ATM (66), a protein critical for TCR signaling-induced proliferation (67), may contribute to this non-Tfh cell–promoting process. Discussion Increased expression of Eef1e1, CD25, and IRF4 and induc- Our results indicate that uncommitted effector Th cells are induced tion of downstream pathways is a plausible explanation for the by low-affinity TCR/p:MHCII interactions. Tfh cells then become increase in Th1 cell formation that occurs as TCR affinity in- Downloaded from the preferred effector cells as TCR affinity increases, until Th1 and creases from moderate to high levels. This mechanism, however, Th17 cells become predominant at higher affinities. The fact that cannot explain the increase in Tfh cell formation that occurs as + XCR1 DCs are potent producers of IL-12 (18), but relatively TCR affinity increases from low to moderate levels. Our results poor producers of p:MHCII complexes, whereas IL-2–consuming indicate that Gbp2, a TCR-induced protein that promoted Tfh ⍺+ SIRP DCs have the opposite properties suggested an extrinsic cell formation, could be involved in this transition. Gbp2 is explanation of these TCR affinity–based effects on Th cell dif- associated with reduced mitochondrial fission (68), which is http://www.jimmunol.org/ + ferentiation. XCR1 DCs could display fewer p:MHCII complexes important for aerobic glycolysis (69–71), and operates at low + than SIRP⍺ DCs, creating a situation in which Th cells with low- levels in Tfh cells (65, 72). Therefore, Gbp2 may promote Tfh + affinity TCRs would be unable to interact with XCR1 DCs cell formation by keeping aerobic glycolysis in check. An al- thereby receive IL-12 and become Th1 cells, but could still in- ternative hypothesis is that Gbp2 represses Th1 cell differenti- + teract with SIRP⍺ DCs to become Tfh cells. There was potential ation, and high TCR affinity allows T cells to overcome this + for this model to be correct because we confirmed that XCR1 inhibition. b + DCs present fewer P5R:I-A complexes than SIRP⍺ DCs 2 d Together, our results suggest a general model in which low TCR after L. monocytogenes/P5R infection, the time frame when Tfh affinity and signaling induces Gbp2, thereby promoting Tfh cell by guest on October 1, 2021 cell differentiation is initiated. However, we found medium- and differentiation, whereas high TCR affinity and signaling drives high-affinity Th cells equally interacted with each DC subset, Eef1e1 and CD25, which represses the Tfh cell fate and promotes + + 20% with SIRP⍺ DCs, and 15% with XCR1 DCs. Thus, the non-Tfh cell formation. The type of non-Tfh cells would then + frequency of interaction with XCR1 DCs cannot explain the depend on the type of cytokines produced by the innate immune greater tendency of high-affinity Th cells to produce non-Tfh system. For example, intranasal infection, which elicits IL-6 and cells in this system. TGF-b production by cervical lymph node DCs (73), would favor We found better evidence in favor of a Th cell intrinsic Th17 cell differentiation, whereas i.v. infection, which stimulates mechanism. The CD25 component of the IL-2R was an attractive IL-12 production by splenic XCR1+ DCs (18), would bias to- mediator of such an effect because its expression is proportional to ward Th1 cell differentiation. This mechanism would ensure TCR signaling (7, 10, 13, 14) and biases differentiation by that polyclonal populations of epitope-specific naive Th cells, inhibiting Tfh cell formation through Blimp1 induction (12). We which always contain a spectrum TCR affinities (26), generate found that high TCR affinity positively correlated with stronger effector cells capable of promoting humoral and cellular im- TCR signaling, maintenance of CD25 expression, and Th1 cell munity suited to clearance of the microbe. The tendency of differentiation. Longer expression of the IL-2R would provide moderate-affinity T cells to become Tfh cells may foster more time for IL-2 binding and induction of the STAT5-regulated affinity maturation by only allowing B cells with the highest- Tfh cell repressor Blimp1 (11, 12). DiToro et al. (56) recently affinity Igs to garner T cell help, whereas diversion of high- showed that the capacity to produce IL-2 is also a key factor in the affinity T cells to become myeloid cell helpers would ensure Tfh/Th1 cell bifurcation process. They found that Th1 cells are that the helped cells achieve the optimal state of microbicidal derived from precursors that express CD25 and experience strong activity. STAT5 signaling, but do not produce their own IL-2, whereas the Tfh cells are derived from precursors that produce the IL-2 used by the CD25hi Th1 cell precursors, but do not respond to IL-2 Acknowledgments because of low IL-2R expression. Our result that Th1 cells express We thank J. Walter, C. Ellwood, the University of Minnesota Center for more CD25 than Tfh cells is consistent with this model. TCR Immunology Imaging Core, the University of Minnesota Imaging Centers, affinity likely influences the non-Tfh/Tfh cell balance by deter- and the University of Minnesota Flow Cytometry Resource for technical assistance; D. F. Voytas and J. J. Belanto for helping generate the mining how many precursor cells respond to IL-2. This process CRISPR/Cas9 system; and M. Y.Gerner for suggestions on histo-cytometry. may also operate during type 2 immune responses because the population of house dust mite allergen p:MHCII-specific Th cells induced by allergen inhalation is comprised of Th2 and Tfh cells Disclosures (57), and the Th2 cells are CD25 dependent (58–60). The authors have no financial conflicts of interest. 10 TCR AFFINITY–BASED CONTROL OF Th CELL FATE

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