Specific T Cells Ex Vivo − Dextramers Identify Human Lipopeptide

Cutting Edge: CD1a Tetramers and Dextramers Identify Human Lipopeptide− Specific T Cells Ex Vivo

This information is current as Anne G. Kasmar, Ildiko Van Rhijn, Kelly G. Magalhaes, of October 1, 2021. David C. Young, Tan-Yun Cheng, Marie T. Turner, Andre Schiefner, Ravi C. Kalathur, Ian A. Wilson, Mugdha Bhati, Stephanie Gras, Richard W. Birkinshaw, Li L. Tan, Jamie Rossjohn, John Shires, Søren Jakobsen, John D. Altman and D. Branch Moody

J Immunol 2013; 191:4499-4503; Prepublished online 2 Downloaded from October 2013; doi: 10.4049/jimmunol.1301660 http://www.jimmunol.org/content/191/9/4499 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2013/10/02/jimmunol.130166 Material 0.DC1 References This article cites 22 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/191/9/4499.full#ref-list-1 by guest on October 1, 2021 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

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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology

Cutting Edge: CD1a Tetramers and Dextramers Identify Human Lipopeptide–Specific T Cells Ex Vivo ,† Anne G. Kasmar,* Ildiko Van Rhijn,* Kelly G. Magalhaes,*x David C. Young,*x Tan-Yun Cheng,* Marie T. Turner,‡ Andre Schiefner, ,1 Ravi C. Kalathur, x,{ ‖ ‖ ‖ Ian A. Wilson,‖ Mugdha‖ Bhati, Stephanie Gras, Richard W. Birkinshaw, Li L. Tan, Jamie Rossjohn, ,# John Shires,** Søren Jakobsen,†† John D. Altman,** and D. Branch Moody* Human CD1a mediates foreign Ag recognition by a of a few human T cell clones propagated long-term in vitro T cell clone, but the nature of possible TCR interac- (4, 5). The mycobacterial lipopeptide dideoxymycobactin tions with CD1a/lipid are unknown. After incubating (DDM) is the only well-characterized foreign Ag in the CD1a CD1a with a mycobacterial lipopeptide Ag, dideoxymy- system and has served as a model to understand the specificity Downloaded from cobactin (DDM), we identified and measured binding and mechanism of action of CD1a-restricted T cells in vitro (4, 6, 7). Building on the success of human MHC class I to a recombinant TCR (TRAV3/ TRBV3-1, KD of 100 mM). Detection of ternary CD1a/lipid/TCR tetramers to quantitatively track fresh human T cells ex vivo interactions enabled development of CD1a tetramers (8), CD1 tetramers (9–16) capture lipid-reactive T cells as and CD1a multimers with carbohydrate backbones populations for study ex vivo. Dextramers rely on the same http://www.jimmunol.org/ (dextramers), which specifically stained T cells using principle, but they use higher order multimers that allow a mechanism that was dependent on the precise stereo- detection of rare, lower affinity T cells (17). In this study, we investigated whether human TCRs directly bind to CD1a/ chemistry of the peptide backbone and was blocked lipopeptide to develop tetramers and dextramers that iden- with a soluble TCR. Furthermore, sorting of human tify polyclonal T cell populations, which recognize foreign Ag T cells from unrelated tuberculosis patients for bright bound to CD1a. DDM-dextramer staining allowed recovery of T cells that were activated by CD1a and DDM. These studies Materials and Methods

demonstrate that the mechanism of T cell activa- by guest on October 1, 2021 Generation of soluble CD1a proteins tion by lipopeptides occurs via ternary interactions of CD1a/Ag/TCR. Furthermore, these studies demon- We produced soluble biotinylated CD1a monomers in lentivirus-transduced HEK293T cells at the National Institutes of Health Tetramer Core Facility strate the existence of lipopeptide-speciﬁc T cells in (Emory University, Atlanta, GA) (14, 16). humans ex vivo. The Journal of Immunology, 2013, 191: 4499–4503. Loading CD1a monomers with DDM DDM with deﬁned stereochemistry (DDM 1S,3R or DDM 1R,3S) (7) was incubated with CD1a monomers and complexed with streptavidin coupled umans express four CD1 Ag-presenting molecules, to allophycocyanin. Staining was optimized by analyzing T cells with CD1a CD1a, CD1b, CD1c, and CD1d (1). Whereas the tetramers generated after loading under varied conditions (Supplemental Fig. H basic biology of CD1d and NKT cells has been 1). Optimal staining was seen with DDM solubilized in DMSO, sonicated extensively studied in mice lacking either CD1d or invariant into 30-fold excess 50 mM sodium citrate plus 1% CHAPS at pH 6 for 2 min, incubated at 42˚C for 1 h, and then added at 40-fold molar excess to NKT TCRs (2), CD1a proteins have been deleted from the CD1a monomers and incubated in a 37˚C water bath for 1 h prior to neu- murine genome (3), effectively limiting analysis to the study tralization to pH 7.4 with 2 ml Tris (pH 9).

*Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Fund Program in Translational Research (to D.B.M.), as well as by National Institutes of Harvard Medical School, Boston, MA 02115; †Department of Infectious Diseases and Health Grants T-32 AI 007306-22, T-32 AR 007530-23, K08 AI089858 (to A.G.K.), Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht, R01 AI49313, R01 AR048632 (to D.B.M.), and R01 A1042266 (to I.A.W.) and a grant The Netherlands; ‡Tuberculosis Treatment Unit, Lemuel Shattuck Hospital, Jamaica from the Nederlands Wetenschappelijk Onderzoek (Meervoud 836.08.001) (to I.V.R.). x Plain, MA 02130; Department of Integrative Structural and Computational Biology, S.G. is supported by an Australian Research Council Future Fellowship. J.R. is supported { The Scripps Research Institute, La Jolla, CA 92037; Skaggs Institute for Chemical by a National Health and Medical Research Council Australia Fellowship. ‖ Biology, The Scripps Research Institute, La Jolla, CA 92037; Department of Biochem- Address correspondence and reprint requests to D. Branch Moody, Division of Rheu- istry and Molecular Biology, School of Biomedical Sciences, Monash University, Clay- matology, Allergy and Immunology, Brigham and Women’s Hospital, Smith Building ton, Victoria 3800, Australia; #Institute of Infection and Immunity, Cardiff University, Room 514, 1 Jimmy Fund Way, Boston MA 02115. E-mail address: bmoody@partners. School of Medicine, Cardiff CF14 4XN, United Kingdom; **Emory Vaccine Center, org Atlanta, GA 30329; and ††Immudex, 2100 Copenhagen, Denmark The online version of this article contains supplemental material. 1Current address: Technische Universita¨tMu¨nchen, Munich, Germany. Abbreviation used in this article: DDM, dideoxymycobactin. Received for publication July 17, 2013. Accepted for publication August 29, 2013. This work was supported by grants from the Howard Hughes Medical Institute Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 KwaZulu-Natal Research Institute for Tuberculosis and HIV (to A.G.K. and D.B.M.), the Harvard University Global Health Initiative (to A.G.K.), the Burroughs Wellcome www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301660 4500 CUTTING EDGE: CD1a MULTIMERS DETECT HUMAN ab T CELLS

CD1a tetramer and dextramer staining of clones CD1a tetramers were generated and validated by staining the DDM-reactive ab T cell clone CD8-2 (18) and using methods similar to those reported for CD1b tetramers (14). We generated dextramers by diluting CD1a complexes to 0.1 mg/ml in PBS prior to incubation with 2 ml allophycocyanin-labeled dextramer backbone for 30 min at room temperature in the dark, followed by staining of T cells as previously reported (17). For dual staining with TCR Abs, TRBV3-1 (Beckman Coulter) was added after tetramer staining for the last 15 min of incubation. Cells were acquired on a FACSCanto II flow cytometer (Beckton Dickinson) and analyzed using FlowJo (Tree Star) software in the presence or absence of mAbs or recombinant TCRs. TCR affinity measurements The cDNAs of the a- and b-chains of the CD1b-restricted TCR LDN5 and the CD1a-restricted, DDM-specific TCR CD8-2 were produced as previously described (14, 19). Loaded CD1a-DDM was coupled to research-grade streptavidin-coated chips. Increasing concentrations of the CD8-2 TCR (0– 533 mM) were injected over all flow cells. BIAevaluation version 3.1 software (Biacore) was used to fit the data to the 1:1 Langmuir binding model, and the equilibrium data were analyzed with the Prism program (GraphPad Soft- ware).

Dextramer staining of human PBMCs Downloaded from Work with human subjects was overseen by the Institutional Review Boards of the Lemuel Shattuck Hospital (00000786), Partners Healthcare (2002-P- 000061), and the Harvard Committee on Microbiologic Safety (08-184). PBMCs were treated with dextramers at a 1:100 dilution for 15 min at room temperature followed by 15 min at 4˚C, after which cells were washed and

incubated with violet viability dye, CD3-FITC, CD14-PerCP-Cy5.5, and http://www.jimmunol.org/ CD19-PerCP-Cy5.5 for 15 min at 4˚C. Unfixed dextramer+ cells were sorted using a FACSAria flow cytometer and expanded by stimulation with anti- CD3 (30 ng/ml) in the presence of irradiated feeder cells and IL-2 (2 nM). After 3 wk, clones were analyzed for binding CD1a dextramers and were FIGURE 1. CD1a tetramers and dextramers stain human T cells. (A) DDM tested for DDM recognition in an ELISPOT assay (20). is a biosynthetic precursor to mycobactin siderophores composed of a fatty acyl tail and a peptide backbone that contains four chiral centers (1–4), which Results and Discussion are in the S or R configuration as indicated. Natural DDM occurs as the Generation of antigenic CD1a/lipopeptide complexes 1S,3R diastereomer. The nonstimulatory DDM analog is presumed to be B We generated transmembrane-truncated soluble biotinylated a mixture of 1S,3R and 1R,3S diastereomers (7). ( ) Tetramerizable CD1a monomers were bound to streptavidin-coated plates, treated with Ag over- by guest on October 1, 2021 CD1a proteins based on methods previously developed for night at 37˚C, and used to activate IFN-g release by the CD1a-restricted MHC, CD1d, CD1b, and CD1c (8, 12, 14, 16). To test the human T cell line CD8-2 (mean 6 SD). (C) The CD8-2 T cell line, which folding and Ag-presenting function of this new CD1a con- contains cells with the DDM-specific TCR b-chain TRBV3-1, as well as cells struct, it was bound to streptavidin plates, treated with a with other TCRs, was stained with CD1a tetramers loaded with the indicated synthetic DDM, and used to activate T cells (7). The synthetic DDM isomer and TRBV3-1–specific mAb. (D) CD8-2 T cells were stained Ag recapitulates the naturally occurring S or R stereochemistry by CD1a dextramers loaded with the indicated DDM isomer followed by TCR anti–Vb-chain Ab (TRBV3-1). Data in (B)–(D) are representative among the four stereocenters in the natural DDM peptide, of three or more experiments. including S and R stereochemistry at positions 1 and 3 present in M. tuberculosis DDM (DDM 1S,3R) (Fig. 1A) (7). This lipopeptide activated the CD1a-restricted human T cell line the clonotypic TCR that defines CD8-2. These data estab- CD8-2 in a dose-dependent manner. No activation was seen lished a working CD1a tetramer and strongly supported the in response to DDM with the opposite stereochemical con- model of direct binding of an ab TCR to CD1a. To further figuration at positions 1 and 3 (DDM 1R,3S) or a synthetic increase the avidity of interaction, we developed CD1a dex- analog that deviated from the optimal natural DDM based on tramers that are composed of 10–14 CD1a monomers on a fully saturated acyl chain and serine substituting for a-methyl a flexible, fluorescently labeled dextran backbone (17). We ob- serine (DDM analog; Fig. 1B). Thus, soluble CD1a monomers served bright, selective, and highly reproducible staining of were properly folded and were sufficient to present a lip- the TRBV3-1 subset of CD8-2 T cells (Fig. 1D) with DDM opeptide Ag to human T cells. (1S,3R)-loaded dextramers. Thus, dextramers provided a sec- ond reagent for probing the interaction of CD1a with T cells. CD1a tetramers and dextramers stain T cells ab After optimizing loading conditions based on pH, time, tem- CD1a multimers bind to the TCR perature, and solvent variables (Supplemental Fig. 1), we CD1a-DDM tetramer and dextramer binding to TRBV3-1+ observed that DDM (1S,3R)-treated CD1a tetramers selec- T cells strongly implicated a cognate model in which a ternary tively stain T cells expressing the clonotypic TRBV3-1 TCR, interaction of CD1a/lipid binds to the TCR. We performed but not other T cells (Fig. 1C). T cells were not stained by experiments to directly test binding between lipopeptide/ CD1a tetramers that were not exposed to lipids or tetramers CD1a complexes and the clonotypic human ab TCR ver- treated with the two nonantigenic DDMs that substantially sus all other surface receptors. Preincubation of dextramers mimic DDM (1S,3R). Thus, tetramer staining was dependent with anti-CD1a Ab blocked staining to background (Fig. 2A), on the structure of the added lipopeptide and was specific for confirming that staining was mediated by CD1a. We gener- The Journal of Immunology 4501

FIGURE 2. Tetramer and dextramer staining demonstrates a trimolecular interaction among CD1a, DDM, and the clonotypic TCR. (A) The CD8-2 T cell line was stained with CD1a dextramers that were preincubated with isotype control Ab or anti-CD1a Ab (10 mg/ml). (B) Soluble TCR a-chains with hexahistidine tags and b-chains with Strep-tag II were formed into soluble TCR dimers. (C) Loaded and unloaded tetramers were preincubated with 50-fold molar excess of soluble TCR. Data are representative of three or more experiments. (D) CD1a monomers were afﬁxed to streptavidin-coated chips, loaded with DDM, and then treated with soluble CD8-2 TCRs at the indicated concentrations to measure a dissocia- tion constant of 95.78 6 13.51 mM. Data are representative of two experiments; error bars are shown for each datum point (mean 6 SEM). Downloaded from

ated soluble TCRs containing the TCR a-andb-chains from CD1a-expressing APCs treated with DDM (Fig. 4). Three CD8-2 (ab heterodimers encoded by the TRAV3-1 and of the clones express CD8 and the other five express CD4. TRBV3-1 variable regions) or analogous constructs from the We therefore conclude that CD1a- and DDM-reactive T http://www.jimmunol.org/ CD1b-restricted T cell LDN5 (composed of the TRAV17 cells are present as populations among genetically diverse and TRBV4-1 variable regions) (Fig. 2B). Preincubation with donors. soluble LDN5 TCR minimally impacted staining, whereas Overall, these results distinguish between indirect and cog- preincubation with soluble CD8-2 TCR blocked T cell stain- nate TCR interaction models, illustrating that direct CD1a/ ing to background levels (Fig. 2C). Thus, CD1a-DDM staining DDM/TCR interactions are the mechanism of T cell activa- ofcellsismediatedbytheab TCR. tion by CD1a and lipopeptide. These studies extend prior We then measured the affinity of interaction between soluble work describing polyclonal responses to lipoprotein mixtures transmembrane-truncated CD8-2 TCR and CD1a proteins alone or pretreated with DDM using surface plasmon reso- by guest on October 1, 2021 nance (Fig. 2D). No binding was seen to CD1a alone; the 6 determined KD was 95.78 13.51 mM. This affinity is sig- nificantly lower than that of TCRs recognizing a-galactosyl ceramide-CD1d (,1 mM) and glucose monomycolate-CD1b (∼1 mM) (15)) but approximates the affinity of NKT TCRs recognizing b-linked glycolipids (21).

CD1a dextramers detect human lipopeptide–speciﬁc T cells ex vivo To determine whether DDM-reactive T cells exist as cell populations ex vivo, PBMCs from subjects with active tuberculosis or positive tuberculin skin tests were stained with dextramers treated with DDM (1S,3R) and then gated on 2 2 CD3+CD14 CD19 live lymphocytes (Supplemental Fig. 2A). Rare CD3+ cells were identiﬁed with the DDM-loaded dextramer among four subjects with mycobacterial exposure (Fig.3,SupplementalFig.2B).Thisresultsuggestedthat DDM-reactive T cells are present in the blood of unrelated human donors. To determine whether the rare dextramer+ cells recognize CD1a and DDM, T cell yields were increased through leukapheresis (subject C58) or ex vivo expansion using anti- CD3 Ab and IL-2 (subject A32). We then used DDM- treated dextramers to sort cells (Supplemental Fig. 2C), cloned them using limiting dilution, and tested them for reactivity FIGURE 3. CD1a dextramers stain polyclonal cells ex vivo. PBMCs from one subject with active tuberculosis (subject A24) as well as three tuberculin in ELISPOT assays. Dextramer-based sorting generated many skin test–positive subjects (subjects A22, A32, and C58) were stained with T cell clones that were brightly stained using DDM-loaded CD1a dextramers in addition to CD3-FITC, CD14-PerCP-Cy5.5, CD19- dextramers, and these clones secreted TNF-a (clones 3, 6, PerCP-Cy5.5, and violet viability dye, after which they were gated on live 9, 21) or IFN-g (clones P1, P5, P7, P9) in response to lymphocytes. 4502 CUTTING EDGE: CD1a MULTIMERS DETECT HUMAN ab T CELLS Downloaded from http://www.jimmunol.org/

FIGURE 4. CD1a dextramers detect human lipopeptide–speciﬁc T cells. T cell clones derived from tuberculin skin test–positive subjects A32 (A) or C58 (B) were originally obtained by sorting based on CD1a-DDM dextramer binding as shown in Supplemental Fig. 2B. Clones were harvested and stained with CD1a dextramers as in Fig. 1. Positively staining clones were incubated with K562 APCs transfected with empty vector or the noted CD1 isoform with or without1mM DDM (mean 6 SD) in an ELISPOT assay. by guest on October 1, 2021

(18) and lipopeptide-specific T cell clones (22) to prove that Molecular mechanism of lipopeptide presentation by CD1a. Immunity 22: 209–219. lipopeptide-reactive T cells exist in the in vivo state. More 7. Young, D. C., A. Kasmar, G. Moraski, T. Y. Cheng, A. J. Walz, J. Hu, Y. Xu, generally, generation of working CD1a multimers completes G. W. Endres, A. Uzieblo, D. Zajonc, et al. 2009. Synthesis of dideoxymycobactin antigens presented by CD1a reveals T cell fine specificity for natural lipopeptide the set of tools needed to study human CD1-restricted T structures. J. Biol. Chem. 284: 25087–25096. cells, allowing determination of the phenotype and func- 8. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G. McHeyzer- tion of T cells recognizing CD1a bound to self and foreign Williams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274: 94–96. Ags. 9. Benlagha, K., A. Weiss, A. Beavis, L. Teyton, and A. Bendelac. 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191: 1895–1903. 10. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, Disclosures C. R. Wang, Y. Koezuka, and M. Kronenberg. 2000. Tracking the response of S.J. works for Immudex, the company that makes the dextramers used in this natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192: study to discover lipopeptide-specific T cells. The other authors have no finan- 741–754. 11. Karadimitris, A., S. Gadola, M. Altamirano, D. Brown, A. Woolfson, P. Klenerman, cial conflicts of interest. J. L. Chen, Y. Koezuka, I. A. Roberts, D. A. Price, et al. 2001. Human CD1d- glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl. Acad. Sci. USA 98: 3294–3298. 12. Gumperz, J. E., S. Miyake, T. Yamamura, and M. B. Brenner. 2002. Functionally References distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer 1. Yu, C. Y., and C. Milstein. 1989. A physical map linking the five CD1 human staining. J. Exp. Med. 195: 625–636. thymocyte differentiation antigen genes. EMBO J. 8: 3727–3732. 13. Lee, P. T., K. Benlagha, L. Teyton, and A. Bendelac. 2002. Distinct functional 2. Bedel, R., J. L. Matsuda, M. Brigl, J. White, J. Kappler, P. Marrack, and L. Gapin. lineages of human Va24 natural killer T cells. J. Exp. Med. 195: 637–641. 2012. Lower TCR repertoire diversity in Traj18-deficient mice. Nat. Immunol. 13: 14. Kasmar, A. G., I. van Rhijn, T.-Y. Cheng, M. Turner, C. Seshadri, A. Schiefner, 705–706. R. C. Kalathur, J. W. Annand, A. de Jong, J. Shires, et al. 2011. CD1b tetramers 3. Miller, M. M., C. Wang, E. Parisini, R. D. Coletta, R. M. Goto, S. Y. Lee, bind ab T cell receptors to identify a mycobacterial glycolipid-reactive T cell D. C. Barral, M. Townes, C. Roura-Mir, H. L. Ford, et al. 2005. Characterization repertoire in humans. J. Exp. Med. 208: 1741–1747. of two avian MHC-like genes reveals an ancient origin of the CD1 family. Proc. 15. Van Rhijn, I., A. Kasmar, A. de Jong, S. Gras, M. Bhati, M. E. Doorenspleet, Natl. Acad. Sci. USA 102: 8674–8679. N. de Vries, D. I. Godfrey, J. D. Altman, W. de Jager, et al. 2013. A conserved 4. Moody, D. B., D. C. Young, T. Y. Cheng, J. P. Rosat, C. Roura-Mir, human T cell population targets mycobacterial antigens presented by CD1b. Nat. P. B. O’Connor, D. M. Zajonc, A. Walz, M. J. Miller, S. B. Levery, et al. 2004. Immunol. 14: 706–713. T cell activation by lipopeptide antigens. Science 303: 527–531. 16. Ly, D., A. G. Kasmar, T.-Y. Cheng, A. de Jong, S. Huang, S. Roy, A. Bhatt, 5. Manolova, V., M. Kistowska, S. Paoletti, G. M. Baltariu, H. Bausinger, D. Hanau, R. P. van Summeren, J. D. Altman, W. R. Jacobs, Jr., et al. 2013. CD1c tetramers L. Mori, and G. De Libero. 2006. Functional CD1a is stabilized by exogenous detect ex vivo T cell responses to processed phosphomycoketide antigens. J. Exp. lipids. Eur. J. Immunol. 36: 1083–1092. Med. 210: 729–741. 6.Zajonc,D.M.,M.D.M.Crispin,T.A.Bowden,D.C.Young,T.-Y.Cheng, 17. Batard, P., D. A. Peterson, E. Deveˆvre, P. Guillaume, J.-C. Cerottini, D. Rimoldi, J. Hu, C. E. Costello, P. M. Rudd, R. A. Dwek, M. J. Miller, et al. 2005. D. E. Speiser, L. Winther, and P. Romero. 2006. Dextramers: new generation of The Journal of Immunology 4503

fluorescent MHC class I/peptide multimers for visualization of antigen-specific 20. de Jong, A., V. Penã-Cruz, T.-Y. Cheng, R. A. Clark, I. Van Rhijn, and CD8+ T cells. J. Immunol. Methods 310: 136–148. D. B. Moody. 2010. CD1a-autoreactive T cells are a normal component of the 18. Rosat, J.-P., E. P. Grant, E. M. Beckman, C. C. Dascher, P. A. Sieling, human ab T cell repertoire. Nat. Immunol. 11: 1102–1109. D. Frederique, R. L. Modlin, S. A. Porcelli, S. T. Furlong, and M. B. Brenner. 21. Pellicci, D. G., A. J. Clarke, O. Patel, T. Mallevaey, T. Beddoe, J. Le Nours, 1999. CD1-restricted microbial lipid antigen-specific recognition found in the A. P. Uldrich, J. McCluskey, G. S. Besra, S. A. Porcelli, et al. 2011. Recognition of CD8+ ab T cell pool. J. Immunol. 162: 366–371. b-linked self glycolipids mediated by natural killer T cell antigen receptors. Nat. 19.Gras,S.,S.R.Burrows,L.Kjer-Nielsen,C.S.Clements,Y.C.Liu, Immunol. 12: 827–833. L.C.Sullivan,M.J.Bell,A.G.Brooks,A.W.Purcell,J.McCluskey,and 22. Van Rhijn, I., D. C. Young, A. De Jong, J. Vazquez, T.-Y. Cheng, R. Talekar, J. Rossjohn. 2009. The shaping of T cell receptor recognition by self-tolerance. D. C. Barral, L. Leoń, M. B. Brenner, J. T. Katz, et al. 2009. CD1c bypasses lysosomes Immunity 30: 193–203. to present a lipopeptide antigen with 12 amino acids. J. Exp. Med. 206: 1409–1422. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021