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Specific T Cells Ex Vivo−Dextramers Identify Human Lipopeptide

Cutting Edge: CD1a Tetramers and

D. Branch MoodyRossjohn, John Shires, Søren Jakobsen, John D. Altman andStephanie Gras, Richard W. Birkinshaw, Li L. Tan, Jamie Schiefner, Ravi C. Kalathur, Ian A. Wilson, Mugdha Bhati,David C. Young, Tan-Yun Cheng, Marie T. Turner, Andre Anne G. Kasmar, Ildiko Van Rhijn, Kelly G. Magalhaes,

http://www.jimmunol.org/content/191/9/4499doi: 10.4049/jimmunol.1301660October 2013;

2013; 191:4499-4503; Prepublished online 2J Immunol 

MaterialSupplementary

0.DC1http://www.jimmunol.org/content/suppl/2013/10/02/jimmunol.130166

Referenceshttp://www.jimmunol.org/content/191/9/4499.full#ref-list-1

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Cutting Edge: CD1a Tetramers and Dextramers IdentifyHuman Lipopeptide–Specific T Cells Ex VivoAnne G. Kasmar,* Ildiko Van Rhijn,*,† Kelly G. Magalhaes,* David C. Young,*Tan-Yun Cheng,* Marie T. Turner,‡ Andre Schiefner,x,1 Ravi C. Kalathur,x

Ian A. Wilson,x,{ Mugdha Bhati,‖ Stephanie Gras,‖ Richard W. Birkinshaw,‖

Li L. Tan,‖ Jamie Rossjohn,‖,# John Shires,** Søren Jakobsen,†† John D. Altman,** andD. Branch Moody*

Human CD1a mediates foreign Ag recognition by aT cell clone, but the nature of possible TCR interac-tions with CD1a/lipid are unknown. After incubatingCD1a with a mycobacterial lipopeptide Ag, dideoxymy-cobactin (DDM), we identified and measured bindingto a recombinant TCR (TRAV3/ TRBV3-1, KD of�100 mM). Detection of ternary CD1a/lipid/TCRinteractions enabled development of CD1a tetramersand CD1a multimers with carbohydrate backbones(dextramers), which specifically stained T cells usinga mechanism that was dependent on the precise stereo-chemistry of the peptide backbone and was blockedwith a soluble TCR. Furthermore, sorting of humanT cells from unrelated tuberculosis patients for brightDDM-dextramer staining allowed recovery of T cellsthat were activated by CD1a and DDM. These studiesdemonstrate that the mechanism of T cell activa-tion by lipopeptides occurs via ternary interactions ofCD1a/Ag/TCR. Furthermore, these studies demon-strate the existence of lipopeptide-specific T cells inhumans ex vivo. The Journal of Immunology, 2013,191: 4499–4503.

Humans express four CD1 Ag-presenting molecules,CD1a, CD1b, CD1c, and CD1d (1). Whereas thebasic biology of CD1d and NKT cells has been

extensively studied in mice lacking either CD1d or invariantNKT TCRs (2), CD1a proteins have been deleted from themurine genome (3), effectively limiting analysis to the study

of a few human T cell clones propagated long-term in vitro(4, 5). The mycobacterial lipopeptide dideoxymycobactin(DDM) is the only well-characterized foreign Ag in the CD1asystem and has served as a model to understand the specificityand mechanism of action of CD1a-restricted T cells in vitro(4, 6, 7). Building on the success of human MHC class Itetramers to quantitatively track fresh human T cells ex vivo(8), CD1 tetramers (9–16) capture lipid-reactive T cells aspopulations for study ex vivo. Dextramers rely on the sameprinciple, but they use higher order multimers that allowdetection of rare, lower affinity T cells (17). In this study, weinvestigated whether human TCRs directly bind to CD1a/lipopeptide to develop tetramers and dextramers that iden-tify polyclonal T cell populations, which recognize foreign Agbound to CD1a.

Materials and MethodsGeneration of soluble CD1a proteins

We produced soluble biotinylated CD1a monomers in lentivirus-transducedHEK293T cells at the National Institutes of Health Tetramer Core Facility(Emory University, Atlanta, GA) (14, 16).

Loading CD1a monomers with DDM

DDM with defined stereochemistry (DDM 1S,3R or DDM 1R,3S) (7) wasincubated with CD1a monomers and complexed with streptavidin coupledto allophycocyanin. Staining was optimized by analyzing T cells with CD1atetramers generated after loading under varied conditions (Supplemental Fig.1). Optimal staining was seen with DDM solubilized in DMSO, sonicatedinto 30-fold excess 50 mM sodium citrate plus 1% CHAPS at pH 6 for 2min, incubated at 42˚C for 1 h, and then added at 40-fold molar excess toCD1a monomers and incubated in a 37˚C water bath for 1 h prior to neu-tralization to pH 7.4 with 2 ml Tris (pH 9).

*Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital,Harvard Medical School, Boston, MA 02115; †Department of Infectious Diseases andImmunology, Faculty of Veterinary Medicine, Utrecht University, 3584CL Utrecht,The Netherlands; ‡Tuberculosis Treatment Unit, Lemuel Shattuck Hospital, JamaicaPlain, MA 02130; xDepartment of Integrative Structural and Computational Biology,The Scripps Research Institute, La Jolla, CA 92037; {Skaggs Institute for ChemicalBiology, The Scripps Research Institute, La Jolla, CA 92037; ‖Department of Biochem-istry and Molecular Biology, School of Biomedical Sciences, Monash University, Clay-ton, Victoria 3800, Australia; #Institute of Infection and Immunity, Cardiff University,School of Medicine, Cardiff CF14 4XN, United Kingdom; **Emory Vaccine Center,Atlanta, GA 30329; and ††Immudex, 2100 Copenhagen, Denmark

1Current address: Technische Universitat Munchen, Munich, Germany.

Received for publication July 17, 2013. Accepted for publication August 29, 2013.

This work was supported by grants from the Howard Hughes Medical InstituteKwaZulu-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

Fund Program in Translational Research (to D.B.M.), as well as by National Institutes ofHealth Grants T-32 AI 007306-22, T-32 AR 007530-23, K08 AI089858 (to A.G.K.),R01 AI49313, R01 AR048632 (to D.B.M.), and R01 A1042266 (to I.A.W.) and a grantfrom the Nederlands Wetenschappelijk Onderzoek (Meervoud 836.08.001) (to I.V.R.).S.G. is supported by an Australian Research Council Future Fellowship. J.R. is supportedby a National Health and Medical Research Council Australia Fellowship.

Address correspondence and reprint requests to D. Branch Moody, Division of Rheu-matology, Allergy and Immunology, Brigham and Women’s Hospital, Smith BuildingRoom 514, 1 Jimmy Fund Way, Boston MA 02115. E-mail address: bmoody@partners.org

The online version of this article contains supplemental material.

Abbreviation used in this article: DDM, dideoxymycobactin.

Copyright� 2013 by TheAmerican Association of Immunologists, Inc. 0022-1767/13/$16.00

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CD1a tetramer and dextramer staining of clones

CD1a tetramers were generated and validated by staining the DDM-reactiveab T cell clone CD8-2 (18) and using methods similar to those reported forCD1b tetramers (14). We generated dextramers by diluting CD1a complexesto 0.1 mg/ml in PBS prior to incubation with 2 ml allophycocyanin-labeleddextramer backbone for 30 min at room temperature in the dark, followed bystaining of T cells as previously reported (17). For dual staining with TCRAbs, TRBV3-1 (Beckman Coulter) was added after tetramer staining for thelast 15 min of incubation. Cells were acquired on a FACSCanto II flowcytometer (Beckton Dickinson) and analyzed using FlowJo (Tree Star) soft-ware 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 andthe CD1a-restricted, DDM-specific TCR CD8-2 were produced as previ-ously described (14, 19). Loaded CD1a-DDM was coupled to research-gradestreptavidin-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 theequilibrium data were analyzed with the Prism program (GraphPad Soft-ware).

Dextramer staining of human PBMCs

Work with human subjects was overseen by the Institutional Review Boardsof 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 roomtemperature followed by 15 min at 4˚C, after which cells were washed andincubated with violet viability dye, CD3-FITC, CD14-PerCP-Cy5.5, andCD19-PerCP-Cy5.5 for 15 min at 4˚C. Unfixed dextramer+ cells were sortedusing 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 weretested for DDM recognition in an ELISPOT assay (20).

Results and DiscussionGeneration of antigenic CD1a/lipopeptide complexes

We generated transmembrane-truncated soluble biotinylatedCD1a proteins based on methods previously developed forMHC, CD1d, CD1b, and CD1c (8, 12, 14, 16). To test thefolding and Ag-presenting function of this new CD1a con-struct, it was bound to streptavidin plates, treated with asynthetic DDM, and used to activate T cells (7). The syntheticAg recapitulates the naturally occurring S or R stereochemistryamong the four stereocenters in the natural DDM peptide,including S and R stereochemistry at positions 1 and 3 presentin M. tuberculosis DDM (DDM 1S,3R) (Fig. 1A) (7). Thislipopeptide activated the CD1a-restricted human T cell lineCD8-2 in a dose-dependent manner. No activation was seenin response to DDM with the opposite stereochemical con-figuration at positions 1 and 3 (DDM 1R,3S) or a syntheticanalog that deviated from the optimal natural DDM based ona fully saturated acyl chain and serine substituting for a-methylserine (DDM analog; Fig. 1B). Thus, soluble CD1a monomerswere properly folded and were sufficient to present a lip-opeptide Ag to human T cells.

CD1a tetramers and dextramers stain T cells

After optimizing loading conditions based on pH, time, tem-perature, and solvent variables (Supplemental Fig. 1), weobserved that DDM (1S,3R)-treated CD1a tetramers selec-tively stain T cells expressing the clonotypic TRBV3-1 TCR,but not other T cells (Fig. 1C). T cells were not stained byCD1a tetramers that were not exposed to lipids or tetramerstreated with the two nonantigenic DDMs that substantiallymimic DDM (1S,3R). Thus, tetramer staining was dependenton the structure of the added lipopeptide and was specific for

the clonotypic TCR that defines CD8-2. These data estab-lished a working CD1a tetramer and strongly supported themodel of direct binding of an ab TCR to CD1a. To furtherincrease the avidity of interaction, we developed CD1a dex-tramers that are composed of 10–14 CD1a monomers ona flexible, fluorescently labeled dextran backbone (17). We ob-served bright, selective, and highly reproducible staining ofthe TRBV3-1 subset of CD8-2 T cells (Fig. 1D) with DDM(1S,3R)-loaded dextramers. Thus, dextramers provided a sec-ond reagent for probing the interaction of CD1a with T cells.

CD1a multimers bind to the ab TCR

CD1a-DDM tetramer and dextramer binding to TRBV3-1+

T cells strongly implicated a cognate model in which a ternaryinteraction of CD1a/lipid binds to the TCR. We performedexperiments to directly test binding between lipopeptide/CD1a complexes and the clonotypic human ab TCR ver-sus all other surface receptors. Preincubation of dextramerswith anti-CD1a Ab blocked staining to background (Fig. 2A),confirming that staining was mediated by CD1a. We gener-

FIGURE 1. CD1a tetramers and dextramers stain human T cells. (A) DDM

is a biosynthetic precursor to mycobactin siderophores composed of a fatty

acyl tail and a peptide backbone that contains four chiral centers (1–4), whichare in the S or R configuration as indicated. Natural DDM occurs as the

1S,3R diastereomer. The nonstimulatory DDM analog is presumed to be

a mixture of 1S,3R and 1R,3S diastereomers (7). (B) Tetramerizable CD1a

monomers were bound to streptavidin-coated plates, treated with Ag over-

night at 37˚C, and used to activate IFN-g release by the CD1a-restricted

human T cell line CD8-2 (mean 6 SD). (C) The CD8-2 T cell line, which

contains cells with the DDM-specific TCR b-chain TRBV3-1, as well as cells

with other TCRs, was stained with CD1a tetramers loaded with the indicated

DDM isomer and TRBV3-1–specific mAb. (D) CD8-2 T cells were stained

by CD1a dextramers loaded with the indicated DDM isomer followed by

TCR anti–Vb-chain Ab (TRBV3-1). Data in (B)–(D) are representative

of three or more experiments.

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ated soluble TCRs containing the TCR a- and b-chains fromCD8-2 (ab heterodimers encoded by the TRAV3-1 andTRBV3-1 variable regions) or analogous constructs from theCD1b-restricted T cell LDN5 (composed of the TRAV17and TRBV4-1 variable regions) (Fig. 2B). Preincubation withsoluble LDN5 TCR minimally impacted staining, whereaspreincubation with soluble CD8-2 TCR blocked T cell stain-ing to background levels (Fig. 2C). Thus, CD1a-DDM stainingof cells is mediated by the ab TCR.We then measured the affinity of interaction between soluble

transmembrane-truncated CD8-2 TCR and CD1a proteinsalone or pretreated with DDM using surface plasmon reso-nance (Fig. 2D). No binding was seen to CD1a alone; thedetermined KD was 95.78 6 13.51 mM. This affinity is sig-nificantly lower than that of TCRs recognizing a-galactosylceramide-CD1d (,1 mM) and glucose monomycolate-CD1b(∼1 mM) (15)) but approximates the affinity of NKT TCRsrecognizing b-linked glycolipids (21).

CD1a dextramers detect human lipopeptide–specific T cells ex vivo

To determine whether DDM-reactive T cells exist as cellpopulations ex vivo, PBMCs from subjects with active tu-berculosis or positive tuberculin skin tests were stained withdextramers treated with DDM (1S,3R) and then gated onCD3+CD142CD192 live lymphocytes (Supplemental Fig.2A). Rare CD3+ cells were identified with the DDM-loadeddextramer among four subjects with mycobacterial exposure(Fig. 3, Supplemental Fig. 2B). This result suggested thatDDM-reactive T cells are present in the blood of unrelatedhuman donors.To determine whether the rare dextramer+ cells recog-

nize CD1a and DDM, T cell yields were increased throughleukapheresis (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), clonedthem using limiting dilution, and tested them for reactivityin ELISPOT assays. Dextramer-based sorting generated manyT cell clones that were brightly stained using DDM-loadeddextramers, and these clones secreted TNF-a (clones 3, 6,9, 21) or IFN-g (clones P1, P5, P7, P9) in response to

CD1a-expressing APCs treated with DDM (Fig. 4). Threeof the clones express CD8 and the other five express CD4.We therefore conclude that CD1a- and DDM-reactive Tcells are present as populations among genetically diversedonors.Overall, these results distinguish between indirect and cog-

nate TCR interaction models, illustrating that direct CD1a/DDM/TCR interactions are the mechanism of T cell activa-tion by CD1a and lipopeptide. These studies extend priorwork describing polyclonal responses to lipoprotein mixtures

FIGURE 2. Tetramer and dextramer staining demonstrates

a trimolecular interaction among CD1a, DDM, and the clo-

notypic 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 experi-

ments. (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).

FIGURE 3. CD1a dextramers stain polyclonal cells ex vivo. PBMCs from

one subject with active tuberculosis (subject A24) as well as three tuberculin

skin test–positive subjects (subjects A22, A32, and C58) were stained with

CD1a dextramers in addition to CD3-FITC, CD14-PerCP-Cy5.5, CD19-

PerCP-Cy5.5, and violet viability dye, after which they were gated on live

lymphocytes.

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(18) and lipopeptide-specific T cell clones (22) to prove thatlipopeptide-reactive T cells exist in the in vivo state. Moregenerally, generation of working CD1a multimers completesthe set of tools needed to study human CD1-restricted Tcells, allowing determination of the phenotype and func-tion of T cells recognizing CD1a bound to self and foreignAgs.

DisclosuresS.J. works for Immudex, the company that makes the dextramers used in this

study to discover lipopeptide-specific T cells. The other authors have no finan-

cial conflicts of interest.

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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)

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