of March 29, 2018.This information is current as

EpitopeThy-1/CD90 Bearing the 6C10 CarbohydrateRecognized by Increased Expression of

T Cell Maturation+Peripheral CD4

R. Hardy and Kyoko HayakawaMing Gui, David L. Wiest, Jin Li, Dietmar Kappes, Richard

http://www.jimmunol.org/content/163/9/47961999; 163:4796-4804; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/9/4796.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Peripheral CD41 T Cell Maturation Recognized by IncreasedExpression of Thy-1/CD90 Bearing the 6C10 CarbohydrateEpitope1

Ming Gui, David L. Wiest, Jin Li, Dietmar Kappes, Richard R. Hardy, and Kyoko Hayakawa 2

The SM6C10 IgM autoantibody recognizes a surface determinant, 6C10, that is highly expressed on all immature thymocytes. Incontrast, its expression on peripheral T cells appears developmentally regulated, i.e., absent from most naive T cells in spleen ofneonatal mice, but expressed on 40–80% of naive CD41 T cells in adult. In this paper, we demonstrate that SM6C10 recognizesa carbohydrate epitope on the Thy-1 glycoprotein using immunoprecipitation analysis, by binding to affinity-purified Thy-1 in anELISA, and by sensitivity to N-glycosidase-F treatment. Retroviral Thy-1 gene transduction experiments into Thy-12 variant Tcell lines and a pro-B cell line provide evidence that 6C10 glycosylated Thy-1 expression is not restricted to T cells but dependson the recipient cell. Therefore, differences in 6C10 levels among Thy-11 T cells in mice likely reflect developmental regulationof posttranslational modification of the Thy-1 glycoprotein. The ability of naive CD41 T cells to respond to anti-Thy-1 stimulationincreases from neonate to adult, and 6C102 naive cells from adult mice respond poorly compared with 6C101 cells, similar to thecells in neonatal mice. These results suggest that there is functional maturation by peripheral CD41 T cells that coincides with6C10 glycosylated Thy-1 up-regulation, and natural autoantibody recognizes this 6C10 carbohydrate epitope.The Journal ofImmunology,1999, 163: 4796–4804.

T he development and differentiation of T cells is accom-panied by alteration of surface Ag expression, generatingsignificant heterogeneity in the periphery (1–7). Although

most commonly recognized phenotypic heterogeneity amongCD41 or CD81 T cells is a result of antigenic activation (5), thereare several surface proteins expressed differentially in the pool ofcells considered to be mature Ag nonexposed T cells (“naive” Tcells). In mice, these surface proteins include several GPI-linkedmolecules, such as Qa-2 (8), Ly-6C (6, 9), Ly-6A/E (6, 10), andthe Thy-1/CD90-dependent 6C10 (3, 11–13). GPI-linked proteinsare clustered in specialized glycolipid-enriched membrane mi-crodomains called caveolae (14–18). Because several GPI-linkedproteins associate withsrc-family tyrosine kinases, p56lck andp59fyn, and cross-linking of these proteins leads to tyrosine phos-phorylation of intracellular substrates (19–21), resulting in activa-tion (22–25), they could function as signaling molecules on T cellsor could modulate TCR-mediated cell activation (22, 26–30).Thus, there is increasing interest in the possibility that such phe-notypic heterogeneity reflects functional diversification of periph-eral T cells, predetermining cell fate following activation.

6C10 is of particular interest due to its association with CD41

T cell function and with the GPI-linked protein Thy-1. The 6C10determinant is recognized by the mouse IgM monoclonal anti-thymocyte/T cell autoantibody SM6C10 as a natural autoantibody(11). 6C10 expression is dependent on surface Thy-1/CD90 gly-coprotein expression, and it is found at high levels on immature

thymocytes of all inbred mouse strains, similar to Thy-1 (11).However, although Thy-1 is expressed by nearly all T cells in thespleen or lymph nodes, 6C10 is expressed only by a fraction ofperipheral T cells, mostly by CD41 T cells, and at lower level.Thus, in adult mice, 40–80% of CD41 native T cells in spleen orlymph nodes are 6C101. Interestingly, this 6C101 CD41 T cellpopulation shows generally higher levels of several GPI-linkedmolecules (6) and greater responsiveness. Both 6C102 and 6C101

CD41 naive T cells in the adult can proliferate and secrete IL-2after activation (3). However, 6C101 cells respond higher than6C102 cells in certain stimulation systems, such as by superanti-gen (31), by Con A in the presence of resting accessory B cells (3),or, as will be shown in this paper, by cross-linking Thy-1. Fur-thermore, 6C101 is an obligatory phenotype for long-term mem-ory Th cells (3), and it is absent from cells rendered anergic (3, 31).Thus, 6C10 is a unique phenotypic marker that distinguishes thefunctional status of CD41 T cells both before and after activation.

Our recent observation of a complete lack of 6C101 cells inThy-1 knockout mice demonstrated that the Thy-1 glycoprotein isabsolutely required for 6C10 expression in mice (32). However, afraction of peripheral Thy-11 T cells lacks 6C10, and 6C10 isabsent from peripheral T cells in neonates even though they ex-press Thy-1 protein (6). Furthermore, 6C10 is not detectable onbrain cells, activated NK cells, or on rat thymocytes, despite a highlevel of Thy-1 protein expression by all of these cell types (Ref.11, and our unpublished observations). Thus, whereas 6C10 ex-pression requires Thy-1, it is limited to certain Thy-11 cell types,depending on tissue, species, and age.

Because of the difficulty to immunoprecipitate 6C10 from celllysates using nonionic detergent such as Nonidet P-40 with thisIgM autoantibody, and because of some discordance between6C10 and Thy-1 surface expression, it has remained unclearwhether 6C10 is an epitope on Thy-1 or else resides on a moleculedistinct from, but associated with, Thy-1. Interestingly, previousimmunoprecipitation analysis using anti-thymocyte autoantibodies

Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111

Received for publication May 28, 1999. Accepted for publication August 19, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a grant from the National Institutes of Health (CAAI31412).2 Address correspondence and reprint requests to Dr. Kyoko Hayakawa, Institute forCancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia,PA 19111. E-mail address: k_hayakawa@fccc.edu

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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that cross-block with SM6C10 and that show similar T cell reac-tivity suggested the presence of a 100-kDa molecule noncovalentlyassociated with Thy-1 (33, 34). Because the issue of how GPI-anchored proteins such as Thy-1, associated only with the outerleaflet of the plasma membrane, can transduce signal into the cy-toplasm remains unsettled, the possibility of transmembrane mol-ecules associated with GPI-linked molecule(s) is a recurring at-tractive hypothesis, and this p100 molecule was proposed as apotential candidate to explain functional association (35). It wastherefore critical to determine whether SM6C10 autoantibody re-acts with p100 (or another Thy-1-associated protein), rather thandirectly with the Thy-1 epitope. In this study, we have attemptedto discriminate between these possibilities and also to understandwhy 6C10 serves to mark the functional status of CD41 T cells.

Materials and MethodsMice

C57BL/6JN (B6), BALB/c, and B6.Thy-12/2 (36) mice were bred andmaintained in the Institute for Cancer Research Animal Facility. Pigeoncytochromec/I-Ek specific TCRab transgenic mice, TCR “AND” (37),were purchased from The Jackson Laboratory (Bar Harbor, ME) and con-tinuously crossed with Rag-22/2 mice, selecting offspring expressingtransgene TCR and lacking B cells in the peripheral blood (by immuno-fluorescence analysis), to obtain transgene-expressing mice on a Rag-22/2

background.

Cell lines

EL-4 was obtained from the American Type Culture Collection (Manassas,VA) and maintained in Opti-MEM (Life Technologies, Rockville, MD)medium. AKR1 and AKR1-d.6.6 (38) were provided by Dr. R. Hyman(Salk Institute, La Jolla, CA) and maintained in Dulbecco’s medium (11).The mouse pro-B cell line ret2/0 was maintained in RPMI 1640 medium,as described previously (39).

Abs

Rat anti-mouse Thy-1, G7 (22), was purchased from PharMingen (SanDiego, CA). Rat Abs to mouse Thy-l.2 (30-H12), heat stable Ag (HSA)/CD24 (30F1), T200/CD45 (30F11), IgM (331.12), CD3e (500A-A2), L-se-lectin/CD62L (MEL-14), CD4 (GK1.5), IL-2 (JES6-1A12 and JES6-5H4),and mouse anti-ratk (MAR18.5) were purified from ascites and fluoro-chrome coupled for flow cytometry or used for ELISA. Rat anti-mouseIgM, M41, was purified by a protein G affinity column (Pierce, Rockford,IL), and the mouse IgM/k Abs, SM6Cl0 and SM3G11, were purified fromascites by protein L affinity columns (ACTIgen, Cambridge, U.K.) accord-ing to the manufacturer’s instruction. Purified TEPC183 was purchasedfrom Sigma (St. Louis, MO). In some experiments, SM6C10 was used inascites form together with other ascites controls. Control hybridomas,SM3G11, SM4E9, and SM5D8 were all derived from the same cell fusionexperiment as SM6C10 (11). Their IgM titers in ascites were all equalizedbased on an anti-m/anti-k ELISA sandwich assay to measure IgM content.

Immunochemical analysis

Cell surface biotin-labeling, immunoprecipitation (i.p.), electrophoresis,and enhanced chemiluminescence procedures have all been described else-where (40). In brief, 2–103 107 cells were incubated with Sulfo-NHS-Biotin (Pierce) at 1 mg/ml in HBSS for 30 min at 4°C and then washedwith HBSS with 25 mM lysine. Live biotinylated cells (.98%) were har-vested by overlay of lympholyte M and then lysed in 1% digitonin (Wako,Richmond, VA) lysis buffer in Tris (pH 7.4) with a protease inhibitormixture at 23 107 cells/ml for 30 min at 4°C; subsequently, supernatantwas obtained by centrifugation at 12,000 rpm for 15 min. For i.p. of Thy-1or 6C10, MAR (for Thy-1), or anti-IgM (M41, for 6C10) was first coupledto Sepharose 4B beads (Sigma). A 20-ml-coupled bead aliquot was incu-bated with 5mg of Ab or 1ml of ascites, and then washed before incubationwith 25–50ml of cell lysate. After lysate incubation for 4 h at 4°Cwithrotation, beads were washed and resuspended in sample buffer for disso-

ciation, and supernatant was applied to one-dimensional SDS-PAGE ortwo-dimensional electrophoresis (2-DE; NEPHGE/SDS-PAGE) (3) proce-dure. After electrophoresis, gels were blotted onto membrane (polyvinyli-dene fluoride), blocked with 5% nonfat dry milk (Bio-Rad, Hercules, CA)in PBS containing 0.4% Tween 20 (Sigma) for 1 h at room temperature(RT), incubated with HRP-coupled streptavidin conjugate (Southern Bio-technology Associates, Birmingham, AL) for 1 h at RT,washed exten-sively, and then incubated with Supersignal Western Blotting Substrate(Pierce) for luminescence development. Luminescence was detected byexposure of Kodak (Rochester, NY) x-ray film for various times (from 5 sto 15 min) for intensity comparison. Rainbow-colored high m.w. proteinmarkers (Amersham, Arlington Heights, IL) and biotinylated broad rangeprotein markers (New England BioLabs, Beverly, MA) were used as m.w.standards. OVA and carbonic anhydrase from Sigma were used to mark pIpoints on 2-DE. PeptideN-glycosidase-F (PNAGase-F; Oxford Glyco-Systems, Rosedale, NY) treatment of lysate was conducted according tomanufacturer’s recommendation.

Thy-1 glycoprotein affinity purification

Thy-1 affinity purification was according to the method previously de-scribed by Chang et al. (41) with some modification. In brief, lysate wasmade from 13 109 thymocytes from either B6 or B6.Thy-12/2 mice at2 3 108 cells/ml in Nonidet P-40 lysis buffer (1% Nonidet P-40 in 50 mMTris-saline (pH 7.4), protease inhibitors, and 10 mM iodoacetamide) on icefor 45 min. Lysate was incubated with 1.5 ml of an anti-Thy-1.2- (30-H12)coupled Sepharose 4B slurry for 2 h at 4°Cwith rotation and then packedinto a column. After extensively washing the column with 0.1% NonidetP-40, bound material was eluted with 0.2 M glycine (pH 2.8). The 0.5-mlfractions were immediately neutralized by addition of 50ml of 2 M Tris(pH 8.0). A pool of the first eight fractions was immediately applied to aCentricon-10 (Amicon, Beverly, MA) for concentration, followed by two“resuspensions” with 0.01 M PBS (pH 7.2), thus finally obtaining a 0.5-mlconcentrated eluate in PBS. To examine the protein(s) in such eluates,samples were applied to 12% SDS-PAGE minigel and silver-stained. Pu-rification of HSA/CD24 from B6 thymocytes Nonidet P-40 lysate wasconducted by using a 30F1-coupled affinity column.

ELISA assay for detection of Thy-1/6C10

ELISA was conducted in a 96-well Immunoplate (Nalge Nunc Interna-tional, Roskilde, Denmark) as described previously, with some modifica-tion. For direct plate coat of Thy-1, 30–50ml of eluate was used for platecoating, adjusted to 100ml with PBS, by overnight incubation at 4°C.Coated wells were then blocked with 3% OVA, 30 min at RT, beforesubsequent steps with biotinated anti-Thy1.2(30H12) and alkaline phos-phatase (AP)-coupled avidin incubation. For ELISA sandwich assay, anti-Thy-1 (or anti-HSA) was coated at 1mg/ml overnight at 4°C, followed byblocking with 3% OVA at RT, and then incubated with 30–50m1 of Thy-1containing eluate overnight at 4°C. After washing, wells were againblocked with OVA, incubated with SM6C10 Ab (2mg/ml) at RT for 1.5 h,and subsequently incubated with biotinated anti-IgM and AP-avidin.

Immunofluorescence staining and multicolor flow cytometryanalysis and sorting

Four-color flow cytometry analysis and sorting were conducted using aFACStarPlus(Becton Dickinson, San Jose, CA) as described previously (3).

Anti-Thy-1 stimulation

Anti-Thy-1 stimulation was done in 96-well, U-bottomed plate (Coster,Cambridge, MA). Then 2–53 104 cell sorter-purified CD41 T cells, orsubsets, were incubated with G7 with or without PMA (Sigma) at differentconcentrations as described in the experiments. The 24-h culture superna-tant was harvested and tested for the presence of IL-2 by ELISA sandwichassay by using JES6-1A12 and biotin-JES6-5H4, in combination. MouserIL-2 (R&D Systems, Minneapolis, MN) and supernatant from IL-2 se-creting cell line, 16H (42), were used as IL-2 standards. An OD of 0.5corresponds to;150 pg/ml. Cells were cultured for 6 more days, har-vested, and then counted by passing through the cell sorter. Live cells werediscriminated by light scatter and by propidium iodide exclusion.

Retrovirus-mediated Thy1.2 gene transduction

The Thy-1-coding sequence was amplified by PCR usingTaqpolymerasefrom cDNA made using RNA isolated from T cells with primers engi-neered to containEcoRI andXhoI restriction sites (sense 59-GGAATTCATGAACCCAGCCATCAGCGTC-39, anti-sense 59-CCGCTCGAGTCACAGAGAAATGAAGTCCAGG-39). The 505-nt-amplified fragment

3 Abbreviations used in this paper: HSA, heat stable Ag; i.p., immunoprecipitation;2-DE, 2-dimensional electrophoresis; NEPHGE, nonequibrium pH gradient electro-phoresis; MAR, mouse anti-ratk Ab; PNGase-F, peptideN-glycosidase-F; AP, alka-line phosphatase; GFP, green fluorescent protein; RT, room temperature; pI, isoelec-tric point.

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was cloned into TA vector (Invitrogen, San Diego, CA) and verified bysequence analysis. TheEcoRI/XhoI fragment released by double restrictiondigest was gel-purified and then ligated withEcoRI/XhoI cut pBMN-IRES-EGFP retroviral vector provided by Dr. G. Nolan (Stanford University,Palo Alto, CA). Clones containing appropriate size insert (released by dou-ble digest withEcoRI/XhoI) were again sequenced for verification. Purifiedplasmid was introduced into the Phoenix ecotropic packaging line (pro-vided by Dr. G. Nolan) (43) by lipofection (LipofectAMINE, Life Tech-nologies) following the manufacturer’s protocol; 1 day later, recipientcell lines were added together with polybrene (44) to facilitate viral entry.After 2 days of coculture, the infected cells were suspended by gentleagitation, washed with staining medium, and then stained with anti-CD4(for AKR1-d.6.6) or AA4.1 (for ret2/0) to distinguish them from packagingcells. CD41 or AA4.11 cells expressing green fluorescent protein (GFP)were sorted by flow cytometry directly into wells containing culture me-dium. After expansion in culture, cells were reanalyzed to reveal.95%GFP positive cells.

ResultsInduction of 6C10 expression and up-regulation of Thy-1 as aindication of peripheral CD41 T cell development.

Although 6C10 is highly expressed on all immature CD4181

cells, it is down-modulated on more mature CD41 single positiveHSA2 T cells in the thymus of both neonates and adults (Fig. 1A,left two panels). CD41 T cells in the spleen of neonatal mice aremostly 6C102 (Fig. 1A, right panel, thin line) similar to thymicHSA2 CD41 T cells. In contrast, the splenic CD41 T cells estab-lished in adult animals are predominantly 6C101 (40–80%) (Fig.1A, right panel, thick line), as we have previously described (3).To test whether this 6C10 phenotypic change in the periphery is adevelopmental event independent of antigenic influence, we ana-lyzed CD41 T cells in spleens of Rag-22/2 TCRab “AND” trans-genic mice that express a monomorphic TCR, precluding possibleactivation by self-Ag. CD41 T cells in these transgenic mice alsoexhibited an age-dependent increase of 6C10 levels (Fig. 1B, left),verifying that this occurs in the naive T cell pool. These naive6C102 CD41 T cells in both neonates and adults of transgenicmice express substantial levels of Thy-1. However, Thy-1 levelson peripheral CD41 T cells increase with age, correlated with

6C10, reaching highest Thy-1 levels on 6C101 CD41 T cells inthe adult (Fig. 1B,right two panels). This result suggests that theheterogeneity of 6C10 expression among CD41 naive T cells inadult is due to continuing CD41 T cell development in the periph-ery, up-regulating Thy-1 and inducing 6C10.

SM6C10 immunoprecipitates a 26- to 29-kDa species bySDS-PAGE

The correlated 6C10 and Thy-1 expression in Fig. 1B may meaneither that 6C10 is a Thy-1 epitope or that it is expressed on aThy-1 associated molecule whose expression depends on surfaceThy-1. To distinguish between these, we conducted i.p. analyses.Although i.p. of 6C10 has been difficult with Nonidet P-40 lysate,we found that digitonin lysate allows specific i.p. Thy-11 T celllines, thymocytes, and Thy-12 mutants were surface biotinylatedand digitonin lysates were prepared for i.p. All Thy-12 (Thy-1o)cells are surface 6C102 by immunofluorescence staining (11, 32).Fig. 2A, a–i, shows 10% SDS-PAGE analysis of T cell lines. Spe-cific i.p. of Thy-1 from EL-4 (Thy-1.2) and AKR1 (Thy-1.1) withnon-allele-specific or allele-specific Ab revealed a 25- to 30-kDaspecies under reducing conditions by SDS-PAGE that was notfound with the Thy-12 variant AKR1-d. We could detect a specificband with SM6C10 from AKR1 in the same region, albeit weakly(requiring longer exposure), but not with AKR1-d, nor with controlIgM (Fig. 2A, j–o). Some background in the region (and alsoaround 55–70 kDa with variable intensity in samples) withoutSM6C10 Ab was also observed in the absence of lysate (data notshown), apparently due to nonspecific avidin binding to releasedimmobilized Ab.

Fig. 2Bshows thymocyte i.p. results, where Thy-1 was readilydetectable by anti-Thy-1 (Fig. 2Bb). A species in the 26- to 29-kDaregion was also immunoprecipitated with SM6C10 from thymo-cyte digitonin lysates at levels;10- to 20-fold less than obtainedwith anti-Thy-1 (Fig. 2B, c vs e). Importantly, as shown by thenext panel, this species was absent from i.p. of Thy-12 thymocytes(Fig. 2Bj), which showed only the background band seen with

FIGURE 1. Re-expression of 6C10 during peripheralCD41 T cell development coordinate with Thy-1 up-regulation, shown by immunofluorescence analysis.A,Thymocytes and spleen cells from 6-day neonatal (thinline) and 3-mo adult (thick line) C57BL/6 mice werestained with fluorescent-labeled Abs (3); the fluoresce-in-SM6C10 histograms of gated cell fractions areshown.B, Spleen cells from Rag-22/2 “AND” TCR ab1-wk neonatal and 3-mo adult transgenic mice werestained and analyzed for 6C10 and Thy-1.2 expressionon CD41 T cells. CD41 T cells represent 0.4% and 17%of splenocytes in neonates and in adult, respectively.

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control IgM Abs (Fig. 2B,h andi) or immobilized SM6C10 alonewithout lysate (Fig. 2Bl). No other specific bands were observedover the range from 10 to 220 kDa in SDS-PAGE (7%, 10%, and15%) analysis. Pre-absorption of 6C101 lysate with anti-Thy-1-coupled MAR beads, but not with uncoupled MAR beads, elimi-nated the 26- to 29-kDa band (Fig. 3), demonstrating association oridentity of this material with the Thy-1 molecule. The use of oc-tylglucoside in cell lysis, which prevents formation of detergent-resistant complexes by GPI-anchored proteins (45), again gaveSM6C10 specific immunoprecipitation at the same location with-out generation of new specific bands (data not shown).

SM6C10 binds to affinity-purified Thy-1

Because the above experiments leave open the possibility thatSM6C10 binds to a Thy-1-associated molecule that is not effi-ciently biotinylated, we conducted additional experiments to dem-onstrate its direct binding to Thy-1. For this purpose, we affinity-purified Thy-1 from Nonidet P-40 thymocyte lysate and performedan ELISA using Thy-1 glycoprotein as the target. As Thy-12 con-trols, we used: 1) material eluted from an anti-Thy-1 column usingThy-12 thymocyte lysate (Thy-1o) and 2) HSA/CD24 purifiedfrom Thy-11 thymocyte lysate using an anti-HSA column. Wechose HSA because, like Thy-1, it is a GPI-linked glycoprotein

expressed by all immature thymocytes. Silver staining of SDS-PAGE analysis of purified material from Thy-11 thymocytesshowed a single specific band in the Thy-1 region, which is lackingfrom Thy-1o material (Fig. 4). ELISA confirmed the presence ofThy-1 in this material from Thy-11 thymocytes (Fig. 5A, upper).Of importance, SM6C10 showed greatest binding to wells coatedwith anti-Thy-1-purified material from thymocytes (Fig. 5A, low-er). Furthermore, as Fig. 5B shows, SM6C10 exhibited specificbinding in a sandwich assay using anti-Thy-1-coated wells, but notwith isotype control Abs or control eluate, demonstrating thatSM6C10 specifically binds to the same molecule (or molecularcomplex) recognized by anti-Thy-1. Considering our SDS-PAGE

FIGURE 2. Thy-1-dependent detection of 26- to 29-kDa species with SM6C10. Data are from a 10% SDS-PAGE analysis of T cell digitonin lysate i.p.A, T cell line lysates, Thy-1 phenotype as marked, were immunoprecipitated with rat Abs plus MAR beads (a–i) or MAR beads alone (2), or mouse IgMAbs plus anti-IgM beads (j–o) or anti-IgM beads alone (2). Exposure times fora–i and j–o are 15 s and 1 min, respectively.B, Thymocyte lysates fromB6 (Thy-11) or B6. Thy-12/2 (Thy-1o) mice were immunoprecipitated as described inA. p, Twenty times less lysate was used.a–f andg–l were from twoseparate experiments. Each exposure time was 1 min except for anti-CD45 (10 s). Numbers on the left indicate m.w. markers (kDa).

FIGURE 3. Elimination of the SM6C10 20- to 29-kDa band after pre-absorption with anti-Thy-1-coupled beads. Thymocyte lysate was first titratedfor Thy-1 absorption efficiency by varying lysate amount, to determine anappropriate ratio. After incubation of lysate with anti-Thy-1-coupled MARbeads (or with uncoupled MAR beads) for 30 min on ice, supernatant wasdivided for each immunoprecipitation as shown.

FIGURE 4. Silver staining of 12% SDS-PAGE of anti-Thy-1 affinity-purified material. Anti-Thy-1 column eluates from B6 (Thy-11) or B6.Thy-12/2 (Thy-1o) thymi were applied to 12% SDS-PAGE minigel usingreducing conditions. The arrow indicates material corresponding to theThy-1 glycoprotein. Two bands at 50–70 kDa present in all lanes, includ-ing a lane without sample loading (2), were caused by reducing agent.

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analysis of purified material that shows a single predominant bandof m.w. equivalent to Thy-1, we conclude that SM6C10 recognizesa determinant on Thy-1, without any associated species.

The 6C10 carbohydrate epitope is expressed on several Thy-1glycoforms

The Thy-1 glycoprotein contains a high proportion of carbohydratechains at threeN-linked glycosylation sites, with variable com-plexity at each site (46–48). Thus, in 2-DE (NEPHGE/SDS-PAGE) analysis (Fig. 6), Thy-1 from thymocyte lysate migrates assix to eight distinct isoelectric focussing species (Fig. 6a), as ev-ident from comparison to the lysate negative control (Fig. 6c) (49).SM6C10 immunoprecipitated Thy-1 from thymocyte lysate with asimilar extent of charge heterogeneity (Fig. 6b). Treatment ofThy-1 with PNGase-F generated a homogeneous 15-kDa polypep-tide by elimination of carbohydrate (Fig. 7b), whereas SM6C10 nolonger bound to such PNGaseF-treated Thy-1 (Fig. 7d). Thus,

FIGURE 5. SM6C10 binding to purified Thy-1 in an ELISA assay.A,Affinity-purified Thy-1 or HSA material from B6 thymocytes, or mockanti-Thy-1 material prepared from Thy-12/2 thymocytes (Thy-1o) wereplate-coated for ELISA assay. Levels of binding were tested by biotin-anti-Thy-1.21 AP-avidin (upper) or SM6C10 (or control TEPC183)1biotin-anti-IgM1 AP-avidin (lower).p, Subtracted OD data based on con-trol TEPC183 binding to each coated plate, due to nonspecific IgM Abbinding to the coated plates. Purified Abs were used in all groups.B, Plateswere precoated with anti-Thy-1 (G7) or anti-HSA (30F1), both rat IgG2c,and subsequently incubated with purified Thy-1 or HSA material. Afterwashing, plates were then tested for reactivity to either purified SM6C10 orcontrol TEPC 183 IgM, developed by biotin-IgM1 AP-avidin. The OD1 h after addition of substrate is shown.

FIGURE 6. 6C101 Thy-1 molecules in thethymus exhibit multiple isofocusing points sim-ilar to total thymocyte Thy-1 molecules. The2-DE (NEPHGE/SDS-PAGE, 15%) analysis ofanti-Thy-1.2 or SM6C10 immunoprecipitates.aandb, Digiton lysate;c andd, Nonspecific bind-ing by avidin to the reagents without lysate ad-dition. Two circles ina and b indicate markerlocation, OVA (m.w. 45 kDa, pI 5.1) and car-bonic anhydrate (m.w. 29 kDa, pI 7.0). Exposuretime points 30 s (aandc) and 1 min (bandd).

FIGURE 7. Peptide N-glycosidase F treatment of Thy-1 abolishesSM6C10 i.p. Digitonin thymocyte lysate was treated with PNGase andimmunoprecipated with anti-Thy-1.2 or SM6C10. A 15% SDS-PAGEanalysis is shown.

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6C10 is a carbohydrate epitope present on several Thy-1 glyco-forms on thymocytes.

Recipient cell dependent induction of 6C10 by Thy-1 genetransduction

Although 6C10 is expressed by all immature Thy-11 thymocytes,it is limited to certain Thy-11 cell types in the periphery and is notexpressed by brain Thy-1, suggesting cell type or differentiationstage specific glycosylation. We have previously shown that Thy-1gene transfection into a Thy-12 variant T cell lines restores 6C10expression (11). As Fig. 8,left, shows, retroviral Thy-1 gene trans-duction of the mouse pro-B ret2/0 line, originally 6C102 Thy-12,resulted in 6C10 expression that was correlated with Thy-1, similarto most Thy-11 T cell lines. Thus, 6C10 glycosylation is not re-stricted to T lineage cells. In turn, we found that some T cell lines

failed to express 6C10 upon expression of Thy-1, reminiscent ofthe neonatal CD41 T cell phenotype. Using the same retroviralThy-1 transduction system, infection of the AKR1-d.6.6 T lym-phoma, a spontaneous Thy-12 variant defective for expression ofseveral surface Ags, resulted in a high level of surface Thy-1 ex-pression but without detectable 6C10, and this lack of 6C10 wasmaintained in culture for.3 mo (Fig. 8,right). These data supportthe idea that 6C10 expression requires specific glycosylation ma-chinery that is dependent on the cell’s developmental/differentia-tion stage and that such glycosylation can occur in non-T cells.

Functional maturation by CD41 T cells in association with6C101 phenotype

To assess whether 6C101 CD41 T cells exhibit functional matu-rity more than 6C102 cells, we purified 6C102 3G111 and 6C101

3G111 naive CD41 T cell fractions from adult spleen (Fig. 9A)and tested for IL-2 release and proliferation using various doses ofanti-Thy-1 (G7) and PMA stimulation, a system to quantitate Tcell responsiveness independent of accessory cells. More than 90–95% of cells were Thy-11 in both fractions. As Fig. 9B shows,6C101 cells consistently showed higher IL-2 release and prolifer-ation in comparison to 6C102 cells under all conditions employed.Anti-Thy-1-mediated cell aggregation (50) was also greater with6C101 T cells compared with 6C102, suggesting that proximalanti-Thy-1 cross-linking events (51) also differ (not shown).

Using conditions that yielded the strongest anti-Thy-1 response,splenic naive 3G111 CD41 T cells purified from neonates (1 wk)and from adults (3 mo) of wild-type B6 and Rag-22/2 TCRab“AND” transgenic mice were compared for IL-2 secretion. As Fig.9C shows, neonatal naive CD41 T cells from both wild-type andtransgenic mice responded poorly to anti-Thy-1 stimulation, com-pared with adult CD41 T cells. Thus 6C102 cells show a consis-tently lower response to anti-Thy-1 activation. Levels of TCR/CD3and CD45, which could potentially influence anti-Thy-1-mediatedIL-2 secretion (27, 52), were comparable between 6C102 and6C101 naive cells (not shown). These results suggest that there ismaturation that coincides with 6C10 glycosylated Thy-1 up-regu-lation by peripheral CD41 T cells, generating functional and phe-notypic heterogeneity.

FIGURE 8. Recipient cell line dependence for induction of 6C10by retroviral Thy-1 gene transduction. Immunoflurescence analysis of6C10 and Thy1.2 expression before and after Thy-1 transduction intoAKR1-d.6.6 or ret2/0 cell lines. Infected cells all express high levels ofGFP. The 6C10 level was measured by using PE-coupled anti-IgM as asecond step Ab together with allophycocyanin-coupled anti-Thy-1.2.

FIGURE 9. Higher responsiveness by 6C101 cells than 6C102 cells in anti-Thy-1 stimulation of naive CD41 T cells. A, Three-month-old BALB/cspleen cells were four-color stained for CD4, 6C10, 3G11/GD1c, and CD62L, and then 6C101 and 6C102 naive CD41 T cells were purified for in vitrostimulation assay (sorting gates are boxed). The majority of 3G111 cells were also CD62L1. The 3G111 CD62L1 cells were sorted to purify naive T cells.These were also.95% CD45RB1 as we have previously described (6).B, Dose responses to anti-Thy-11 PMA stimulation as assessed by IL-2 secretion(1-day supernatant) and proliferation (1-wk-cultured cells).f, 6C101 cells 1 5 mg/ml of G7;F, 6C101 cells 1 0.5 mg/ml of G7;Œ, 6C102 cells 1 5mg/ml of G7; andl, 6C102 cells1 0.5 mg/ml of G7. An average of duplicate cultures is shown. Total cell numbers and viability were also consistentlyhigher in 6C101 cell cultures. PMA alone did not elicit IL-2 and proliferation. Representative data of three experiments.C, CD41 3G111 CD62L1 T cellsin C57BL/6 and Rag-22/2 TCRab “AND” mice were purified from 1-wk- or 3-mo-old mouse spleen and stimulated with G7 (5mg/ml) 1 PMA (0.01mg/ml).

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DiscussionNatural IgM autoantibodies recognizing Thy-1 associated Ag(s)appear to be common (11, 53–57). However, chemical character-ization of relevant self-Ags has been hampered for a long time bydifficulties in obtaining clear immunoprecipitates with such IgMautoantibodies. In this paper, we used an alternative approach, pu-rification of Thy-1, in combination with immunoprecipiation, todemonstrate that 6C10 is a carbohydrate epitope expressed on theThy-1 glycoprotein. SM6C10 binds to affinity-purified Thy-1 gly-coprotein as demonstrated by a “sandwich” ELISA. Lack of spe-cific bands other than the 26- to 29-kDa region corresponding toThy-1, and disappearance of the 26- to 29-kDa band by anti-Thy-1pre-absorption in SDS-PAGE analyses, indicate that there is nodistinct 6C10-reactive protein associated with Thy-1 under theconditions of our analysis. Analysis showing sensitivity of the6C10 determinant to periodate treatment led us previously to sug-gest that this epitope has a carbohydrate nature (11); this is con-firmed here by loss of reactivity after PNGase-F treatment. Fur-thermore, 6C101 cells are absent in Thy-1 gene knockout mice (aswe have recently shown) (32). Thus, the Thy-1 glycoprotein mustbe the predominant molecule that carries the 6C10 determinant inmice. Carbohydrates expressed on glycoproteins and/or glycolip-ids are frequent targets of natural autoantibodies (58), and we showin this paper that this also holds for the Thy-1 glycoprotein.

Thy-1/CD90 is a ubiquitous neural glycoprotein in most species,with extensive glycosylation, and is a major cell-surface glyco-protein of thymocytes in mice and rats (46). Whereas the polypep-tide backbone is identical for Thy-1 in brain and thymus, the car-bohydrate composition differs significantly (46, 47). Speciesspecific glycosylation of Thy-1 is also well-documented (47, 59).Thus, the lack of 6C10 epitope expression in brain or on rat Thy-1is not surprising. T cell differentiation associated differences inThy-1 glycosylation have been also described previously. De-creased branching of the carbohydrate chains of Thy-1 with in-creased sialic acid addition were shown to accompany T cell dif-ferentiation from thymocytes to peripheral T cells (49, 60, 61).6C10 becomes undetectable during maturation in the thymus, con-sistent with this differentiation related change in glycosylation.However, we found that 6C10 is re-expressed by peripheral Tcells, specifically on adult CD41 T cells. Our 2-DE analysis didnot reveal specific association of 6C10 with acidic Thy-1 speciesand 6C10 seems relatively resistant to neuraminidase treatment(34), suggesting that the determinant may not be a specific sialy-lated form of Thy-1. Additionally, 6C10 expression was not af-fected by deficiency in either biosynthesis of complex oligosac-charides or fucose addition (our unpublished results, and fromanalysis of BW5147.PHAR and BW5147.PLR (62, 63), respective-ly). These results suggest that the 6C10 epitope shared betweenthymocyte and peripheral T cell Thy-1 glycoprotein may be ex-pressed on various Thy-1 glycoforms.

The question arises as to whether the re-expression of 6C10 onperipheral CD41 T cells is due to a quantitative increase in Thy-1glycoprotein bearing a similar ratio of glycoforms or, instead, re-flects a shift in glycoform expression on a subset of Thy-1. That is,considering the close correlation between Thy-1 and 6C10 epitopelevels, one might ask whether the apparent lack of 6C10 on someT cells might simply reflect a lower staining efficiency by SM6C10compared with anti-Thy-1 peptide Abs. Answering this questionby immunochemical means has been difficult because of the com-bination of lower Ag level in the periphery and lower Ab affinity.However, our retroviral transduction data revealed that T cells ex-pressing even high levels of Thy-1 can lack 6C10, arguing againstthis interpretation. Of interest, this lack of 6C10 was observed in

the Thy-1 gene transduced AKR1-d.6.6 T lymphoma. This lym-phoma is a spontaneous variant of the original Thy-1 deficientAKR1-d, which shows several additional defects in expression ofsurface Ags, including absence of CD45 (R. Hyman, unpublishedobservations). Thy-1 transfection into the original AKR1-d lineresults, in contrast, in 6C101 Thy-1 expression as we have previ-ously shown (11). This prompts us to suggest that 6C102 Thy-11

T cells in mice, whether CD41 or CD81, truly lack the specific6C10 epitope due to alternative posttranslational modification, butthat CD41 T cells in particular regain the ability to synthesizeand/or express 6C101 Thy-1 glycoform(s) during peripheraldevelopment.

Although it has been known that most CD41 T cells in thethymus are not fully functionally competent despite their highTCR/CD3 expression (8, 64), it has not been clear whether theCD41 T cells newly arrived in peripheral sites are all equallyfunctionally mature. We found that 6C102 CD41 naive T cells inneonatal mouse spleen of transgenic mice with monomorphicTCR, consisting of cells newly exported from thymus, exhibitlower responsiveness to anti-Thy-1 stimulation compared with6C101 cells in adult mice. Aside from this ontogenic difference,we have previously described a pattern of 6C102 to 6C101 tran-sition by analyzing CD41 T cells reconstituted by transfer of adultbone marrow cells into adult recipients (6); thus, this phenotypicchange is not due to ontogenic differences in cell microenviron-ment. Rather, our data indicate that postthymic maturation contin-ues in the periphery for CD41 T cells as a programmed process,including up-regulation of 6C101 Thy-1 and gain of function.

Why do 6C101 CD41 T cells respond better to anti-Thy-1 (andother) activation? Our data clearly exclude the possibility that6C10 recognizes novel transmembrane signaling molecule. Acqui-sition of signaling capability in B cell or human T cell lines bytransfection of the mouse Thy-1 gene has been reported previ-ously, in which anti-Thy-1 cross-linking resulted in a calcium in-flux (26). In this work, it was suggested that if any transmembranemolecule was required for anti-Thy-1 signaling, then such a mol-ecule must be conserved between species and be ubiquitously dis-tributed. We show in this paper that 6C10 is a carbohydrate epitopeof Thy-1, and that 6C101 Thy-1 can be induced in non-T cellssuch as in pro-B cells by Thy-1 gene transduction, demonstratingthat non-T cells can express the 6C101 Thy-1 glycoform. Thisresult strongly suggests that 6C101 Thy-1 alone is important forcells to signal via anti-Thy-1 cross-linking.

Because the GPI-anchor is located entirely within the outer leaf-let of the cell membrane bilayer, GPI-linked proteins possess in-creased mobility compared with transmembrane proteins, resultingin their rapid lateral redistribution. This has been considered asignificant feature in cell-cell adhesion, and in transduction of ex-tracellular stimuli (15). Internalization and concentration of GPI-linked small molecules appears to occur at a unique site, the caveo-lae (16). Caveolae are a specialized detergent-resistant membranecompartment where many signaling molecules appear to be con-centrated, such as Src family tyrosine kinases (17, 18, 45, 65) andphosphatidylinositol bisphosphate (PIP2) (66). Thus, one proposedmodel has been the coupling of GPI-linked internalized moleculeswith the intracellular-signaling machinery at the caveolae (30). Be-cause subtle differences in the carbohydrate side chains influencethe physiological properties of plasma glycoproteins (59), it is pos-sible that 6C101 Thy-1 glycoforms may have an advantage in thislateral redistribution and invagination process, resulting in en-hanced signal transduction.

Alternatively, higher anti-Thy-1 responsiveness by the 6C101

Thy-1 cells may simply reflect functional maturity of CD41 Tcells. “Mature” CD41 T cells may possess altered biosynthesis of

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glycosidases and/or glycosyltransferases, resulting in changes insurface phenotype, such as production of 6C10 glycosylatedThy-1, and an altered signaling mechanism. Considering such Tcell “maturation” may be more important in terms of understand-ing increasing T cell function in the immune system from newbornto adult. Besides their gain of 6C10, CD41 T cells in adults alsoexpress higher levels of Qa2 and Ly6A/E when compared withneonates (Ref. 8, and our unpublished observations). As recentdata suggests, dynamic accretion of activated Ag receptor com-plexes rafting to the signaling molecule rich caveoli appears toconstitute an important step in T cell activation (65, 67). Thus,coordinate up-regulation of GPI-linked glycoproteins in matureCD41 T cells may provide increased opportunity for signalinginteractions, influencing the fate of cells during activation, eitherpositively or negatively.

In summary, our work demonstrates that the 6C10 determinant,recognized by a natural autoantibody, is a carbohydrate epitope onthe GPI-anchored glycoprotein Thy-1. Significantly, we show inthis paper that the functional and phenotypic heterogeneity amongnaive CD41 T cells is due to continuing maturation of CD41 Tcells in the periphery, coincident with 6C10 glycosylated Thy-1up-regulation. Whether the 6C10 carbohydrate epitope on Thy-1has direct significance in T cell function or is simply an associatedsurface phenotype remains to be determined. A potential role fornatural anti-T cell autoantibody in T cell activation is another in-triguing question to be answered. Nevertheless, our study intro-duces the concept of peripheral T cell maturation, demonstratingthat most CD41 T cells in the neonate and many of the CD41 Tcells in the adult have yet to reach full functional competence.Thus, it is important to consider that differences in the CD41 Tcells, in addition to differences in APCs (68), contribute to dis-tinctions between neonatal and adult immunity, in terms of toler-ance induction, memory T cell generation, and generation of dis-tinct effector cell types. Molecular understanding of maturationand determining why the capacity to express certain glycosylatedThy-1 is retained by memory Th cells but not by anergic T cells (3,31) are important subjects for future investigation.

AcknowledgmentsWe thank Drs. R. Hyman (The Salk Institute, San Diego, CA) for Thy-12/2

mutant cell lines, G. Nolan (Stanford University, Palo Alto, CA) for thePhoenix ecotropic packaging line and the retroviral vector, J. Silver (NorthShore University Hospital, Manhasset, NY), and C. L. Stewart (NationalCancer Institute-Frederick Cancer Research and Development Center,Frederick, MD) for Thy-12/2 mice. We also thank J. Dashoff and S. A.Shinton for expert technical assistance.

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