Of T Cells /CD8 Interaction on the Surface Ε TCR/CD3 Monomeric Class I Molecules Mediate

Monomeric Class I Molecules Mediate TCR/CD3 ε/CD8 Interaction on the Surface of T Cells

This information is current as Matthew S. Block, Aaron J. Johnson, Yanice of September 25, 2021. Mendez-Fernandez and Larry R. Pease J Immunol 2001; 167:821-826; ; doi: 10.4049/jimmunol.167.2.821 http://www.jimmunol.org/content/167/2/821 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Monomeric Class I Molecules Mediate TCR/CD3⑀/CD8 Interaction on the Surface of T Cells

Matthew S. Block, Aaron J. Johnson, Yanice Mendez-Fernandez, and Larry R. Pease1

Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using ﬂuorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3⑀ is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular

activation. The Journal of Immunology, 2001, 167: 821–826. Downloaded from

he CD8 molecule plays a critical role in positive selection both studies the binding of soluble class I-CD8 complexes to sur- and activation of T cells that recognize peptide Ags pre- face-bound TCR could be inferred from the observed changes in T sented in the context of MHC class I molecules (1). It has plasmon resonance, indicating that it is sterically possible for class been known for several years that CD8 binds directly to class I, I molecules to be bound simultaneously by TCR and CD8. and various mechanisms have been proposed to explain the aug- Although binding of both CD8 and TCR to the same class I http://www.jimmunol.org/ mentation of T cell responses to Ag by CD8. One early hypothesis molecule has not been directly demonstrated on the surface of was that CD8, along with its class II-recognizing counterpart CD4, living T cells, several indirect methods have suggested that mo- serves primarily as an accessory molecule, enhancing T cell-APC nomeric class I binding and triggering of T cells requires the pres- interactions by binding to MHC molecules on the surface of APC ence of CD8. Using photoafﬁnity labeling, Luescher et al. (7) dem- without discriminating whether the MHC molecules are those rec- onstrated that labeling of a class I-restricted CTL clone with a ognized by the TCR (2). It is now known that augmentation of T soluble class I monomer could be inhibited by mAbs to either CD8 cell activation by CD8 requires that CD8 be able to bind to the or the CD8-binding ␣3 domain of class I. Additionally, Delon et al. same species of class I molecule that is recognized by the TCR, (8) showed that Ca2ϩ signaling induced by soluble class I mono-

suggesting that CD8 acts as a coreceptor rather than as an acces- mers was dependent on intact interactions between CD8 and class by guest on September 25, 2021 sory molecule (3, 4). An attractive hypothesis is that TCR and CD8 I. Both of these findings support the hypothesis that CD8 and the simultaneously bind the same class I molecule on the surface of TCR bind simultaneously to class I molecules. the APC or target cell, providing a mechanism for associating the Several groups have demonstrated cell surface interactions be- CD8-linked kinase Lck with target sites on CD3 components of tween members of the TCR/CD3 complex and the coreceptors the TCR complex. However, simultaneous binding of class I CD4 and CD8 on murine T cells and clones. Immunoprecipitation molecules by TCR and CD8 has not been demonstrated directly on studies have revealed that the majority of CD3 ␦-chains and a the surface of cells. Here we report direct evidence that class I minority of TCR␤-chains and CD3␥-, ⑀-, and ␨-chains associate molecules mediate TCR/CD8 association on the surface of T cells. with CD4 or CD8 on resting T cells (9, 10). However, there are Furthermore, we show that ligation of monomeric class I mole- conflicting reports regarding which interactions between the core- cules by T cells causes enhanced association of CD3⑀ and CD8. ceptors and TCR/CD3 are affected during T cell activation. Osono Simultaneous binding of soluble CD8 and TCR to the same et al. (11) reported that upon activation with anti-TCR␤ Abs, in- MHC molecule in vitro has been shown by surface plasmon res- teractions between CD3␥-, ␦-, and ⑀-chains and coreceptor mole- onance studies. One group reported an enhanced affinity of the cules remain unchanged, whereas CD3␨-CD4/8 association is aug- TCR for soluble class I molecules upon binding of the class I mented during activation. In contrast to this, Anel et al. (12) molecules to soluble CD8 (5). While this reported change in bind- showed enhanced CD3⑀-CD8 association upon Con A activation. ing affinity might be taken as evidence of simultaneous binding of Of note, Thome et al. (13) showed enhanced TCR-CD8 association CD8 and the TCR to the same class I molecule, a second report upon activation in a human T cell line. This association is depen- argued that no such affinity changes could be seen under similar dent on the activity of the tyrosine kinase Lck. conditions (6). However, pertinent to the present discussion, in Fluorescence resonance energy transfer (FRET)2 is a property of fluorochromes readily adaptable to the study of proximity between molecules. When certain fluorochromes are brought into close Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, Ͻ Rochester, MN 55905 proximity ( 80 Å), they interact such that a fluorochrome that has Received for publication November 27, 2000. Accepted for publication May 7, 2001. been excited (the donor) can transfer energy to a second fluorochrome (the acceptor), causing it to fluoresce (14). Detection of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Larry R. Pease, Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, 200 First Street 2 Abbreviations used in this paper: FRET, fluorescence resonance energy transfer; SW, Rochester, MN 55905. E-mail address: [email protected] LN, lymph node.

enhanced acceptor fluorescence when only the donor has been di- Table I. Comparison of compensated vs uncompensated flow cytometry rectly excited indicates that the donor and acceptor fluorochromes readings are very close to one another. Although several groups have stud- ied molecular interactions on cell surfaces using FRET (15–18), Uncompensated Compensated Signal Staining Regimen MFIa MFI here we employ novel combinations of commonly available fluorescent markers to assess interactions between T cell surface FL2 Kb/SIYR 810 821 proteins. Kb/SIYR and anti-CD8 APC 735 752 FL3 Kb/SIYR 188 4 Anti-CD8-APC 15 5 b Materials and Methods K /SIYR and anti-CD8 APC 313 115 FL4 Anti-CD8-APC 902 920 Mice Kb/SIYR and anti-CD8 APC 877 884

2C TCR␣␤ transgenic mice were originally described by D. Loh (19) and a FL, Fluorescence; MFI, mean fluorescence intensity. have been maintained at the Mayo Clinic (Rochester, MN). OT-1 TCR␣␤ transgenic mice (C57BL/6-TgN(TcrOva)1100 Mjb) were obtained from The Jackson Laboratory (Bar Harbor, ME). All experiments were performed in compliance with institutional and National Institutes of Health Results guidelines for animal care and use. Allophycocyanin is an efficient FRET acceptor in combination with FITC or PE as FRET donors Monomers and tetramers Allophycocyanin is a widely available phycobiliprotein that is Downloaded from Kb/SIYR and Kb/OVA monomers and tetramers were prepared as previ- maximally excited by light of 615–655 nm (but not 488 nm), and ously described (20). The expression vector for the production of Kb tet- emits most efficiently at wavelengths above 650 nm (Fig. 1) (24). ramers and monomers was generated by site-directed mutagenesis of Kb FITC and PE both absorb light efficiently at 488 nm, and fluoresce cDNA and cloned into pET23 (Novagen, Madison, WI) for expression of at wavelengths that overlap the excitation range for allophycocya- protein in Escherichia coli. The human ␤ -microglobulin construct was 2 nin. The large size of phycobiliproteins and the existence of mul- described previously (21). SIYR (SIYRYYGL) and OVA (SIINFEKL) peptides were generated at the Mayo Protein Core Facility. tiple fluorescent moieties within each molecule make PE and allophycocyanin unsuitable for precise measurements of distance http://www.jimmunol.org/ PP1 treatment using FRET. However, quantitation of distances between fluoro- phores is not necessary to effectively demonstrate that cell surface Cells were incubated in 20 ␮M PP1 (22) (Biomol, Plymouth Meeting, PA) molecules are or are not associating with one another. Thus, allo- ␮ at 37°C for 30 min and stained on ice in the presence of 20 M PP1. phycocyanin paired with either FITC or PE is an ideal set of re- Flow cytometry agents to study cell surface interactions qualitatively by FRET. Using a standard two-laser flow cytometry system, allophycocya- The mAbs anti-CD8-allophycocyanin (53.6.7), anti-CD4-allophyco- nin fluorescence can be assessed both when directly excited cyanin (RM4-5), anti-V␣2-PE (B20.1), and anti-CD3⑀-PE (145-2C11) (aligned with the 635-nm laser) and when indirectly excited by were obtained from BD PharMingen (San Diego, CA). CT-CD8a by guest on September 25, 2021 (anti-CD8) was purchased from Caltag (Burlingame, CA). The anti-2C FRET (aligned with the 488-nm laser). Allophycocyanin fluo- clonotypic mAb 1B2 (23) was conjugated to PE using a Phycolink kit resces in a range similar to that of the dyes PerCP and Red613; from Prozyme (San Leandro, CA). Unconjugated polyclonal anti-IgG thus, allophycocyanin fluorescence due to FRET can be measured was obtained from ICN Biomedicals (Costa Mesa, CA). FITC- using standard detectors normally assigned to PerCP or Red613 conjugated anti-IgG was purchased from Accurate Chemical and Sci- entific (Westbury, NY). PE-conjugated anti-IgG was obtained from Serotec (Raleigh, NC). Lymph node (LN) cells and thymocytes were isolated, and approximately 2 ϫ 106 cells/sample were used. Cells were incubated with 20 ␮g/ml Ab or tetramer or with 200 ␮g/ml monomer for 20 min on ice. Reagents were diluted in HBSS containing 10 g/l BSA and 0.2 g/l sodium azide. After incubation with Abs or tetramers, cells were washed three times in HBSS/BSA/azide. Cells incubated with monomers were fixed immediately after incubation. Paraformal- dehyde was added directly to the monomer-cell mixture. Cells were fixed in 2% paraformaldehyde. FACS analyses were performed by the Mayo Flow Cytometry Core Facility on a FACSCaliber (BD Bio-

sciences, Franklin Lakes, NJ), and data collected as log10 fluorescence were analyzed using CellQuest (BD Biosciences). FL3 (FRET) signals were compensated by subtracting 28.8% of FL2 signal strength to correct for bleed-over of signals from PE into FL3. An example of the uncompensated and compensated mean fluorescence intensities detected in FL2, FL3, and FL4 is shown for CD8ϩ cells stained with Kb/SIYR-PE, anti-CD8-allophycocyanin, or both Kb/SIYR-PE and anti- CD8-allophycocyanin (Table I). Comparable FRET increases (gains in the FL3 channel) were detected using compensated (115–4 ϭ 111 arbitrary mean fluorescence intensity units) or uncompensated (313– FIGURE 1. Excitation and Emission Spectra for FITC, PE, and allo- 188 ϭ 124 arbitrary mean fluorescence intensity units) conditions. phycocyanin. a, Overlay of excitation and emission spectra of FITC (solid line, excitation; dashed line, emission) and allophycocyanin (gray line, Analytical gel filtration excitation; dotted line, emission). Arrow, excitation at 488 nm. Wave- lengths greater than 670 nm (detected by FL3 on FACSCalibur (BD Bio- Size exclusion gel filtration analysis was performed by the Mayo Protein sciences)) are shaded gray. FITC, but not allophycocyanin, is excited di- Core Facility on a Superdex 200 10/30 column (Amersham Pharmacia Biotech, Piscataway, NJ) using a buffer flow rate of 0.5 ml/min. PBS was rectly at 488 nm, whereas allophycocyanin, but not FITC, fluoresces at used as the buffer, and samples were run at room temperature. Samples of wavelengths detected by FL3. b, Overlay of spectra for PE (excitation is Kb/SIYR tetramer and monomer were diluted to 200 ␮g/ml in PBS, and shown by a line, emission is shown by a dashed line) and allophycocyanin 100 ␮l of each sample was injected into the column. The relative protein (gray line, excitation; dotted line, emission). Only PE is excited at 488 nm, concentration was determined by measuring absorbance at 280 nm. and only allophycocyanin fluoresces in the FL3 range. The Journal of Immunology 823

(670 long-pass filter; Fig. 1, shaded regions). Importantly, neither FITC nor PE fluoresces appreciably at wavelengths Ͼ670 nm (Fig. 1), and any bleed-over present can be controlled by compensation. To demonstrate that FRET occurs between FITC-allophycocyanin and PE-allophycocyanin donor-acceptor fluorochrome pairs, we labeled cells with an allophycocyanin-conjugated Ab, then used anti-IgG Abs conjugated to donor fluorochromes to bring together donor and acceptor (allophycocyanin) fluorochromes. We stained OT-1 LN cells with allophycocyanin-conjugated 53.6.7 mAb, which recognizes CD8, followed by Abs against mouse IgG, either unconjugated or conjugated to FITC or PE. The 53.6.7 rat mAb was bound by all three anti-mouse-IgG Abs. Fluorescence at 670ϩ nm upon excitation at 488 nm (FL3) was found only when FITC- or PE-conjugated anti-IgG was used (Fig. 2). Furthermore, when fluorochromes were conjugated to reagents that were not FIGURE 3. MHC class I-peptide tetramers recruit CD8 to the TCR. a and b, Samples of 2C LN cells were stained with Kb/SIYR-PE tetramer expected to interact, such as CD4 and class I-restricted TCRs, no b FRET was observed despite the presence of both fluorochromes on (light gray), K /OVA-PE tetramer (dark gray), or 1B2-PE (dotted line), each followed by staining with 53.6.7-allophycocyanin (anti-CD8). An ad- the same cell (Fig. 4, b and d, dotted and bold lines). ditional sample of 2C cells was incubated with both anti-CD8 reagents, b Downloaded from MHC class I-peptide tetramers recruit CD8 to the TCR CT-CD8a and 53.6.7-allophycocyanin, then stained with K /SIYR-PE tetramer (thin line). All cells were analyzed for staining with tetramer-PE or T cells that react against a particular peptide-MHC complex can be 1B2-PE (a) and for FRET (b). 2C cells stained efficiently with Kb/SIYR effectively identified by their ability to bind to tetramers of soluble and 1B2 (a), but only exhibited FRET when bound by Kb/SIYR (b). Fur- MHC complexed with the reactive peptide (25). To demonstrate thermore, when interactions between CD8 and class I molecules were that class I tetramers bind CD8 and recruit it to the TCR, we blocked with CT-CD8a, FRET did not occur (b) despite the presence of Kb/SIYR-PE on the cells (a). c and d, Samples of OT-1 LN cells were http://www.jimmunol.org/ stained LN cells from 2C and OT-1 TCR-transgenic mice (specific b for Kb/SIYR and Kb/OVA, respectively) with 53.6.7-allophyco- stained with 53.6.7-allophycocyanin along with K /SIYR-PE tetramer (light gray), Kb/OVA-PE tetramer (dark gray), anti-V␣2 (B20.1) conju- cyanin (a CD8-specific mAb) and PE-conjugated tetramers. With b ϩ gated to PE (dashed line), or CT-CD8a and K /OVA-PE tetramer (bold both 2C and OT-1 CD8 cells, incubation with the relevant tet- line). Again, cells were analyzed for both staining with tetramer-PE or ramer stained the cells with PE and brought CD8 into close prox- anti-V␣2-PE (c) and FRET (d). OT-1 cells stained efficiently with Kb/OVA imity with the TCR, as evidenced by allophycocyanin fluorescence and anti-V␣2, but CT-CD8a abrogated binding of Kb/OVA (c). FRET upon excitation at 488 nm (Fig. 3, shaded histograms). Tetramers occurred when cells were stained with Kb/OVA, but not anti-V␣2(d). containing an irrelevant peptide did not label the CD8ϩ cells or Because CT-CD8a blocked Kb/OVA binding, no FRET activity was ϩ induce FRET, demonstrating that binding to the TCR is an essen- observed, as expected (d). Events were gated for CD8 live cells. tial step in the recruitment of CD8. To show that recruitment of by guest on September 25, 2021 CD8 to the TCR is dependent on the presence of MHC molecules, we stained LN cells with 1B2 or anti-V␣2, mAbs that recognize can be efficiently induced by artificial cross-linking with poly- the 2C and OT-1 TCRs, respectively (23, 26). PE-conjugated 1B2 clonal anti-IgG (data not shown). Therefore, the substantial levels and anti-V␣2 bound effectively to their respective TCRs (Fig. 3, a of FRET that occur upon tetramer binding are indicative of tet- and c), but were unable to recruit CD8 and produced minimal ramer-dependent association of CD8 and the TCR. FRET (Fig. 3, b and d). The augmentation of the FRET signals The anti-CD8 mAb 53.6.7 enhances the ability of MHC tetram- produced by the interaction between tetramers and anti-CD8 com- ers to bind to T cells, but other anti-CD8 mAbs, including CT- pared with those produced by anti-TCR Abs and anti-CD8 implies CD8a, impair tetramer binding (27). CT-CD8a and 53.6.7 bind to that in the presence of tetramers, CD8 and the TCR are colocalized distinct epitopes on CD8␣ and do not inhibit one another from and are not randomly distributed on the T cell membrane. The binding (data not shown). To determine whether recruitment of inefficient induction of FRET by 1B2 and anti-V␣2 cannot be at- CD8 to the TCR by MHC class I tetramers is dependent on binding tributed to an intrinsic inability of these mAbs to interact with of CD8 by the MHC, we incubated 2C and OT-1 LN cells with anti-CD8-allophycocyanin, because FRET between these reagents CT-CD8a and 53.6.7-allophycocyanin, followed by relevant tetramer. As has been reported previously, preincubation with CT- CD8a abrogated binding of Kb/OVA to OT-1 CD8ϩ LN cells, but only moderately reduced binding of Kb/SIYR to 2C CD8ϩ cells (23) (Fig. 3, a and c). However, blockade of the CD8-MHC interaction with CT-CD8a dramatically reduced FRET acceptor fluorescence on 2C cells (Fig. 3b). This indicates that CD8 recruitment to the TCR is dependent on MHC-CD8 binding.

MHC class I-induced FRET is observed only with the CD8 coreceptor FIGURE 2. Allophycocyanin is an efficient FRET acceptor in combi- To demonstrate that MHC class I-mediated recruitment of core- nation with FITC or PE as FRET donors. OT-1 LN cells were stained with ceptor to the TCR is specific for CD8 and not CD4, we stained 2C allophycocyanin-labeled anti-CD8 mAb (53.6.7), washed, and incubated b b with polyclonal anti-mouse IgG, either unconjugated (solid line), FITC- and OT-1 thymocytes with PE-conjugated K /SIYR or K /OVA tetramers as well as allophycocyanin-conjugated anti-CD8 or anti- conjugated (dotted line), or PE-conjugated (dashed line). Stained cells were ϩ analyzed for FRET by detection at FL3. Both FITC and PE were efficient CD4 mAbs. CD8 thymocytes (mostly double-positive cells) that FRET donors, as indicated by their ability to enhance the FL3 signal. were costained with 53.6.7-allophycocyanin and the relevant tet- Events shown were gated for CD8ϩ (FL4 signal) live cells. ramer produced an enhanced FRET acceptor signal. However, 824 CLASS I MOLECULES MEDIATE TCR/CD3⑀/CD8 INTERACTION

CD4ϩ thymocytes (again, mostly double-positive cells) labeled with CT-CD8a blocked Kb/SIYR-mediated FRET signaling (Fig. with relevant tetramer and anti-CD4-allophycocyanin did not pro- 5, b and d). Maximal cross-linking of anti-CD8 and anti-CD3⑀ duce an acceptor signal despite the fact that the CD4-labeled thy- using anti-IgG induced approximately the same intensity of FRET mocytes bound relevant tetramer at approximately equal levels with CD8-labeled thymocytes (Fig. 4). This difference in FRET signal despite similar levels of anti-CD4 and anti-CD8 implies that the association of tetramer with CD8 is far greater than that expected due to random distribution of the TCR and CD8.

TCR and CD8 receptors bind the same MHC ligand, assembling TCR/CD3/CD8 complexes To this point, the experiments shown have used multivalent MHC tetramers to assess TCR/CD8 interactions and have not addressed directly the question of whether CD8 and TCR molecules bind to the same class I molecule. To address this question, we stained 2C LN cells with anti-CD3⑀-PE and 53.6.7-allophycocyanin (anti- CD8), then washed unbound Abs away. The stained 2C cells were then incubated with unlabeled monomeric Kb/SIYR or Kb/OVA.

Immediately after incubation with monomer, the cells were fixed Downloaded from with paraformaldehyde to preserve any weak interactions between the class I monomers and the T cells. As positive controls, we incubated stained cells with unlabeled Kb/SIYR tetramers or anti- IgG polyclonal Abs, reagents expected to efficiently assemble TCR/CD8 complexes. As a negative control, the 2C cells were b incubated with irrelevant (K /OVA) tetramers or monomers. http://www.jimmunol.org/ Kb/SIYR tetramers and monomers, but not Kb/OVA tetramers or monomers, induced indirect allophycocyanin fluorescence on CD3ϩ8ϩ 2C T cells (Fig. 5, a and c). Since Kb/OVA monomers were unable to induce FRET, we conclude that the FRET signal is not due to weak nonspecific interactions that were inadvertently preserved through immediate fixation. As before, preincubation FIGURE 5. TCR and CD8 bind the same MHC ligand, assembling TCR/CD3/CD8 complexes. a, 2C LN cells were labeled with anti- CD3⑀-PE (145-2C11) and anti-CD8-allophycocyanin (53.6.7), washed, incubated with unlabeled Kb/SIYR (bold line) or Kb/OVA (light gray) tet- by guest on September 25, 2021 ramers, then washed and fixed in 2% paraformaldehyde before analysis for FRET. Kb/SIYR, but not Kb/OVA tetramer, is able to bring together CD8 and CD3⑀, as indicated by FRET. b, 2C LN cells were labeled with anti- CD3⑀-PE and 53.6.7-allophycocyanin (bold line) or with anti-CD3⑀-PE, 53.6.7-allophycocyanin, and CT-CD8a (dark gray); washed; incubated with unlabeled Kb/SIYR tetramer; washed again; and fixed. CT-CD8a abrogated the FRET induced by Kb/SIYR tetramer, demonstrating a require- ment for CD8 binding to the MHC tetramer for TCR/CD3/CD8 complex assembly. c and d, Cells were stained as in a and b, except that in this case Kb/SIYR or Kb/OVA monomers were used to assemble the TCR/CD3/CD8 complexes, and the treated cells were fixed immediately after incubation with the monomers. As with the tetramers, monomers of Kb/SIYR (bold line) induced FRET, indicating TCR/CD3/CD8 complex formation, while Kb/OVA monomers (light gray) did not (c). As before, CT-CD8a blocked FIGURE 4. MHC class I-induced FRET is observed only with the CD8 Kb/SIYR-mediated association and FRET (dark gray, d). e, Cells were coreceptor. a and b, 2C thymocytes (predominantly CD4/CD8 double- stained with anti-CD3⑀-PE and anti-CD8-allophycocyanin (53.6.7), then positive cells) were stained with Kb/SIYR-PE tetramer and anti-CD8 incubated with FACS medium (dotted line) or anti-IgG (line). Cross-link- (53.6.7) conjugated to allophycocyanin (light gray), Kb/OVA-PE tetramer ing the PE- and allophycocyanin-linked Abs with anti-IgG produced FRET and anti-CD8-allophycocyanin (dark gray), or Kb/SIYR-PE tetramer and with an efficiency similar to that of Kb/SIYR tetramers and monomers. anti-CD4 (RM4–5) conjugated to allophycocyanin (dotted line). Cells Because the magnitude of FRET induced by treatment of cells with MHC were analyzed for tetramer staining (a) and FRET (b). Kb/SIYR tetramer monomers (c and d) was equivalent to levels induced by treatment with bound 2C thymocytes at comparable levels when costained with either MHC tetramers (a and b) and with anti-IgG (e), we conclude that the extent anti-CD4 or anti-CD8, but cells only exhibited FRET when anti-CD8 was of TCR/CD3/CD8 complex assembly by all three treatment schemes was bound. Kb/OVA tetramer did not stain 2C thymocytes and did not induce equivalent. f, Cells were incubated in the presence of 20 ␮M PP1 (light FRET (a and b). c and d, OT-1 thymocytes were stained with Kb/SIYR-PE gray) or medium (bold line) before and during staining, then stained with tetramer and anti-CD8-allophycocyanin (light gray), Kb/OVA-PE tetramer anti-CD3⑀-PE and anti-CD8-allophycocyanin (53.6.7) and incubated with and anti-CD8-allophycocyanin (dark gray), or Kb/OVA-PE tetramer and Kb/SIYR monomers, as in c and d. The presence of PP1 at concentrations anti-CD4-allophycocyanin (bold line). As in a and b, cells were analyzed sufficient to block Lck activity did not affect TCR/CD3/CD8 complex as- for tetramer staining (c) and FRET (d). Kb/OVA tetramer (but not Kb/SIYR sembly. Events were gated for CD3ϩ8ϩ live cells. g, Monomeric (gray) or tetramer) stained the thymocytes in the presence of either anti-CD8 or tetrameric (bold line) preparations of soluble Kb/SIYR were analyzed for anti-CD4 (c), but only induced FRET when the cells were costained with the presence of aggregates by analytical gel filtration. No aggregates were anti-CD8 (d). Events shown were gated for allophycocyaninϩ (i.e., CD4ϩ found in the monomeric preparation, while the tetrameric preparation con- or CD8ϩ) live cells. tained higher m.w. species. The Journal of Immunology 825 as Kb/SIYR tetramers and monomers (Fig. 5e). FRET intensity is plex is the result of binding of CD8 and TCR molecules to the a function of both the efficiency of formation of donor-acceptor same MHC molecule. complexes and the degree of proximity between the complexed Our experiments have shown FRET interactions between allo- donor and acceptor fluorochromes. Thus, although the absolute phycocyanin-labeled anti-CD8 mAbs and both PE-labeled tetram- intensity of FRET observed using anti-CD3⑀-PE as the donor ers and PE-labeled anti-CD3⑀ mAbs. The PE-tagged reagents are reagent (Fig. 5) is less than that induced by Kb/SIYR-PE (Fig. 3b), likely to position their fluors at different distances from the cell we conclude that the efficiency of TCR/CD3/CD8 complex for- membrane and from the allophycocyanin-tagged anti-CD8 mAb. mation is the same in both cases, because monomer induced FRET We do not know the precise location of either the PE or allophy- is as efficient as tetramer- and anti-IgG-induced FRET. To verify cocyanin fluors on the Abs or tetramers, nor do we know to what that our Kb/SIYR preparation is indeed monomeric, we recharac- extent these fluors can move about once the reagents are bound to terized our preparation by analytical gel filtration. No aggregates cell surface molecules on T cells. Thus, it is impossible to pre- were detected in the monomeric preparation, while our multimeric cisely state the relative positions of TCR, CD3⑀, and CD8 during b preparation of K /SIYR did contain higher m.w. species as ex- class I ligation based on our data. However, we can demonstrate pected (Fig. 5g). Furthermore, washing of stained cells incubated that class I-dependent interactions occur between the TCR and with monomers before fixation failed to produce an augmented CD8 as well as between CD3⑀ and CD8. FRET signal (data not shown). Since tetramers have sufficient All our experiments used the anti-CD8 mAb 53.6.7 to detect avidity to remain bound during washing, it is implicit that our interactions between CD8 and CD3⑀ or the TCR. We used this preparation contains only low avidity monomers. Thus, a single mAb because other available Abs against CD8, such as CT-CD8a, soluble MHC class I-peptide complex is able to bind to both CD8 block its ability to bind to class I. However, it is possible that Downloaded from and the TCR, thereby recruiting CD8 to the TCR/CD3 complex. ligation of CD8 with 53.6.7 might alter the ability of CD8 to in- As mentioned previously, CD8 and the TCR can associate upon teract with either the TCR or class I. In fact, photoaffinity exper- Ab cross-linking in the absence of class I ligation (13, 28). Thome iments show that addition of 53.6.7 augments the ability of class I et al. (13) coimmunoprecipitated CD8 with the TCR after activa- to bind to a CD8ϩ T cell (7). However, in our hands the presence tion of hybridoma cells with anti-CD3 Abs. As there is no MHC of 53.6.7 only minimally affects the affinity of tetramers for thy- ligand in the activation step in their experiment, the association of mocytes (Fig. 4, a and c). Furthermore, even in the absence of http://www.jimmunol.org/ CD8 with the TCR complex could not have been mediated by 53.6.7, Kb/OVA tetramers require CD8 ligation to bind to OT-1 simultaneous binding of the T cell surface coreceptors to the same cells, while Kb/SIYR tetramer binding to 2C cells is augmented by target molecule. Rather, some other explanation, such as redistri- intact CD8-class I interactions (27). Thus, while 53.6.7 may affect bution of the molecules into the same membrane compartment, the interaction of CD8 with class I or the TCR, it is unlikely that followed by physical associations of the cytoplasmic components the observed class I-dependent CD8-TCR interactions require the of CD8-lck and the TCR complex must be responsible for the presence of 53.6.7. observed coimmunoprecipitation of the two complexes. Several reports indicate that a minor population of CD8 mole- Our experiments, which were conducted on ice and in an azide-

cules on resting T cells interacts with the TCR or members of the by guest on September 25, 2021 containing medium, imply that when the TCR is ligated by MHC, CD3 complex, as evidenced by immunoprecipitation (9, 10). Al- it associates with CD8 in an ATP-independent fashion. To test though we show that interactions between TCR and CD8 (Fig. 3) formally whether Src family kinase activity is required for MHC- and between CD3⑀ and CD8 (Fig. 5) are dramatically enhanced by induced TCR/CD8 interaction, we preincubated 2C LN cells with the Src family kinase inhibitor PP1 before and during staining, class I ligation, our results do not exclude a basal level of TCR/ then stained the cells with anti-CD3⑀-PE and 53.6.7-allophycocya- CD8 association. In fact, while the anti-TCR reagents 1B2-PE and ␣ nin, followed by monomeric Kb/SIYR. The presence of PP1 at V 2-PE did not produce nearly as much FRET as did PE-conju- levels that completely inhibit Src kinase activity in T and NK cells gated tetramers, they did induce an FL3 signal distinguishable had no impact upon the interaction between TCR and CD8 (Fig. from background (Fig. 3). This could be interpreted as indicative 5f). This demonstrates that CD8 can be recruited by MHC to the of a low level interaction between the TCR and CD8. In fact, while TCR complex independent of cellular activation or phosphoryla- the dominant view in the literature is that CD8 is primarily not tion of receptor components. associated with the TCR until Ag ligation, some have argued that the majority of coreceptor molecules on resting T cell are associ- Discussion ated with members of the CD3 complex (10). Under this model, We have demonstrated the feasibility of detecting molecular asso- ligation of CD8 and the TCR by class I molecules would not cause ciations on the surface of living T cells using the principal of recruitment of CD8 to the TCR, but simply a rearrangement of the FRET. Surprisingly, the fluorochromes we used, PE and allophy- TCR/CD3 complex, such that the orientations of the TCR and CD8 cocyanin, have not been used together in the literature to assess change relative to one another. Our data do not exclude either molecular proximity despite the existence of ample commercial model; however, they show that class I ligation is capable of me- sources and the convenient availability of standard detection sys- diating such a recruitment or rearrangement. tems to measure FRET using these fluorochromes. PE and allo- Since the interaction between the TCR and CD8 upon class I phycocyanin are an efficient donor-acceptor pair and are excellent binding occurs in cells poisoned with azide and in those with reagents to probe live cells for interactions between cell surface blocked kinase function, no intracellular activation signals are re- proteins. quired for this association. CD8 and the TCR each associate with Using FRET, we have directly demonstrated that CD8 and the the same MHC molecule based on their combined molecular avid- TCR make intimate contact on the surface of T cells upon binding ity for class I, leading to the assembly or rearrangement of the to MHC class I molecules. Soluble MHC-peptide monomers alone TCR/CD3/CD8 complex. As FRET was observed between re- can induce this association. Even though bivalent Abs were used to agents targeting CD8 and CD3⑀, it is evident that complex assem- detect the relative positions of CD3⑀ and CD8, soluble monomeric bly brings Lck into close proximity with CD3⑀, one of several class I molecules only bind a single molecule each of TCR and members of the CD3 complex shown to be dependent on Lck for CD8. This implies that the interaction of CD8 with the TCR com- phosphorylation. Recruitment of CD8-lck to CD3⑀ would enhance 826 CLASS I MOLECULES MEDIATE TCR/CD3⑀/CD8 INTERACTION cellular activation by targeting CD3⑀ for phosphorylation and sub- antigen receptor complex after ligand binding: analyses by co-precipitation pro- sequent use as a docking molecule. Although we have demon- files. Scand. J. Immunol. 45:487. 12. Anel, A., M. J. Martinez-Lorenzo, A.-M. Schmitt-Verhulst, and C. Boyer. 1997. strated CD8-TCR interactions that are class I dependent and Lck Influence on CD8 of TCR/CD3-generated signals in CTL clones and CTL pre- independent, others have shown that cross-linking of CD3 in the cursor cells. J. Immunol. 158:19. lck absence of class I is able to mediate the formation of complexes 13. Thome, M., V. Germain, J. P. DiSanto, and O. Acuto. 1996. The p56 SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/ between the TCR and CD8 (13) or CD4 (18), and that these in- CD3/␨ complex. Eur. J. Immunol. 26:2093. teractions depend on the intact coupling of Lck to the relevant 14. Cantor, C. R. 1980. Biophysical Chemistry. C. R. Cantor and P. R. Schimmel, coreceptor. Our findings do not exclude a model in which TCR/ eds. Freeman, San Francisco. 15. Matko, J., and M. Edidin. 1997. Energy transfer methods for detecting molecular CD3 complexes interact with CD4 or CD8 in the absence of MHC clusters on cell surfaces. 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