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 Supplementary http://www.jimmunol.org/content/suppl/2019/03/08/jimmunol.180160 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 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.