TCR Revision Generates Functional CD4+ T Cells J. Scott Hale, Maramawit Wubeshet and Pamela J. Fink This information is current as J Immunol 2010; 185:6528-6534; Prepublished online 22 of September 23, 2021. October 2010; doi: 10.4049/jimmunol.1002696 http://www.jimmunol.org/content/185/11/6528 Downloaded from References This article cites 36 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/185/11/6528.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 September 23, 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology TCR Revision Generates Functional CD4+ T Cells J. Scott Hale, Maramawit Wubeshet, and Pamela J. Fink CD4+Vb5+ peripheral T cells in C57BL/6 mice respond to encounter with a peripherally expressed endogenous superantigen by undergoing either deletion or TCR revision. In this latter process, cells lose surface Vb5 expression and undergo RAG-dependent rearrangement of endogenous TCRb genes, driving surface expression of novel TCRs. Although postrevision CD4+Vb52TCRb+ T cells accumulate with age in Vb5 transgenic mice and bear a diverse TCR Vb repertoire, it is unknown whether they respond to homeostatic and antigenic stimuli and thus may benefit the host. We demonstrate in this study that postrevision cells are functional. These cells have a high rate of steady-state homeostatic proliferation in situ, and they undergo extensive MHC class II-dependent lymphopenia-induced proliferation. Importantly, postrevision cells do not proliferate in response to the tolerizing superantigen, implicating TCR revision as a mechanism of tolerance induction and demonstrating that TCR-dependent activation of postrevision cells is not driven by the transgene-encoded receptor. Postrevision cells proliferate extensively to commensal b bacterial Ags and can generate I-A –restricted responses to Ag by producing IFN-g following Listeria monocytogenes challenge. Downloaded from These data show that rescued postrevision T cells are responsive to homeostatic signals and recognize self- and foreign peptides in the context of self-MHC and are thus useful to the host. The Journal of Immunology, 2010, 185: 6528–6534. eveloping T cells within the thymus undergo rigorous tional selective force that maintains useful components of the peri- selection to ensure the usefulness and safety of mature pheral T cell repertoire (reviewed in Ref. 2). T cells bearing rearranged ab TCRs. Thymocytes at the Mechanisms in the lymphoid periphery exist to keep in check http://www.jimmunol.org/ D2 2 CD4 CD8 double-negative stage undergo RAG-mediated TCRb self-reactive peripheral T cells that elude negative selection in gene rearrangement, and generation of a functional TCRb-chain the thymus. In Vb5 transgenic (Tg) mice, Vb5+CD4+ T cells in induces proliferation and CD4 and CD8 coreceptor expression. At the lymphoid periphery encounter an endogenous superantigen the CD4+CD8+ double-positive stage, a second wave of RAG- encoded by a defective mouse mammary tumor virus (Mtv) and dependent rearrangement, this time at the TCRa locus, gener- are tolerized to this self-Ag by several means. Mtv superantigen ates rearrangements that encode TCRa-chains that pair with the drives deletion of most CD4+ T cells, resulting in a severe age- already available TCRb-chains, and these TCRab pairs are tested dependent decline in the CD4/CD8 ratio (3, 4). Mtv drives some for interactions with self-MHC molecules. Useful affinity for self- cells to undergo TCR revision, through which CD4+ T cells lose MHC class II (MHC II) and class I molecules drives positive surface Vb5 expression, re-express RAG, and undergo TCRb by guest on September 23, 2021 selection, upregulation of TCR surface expression, and commit- gene rearrangement, resulting in the age-dependent accumulation ment to the CD4 and CD8 T cell lineages, respectively. Insuf- of a rescued population of postrevision CD4+Vb52TCRb+ T cells ficient interactions with MHC result in death by neglect. Positively (5, 6). The expression of RAG (5, 7–18) and the presence of TCR selected CD4 single-positive and CD8 single-positive thymocytes Vb-(DJ)b or Va-Da recombination intermediates (5, 7, 11, 13–17) test their TCRs against a broad array of self-MHC–self-peptide in peripheral T cells, as well as a blockade in the generation of ligands for self-reactivity, and those cells that recognize self are postrevision CD4+Vb52TCRb+ T cells when Rag2 is condition- removed from the repertoire by apoptosis (reviewed in Ref. 1). ally deleted in postthymic mature Vb5+ T cells (19), unequivo- After thymic exit, basal TCR signaling in the lymphoid periphery cally identify TCR revision as a process that targets mature CD4+ promotes survival and homeostatic proliferation and is an addi- Vb5+ cells in the lymphoid periphery for RAG-dependent gen- eration of new TCRs (reviewed in Ref. 20). Postrevision T cells express a diverse TCRb repertoire with TCRs characterized by shortened CDR3 loops resulting from fewer N nucleotides (18). Department of Immunology, University of Washington, Seattle, WA 98195 Given the specialized selecting environment of the thymus, it is Received for publication August 9, 2010. Accepted for publication September 24, unknown whether cells generated through TCR revision in the 2010. lymphoid periphery have been selected for appropriate T cell 2 This work was supported by the National Institutes of Health (Grant RO1 AG 13078 function that could benefit the host. Although postrevision Vb5 to P.J.F.), the Cancer Research Institute’s Predoctoral Emphasis Pathway in Tumor T cells proliferate in response to TCR cross-linking (5) and con- Immunology Program (to J.S.H.), and the National Cancer Institute Basic and Cancer Immunology (Grant T32CA09537 to J.S.H.). tain a population of IL-17–producing cells following PMA plus The content of this work is solely the responsibility of the authors and does not ionomycin stimulation (21), their homeostatic potential and im- necessarily represent the official views of the National Institutes of Health, the munocompetence remain untested. In this study, we assessed the Cancer Research Institute, or the National Cancer Institute. ability of postrevision cells to respond to homeostatic factors in- Address correspondence and reprint requests to Dr. Pamela J. Fink, University of cluding self-MHC and foreign Ag following bacterial challenge of Washington, 1959 NE Pacific Street, Health Sciences Building I607H, Seattle, WA 98195. E-mail address: pfi[email protected] Vb5 Tg mice. Our data indicate that postrevision T cells require Abbreviations used in this paper: B6, C57BL/6; iono, ionomycin; LIP, lymphopenia- TCR–MHC interactions for maximal lymphopenia-induced pro- induced proliferation; LLO, listeriolysin O; MHC II, MHC class II; MLN, mesenteric liferation (LIP) and the generation of Ag-specific self-MHC– lymph node; Mtv, mammary tumor virus; nonTg, nontransgenic; Tg, transgenic; restricted effector T cell responses to bacterial Ag. Additionally, unstim, untreated. postrevision cells possess no residual reactivity to Mtv super- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 antigen, showing that TCR signaling occurs through the revised www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002696 The Journal of Immunology 6529 TCR and not the transgene-encoded receptor, further defining TCR cultured for 3 d at 37˚C and 7% CO2, followed by surface staining and flow revision as a mechanism of peripheral T cell tolerance. cytometric analysis. Listeria monocytogenes infections Materials and Methods Wild-type L. monocytogenes was grown in brain–heart infusion broth and Mice measured by OD (A600) at midlog growth phase. The culture was diluted in sterile PBS and 200 ml containing 2000 CFU injected into the lateral tail Vb5 Tg and nontransgenic (nonTg) littermates on the C57BL/6 (B6) vein. background were bred under specific-pathogen free conditions at the University of Washington (Seattle, WA). Mice on the B6 background carry Intracellular cytokine staining the endogenous proviral genes encoding Mtv-8, -9, -17, and -30. B6 Ly5.2+ a b + 6 and B6.SJL (B6.SJL-Ptprc Pepc /BoyJ) Ly5.1 mice and their F1 gener- To detect IFN-g–producing T cells from uninfected mice, 2 to 3 3 10 ation (Ly5.1+Ly5.2+) were purchased from The Jackson Laboratory (Bar splenocytes were incubated in 96-well plates for 6 h at 37˚C in RP10 in the Harbor, ME) or bred in-house. B6.PL-Thy1a/CyJ (Thy-1.1+) mice were presence of monensin (GolgiStop, BD Pharmingen) either in the absence 2 2 kindly provided by M.K. Kaja (University of Washington). CD4 / (22) or presence of 0.7 mM ionomycin plus 50 ng/ml PMA (both from Sigma- 2 2 and I-Ab / (23) mice on the B6 background were originally purchased Aldrich). For detection of L. monocytogenes-specific CD4+ T cells, 2 2 2 2 from The Jackson Laboratory. Rag2 / (hereafter Rag / ) mice on the B6 splenocytes from mice infected 7 d previously were incubated in 96-well background were provided by D. Stetson (University of Washington). To plates for 5 h at 37˚C in RP10 in the presence of monensin and either in the induce lymphopenia in recipient mice, whole-body sublethal irradiation of absence or presence of 1 mg/ml I-Ab–restricted peptide listeriolysin O 650 rad was administered 1 d before cell transfer.