Negative Selection Thymocyte Interactions That Support

Home , Thymocyte

Stable Interactions and Sustained TCR Signaling Characterize Thymocyte− Thymocyte Interactions that Support Negative Selection This information is current as of September 28, 2021. Heather J. Melichar, Jenny O. Ross, Kayleigh T. Taylor and Ellen A. Robey J Immunol 2015; 194:1057-1061; Prepublished online 17 December 2014; doi: 10.4049/jimmunol.1400169 Downloaded from http://www.jimmunol.org/content/194/3/1057

Supplementary http://www.jimmunol.org/content/suppl/2014/12/17/jimmunol.140016 http://www.jimmunol.org/ Material 9.DCSupplemental References This article cites 46 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/194/3/1057.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision by guest on September 28, 2021

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stable Interactions and Sustained TCR Signaling Characterize Thymocyte–Thymocyte Interactions that Support Negative Selection

Heather J. Melichar,1 Jenny O. Ross,1 Kayleigh T. Taylor, and Ellen A. Robey

Negative selection is one of the primary mechanisms that render T cells tolerant to self. Thymic dendritic cells play an important role in negative selection, in line with their ability to induce migratory arrest and sustained TCR signals. Thymocytes themselves display self-peptide/MHC class I complexes, and although there is evidence that they can support clonal deletion, it is not clear whether they do so directly via stable cell–cell contacts and sustained TCR signals. In this study, we show that murine thymocytes can support surprisingly efficient negative selection of Ag-specific thymocytes. Furthermore, we observe that agonist-dependent thymocyte– thymocyte interactions occurred as stable, motile conjugates led by the peptide-presenting thymocyte and in which the trailing Downloaded from peptide-specific thymocyte exhibited persistent elevations in intracellular calcium concentration. These data confirm that self-Ag presentation by thymocytes is an additional mechanism to ensure T cell tolerance and further strengthen the correlation between stable cellular contacts, sustained TCR signals, and efficient negative selection. The Journal of Immunology, 2015, 194: 1057–1061.

he importance of T cell tolerance is evidenced by the fact express both MHC class I and II, thymocytes potentially present that its breakdown leads to devastating autoimmune dis- self-peptides to other thymocytes and participate in their selection T eases. Thus, there exist several overlapping mechanisms to (14–17). It is well-established that thymocyte–thymocyte inter- http://www.jimmunol.org/ ensure that self-reactive T cells are eliminated or held in check. actions promote the development of certain T cell populations. For Clonal deletion of autoreactive T cells in the thymus is a major example, NKT cell development relies on CD1d ligands expressed component in the establishment of central tolerance. Early studies by cortical thymocytes (18–20). In addition, expression of MHC showed that hematopoietic cells are important for efﬁcient deletion class II molecules by human thymocytes plays a role in positive of self-reactive thymocytes, whereas thymic stromal cells, in- selection of other, nonconventional, PLZF-expressing CD4+ cluding thymic epithelial cells, are less effective at inducing de- T cells (21–24). Although earlier studies have also suggested that letion (1–4). Of the hematopoietic cells in the thymus, it is clear thymocyte-speciﬁc Ag can induce negative selection (25–28), the

that dendritic cells (DCs) play a particularly important role in mechanism of deletion and the cellular dynamics involved in this by guest on September 28, 2021 negative selection of self-reactive thymocytes (5, 6). We have process have not yet been addressed. previously shown that thymic DCs bearing negative selecting To explore the contribution of Ag presentation by thymocytes to peptide provide cognate thymocytes with a strong “stop signal” the development of central tolerance, we determined the efficiency and sustained TCR signaling to induce cell death, whereas pre- of negative selection when high-affinity Ag presentation is re- sentation of the same peptide by thymic stromal cells provides stricted to thymocytes and characterized the resulting thymocyte– a weaker “stop signal” correlating with less efficient negative thymocyte interactions. In this study, we describe the rather effi- selection (7). Other hematopoietic cell types, including B cells and cient negative selection supported by thymocytes bearing cognate activated T cells, have also been implicated in negative selection, peptide and the prolonged cell–cell contacts and sustained calcium although the efficiency of negative selection and the nature of the signaling that typify these interactions. cellular interactions involved has not been addressed (8–13). The most abundant cells in the thymus are thymocytes. Because Materials and Methods murine thymocytes express MHC class I, and human thymocytes Thymocytes and thymic slices Mice were housed in an American Association of Laboratory Animal Care– Division of Immunology and Pathogenesis, Department of Molecular and Cell Biol- approved facility at the University of California, Berkeley under specific ogy, University of California, Berkeley, Berkeley, CA 94720 pathogen-free conditions, and all procedures were approved by the Animal 1H.J.M. and J.O.R. contributed equally to this work. Care and Use Committee. Thymus tissue was harvested from wild-type (WT) C57BL/6 mice (The Jackson Laboratory), b2m2/2 mice (Taconic, Received for publication January 23, 2014. Accepted for publication November 16, 2014. Germantown, NY), MHC class I and II–deficient Ly5.1/Ly5.2 mice (MHCko, Abb-b2m2/2) (Taconic), and OT1tg Rag22/2b2m2/2 mice at This work was supported by California Institute of Regenerative Medicine Postdoc- 3–6 wk of age or from radiation chimeras at 5–12 wk after reconstitution toral Training Grant TG2-01164 (to H.J.M.) and National Institutes of Health Grant where 1–5 3 106 OT1tg Rag22/2 Ly5.1 bone marrow was transferred into AI064227-07 (to E.A.R.). lethally irradiated MHCko hosts. Vibratome-cut thymic slices, 400–500 Address correspondence and reprint requests to Prof. Ellen A. Robey, Division of mm thick, were prepared as previously described (29), and 1 3 106 cells of Immunology and Pathogenesis, Department of Molecular and Cell Biology, Univer- each thymocyte population were added to each thymic slice as indicated. sity of California, Berkeley, 142 Life Sciences Addition #3200, Berkeley, CA 94720. Thymic tissue was dissociated into single-cell suspensions with glass tissue E-mail address: [email protected] homogenizers. The online version of this article contains supplemental material. Abbreviations used in this article: cTEC, cortical thymic epithelial cell; DC, dendritic Cell labeling and Ag loading cell; DP, double-positive; OT1tg, OT1 TCR transgenic Rag22/2; WT, wild-type. Cells were loaded with Indo-1LR (TEFLabs), a ratiometric calcium indi- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 cator dye, for 90 min at 37˚C, washed, and incubated an additional 60 min www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400169 1058 THYMOCYTE–THYMOCYTE INTERACTIONS SUPPORT NEGATIVE SELECTION

at 37˚C prior to addition to thymic slices. Thymocytes were loaded with 2 mM SNARF (Life Technologies), 1 mM Cell Proliferation Dye eFluor 670, or 2 mM Cell Proliferation Dye eFluor 450 (eBioscience), for 10 min at 37˚C and washed three times in complete DMEM. Thymocyes were incubated in the presence or absence of 1 nM OVA257–264 peptide (Anaspec) for 20 min at 37˚C and washed three times to remove unbound peptide prior to addition to thymic slices. Flow cytometry Single-cell thymocyte suspensions were incubated with a fixable viability dye eFluor506 in PBS and subsequently stained with anti-mouse Abs to the following: Ly5.1-PE, Ly5.2-FITC, CD4-PE-Cy7, CD8-eFluor450, CD69- PerCP, and CD24-allophycocyanin (eBioscience). Data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star). Statistical significance between groups was determined by performing one-way ANOVA and Tukey post hoc tests using Prism software. Two-photon microscopy Thymic slices were adhered to glass coverslips with tissue glue and maintained in oxygenated phenol-red free DMEM at 37˚C during imag-

ing on a custom-built upright two-photon microscope with a 320/0.95 Downloaded from Olympus objective and Spectra-Physics MaiTai laser tuned to 750 nm. Fluorescent signals were separated with 440- and 510-nm dichroic mirrors and 400/45, 480/50, and 605/75 bandpass filters. Images were acquired every 20 s for 20 min with 3 mm z-steps over an area of 172 3 143mm using custom software. Imaging volumes were recorded at a depth of .70 mm beneath the cut site. Cell coordinates and fluorescence intensity parameters were processed using Imaris software (Bitplane Scientific Software), and data converted to flow cytometry-like files using custom http://www.jimmunol.org/ DISCit software for further analysis with FlowJo software (Tree Star) (30). 2+ 2+ For analysis of intracellular calcium levels ([Ca i]), the ratio of Ca bound to unbound dye intensity was calculated. Results Thymocytes can serve as peptide-presenting cells to support efficient negative selection To determine whether thymocytes presenting agonist peptide support negative selection, we examined the development and be- by guest on September 28, 2021 havior of thymocytes in the presence of Ag-bearing thymocytes within thymic slices. Thymic slices maintain the three-dimensional architecture of the thymus including an intact cortex and medulla and chemokine gradients that direct added thymic subsets to their appropriate niche (31, 32). We mixed labeled preselection OT1 TCR transgenic Rag22/2 (OT1tg) CD4+CD8+ (double-positive [DP]) thymocytes with labeled b2m2/2 thymocytes (to serve as an internal control) on nonselecting (b2m2/2) thymic slices and allowed them to migrate into the tissue (Fig. 1A). Subsequently, we added WT (b2m+/+) thymocytes that had been preloaded with the agonist OVA257–264 (OVA) peptide to serve as APCs. WT thymocytes without added peptide served as a control. After 3 h, substantial CD69 upregulation was apparent on OT1tg thymocytes in the presence of OVA-loaded thymocytes, indicative of TCR FIGURE 1. Thymocytes bearing agonist peptide efficiently support signaling (Fig. 1B). However, no significant upregulation of CD69 negative selection. Preselection cell proliferation dye eFluor670-labeled was detected in the presence of WT thymocytes without added OT1tg DP thymocytes were mixed 1:1 with cell proliferation dye eFluor450- peptide, although they presumably express low-affinity endoge- labeled b2m2/2 thymocyte population as an internal reference, overlaid on 2 2 nous peptides. Moreover, no CD69 upregulation was observed b2m / thymic slices, and allowed to migrate into the tissue for 2 h. 2 2 when thymocytes were loaded with low-potency OVA peptide Thymic slices were washed, and unlabeled WT or b2m / thymocytes preloaded with or without 1 nM OVA peptide were added. After an ad- variant Q7, implying that recognition of peptide on thymocytes ditional 2 h, thymic slices were washed, and thymocytes were harvested at requires high-affinity TCR binding (data not shown). By 24 h, either 3 or 24 h after the addition of the peptide-loaded thymocyte population. (A) Flow cytometry gating strategy to distinguish b2m2/2 thymocyte internal reference cells versus OT1tg DP thymocytes from endogenous OT1tg DP cells to internal control population 24 h after addition of WT or cells. (B) Representative flow cytometry plots of CD24 and CD69 on live b2m2/2 thymocytes with or without OVA peptide. Each dot represents an OT1tg DP thymocytes 3 h after the addition of WT thymocytes with or individual thymic slice, and lines represent the mean. Data from one without OVA peptide. (C) Representative flow cytometry plots of live representative experiment of at least two. *p , 0.05. (E) Representative OT1tg DP thymocytes 24 h after the addition of WT thymocytes with or flow cytometry plots of thymocyte preparations prior to addition to thymic without OVA peptide. Flow cytometry plots are representative of triplicate slices. CD11c and MHC II on live, Lin2 cells (CD4, CD3, TCRb, CD19, samples from one experiment of at least two. (D) Normalized ratio of live and NK1.1) from indicated populations. The Journal of Immunology 1059 there was a significant decrease in the proportion of OT1tg cells motile conjugates with OVA-bearing thymocytes. In these con- cultured with OVA-loaded thymocytes (Fig. 1C). By comparing the jugates, the OVA-bearing thymocyte migrated in the lead, appar- number of OT1tg thymocytes remaining in the slice relative to the ently dragging an OT1tg thymocyte behind (Fig. 2A, Supplemental internal reference population, we estimate that ∼80% of OT1tg Video 1). These interactions are reminiscent of examples of mature DP thymocytes had been deleted by 24 h (Fig. 1D). No deletion T-B cell contacts and CTL-target B cell conjugates observed within was evident when b2m2/2 thymocytes were used as APCs, con- intact lymph nodes (33–35) and suggest that the OT1tg thymocytes firming that negative selection was not due to carryover of OVA are “arrested” in tight contact with the peptide-presenting thymo- peptide and presentation by residual MHC class I on thymic slice- cytes, whereas the peptide-presenting thymocytes continue to resident DCs (Fig. 1D). Surprisingly, the extent of deletion in- migrate. duced using thymocytes as APCs is similar to previous reports Of the 13 examples of OT1tg thymocytes in conjugates that we using DCs as APCs with high-affinity ligands (7). The purity of observed, 9 remained in contact during the entire time that they the overlaid thymocyte populations suggests that the observed remained in the imaging volume (7–20 min), whereas 4 OT1tg levels of negative selection are not due to Ag-loaded DC con- thymocytes encountered a peptide-loaded thymocyte and formed tamination (Fig. 1E). Thus, thymocytes can serve as APCs to a conjugate during the imaging run (Fig. 2B). We did not observe present negative selecting ligands to other thymocytes on non- any examples of conjugate dissociation. The observation that selecting (b2m2/2) thymic slices. thymocytes presenting agonist peptide support sustained interactions with cognate thymocytes and result in negative selection Thymocytes bearing negative selecting peptide support stable adds to our previous data to suggest that stable cell–cell inter- interactions actions support efficient negative selection (7). Downloaded from We have previously shown that high-affinity peptide presentation Given that only 5% of OT1tg thymocytes engaged with peptide by DCs but not cortical thymic epithelial cells (cTECs), supports bearing thymocytes at the 2- to 4-h time point, it is surprising that stable thymocyte interactions, sustained TCR signaling, and effi- 80% of OT1tg thymocytes are deleted by 24 h. However, because cient negative selection (7). To investigate the nature of cellular the OT1tg thymocytes and the peptide presenting thymocytes interactions when thymocytes serve as peptide-presenting cells, each make up fewer than 1% of thymocytes in this system, the

we directly examined these interactions using two-photon time- low number of interactions observed during this time period may http://www.jimmunol.org/ lapse microscopy in the cortex of thymic slices. We labeled OVA- simply reflect that time is needed for the OT1tg thymocytes to loaded WT thymocytes (the APC population) with a vital dye and “find” peptide bearing thymocytes. This is consistent with the OT1tg thymocytes with a ratiometric calcium indicator prior to finding that 4 of 13 conjugates formed, whereas 0 of 13 dissoci- addition to thymic slices and imaged 2–4 h after addition of OVA- ated, during the time of imaging. loaded thymocytes. In the absence of OVA peptide, both thymocyte populations migrated freely, and no thymocyte–thymocyte Encounter with thymocytes bearing negative selecting peptide interactions were observed (0 of 199 OT1tg cell tracks). However, leads to sustained low-level TCR signaling when WT thymocytes were preloaded with OVA peptide, we As we have previously shown that efficient negative selection observed ∼5% of OT1tg thymocytes (13 of 265) forming stable is associated with monogamous interactions and sustained TCR by guest on September 28, 2021

FIGURE 2. Thymocytes engage in prolonged, stable interactions with thymocytes presenting negative selecting peptide. (A) Preselection OT1tg DP thymocytes (yellow) were overlaid on b2m2/2 thymic slices in the presence of WT OVA-presenting thymocytes (aqua) and imaged by two-photon microscopy. Images were recorded in the cortex as identified by the density of OT1 thymocytes and the proximity to the thymic capsule. Representative images from a time series at the times indicated are shown. Top panels, Fluorescent images. Bottom panels, Lines indicate cell tracks for the duration of the video with spots overlaid to indicate the location of each cell at the time points indicated above the top panels. (B) Time line indicating the duration of thymocyte–thymocyte contacts of 13 interacting OT1tg thymocytes identified out of 265 OT1 cell tracks, in three imaging runs. 1060 THYMOCYTE–THYMOCYTE INTERACTIONS SUPPORT NEGATIVE SELECTION signaling (7), we characterized TCR signaling in individual cells mus, cTECs do not support deletion of self-reactive thymocytes engaged in thymocyte–thymocyte interactions. We observed sub- even when presenting high-affinity Ag (1–4, 7). One striking 2+ tle but consistent increases in [Ca i] in OT1tg thymocytes difference between cTECs and DCs is the ability of the latter to engaged in conjugates as compared with noninteracting OT1tg provide a strong “stop signal” to promote stable thymocyte–DC thymocytes (Fig. 3A). The OT1tg DP cells engaged in these long- interactions (7). Whether stable conjugate formation is charac- lived thymocyte–thymocyte interactions exhibited modest but teristic of other cellular interactions that promote negative se- 2+ persistent elevated [Ca i] relative to noninteracting OT1tg thy- lection was not known. In this study, we show that thymocytes mocytes (Fig. 3B), and the increase in cytosolic Ca2+ levels was presenting high-affinity Ag can support efficient negative selec- observed shortly after initial engagement (Fig. 3B, track I). Taken tion that is associated with stable thymocyte–thymocyte interactions together, these results suggest that negative selection is associated and sustained Ca2+ signaling. with stable interactions and sustained TCR signals. Thymocytes lack many features of professional APCs, including expression of costimulatory ligands that can promote Discussion negative selection (36–40). Moreover, when confronted by broadly Although a central role for DCs in negative selection of thy- distributed agonist peptide, cortical thymocytes preferentially ar- mocytes is well established, multiple other hematopoietic cell rest adjacent to DCs, implying that DCs induce a more potent types including B cells, activated T cells, and even thymocytes migratory stop signal compared with other peptide presenting themselves are also thought to play a role in the deletion of cells, including thymocytes, in the vicinity (7). Despite this, we autoreactive T cells in the thymus (5, 8–13, 25–28). Despite the find that thymocytes can support prolonged peptide-specific con- diversity of cells that can support negative selection in the thy- tacts with other thymocytes presenting high-affinity ligand and Downloaded from induce negative selection. Interestingly, thymocytes express signaling lymphocyte activation molecule receptors, a family of proteins that stabilize cellular contacts via homotypic interactions and play an essential role in T cell–B cell interactions and thymocyte-driven selection of innate-like T cell subsets (41–47). It

is tempting to speculate that homotypic signaling lymphocyte http://www.jimmunol.org/ activation molecule family interactions may help to stabilize thymocyte–thymocyte contacts that drive negative selection. Our data have relevance for the role of direct versus indirect Ag presentation during negative selection. Thymic DCs are well equipped to directly present MHC class I–associated peptides derived from proteins expressed by the DCs themselves. However, for thymocyte-specific proteins, it is unclear whether peptides are presented directly by thymocytes or whether protein or peptide– MHC complexes are transferred to DCs for presentation. Although by guest on September 28, 2021 we cannot rule out the possibility that Ag-loaded thymocytes transfer peptide–MHC complexes to thymic DCs, the stability of thymocyte–thymocyte interactions and persistent elevated Ca2+ levels strongly support a direct presentation route for thymocyte- driven negative selection in this system. For MHC class II–associated Ags, direct presentation by thymocytes likely does not occur in mice given that mouse thymocytes do not express detectable levels of MHC class II. Moreover, the tolerance of CD8, but not CD4, T cells to thymocyte/T cell– specific expression of an MHC class I protein argues that neither efficient transfer of Ag to DCs nor direct presentation of class II– associated Ags by thymocytes occurs in the mouse system (25– 28). However, it is interesting to consider that in humans direct presentation by thymocytes may play a role in both MHC class I and II tolerance, given that human thymocytes express MHC class FIGURE 3. Thymocytes presenting negative selecting peptide support II (14–17). low sustained TCR signals. (A) The frequency of time points plotted Whether self-peptide–presenting thymocytes share the burden 2+ against the [Ca i] ratio of Indo-1–labeled preselection OT1tg DP thy- of negative selection or act more as a “safety net” during negative mocytes incubated on b2m2/2 thymic slices in the presence of WT thy- selection remains to be determined. Our results indicate that mocytes alone or loaded with 1 nM OVA peptide from two independent thymocytes themselves can support efficient negative selection imaging runs. “Interacting” values are time points that were part of and suggests yet another layer of regulation to prevent self- a thymocyte–thymocyte contact and derived from at least five tracked reactive T cells from leaving the thymus. The thymocyte- OT1tg thymocytes. “Noninteracting” were values from all OT1tg thymo- mediated negative selection we report in this paper may be par- cytes from the same run that were not in a conjugate and that had a similar ticularly relevant for tolerance to thymocyte-specific proteins such overall fluorescence value to the “interacting” tracks. (B) [Ca2+ ] ratios i as TCR. A number of different hematopoietic cell subsets have over time from individual representative interacting OT1tg thymocyte tracks. Track labels correspond to those in Fig. 2B. Line indicates average now been identified that can support negative selection. These 2+ cells are quite diverse, and the characteristics that allow these [Ca i] ratio of noninteracting cells with comparable fluorescence intensity in the unbound Indo-1 channel of the interacting cell. For track I, arrow hematopoietic cells, and not cTECs, to support the stable inter- indicates the timepoint in which the initial thymocyte–thymocyte contact actions necessary to support efficient negative selection remain occurred. to be determined. The Journal of Immunology 1061

Acknowledgments thymocyte-expressed MHC class II selects a distinct T cell population. Immunity 23: 375–386. We thank Ivan Dzhagalov and Joanna Halkias for helpful comments on 25. Jhaver, K. G., P. Chandler, E. Simpson, and A. L. Mellor. 1999. Thymocyte the manuscript. We also thank Paul Herzmark, Seong-ji Han, Brian Weist, antigens do not induce tolerance in the CD4+ T cell compartment. J. Immunol. Nadia Kurd, and Hamlet Chu for technical assistance and suggestions. 163: 4851–4858. 26. Pircher, H., K. P. Muller,€ B. A. Kyewski, and H. Hengartner. 1992. Thymocytes can tolerize thymocytes by clonal deletion in vitro. Int. Immunol. 4: 1065–1069. Disclosures 27. Scho¨nrich, G., G. Strauss, K. P. Muller,€ L. Dustin, D. Y. Loh, N. Auphan, A. M. Schmitt-Verhulst, B. Arnold, and G. J. Ha¨mmerling. 1993. Distinct The authors have no financial conflicts of interest. requirements of positive and negative selection for selecting cell type and CD8 interaction. J. Immunol. 151: 4098–4105. 28. Schulz, R., and A. L. Mellor. 1996. Self major histocompatibility complex class I References antigens expressed solely in lymphoid cells do not induce tolerance in the CD4+ 1. Takeuchi, M., A. Iwasaki, K. Nomoto, and Y. Yoshikai. 1992. Rat thymic epi- T cell compartment. J. Exp. Med. 184: 1573–1578. thelium positively selects mouse T cells with specificity for rat MHC class II 29. Dzhagalov, I. L., H. J. Melichar, J. O. Ross, P. Herzmark, and E. A. Robey. 2012. antigens but fails to induce detectable tolerance in the mouse T cells to the rat Two-photon imaging of the immune system. Curr. Protoc. Cytom. Chapter 12: MHC antigens. Immunobiology 186: 421–434. Unit12 26. 2. von Boehmer, H., and K. Hafen. 1986. Minor but not major histocompatibility 30. Moreau, H. D., and P. Bousso. 2012. A guide to in vivo cytometry. Intravital 1: antigens of thymus epithelium tolerize precursors of cytolytic T cells. Nature 27–31. 320: 626–628. 31. Ehrlich, L. I., D. Y. Oh, I. L. Weissman, and R. S. Lewis. 2009. Differential 3. Webb, S. R., and J. Sprent. 1990. Tolerogenicity of thymic epithelium. Eur. J. contribution of chemotaxis and substrate restriction to segregation of immature Immunol. 20: 2525–2528. and mature thymocytes. Immunity 31: 986–998. 4. van Meerwijk, J. P., S. Marguerat, R. K. Lees, R. N. Germain, B. J. Fowlkes, and 32. Halkias, J., H. J. Melichar, K. T. Taylor, J. O. Ross, B. Yen, S. B. Cooper, H. R. MacDonald. 1997. Quantitative impact of thymic clonal deletion on the A. Winoto, and E. A. Robey. 2013. Opposing chemokine gradients control hu- T cell repertoire. J. Exp. Med. 185: 377–383. man thymocyte migration in situ. J. Clin. Invest. 123: 2131–2142. Downloaded from 5. McCaughtry, T. M., T. A. Baldwin, M. S. Wilken, and K. A. Hogquist. 2008. 33. Azar, G. A., F. Lemaıˆtre, E. A. Robey, and P. Bousso. 2010. Subcellular dy- Clonal deletion of thymocytes can occur in the cortex with no involvement of the namics of T cell immunological synapses and kinapses in lymph nodes. Proc. medulla. J. Exp. Med. 205: 2575–2584. Natl. Acad. Sci. USA 107: 3675–3680. 6. Derbinski, J., and B. Kyewski. 2010. How thymic antigen presenting cells 34. Mempel, T. R., M. J. Pittet, K. Khazaie, W. Weninger, R. Weissleder, H. von sample the body’s self-antigens. Curr. Opin. Immunol. 22: 592–600. Boehmer, and U. H. von Andrian. 2006. Regulatory T cells reversibly suppress 7. Melichar, H. J., J. O. Ross, P. Herzmark, K. A. Hogquist, and E. A. Robey. 2013. cytotoxic T cell function independent of effector differentiation. Immunity 25: Distinct temporal patterns of T cell receptor signaling during positive versus 129–141. negative selection in situ. Sci. Signal. 6: ra92. 35. Okada, T., M. J. Miller, I. Parker, M. F. Krummel, M. Neighbors, S. B. Hartley,

8. Kleindienst, P., I. Chretien, T. Winkler, and T. Brocker. 2000. Functional com- A. O’Garra, M. D. Cahalan, and J. G. Cyster. 2005. Antigen-engaged B cells http://www.jimmunol.org/ parison of thymic B cells and dendritic cells in vivo. Blood 95: 2610–2616. undergo chemotaxis toward the T zone and form motile conjugates with helper 9. Agus, D. B., C. D. Surh, and J. Sprent. 1991. Reentry of T cells to the adult T cells. PLoS Biol. 3: e150. thymus is restricted to activated T cells. J. Exp. Med. 173: 1039–1046. 36. Punt, J. A., W. Havran, R. Abe, A. Sarin, and A. Singer. 1997. T cell receptor + + 10. Tian, C., J. Bagley, D. Forman, and J. Iacomini. 2004. Induction of central (TCR)-induced death of immature CD4 CD8 thymocytes by two distinct tolerance by mature T cells. J. Immunol. 173: 7217–7222. mechanisms differing in their requirement for CD28 costimulation: implications 11. Webb, S. R., and J. Sprent. 1990. Induction of neonatal tolerance to Mlsa for negative selection in the thymus. J. Exp. Med. 186: 1911–1922. antigens by CD8+ T cells. Science 248: 1643–1646. 37. Punt, J. A., B. A. Osborne, Y. Takahama, S. O. Sharrow, and A. Singer. 1994. + + 12. Frommer, F., and A. Waisman. 2010. B cells participate in thymic negative se- Negative selection of CD4 CD8 thymocytes by T cell receptor-induced apo- lection of murine auto-reactive CD4+ T cells. PLoS One 5: e15372. ptosis requires a costimulatory signal that can be provided by CD28. J. Exp. 13. Perera, J., L. Meng, F. Meng, and H. Huang. 2013. Autoreactive thymic B cells Med. 179: 709–713. are efficient antigen-presenting cells of cognate self-antigens for T cell negative 38. Kishimoto, H., and J. Sprent. 1999. Several different cell surface molecules selection. Proc. Natl. Acad. Sci. USA 110: 17011–17016. control negative selection of medullary thymocytes. J. Exp. Med. 190: 65–73. by guest on September 28, 2021 14. Thulesen, S., A. Jørgensen, J. Gerwien, M. Dohlsten, M. Holst Nissen, N. Odum, 39. Kishimoto, H., and J. Sprent. 1997. Negative selection in the thymus includes and C. Ro¨pke. 1999. Superantigens are presented by and activate thymocytes semimature T cells. J. Exp. Med. 185: 263–271. from infants. Exp. Clin. Immunogenet. 16: 226–233. 40. McKean, D. J., C. J. Huntoon, M. P. Bell, X. Tai, S. Sharrow, K. E. Hedin, 15. Gilhus, N. E., and R. Matre. 1983. Development and distribution of HLA-DR A. Conley, and A. Singer. 2001. Maturation versus death of developing double- antigens and Fcg receptors in the human thymus. Acta Pathol. Microbiol. positive thymocytes reflects competing effects on Bcl-2 expression and can be Immunol. Scand. [C] 91: 35–42. regulated by the intensity of CD28 costimulation. J. Immunol. 166: 3468–3475. 16. Marinova, T., I. Altankova, D. Dimitrova, and Y. Pomakov. 2001. Presence of 41. Nichols, K. E., J. Hom, S. Y. Gong, A. Ganguly, C. S. Ma, J. L. Cannons, HLA-DR immunopositive cells in human fetal thymus. Arch. Physiol. Biochem. S. G. Tangye, P. L. Schwartzberg, G. A. Koretzky, and P. L. Stein. 2005. Reg- 109: 74–79. ulation of NKT cell development by SAP, the protein defective in XLP. Nat. 17. Park, S. H., Y. M. Bae, T. J. Kim, I. S. Ha, S. Kim, J. G. Chi, and S. K. Lee. 1992. Med. 11: 340–345. HLA-DR expression in human fetal thymocytes. Hum. Immunol. 33: 294–298. 42. Pasquier, B., L. Yin, M. C. Fondane`che, F. Relouzat, C. Bloch-Queyrat, 18. Bendelac, A. 1995. Positive selection of mouse NK1+ T cells by CD1-expressing N. Lambert, A. Fischer, G. de Saint-Basile, and S. Latour. 2005. Defective cortical thymocytes. J. Exp. Med. 182: 2091–2096. NKT cell development in mice and humans lacking the adapter SAP, the X- 19. Coles, M. C., and D. H. Raulet. 2000. NK1.1+ T cells in the liver arise in the linked lymphoproliferative syndrome gene product. J. Exp. Med. 201: 695–701. thymus and are selected by interactions with class I molecules on CD4+CD8+ 43. Chung, B., A. Aoukaty, J. Dutz, C. Terhorst, and R. Tan. 2005. Signaling lym- cells. J. Immunol. 164: 2412–2418. phocytic activation molecule-associated protein controls NKT cell functions. J. 20. Wei, D. G., H. Lee, S. H. Park, L. Beaudoin, L. Teyton, A. Lehuen, and Immunol. 174: 3153–3157. A. Bendelac. 2005. Expansion and long-range differentiation of the NKT cell 44. Griewank, K., C. Borowski, S. Rietdijk, N. Wang, A. Julien, D. G. Wei, lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. A. A. Mamchak, C. Terhorst, and A. Bendelac. 2007. Homotypic interactions Med. 202: 239–248. mediated by Slamf1 and Slamf6 receptors control NKT cell lineage develop- 21. Choi, E. Y., K. C. Jung, H. J. Park, D. H. Chung, J. S. Song, S. D. Yang, ment. Immunity 27: 751–762. E. Simpson, and S. H. Park. 2005. Thymocyte-thymocyte interaction for efficient 45. Horai, R., K. L. Mueller, R. A. Handon, J. L. Cannons, S. M. Anderson, positive selection and maturation of CD4 T cells. Immunity 23: 387–396. M. R. Kirby, and P. L. Schwartzberg. 2007. Requirements for selection of 22. Choi, E. Y., W. S. Park, K. C. Jung, D. H. Chung, Y. M. Bae, T. J. Kim, conventional and innate T lymphocyte lineages. Immunity 27: 775–785. H. G. Song, S. H. Kim, D. I. Ham, J. H. Hahn, et al. 1997. Thymocytes positively 46. Li, W., M. H. Sofi, S. Rietdijk, N. Wang, C. Terhorst, and C. H. Chang. 2007. select thymocytes in human system. Hum. Immunol. 54: 15–20. The SLAM-associated protein signaling pathway is required for development of 23. Zhu, L., Y. Qiao, E. S. Choi, J. Das, D. B. Sant’angelo, and C. H. Chang. 2013. A CD4+ T cells selected by homotypic thymocyte interaction. Immunity 27: 763– transgenic TCR directs the development of IL-4+ and PLZF+ innate CD4 T cells. 774. J. Immunol. 191: 737–744. 47. Qi, H., J. L. Cannons, F. Klauschen, P. L. Schwartzberg, and R. N. Germain. 24. Li, W., M. G. Kim, T. S. Gourley, B. P. McCarthy, D. B. Sant’Angelo, and 2008. SAP-controlled T-B cell interactions underlie germinal centre formation. C. H. Chang. 2005. An alternate pathway for CD4 T cell development: Nature 455: 764–769.