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Binding to MHCInteraction Recruits CD8 in Absence of High Affinity Xenoreactive TCR:MHC

A. Frelinger, Ettore Appella and Edward J. CollinsJennifer Buslepp, Samantha E. Kerry, Doug Loftus, Jeffrey

http://www.jimmunol.org/content/170/1/373doi: 10.4049/jimmunol.170.1.373

2003; 170:373-383; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2003 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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High Affinity Xenoreactive TCR:MHC Interaction RecruitsCD8 in Absence of Binding to MHC1

Jennifer Buslepp,* Samantha E. Kerry,† Doug Loftus,§ Jeffrey A. Frelinger,†‡ Ettore Appella,§

and Edward J. Collins2*†‡

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2Db complexed with peptidep1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understandmore about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusionbodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate thathuman p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/Db. Yet, T cell bindingand cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs thatblock CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with “CD8-independent” p1049/A2 produces p56lck activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonanceenergy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, theseresults indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHCengagement. The Journal of Immunology, 2002, 169: 373–383.

O n the surface of APCs, CD8� T cells identify Ags pre-sented in the form of class I MHC plus peptide (pMHC)3

(1). Recognition of foreign Ags (due to either nonselfpeptide, MHC, or both) by the TCR causes signaling inside the Tcell, which results in cytokine release, differentiation into cytotoxicT cells, and killing of APCs presenting the foreign Ag. Althoughit is the TCR that is responsible for Ag recognition, additionalaccessory molecules on the surface of the T cell, including theCD8 coreceptor, contribute to the interaction of the T cell with theAPC, and affect T cell activation (2, 3).

The exact mechanism by which the TCR/pMHC interaction re-sults in transmission of signals inside the T cell is unclear. Crystalstructures of TCR with and without class I pMHC indicate a con-formational change does not occur upon binding (4, 5). The �- and�-chains of the TCR are short and contain no recognizable signal-ing motifs, however, the associated CD3� complex contains sev-eral repeats of the immune tyrosine-base activation motif (ITAM)(6). The ITAMs are phosphorylated after TCR engagement ofpMHC Ag, via the Src family kinase p56lck (7). p56lck has beenshown to associate with the coreceptor on the surface of CD4� orCD8� T cells (8). p56lck interacts with the cysteine residues in the

CD8 �-chain (9, 10), and the palmitoylation of both p56lck andCD8 � result in the presence of both proteins in lipid rafts at thecell membrane (11). CD8 makes specific interactions with con-served regions in the �2 and �3 domains of MHC independentlyof the identity of the bound peptide (12, 13). These data have ledto a model for T cell signaling where pMHC Ag interacts with bothTCR and CD8, and this interaction initiates the events that resultin T cell activation (14, 15). But, as plausible as this mechanismfor T cell activation seems, it does not account for the existence offunctional pMHC ligands that are CD8-independent (16–22).

Most studies examining CD8 interactions with class I MHChave used the CD8�� homodimer, but the CD8�� homodimer isfound primarily on intraepithelial T cells in the gut, NK cells, and�� T cells (23, 24). The CD8 isoform found almost exclusively onCD8� �� T cells is the CD8�� heterodimer. Structures of both thehuman and murine CD8�� homodimer cocrystallized with pMHCcomplexes have been determined (12, 13). The structures confirmconclusions from earlier mutagenesis experiments showing thatCD8 binds to class I MHC mostly on the �3 domain and partiallyon the �2 domain (22, 25, 26). Apparently, the CD8 �-chain doesnot significantly change the affinity of CD8 for class I MHC, as theaffinity constant for the binding of CD8�� to pMHC as determinedby surface plasmon resonance (SPR) is �65 �M (27), in the rangeof KD values reported for CD8�� homodimer binding to MHC(11–220 �M) (24).

The function of CD8 binding to class I MHC in T cell activationis still disputed. The predominant function attributed to CD8 is thatof a coreceptor, which increases the avidity of the fairly weakTCR:pMHC interaction (23, 24, 28). However, CD8 has also beenimplicated in cell-cell adhesion (29–31), and the H-2Dk tetramerhas been shown to bind CD8 on naive CD8� T cells independentlyof TCR:pMHC binding (32). Experimental evidence even suggeststhat CD8 is not necessary for functional T cell activation, as somepMHC:TCR interactions have been identified as “CD8-indepen-dent” based on the ability of CTL to lyse target cells in the pres-ence of an anti-CD8 Ab (22, 33). Recent data indicate that T cellslacking CD8 are still able to kill and pMHC tetramers still bind

Departments of *Biochemistry and Biophysics and †Microbiology and Immunology,and ‡Lineberger Comprehensive Cancer Center, University of North Carolina, ChapelHill, NC 27599; and §Laboratory of Cell Biology, National Cancer Institute, NationalInstitutes of Health, Bethesda, MD 20892

Received for publication May 10, 2002. Accepted for publication October 17, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Susan G. Komen Breast Cancer Foundation (toE.J.C.), National Institutes of Health Grant GM67143 (to J.A.F.), and National In-stitutes of Health Training Grant 007273 (to S.E.K.).2 Address correspondence and reprint requests to Dr. Edward J. Collins, Departmentof Microbiology and Immunology, University of North Carolina, CB Number 7290,804 M. E. Jones Building, Chapel Hill, NC 27599. E-mail address: edward_collins@med.unc.edu3 Abbreviations used in this paper: pMHC, peptide MHC; ITAM, immune tyrosine-base activation motif; SPR, surface plasmon resonance; FRET, fluorescence reso-nance energy transfer; CHO, Chinese hamster ovary; RU, resonance unit.

The Journal of Immunology

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00

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(17, 20). Additionally, it has been shown that the presence of CD8is absolutely necessary to trigger T cell calcium responses topMHC (15, 17). Together, these data indicate that some, but notall, T cell functions may occur independently of CD8.

The murine AHIII 12.2 T cell clone recognizes murine class Imolecule H-2Db in complex with a synthetic peptide p1058 (se-quence FAPGFFPYL) (34). This murine CD8� T cell was origi-nally generated by injection of a human B cell into a C57BL/6mouse (35). The clone was shown to be xenoreactive, and re-stricted to (human) class I MHC HLA-A2.1 in complex with pep-tide p1049 (sequence ALWGFFPVL) (35). Our earlier comparisonof the x-ray crystal structures of p1049/A2 and p1027/Db (a p1058variant also recognized by AHIII 12.2) demonstrated that the mo-lecular surfaces recognized by this TCR are not similar (36), thusmolecular mimicry does not play a significant role in the recogni-tion of dissimilar pMHC by the AHIII 12.2 T cell clone. Exactlyhow this TCR recognizes two very different molecular surfaces isstill not known.

In this study, we show affinity and kinetics for binding of therecombinant AHIII 12.2 TCR to syngeneic pMHC (p1058/Db) andxenogeneic pMHC (p1049/A2) ligands by SPR. These experi-ments demonstrate that the affinity of p1049/A2 for the AHIII 12.2TCR is significantly greater than that of p1058/Db. In addition,functional assays with AHIII 12.2 T cells and anti-CD8 Abs showthat xenogeneic p1049/A2 is recognized by the TCR in a CD8-independent fashion, while syngeneic p1058/Db activity is dimin-ished in the presence of anti-CD8 Abs. Additional experimentsprove that the early signaling pathways thought to require CD8coengagement are intact when the T cells are presented with eitherp1049/A2 or p1058/Db. Confocal fluorescence microscopy andfluorescence resonance energy transfer (FRET) flow cytometrywith pMHC tetramers and CD8 Abs reveal TCR and CD8 colo-calize on the T cell membrane even in the absence of CD8 bindingto class I MHC. Thus, binding of pMHC by CD8 is not necessaryfor TCR and CD8 to be brought together and for CD8-associatedsignals to be sent.

Materials and MethodsPeptides

All peptides were synthesized at the National Cancer Institute (Bethesda,MD). Peptides (p1049: ALSGFFPVL, p1058: FAPGFFPYL, MLL:MLLSVPLLL, or FLU: ASNENMETM) were purified to �95% purity byreverse-phase HPLC and their identity was confirmed by matrix-assistedlaser desorption ionization-time-of-flight spectroscopy. Peptides were dis-solved in DMSO at 20 mg/ml by weight. Final peptide concentrations weredetermined by amino acid analysis (Protein Chemistry Laboratory, Depart-ment of Chemistry, University of North Carolina, Chapel Hill, NC).

Cell lines, Abs, and MHC tetramer reagents

The CD8� CTL clone AHIII 12.2, derived from a C57BL/6 mouse (37),was restricted to both HLA-A*0201 (38) and H-2 Db (34). AHIII 12.2 cellswere maintained by weekly stimulation with irradiated HSB stimulatorcells in the presence of IL-2. IL-2 was removed from culture media for atleast 36 h before performing assays for T cell activity. EL-4 cells wereobtained from R. Tisch (Department of Microbiology and Immunology,University of North Carolina). Chinese hamster ovary (CHO)-A2 cellswere a gift from Dr. P. Parham (Department of Microbiology and Immu-nology, Stanford University, Palo Alto, CA)

The �-CD8� (53-6.7), �-CD8� (53-5.8) Abs (conjugated and unconju-gated), and the hamster IgG isotype Ab control were purchased from BDPharMingen (San Diego, CA). The H57–597 anti-C� TCR Ab was purifiedfrom hybridoma supernatant (no. HB-218; American Type Culture Collec-tion, Manassas, VA). The 3A5 �- p56lck Ab (agarose conjugate) was pur-chased from Santa Cruz Biotechnology (Santa Cruz, CA).

MHC tetramers were made as described previously (39). Briefly, solu-bilized class I MHC H chain containing a C-terminal biotinylation se-quence, �2m, and peptide were folded in vitro in a buffer; the crude mixturewas then concentrated in an Amicon ultrafiltration cell (Millipore, Bedford,

MA), and purified by gel filtration (40). Folded MHC was biotinylatedusing BirA enzyme and biotinylation buffer (Avidity, Denver, CO) accord-ing to manufacturer’s specifications. Biotinylated MHC monomers werecomplexed with avidin, avidin-HRP, or avidin-PE (Leinco, St. Louis, MO)to form tetramers, filtered through 0.22 �m, and used in experiments. Theextent of biotinylation was assessed by SDS-PAGE gel as described (41).Octamers were formed by adding �-PE (Sigma-Aldrich, St. Louis, MO) Abto pMHC tetramers as previously described (41). Nonbiotinylated MHCprotein used in SPR experiments was produced as described above, exceptMHC H chain does not contain a biotinylation target sequence as describedpreviously (40).

Construction of TCR expression plasmids, protein expression,and inclusion body preparation

cDNAs for the �- and �-chains of the AHIII 12.2 TCR (D. Loftus, un-published data) were used to construct expression vectors. Residues 1–202and 1–237 of the mature � and � AHIII 12.2 TCR chains, respectively,were cloned into the pLM1 vector (a gift of G. Verdine, Harvard Univer-sity, Cambridge, MA) for expression in Escherichia coli. The expression ofTCR ectodomains in E. coli was as previously described (42). After in-duction, E. coli containing either AHIII 12.2 TCR �- or �-chain werepelleted by centrifugation, and resuspended in buffer containing lysozyme.The cells were lysed by French press, and DNA was degraded by theaddition of DNase. Cells were washed once with detergent buffer and oncewith Tris buffer, then solubilized in guanidium-HCl. Following ultracen-trifugation, protein purity was assessed by SDS-PAGE, and protein con-centration was determined by Bradford assay (Bio-Rad, Hercules, CA).Aliquots were stored at �80°C until use.

Protein folding and purification

TCR was folded as described (42), with a few modifications. Briefly, 70 mgof TCR �-chain and 40 mg of TCR �-chain were combined and brought toa total volume of 30 ml with 10 mM Tris, 6 M guanidine-HCl, 0.2 mMDTT, pH 8.0. Ten milliliters of this solution were injected into 600 ml ofa buffer composed of 1 M L-arginine, 100 mM TrisCl, pH 8.5, 2 mMEDTA, 0.5 mM reduced glutathione, 0.05 mM oxidized glutathione, 0.2mM PMSF, and 120 mg sodium azide. Two additional 10-ml injections ofprotein were added successively in 3–12 h intervals to give a total proteinconcentration of 4 �M. After the final protein injection, the buffer was keptat 10°C for 24 h. The buffer was dialyzed extensively against 10 L of 100mM urea, and finally 10 L of 100 mM urea, 10 mM Tris, pH 7.5. Aftercentrifugation and filtration, the solution was purified and concentrated byDE-52 ion exchange (Whatman, Clifton, NJ) and injected onto a 26/60S300 gel filtration column (Amersham-Pharmacia Biotech, Piscataway,NJ) at 0.5 ml/min in TBS, pH 8.0. The correctly folded TCR eluted at a Mr

of �45,000 kDa. Fractions (5 ml) containing correctly folded TCR wereconcentrated to 200 �M, and stored at �80°C. A typical yield was 7 mgprotein/L of folding buffer (�4%).

ELISA for refolded AHIII 12.2 TCR

An ELISA with soluble TCR and pMHC tetramer has been previouslydescribed (43). Briefly, the anti-C� TCR Ab H57-597 was used to coat thewells of a 96-well ELISA plate at 2 �g/ml. Subsequently, soluble AHIII12.2 TCR was added to the plate at 2 �g/ml, followed by different con-centrations of pMHC tetramers. For this experiment, the avidin used tomake pMHC tetramers was covalently linked to HRP (Sigma-Aldrich) fordetection. Following the addition of 3,3�,5,5�-tetramethylbenzidine (anHRP substrate), the amount of pMHC-tetramer binding was observed atOD405 using a SPECTRAmax 190 plate reader (Molecular Devices,Sunnyvale, CA) and quantitated using SOFTmax Pro 3.1 software.

Soluble TCR binding to cell surface

This experiment was performed as in Ref. 44, with a few modifications.Briefly, T2 cells, which express peptide-deficient HLA-A2 molecules, werepulsed with 1 �M p1049 peptide or a control peptide (MLL), then incu-bated with various concentrations of soluble AHIII 12.2 TCR, followed byFITC labeled-H57-597 (Southern Biotechnology Associates, Birmingham,AL). After washing, median fluorescence was measured using a FACScanflow cytometer.

SPR experiments

Five-thousand resonance units (RUs) of H57-597 (capturing molecule, anti-TCR C� Ab) were covalently bound to a Biacore CM5 sensor chip (Upp-sala, Sweden) using standard amine coupling. Soluble AHIII 12.2 TCR(ligand) was then added to the Ab at a concentration of 67 nM to generate

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300–400 RU of bound TCR. Soluble class I MHC (analyte) was injectedonto the surface at a flow rate of 100 �l/min in a 30-s pulse. TCR and MHCwere removed from the surface with 0.1 M Glycine, 0.5 M NaCl, pH 2.5,and the procedure was repeated until at least three curves were obtained forthe different concentrations of analyte. Curves obtained at each concentra-tion were subtracted from a reference surface that contained Ab alonewithout TCR. After background subtraction, curves were imported intoBIAevaluation 3.0, normalized along the x- and y-axes, and fit globally todetermine kinetic constants. The suitability of the fit was measured basedon �2 values and the appearance of residuals. In all cases, �2 was below 1,residuals were small and random, and the experimental curves visuallymatched the predicted curves. Curves and statistics were calculated fromthe average of at least three curves for each concentration.

Multimer staining of AHIII 12.2 cells

For T cell binding assays, the tetramer or octamer complexes were addedat the indicated concentrations to 2 � 105 AHIII 12.2 T cells. After a45-min incubation at 4°C, cells were washed three times, and analyzed formedian fluorescence by flow cytometry (FACScan; BD Biosciences,Franklin Lakes, NJ). As a negative control in all tetramer or octamer bind-ing experiments, multimers composed of HLA-A2.1 complexed to an ir-relevant peptide (MLL) were used. The median fluorescence of theMLL/A2 octamers or tetramers at each concentration or time point wassubtracted in each experiment. For two-color staining with anti-CD8� andanti-CD8� Abs, tetramer or octamer were added at saturating concentra-tions after staining with the appropriate CD8 Ab for 30 min at 4°C.

Proliferation assay

AHIII 12.2 T cells (3 � 105) were added to each of three wells in a 96-wellflat-bottom plate after being IL-2 starved for at least 36 h. Various con-centrations, from 5 to 0.0005 �g/ml, of pMHC tetramers were then addedto triplicate wells, with or without 5 �g/ml �-CD8 (53-6.7) or an isotypecontrol Ab. Three additional wells receive no tetramer. The plates werethen incubated at 37°C, 5% CO2, for 2 days, or until cells clumped andformed T cell blasts. [3H]Thymidine was then added at 1 �Ci/well. Cellswere again incubated at 37°C, 5% CO2, for 6–18 h, then either frozen at�20°C for future harvesting, or harvested immediately using a multiplesample harvester (Otto Hiller, Madison, WI). Incorporation of [3H]thymi-dine was measured by scintillation counting using a Beckman LS5000counter (Palo Alto, CA).

Cytotoxicity assay

Target cells (2 � 106; CHO, CHO-A2.1, or EL-4) were centrifuged andresuspended in 50 �l of FBS, 0.1 mCi (100 �l) 51Cr, with incubation at37°C, 5% CO2 for 1 h. After several washes with PBS, target cells wereadded at 5 � 103/well to a 96-well round bottom plate containing theindicated amount of peptide. Recombinant human �2m (100 nM) was alsoadded to CHO and CHO-A2.1 cells. 51Cr-labeled target cells and peptidewere incubated at 37°C, 5% CO2 for 1 h. AHIII 12.2 T cells with or withoutCD8� blocking Ab (53-6.7) were then added at 5 � 104 cells/well to targetcells. T cells and target cells were incubated at 37°C, 5% CO2. After 4 h,supernatants were harvested and 51Cr release was measured with a PackardCobra gamma counter (Downers Grove, IL). All assays were performed intriplicate, and the percent specific lysis was calculated as follows: ((exper-imental release � spontaneous release)/(maximum release � spontaneousrelease)) � 100. Spontaneous release is defined as counts per minute re-leased from target cells in the absence of T cells, and maximum release isdefined as counts per minute released from target cells lysed with 2.5%Triton X-100.

Calcium mobilization assay

AHIII 12.2 T cells were loaded with the calcium-specific dye Indo-1-AMas previously described (45). After Indo-1-AM labeling, some cells wereadditionally incubated with anti-CD8� or anti-CD8� FITC-conjugatedAbs. Dye-loaded cells were examined on a MoFlo flow cytometer (Cyto-mation, Fort Collins, CO) for �30 s to generate a baseline, 10 �g of theindicated tetramer complexes were added, and fluorescence was measuredin real time for 9 min. Data was analyzed using the FloJo software package(Treestar, San Carlos, CA).

In vitro kinase assay

Cells (4 � 106 per sample) were resuspended in 100 �l of RPMI 1640 at37°C. The appropriate tetramers and/or Abs were added for 3 min at 37°C,and the cells were lysed for 10 min on ice with 1% Brij-96, 50 mM Tris,pH 7.5, 150 mM NaCl, 1 mM Na3VO4, 10 mM NaF, and protease inhib-itors. After centrifugation, supernatant was precleared and incubated with

agarose-conjugated anti-p56lck for 2 h. After washing, agarose beads wereincubated with 150 �l of kinase buffer (20 �Ci [�-32P]ATP, 25 mMHEPES, pH 7.5, 7.5 mM MgCl, 1.5 mM MnCl, 1 mM Na3VO4, and 20 �gof a peptide corresponding to the C-terminal ITAM of CD3� (ITAMc) (46),for 15 min at 30°C (47). To assess p56lck phosphorylation, beads werepelleted, and equal amounts of the kinase reaction were spotted onto trip-licate circles of P81 phosphocellulose paper (Whatman). After drying, thecircles were washed four times with 1% phosphoric acid, and radioactivitywas measured using the Beckman LS5000 scintillation counter.

Confocal microscopy

AHIII 12.2 T cells (5 � 105) were washed once with PBS and settled for30 min on adherent-coated Colorfrost Plus glass slides (Fisher, Pittsburgh,PA). After blocking for 10–30 min with 10% FBS in PBS, cells werewashed and stained with tetramers (10 �g of p1049/A2, 20 �g of p1058/Db, and MLL/A2) conjugated to Alexa 568 (Molecular Probes, Eugene,OR) or Abs (3 �g of anti-CD8� FITC) for 5 min on ice. After washing,stained cells were fixed with 3% sucrose/0.02% sodium azide/3% parafor-maldehyde in PBS. Slides were mounted with ProLong antifade (MolecularProbes), and examined using a digital deconvolution fluorescence micro-scope (3I, Denver, CO). Data was collected at the same fluorescence in-tensity for each sample, and intensity was renormalized to approximatelythe same magnitude in the final images.

FRET flow cytometry

FRET experiments were performed as described previously (48). Briefly,2 � 105 AHIII 12.2 T cells were stained on ice with 5 �g/ml anti-CD8�-allophycocyanin for 30 min. Cells were washed and the indicated pMHCoctamers conjugated to PE were added at 200 �g/ml, and incubated on icefor an additional 30 min. After further washing, cells were run on a FAC-SCalibur flow cytometer (BD Biosciences). The allophycocyanin was ex-cited between 615 and 655 nm, but was not excited with the 488-nm laseras was PE. However, the excitation spectrum for PE overlaps the absorp-tion spectrum of allophycocyanin allowing for the production of FRET.After stimulation of PE, FRET between PE and allophycocyanin was vi-sualized as an increase in signal in the FL-3 channel, relative to negativecontrol or single-color staining. The FL3 channel was used because thelittle emission from PE at wavelengths �670 nm was easily compensated.Compensation values were almost identical to those published previously(48). To compare the FRET produced from p1058/Db and p1049/A2, theFRET found from p1058/Db was adjusted to the level that would have beenobtained if p1058/Db octamer bound as well as did p1049/A2. Thep1058/Db octamers bound at a consistently smaller fraction (59% of meanchannel fluorescence) than p1049/A2 octamers at the concentrations used.

ResultsAHIII 12.2 TCR produced in vitro binds pMHC

Previous studies with AHIII 12.2 T cells demonstrate that this Tcell clone is xenogeneic and recognizes both human HLA-A2 incomplex with the p1049 (ALWGFFPYL) peptide, and murine H-2Db in complex with the p1058 (FAPGFFPVL) peptide (34, 35). Todetermine the mechanism AHIII 12.2 uses to detect these two dis-parate ligands, biophysical experiments were performed withrTCR produced in vitro in E. coli. To confirm that the rAHIII 12.2TCR was folded properly, its ability to bind p1049/A2 was mea-sured by ELISA. The TCR was captured on an ELISA plate usingan anti-TCR C� Ab, H57-597. Binding was assessed by additionof class I MHC tetramers complexed to avidin-HRP. Thep1049/A2 tetramers bound well to the captured TCR and this re-sponse was dose-dependent (Fig. 1A). Binding of the rTCR wasmeasured on live APCs as a second assay to confirm proper fold-ing of the TCR. T2 cells expressing HLA-A2.1 were incubatedwith p1049 or control peptide, followed by different concentrationsof soluble AHIII 12.2 TCR. TCR binding to surface MHC wasdetected using the anti-TCR C� Ab, H57-597, conjugated to FITC.T2 cells pulsed with p1049 exhibited a dose-dependent increase influorescence due to TCR binding, while those cells pulsed withcontrol peptide did not (Fig. 1B). These data indicate that rAHIII12.2 TCR expressed and refolded in vitro is correctly folded andbiologically active.

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AHIII 12.2 has a higher affinity for p1049/A2 than for p1058/Db

Much of literature on T cell activation implicates either the affinityor half-life of the pMHC/TCR interaction as the trigger for eventsthat result in differentiation into effector T cells. SPR was used tomeasure the binding of rAHIII 12.2 TCR to p1049/A2 or p1058/Db. TCR was immobilized by binding to a sensor chip previouslycoated with an Ab against TCR C� (H57-597). Subsequently, ei-ther p1049/A2 or p1058/Db pMHC complexes were injected atvarious concentrations over the sensor chip. Recombinantp1049/A2 bound well and p1058/Db bound relatively poorly. Lowconcentrations of p1049/A2 generated the same response units ashigh concentrations of p1058/Db (Fig. 2). The calculated affinity ofthe AHIII 12.2 TCR for p1049/A2 was 11.3 �M, �8-fold greaterthan for p1058/Db at 84 �M (Table I). This result is unexpected asCTL assays using AHIII 12.2 T cells demonstrated equal lysis oftarget cells containing A2 or Db (prepulsed with p1049 and p1058,respectively) (34). Even though a disparity exists between the af-finities of these two ligands for the AHIII 12.2 TCR, it is interestingto note that the off-rates for the interaction of either p1049/A2 orp1058/Db with the AHIII 12.2 TCR are similar (Table I).

A potential explanation for differences in affinity, but similarbiological response on cells, is that SPR does not take into accountthe involvement of the CD8 coreceptor. CD8 coordinates bindingof MHC with the TCR to increase the avidity of the TCR:pMHCinteraction. To determine whether the contribution of CD8 to theavidity for pMHC could compensate for the low affinity of

p1058/Db to AHIII 12.2 TCR seen using SPR, we used pMHCtetramers to measure binding of these pMHC to AHIII 12.2 T cells.Thus, the observed tetramer binding to AHIII 12.2 T cells repre-sents the apparent affinity due to TCR:pMHC binding and CD8:pMHC binding. Tetramers composed of p1049/A2 bound to liveAHIII 12.2 T cells, but no binding was detected with p1058/Db

tetramers, as measured by flow cytometry (Fig. 3). Previous stud-ies in our laboratory had indicated that some agonist ligands bindT cells too weakly to be measured by tetramer staining (41). There-fore, we increased the valency of the soluble pMHC ligands usinga mAb to the fluorophore covalently linked to avidin on the tet-ramer, making octamers. Upon this modification, we saw bindingof both p1058/Db and p1049/A2 to the live AHIII 12.2 T cells (Fig.3). As seen in the SPR experiments, p1058/Db octamers boundmore poorly than did p1049/A2 octamers. Therefore, the aviditycontributed by the presence of CD8 does not greatly enhance bind-ing to p1058/Db-presenting cells and does not compensate for thedifferences in affinity between pMHC and TCR observed by SPR.

MHC/TCR binding does not correspond to proliferativeresponse

Although binding of p1058/Db tetramers on the T cell surfacecould not be detected using flow cytometry, AHIII 12.2 T cellslyse either p1049/A2 or p1058/Db-presenting cells with equalefficiency (34). We have previously shown that class I MHCtetramers may stimulate CTL without apparent requirements forcostimulation (49). Tetramers composed of p1049/A2 stimulated

Table I. SPR binding constants to AHIII 12.2 TCR

pMHC On-rate (M�1S�1) Off-rate (s�1) KD (�M)

p1049/A2 26,200 0.295 11.3p1058/Db 6,610 0.538 81.4

FIGURE 1. rAHIII 12.2 TCR is folded properly. A, Refolded AHIII12.2 TCR (2 �g/ml) were captured on anti-C� Ab (H57-597)-coatedELISA plates, followed by addition of p1049/A2 (f) or MLL/A2 (F)pMHC tetramers containing streptavidin-HRP. After addition of 3,3�,5,5�-tetramethylbenzidine substrate, OD405 was measured. B, A2-expressing T2cells were pulsed with 1 �M p1049 (f) or MLL (F) peptide. T2 cells werethen incubated with various concentrations of soluble TCR, washed, andincubated again with FITC-conjugated H57-597 (anti-TCR C�). Afterwashing again, cells were analyzed for median fluorescence intensity usinga FACScan flow cytometer.

FIGURE 2. AHIII 12.2 has a higher affinity for p1049/A2 thanp1058/Db in SPR experiments. H57-597 anti-C� TCR was attached to aBIAcore CM5 chip and used to capture refolded AHIII 12.2 TCR. Using aBIAcore 2000, various concentrations of (A) p1049/A2 and (B) p1058/Db

pMHC proteins were flowed over the surface of the TCR-coated chip, andthe refractive index change was measured. Response curves are shownvertically in the same order as the concentrations indicated to the right.Equilibrium binding, measured by the response of the BIAcore instrumentas a function of concentration, is shown in (C) p1049/A2 and (D) p1058/Db. Curves shown represent the average of at least three curves subtractedfrom a blank surface containing only Ab.

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proliferation of AHIII 12.2 T cells (Fig. 4). Surprisingly, p1058/Db

tetramers stimulated proliferation of AHIII 12.2 T cells equallywell as did p1049/A2 (Fig. 4). These results indicate that eventetramers that bind so poorly as to be undetected by flow cytometrymay produce an agonist response in functional assays. In addition,as seen with peptide-pulsed target cells, A2 and Db cause similarresponses to AHIII 12.2 when late effector responses are measured.

AHIII 12.2 is CD8-independent with respect to p1049/A2 andCD8-dependent with respect to p1058/Db

The CD8 coreceptor, expressed on CD8� T cells, has been shownto increase the avidity of the TCR/pMHC interaction by binding toMHC (24, 31). The AHIII 12.2 CD8� T cell line is derived froma mouse. Murine CD8 binding (from AHIII 12.2 T cells) to humanA2 should be greatly reduced compared with murine CD8 bindingto murine class I MHC (H-2Db) as the residues in murine Db thatinteract with CD8 are different from those in A2. It has been shownthat CD8 preferentially interacts with MHC from the same species(19, 21). Additionally, murine CD8�� does not bind to humanHLA-A2 when examined by SPR (50), and when tested in cell-cell

adhesion assays (51). To confirm that murine CD8 is not involvedin AHIII 12.2 T cell association with human pMHC, binding ofhuman and murine MHC octamers to AHIII 12.2 T cells was mea-sured in the presence and absence of anti-CD8 Abs. Two anti-CD8Abs, one anti-CD8� and one anti-CD8�, were used in these ex-periments. The anti-CD8� Ab has been shown to block associationof CD8 and class I MHC (53-6.8, blocking) (17). Conversely, theanti-CD8� Ab does not block association of CD8 and class I MHC(53-6.7, nonblocking). It does increase binding of multimericpMHC to T cells presumably due to an increase in the local con-centration of CD8 by virtue of the bivalent nature of the Ab (17).We have previously shown that the nonblocking anti-CD8� Abinterferes with proliferation and lysis even though it does not in-terfere with MHC binding to CD8 (49). As predicted, the non-blocking anti-CD8� Ab increases binding for p1058/Db octamersto AHIII 12.2 T cells (Fig. 5A). Nonblocking anti-CD8� Ab doesnot affect p1049/A2 octamer binding. The blocking anti-CD8� Ab,53-5.8, has been shown to impede binding of multimeric class IMHC to T cells (17). If AHIII 12.2 T cells are treated with block-ing anti-CD8� Ab, binding of p1058/Db octamers is completelyabolished. However, p1049/A2 octamer binding is only slightlydecreased in the presence of blocking anti-CD8� Ab. Similar re-sults were observed using either p1049/A2 tetramer or octamer at25 or 37°C (data not shown). These results imply xenoreactivep1049/A2 multimers do not require an association with CD8 tobind AHIII 12.2 T cells.

The role that CD8 plays in the lysis by AHIII 12.2 T cells uponpresentation of p1049/A2 and p1058/Db was examined with a stan-dard CTL assay using A2 or Db target cells pulsed with p1049 orp1058 peptides, respectively. Although the nonblocking anti-CD8� Ab, does not block binding of MHC to T cells (Fig. 5A), ithas been previously shown to inhibit CTL activity (49). The pres-ence of the nonblocking anti-CD8� Ab in a CTL assay withp1049-pulsed CHO-A2 target cells has a very small effect on lysis,while use of the Ab in an assay with EL-4 pulsed p1058 cellsresults in a dramatic reduction in lysis (Fig. 5B). These data con-firm the CD8 independence of xenoreactive p1049/A2 and CD8dependence of p1058/Db seen by multimer binding.

T cell proliferation was tested in the presence of the nonblock-ing anti-CD8� Ab, to determine whether the xenoreactive complexresponded differently from the syngeneic complex. AHIII 12.2 Tcells proliferate equally well when presented with p1049/A2 orp1058/Db tetramers (Fig. 4). However, when nonblocking anti-CD8� is added to AHIII 12.2 T cell culture in addition to pMHCtetramers, proliferation decreases for both p1049/A2 or p1058/Db

tetramers (Fig. 5C). The pattern of decrease is different for the twoligands. For p1049/A2, the effect is a “dampening” of the saturat-ing signal. Nonblocking anti-CD8� has two effects on proliferationin the presence of p1058/Db tetramers; it decreases the saturatingsignal and right-shifts the dose-response curve. The right-shift is aclassic demonstration of CD8 dependence of p1058/Db. The de-crease in the saturating signal in the presence of nonblocking anti-CD8� in both cases is not due to tetramer-induced apoptosis, asAbs against the early apoptosis marker protein, annexin V, do notbind to proliferating T cells. In addition, similar numbers of T cellsdivide in either p1049/A2- or p1058/Db-stimulated T cell culturesas measured by the decrease in CFSE fluorescence over time (datanot shown). Therefore, the decrease in saturating signal in the pres-ence of the nonblocking anti-CD8� Ab is likely due to a decreasein the rate of T cell divisions. These data show that the presence ofnonblocking anti-CD8� Abs do not cause a shift in the dose-re-sponse curve for p1049/A2. Hence, by this assay the xenoreactivecomplex is classically defined as CD8-independent.

FIGURE 3. Multimers of p1049/A2 and p1058/Db bind to AHIII 12.2 Tcells. AHIII 12.2 T cells (2 � 105) were incubated with p1049/A2 tetram-ers (F), p1049/A2 octamers (E), p1058/Db tetramers (�), or p1058/Db

octamers (f) at the indicated concentrations at 4°C for 30 min. After wash-ing, cells were analyzed using a FACScan flow cytometer and medianfluorescence was measured. Median fluorescence intensity was normalizedbetween experiments to the median fluorescence of p1049/A2 octamers atthe highest concentration, after subtraction of the median fluorescence fromirrelevant tetramer.

FIGURE 4. p1058/Db tetramers induce proliferation of AHIII 12.2 Tcells, despite absence of binding as detected by flow cytometry. Tetramerscomposed of p1049/A2 (F), p1058/Db (f), or irrelevant tetramersMLL/A2 (Œ), or FLU/Db (�) were incubated at the indicated concentra-tions with 3 � 105 IL-2-starved AHIII 12.2 T cells. After 24–48 h [3H]thy-midine was added and the cultures were reincubated for an additional 6–12h. [3H]Thymidine incorporation was determined by scintillation counting.Data shown is from one experiment performed in triplicate and is repre-sentative of three separate experiments.

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In summary, anti-CD8 Abs reduce binding, proliferation, andlysis of AHIII 12.2 T cells when presented with syngeneic p1058/Db. These Abs do not impair the response of AHIII 12.2 T cellswhen presented with the xenoreactive p1049/A2. Interestingly,close examination of the effect of the anti-CD8 Abs shows thatthere is a small but reproducible reduction in binding, lysis, andrate of proliferation for the CD8-independent p1049/A2. Thesedata suggest that even though there is no direct binding of the CD8to the class I MHC, CD8 is nearby and involved in signaling in thecell for the xenoreactive ligand.

CD8-independent, xenoreactive responses generate the sameearly signals as CD8-dependent responses

The above experiments examine proliferation and cytolysis whichare late events in T cell activation. The data show that late T cellevents generated by p1049/A2 interactions are similar to thosegenerated by p1058/Db multimers even though p1049/A2 does notbind CD8. These data imply that the early responses do not requireCD8 binding to class I MHC either. Previous experiments havedemonstrated that T cells lacking CD8 expression cannot producea calcium flux, even if the CD8� T cells can still bind pMHCmultimers (17). To determine the effects of anti-CD8 Abs on cal-cium flux by CD8-dependent and CD8-independent pMHC mul-timers, AHIII 12.2 T cells were loaded with the calcium-specificfluorophore, Indo-1AM. Fluorescence from Ca2� binding to Indo-

1AM was measured in real time, immediately after addition ofp1049/A2, p1058/Db, or irrelevant tetramer. Both p1049/A2 andp1058/Db tetramers produced a sustained calcium flux, while theirrelevant tetramer produced no flux (Fig. 6). Calcium mobilizationinduced by the p1058/Db tetramer was delayed and decayed fasterthan did the response to p1049/A2. This difference is likely due tothe poor binding of p1058/Db tetramer (Fig. 3), as a greater pro-portion of AHIII 12.2 T cells responded, and the kinetics of thecalcium flux was faster and slightly more sustained when higherconcentrations of p1058/Db tetramer were used (data not shown).Also, a transient calcium response to weak pMHC ligands, such asseen in this study for p1058/Db, has been demonstrated for someclass II MHC ligands (52). Prior incubation of AHIII 12.2 T cellswith nonblocking anti-CD8� Ab increased the calcium responsefor both p1049/A2 and p1058/Db (data not shown) most likely due tothe bivalence of the Ab as described earlier. As shown for multimerpMHC binding, incubation with blocking anti-CD8� Ab does noteliminate calcium flux after addition of p1049/A2 tetramer, but theanti-CD8� Ab abolished Ca2� mobilization by p1058/Db.

CD8 escorts p56lck to the vicinity of the CD3 ITAMs to initiateT cell activation signals (15, 53). All of the data presented aboveseem to require that p56lck is activated without CD8 binding toclass I MHC. To directly examine the role of CD8 in p56lck sig-naling, the kinase activity of immunoprecipitated p56lck was mea-sured following stimulation of AHIII 12.2 T cells with either

FIGURE 5. AHIII 12.2 is CD8-independent when recognizing xenogeneic p1049/A2, but CD8-dependent when recognizing syngeneic p1058/Db. A, Absto CD8 �-chain abolish binding of p1058/Db octamers, but not p1049/A2 octamers, to the AHIII 12.2 T cell surface. AHIII 12.2 T cells (2 � 105) wereincubated with saturating concentrations of octamers for 30 min at 4°C, followed by the addition of wash buffer, “nonblocking” anti-CD8� FITC, or“blocking” anti-CD8� FITC for 30 min at 4°C. After washing, the T cells were analyzed on a FACScan flow cytometer for median fluorescence. Resultswere normalized to median fluorescence intensity of octamer alone for p1049/A2 and p1058/Db, separately. B, Anti-CD8� reduces the amount of CTL lysisfor p1058-pulsed Db-expressing target cells, but not p1049-pulsed A2-expressing target cells. CHO-A2 (E, F) or EL-4 (�, f) cells were pulsed with p1049or p1058 peptide, respectively, at the indicated concentrations, and mixed with AHIII 12.2 T cells in the presence (open symbols), or absence (closedsymbols), of anti-CD8� Ab. MHC restriction (lack of hamster MHC recognition) is shown by absence of lysis of untransfected CHO cells, and shown tobe peptide-specific by the lack of recognition of irrelevant peptide (data not shown). C, Inclusion of anti-CD8� results in altered in [3H]thymidineincorporation for both p1049/A2- and p1058/Db-stimulated AHIII 12.2 T cells. Proliferation was performed as detailed in Fig. 4, except anti-CD8� Ab wasadded to AHIII 12.2 T cells before incubation with tetramers. Symbols are as in B except for the addition of irrelevant tetramers MLL/A2 (Œ), and FLU/Db

(�). T cells prepulsed with isotype control Ab gave essentially the same results as T cells without Ab (data not shown). Data shown is from one experimentperformed in triplicate and is representative of three separate experiments.

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p1049/A2 or p1058/Db tetramer. p56lck is significantly activated( p � 0.015) when stimulated with p1049/A2 tetramer as comparedwith the irrelevant MLL/A2-tetramer control (Fig. 7). The activityof p56lck is greater in p1049/A2 tetramer-stimulated T cells than inp1058/Db-stimulated T cells. p56lck activity in p1058/Db-stimu-lated cells is not significantly greater than the MLL/A2-tetramerbackground ( p � 0.304). The inability of p1058/Db tetramers tosignificantly activate p56lck may reflect poor TCR:pMHC binding,as weak agonists require longer pMHC:TCR contact times, andhigher concentrations for stimulation of signaling activity (54).Because p1058/Db tetramers cause a calcium flux and proliferationof these T cells, these data imply that either the in vitro kinaseactivity assay is not sensitive enough to show functional differ-ences between p1058/Db tetramer and irrelevant tetramer treat-ment or there is some unknown integrating signal that allows fordownstream events that is separate from p56lck kinase activity.

When AHIII 12.2 T cells are treated with nonblocking anti-CD8� in combination with tetramers, a significant increase inp56lck kinase activity is seen for both p1049/A2 ( p � 0.003) andp1058/Db vs irrelevant tetramer ( p � 0.005). However, whenAHIII 12.2 T cells are treated with blocking anti-CD8� in com-bination with tetramers, lck kinase activity of p1058/Db-stimulatedAHIII 12.2 T cells decreases compared with stimulation with1058/Db tetramer alone or in combination with nonblocking anti-CD8�. Once again, blocking anti-CD8� has little effect on AHIII12.2 T cell stimulation by p1049/A2 tetramer compared with tetrameralone. These data once again demonstrate the CD8 independence ofthe xenoreactive pMHC, and indicate that binding of MHC with CD8is not necessary for signal transduction during T cell activation byp1049/A2. Yet, as seen previously, CD8 engagement by syngeneicp1058/Db is critical for efficient T cell activation.

TCR and CD8 colocalize in the absence of direct pMHC:CD8 binding

AHIII 12.2 T cells proliferate, kill, and signal after presentationwith a CD8-independent ligand, p1049/A2. The kinase that ini-tiates the signal, p56lck, is activated in p1049/A2-tetramer stimu-lated T cells. These data suggest that CD8 is recruited to the im-munological synapse in the absence of binding to class I MHC.Confocal microscopy shows that both p1049/A2 and p1058/Db

tetramers colocalize in discrete patches with the anti-CD8� Ab,

but not in the irrelevant tetramer control (Fig. 8, A–C). Further-more, p1049/A2 tetramers, but not p1058/Db tetramers, colocal-ized with the blocking anti-CD8� Ab (data not shown). In addi-tion, no colocalization was apparent when staining with tetramerand anti-CD90 (Fig. 8D). Colocalization is confirmed with FRETbetween fluorophores conjugated to octameric class I MHC andanti-CD8 Ab (Fig. 9A). Although this particular use of FRET can-not be used to calculate exact distances between the fluorophoresreliably, after corrections for the amount of octamer binding to theT cells, the fluorescence due to p1058/Db octamer is slightly largerthan for p1049/A2. This suggests that the distance between thefluorophores is smaller for p1058/Db than p1049/A2 as expected ifCD8 is binding to Db but not to A2 on AHIII 12.2 T cells (Fig. 9B).

DiscussionThe murine AHIII 12.2 T cell recognizes both murine H-2Db andhuman HLA-A2. The mechanism that the T cell uses to identifythese structurally diverse ligands is not understood. AHIII 12.2 Tcells efficiently kill either A2- or Db-presenting cells when pulsedwith p1049 or p1058 peptides, respectively. Based on the differ-ences in the murine and human class I MHC in the CD8-binding

FIGURE 6. AHIII 12.2 T cells produce calcium mobilization in responseto both p1049/A2 and p1058/Db tetramers. IL-2-starved AHIII 12.2 T cellswere loaded with Indo-1-AM, and background fluorescence was examined ona MoFlo cell sorter for 30 s to determine the baseline (from time 0 to the breakin the curve). The T cells were stimulated by the addition of 10 �g ofp1049/A2 (gray lines) or p1058/Db (black lines). As calcium increases in thecytoplasm, it binds to the Indo-1-AM and an increase in fluorescence is ob-served. Experiments where anti-CD8 Ab was added to T cells before stimu-lation are denoted as thin lines for p1049/A2 (thin gray curve) and p1058/Db

(thin black curve). The dashed line indicates calcium flux with the irrelevantMLL/A2 tetramer, and has been slightly offset from the other curves for clar-ity. Calcium concentrations on the y-axis were determined from a standardcurve with known calcium concentrations as in Ref. 66, and all curves indicatethe median of the responding cell population.

FIGURE 7. CD8-independent, xenoreactive pMHC stimulates greaterp56lck activity than does CD8-dependent pMHC. IL-2-starved AHIII 12.2T cells (4 � 106) were stimulated for 3 min with the indicated tetramersand Abs; the cells were lysed with detergent. Incorporation of [�-32P]ATPtransfer to ITAMc peptide from CD3� (46) was tested using immunoprecipi-tated p56lck. The increase in p56lck activity after p1049/A2 stimulation is sig-nificant (p � 0.015) compared with MLL/A2, while the response of p1058/Db

is not significantly above the response to irrelevant tetramer (MLL/A2).

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region (51), we expected that the xenoreactive response would beindependent of CD8. We decided to confirm the CD8 indepen-dence of the xenoreactive response using functional and biophys-ical assays. Interestingly, our results confirm that the xenoreactiveresponse is CD8-independent as classically defined, but the re-sponse uses CD8-associated signals.

Using recombinant proteins and SPR, we show that the bindingaffinity of p1049/A2 is about eight times greater than p1058/Db forAHIII 12.2 TCR. The difference in affinity is primarily due to amuch slower on-rate of the murine complex (Table I). Biologi-cally, the apparent difference in affinity may be illusory becausep1058/Db binds to CD8 in addition to the TCR when the T cellbinds to the target cell. If the binding of CD8 compensated for the

lack of affinity of the p1058/Db complex for the AHIII 12.2 TCRcompared with p1049/A2, tetramers composed of the two com-plexes should bind equally well to the T cells, but p1058/Db tet-ramers do not bind by flow cytometry and yet p1049/A2 tetramersbind well. Surprisingly, even though the affinities are significantlydifferent, p1058/Db and p1049/A2 have similar dissociation ratesfrom AHIII 12.2 TCR. Because the T cell recognizes cells pre-senting p1058/Db as well as they recognize cells presenting p1049/A2, these data appear to support the idea that the dissociation rateof the complex controls T cell activation.

Most of the allo- and xenogeneic TCR examined have beenclassified as CD8-independent (16–21) and all CD8-independentTCR studied bind their ligands with abnormally high affinity (20,

FIGURE 8. CD8 and TCR associate even in the absence of pMHC:TCR binding. AHIII 12.2 T cells were stained on glass sides with anti-CD8� FITC(green) and Alexa 568-conjugated tetramers (red) or allophycocyanin conjugated anti-CD90 (red). Left column contains tetramer or anti-CD90 fluorescencealone, middle column contains anti-CD8� fluorescence alone, and colocalization is yellow in images in the right column. A, p1049/A2 tetramers; B,p1058/Db tetramers; C, MLL/A2 tetramers; D, anti-CD90.

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55). The 2C TCR allo-response to p2Ca/Ld is CD8-independent(17, 20) with a KD around 3 �M. However, 2C has an �10-foldlower affinity and is CD8-dependent when presented with synge-neic SIYR/Kb (56). BM3.3 is CD8-independent during recognitionof pBM1/Kb (57) with an affinity of �2.5 �M (55). Affinity con-stants of syngeneic pMHC:TCR interactions are usually in therange of 20–100 �M (56–59). The HLA-A2-restricted TCR,Jm22z, has a KD of �20 �M for its syngeneic pMHC ligand andcould not function independently of CD8 (50). Taken together withour results, these data imply that above a certain threshold affinity,a direct interaction between CD8:pMHC is no longer necessary.As most CD8-independent, high affinity pMHC ligands are allo- orxenoreactive, it is likely that these cross-reactive TCR would havebeen eliminated by negative selection if they had undergone T celldevelopment upon their allo- or xenoreactive MHC. Thus, theremust be selective pressure during T cell development for low af-finity, CD8-dependent syngeneic ligands (60) to reduce the poten-tial for autoreactive T cells. Abs also contact their ligands withhigh affinity, and stimulation of T cells with TCR- or CD3-specificAbs initiates T cell activation (61, 62). Thus, our findings agreewell with fluorescence microscopy studies that show cocapping ofCD8 and TCR with anti-CD8� and anti-TCR Abs (63), and im-munoprecipitation experiments that demonstrate an association be-tween CD8 and TCR/CD3 after anti-CD3 stimulation (53).

Two Abs were used to explore the CD8 dependence of murineAHIII 12.2 when presented with p1058/Db and p1049/A2. Multi-mer binding, cytolysis, proliferation, calcium mobilization, and lckactivity were examined in the presence and absence of these Abs.The anti-CD8� Ab (53-5.8) has been shown to block binding ofCD8 to MHC (17). This Ab slightly reduced binding of p1049/A2

multimers to AHIII 12.2 T cells and decreased calcium flux sim-ilarly. However, the anti-CD8� Ab completely abrogates binding,abolished calcium flux, and greatly reduced lck activity when stim-ulated with p1058/Db. Conversely, the anti-CD8� Ab (53-5.7)does not block binding of CD8 to MHC, but it has been shown toblock proliferation and lysis (49). Presumably, this blockage has todo with disruption of an optimal complex of proteins at the im-munological synapse. Treatment with the anti-CD8� Ab improvesbinding of p1058/Db, presumably because of an aggregation ofCD8 receptors by the bivalent Ab. Because murine CD8 does notbind to A2, we would not expect an increase in binding ofp1049/A2 and that is indeed the case. In the presence of anti-CD8�, we see decreased AHIII 12.2, proliferation, but increasedcalcium flux (data not shown) and lck activity when presented withp1058/Db and p1049/A2. The only difference we see is with re-spect to cytolysis. The anti-CD8� Ab had no effect on CTL rec-ognition of p1049/A2, but significantly decreased recognition ofp1058/Db. These data confirm that AHIII 12.2 is CD8-independentby the classical definition when presented with p1049/A2 andCD8-dependent when presented with p1058/Db.

CD8 mobilizes to the area of TCR capping during T cell acti-vation (62). These areas may be called foci when treating withtetramers, but on cells presented with competent APCs they wouldbe fully functional immunological synapses. The mechanism thatCD8 uses to migrate to the synapse is not clear. CD8� contains apalmitylation target sequence such that CD8 is likely partitionedinto lipid microdomains (11, 63). During recognition of CD8-de-pendent ligands, CD8 could be recruited by either actively bindingto class I MHC or CD8 may be recruited by mass action, literallyby being associated with the other proteins because of its locationin the lipid microdomain. Because CD8 is recruited to the fociwithout binding to MHC, our data suggest that CD8 is initiallyrecruited nonspecifically by virtue of its location in a lipid mi-crodomain. In the CD8-dependent case such as p1058/Db, CD8 isrecruited to and remains in the synapse by virtue of associationwith pMHC, and also probably because of continued recruitmentsignals. This would suggest that CD8 recruitment is dependent onthe strength of the signals that stimulate aggregations of lipid mi-crodomains. As CD8 recruitment also seems dependent on thestrength of the binding between pMHC and TCR (data presented inthis study and S. E. Kerry and J. A. Frelinger, unpublished obser-vations), this would suggest that the lipid domain aggregationmechanisms are dependent on the strength of the interaction. Weare testing this hypothesis using a set of CD8-independent pMHC:TCR partners of varying affinities.

Coligation of CD8 and TCR to pMHC leads to p56lck activation(14, 15) as one of the initial signaling events in T cell activation.Calcium flux does not occur when CD8 is absent from the T cellsurface (15, 17). This requirement may be seen using anti-CD3Abs. T cell activation upon anti-CD3 stimulation is dependent onCD8/p56lck association, as mutation of the p56lck-binding site inthe CD8 �-chain results in a loss of calcium flux and protein ty-rosine phosphorylation (64). In this study, we show that xenoge-neic pMHC elicits p56lck kinase activity typical of T cell activa-tion, in the absence of binding between pMHC and CD8. Inaddition, we show that there is not necessarily a correspondencebetween lck activity and calcium mobilization. Treatment of AHIII12.2 T cells with p1058/Db tetramer results in a well-defined cal-cium mobilization even if it is slower and weaker than the responseto p1049/A2. However, lck activity generated by p1058/Db is un-distinguishable from the activity engendered by irrelevant tetramer.Because the time frames of the experiments are comparable, thereappear to be two possible explanations. Either the reproducibility ofthe lck kinase assay is such that we cannot distinguish between an

FIGURE 9. FRET between CD8 and CD8-independent pMHC com-plexes show that CD8 is recruited to the TCR location in the absence ofMHC binding. A, Octamers composed of p1049/A2 (striped histogram) orp1058/Db (open histogram) produce FRET (as measured by the appearanceof FL3 fluorescence after stimulation of PE at (488 nm) to cells incubatedwith both APC-conjugated anti-CD8� Abs and pMHC conjugated to PE).Irrelevant tetramers, MLL/A2 (gray histogram) do not induce FRET. TheFRET produced by AHIII 12.2 T cells alone, T cells with octamer only,anti-CD8� Ab only, and anti-CD8� plus anti-CD3, were similar to FRETproduced by irrelevant MLL/A2 (data not shown). B, FRET for p1049/A2,p1058/Db, and MLL/A2 normalized for levels of tetramer binding as de-scribed in Materials and Methods.

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active signal and background or there is another signal that connectsTCR:MHC complex and calcium mobilization.

Our visual, biophysical and functional data all suggest that theCD8 coreceptor may be recruited in the absence of binding toMHC. The question becomes: what controls CD8 recruitment tothe synapse? We suggest that it may be aggregation of lipid mi-crodomains, because the instance in which we observe recruitmentwithout binding is with a high affinity interaction between TCRand pMHC. In the instance where we observe low affinity, thesignal to aggregate microdomains is not maintained and CD8 bind-ing to MHC is required to keep CD8 in the foci. Therefore, wepropose that high affinity TCR:pMHC binding obviates the re-quirement for CD8:pMHC binding to keep CD8 in the foci. Thisidea is supported by work with mutant TCR that have a higheraffinity for pMHC (65). The rationale for the existence of the twomechanisms, CD8-dependent and CD8-independent, is not yetclear. Additionally, as the data presented in this study suggest thathigh affinity TCR:pMHC engagement occurs predominantly in thecase of allo- or xeno-reactive ligands, it is possible that this high-affinity, CD8-independent activation mechanism presents differentsignals that may be used as a means to identify MHC ligands destinedfor removal by negative selection during T cell development.

AcknowledgmentsWe gratefully acknowledge the excellent technical assistance ofCarrie Barnes, Dr. Larry Arnold, and the Flow Cytometry Facility(Department of Microbiology and Immunology, University ofNorth Carolina) for assistance in the calcium flux measurements,and helpful discussions with the members of the Frelinger andCollins laboratories.

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2001. Co-stimulation and counter-stimulation: lipid raft clustering controls TCRsignaling and functional outcomes. Semin. Immunol. 13:115.

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