Monomeric Class I Molecules Mediate
TCR/CD3 ε/CD8 Interaction on the Surface
of T Cells
This information is current as
of June 18, 2017.
Matthew S. Block, Aaron J. Johnson, Yanice
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
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References
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.
Department of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic,
Rochester, MN 55905
Received for publication November 27, 2000. Accepted for publication May 7, 2001.
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
SW, Rochester, MN 55905. E-mail address: [email protected]
Copyright © 2001 by The American Association of Immunologists
both studies the binding of soluble class I-CD8 complexes to surface-bound TCR could be inferred from the observed changes in
plasmon resonance, indicating that it is sterically possible for class
I molecules to be bound simultaneously by TCR and CD8.
Although binding of both CD8 and TCR to the same class I
molecule has not been directly demonstrated on the surface of
living T cells, several indirect methods have suggested that monomeric class I binding and triggering of T cells requires the presence of CD8. Using photoaffinity labeling, Luescher et al. (7) demonstrated that labeling of a class I-restricted CTL clone with a
soluble class I monomer could be inhibited by mAbs to either CD8
or the CD8-binding ␣3 domain of class I. Additionally, Delon et al.
(8) showed that Ca2⫹ signaling induced by soluble class I monomers was dependent on intact interactions between CD8 and class
I. Both of these findings support the hypothesis that CD8 and the
TCR bind simultaneously to class I molecules.
Several groups have demonstrated cell surface interactions between members of the TCR/CD3 complex and the coreceptors
CD4 and CD8 on murine T cells and clones. Immunoprecipitation
studies have revealed that the majority of CD3 ␦-chains and a
minority of TCR␤-chains and CD3␥-, ⑀-, and ␨-chains associate
with CD4 or CD8 on resting T cells (9, 10). However, there are
conflicting reports regarding which interactions between the coreceptors and TCR/CD3 are affected during T cell activation. Osono
et al. (11) reported that upon activation with anti-TCR␤ Abs, interactions between CD3␥-, ␦-, and ⑀-chains and coreceptor molecules remain unchanged, whereas CD3␨-CD4/8 association is augmented during activation. In contrast to this, Anel et al. (12)
showed enhanced CD3⑀-CD8 association upon Con A activation.
Of note, Thome et al. (13) showed enhanced TCR-CD8 association
upon activation in a human T cell line. This association is dependent on the activity of the tyrosine kinase Lck.
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
proximity (⬍80 Å), they interact such that a fluorochrome that has
been excited (the donor) can transfer energy to a second fluorochrome (the acceptor), causing it to fluoresce (14). Detection of
2
Abbreviations used in this paper: FRET, fluorescence resonance energy transfer;
LN, lymph node.
0022-1767/01/$02.00
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T
he CD8 molecule plays a critical role in positive selection
and activation of T cells that recognize peptide Ags presented in the context of MHC class I molecules (1). It has
been known for several years that CD8 binds directly to class I,
and various mechanisms have been proposed to explain the augmentation of T cell responses to Ag by CD8. One early hypothesis
was that CD8, along with its class II-recognizing counterpart CD4,
serves primarily as an accessory molecule, enhancing T cell-APC
interactions by binding to MHC molecules on the surface of APC
without discriminating whether the MHC molecules are those recognized by the TCR (2). It is now known that augmentation of T
cell activation by CD8 requires that CD8 be able to bind to the
same species of class I molecule that is recognized by the TCR,
suggesting that CD8 acts as a coreceptor rather than as an accessory molecule (3, 4). An attractive hypothesis is that TCR and CD8
simultaneously bind the same class I molecule on the surface of
the APC or target cell, providing a mechanism for associating the
CD8-linked kinase Lck with target sites on CD3 components of
the TCR complex. However, simultaneous binding of class I
molecules by TCR and CD8 has not been demonstrated directly on
the surface of cells. Here we report direct evidence that class I
molecules mediate TCR/CD8 association on the surface of T cells.
Furthermore, we show that ligation of monomeric class I molecules by T cells causes enhanced association of CD3⑀ and CD8.
Simultaneous binding of soluble CD8 and TCR to the same
MHC molecule in vitro has been shown by surface plasmon resonance studies. One group reported an enhanced affinity of the
TCR for soluble class I molecules upon binding of the class I
molecules to soluble CD8 (5). While this reported change in binding affinity might be taken as evidence of simultaneous binding of
CD8 and the TCR to the same class I molecule, a second report
argued that no such affinity changes could be seen under similar
conditions (6). However, pertinent to the present discussion, in
CLASS I MOLECULES MEDIATE TCR/CD3⑀/CD8 INTERACTION
822
enhanced acceptor fluorescence when only the donor has been directly excited indicates that the donor and acceptor fluorochromes
are very close to one another. Although several groups have studied molecular interactions on cell surfaces using FRET (15–18),
here we employ novel combinations of commonly available fluorescent markers to assess interactions between T cell surface
proteins.
Materials and Methods
Mice
2C TCR␣␤ transgenic mice were originally described by D. Loh (19) and
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
guidelines for animal care and use.
Monomers and tetramers
b
b
PP1 treatment
Cells were incubated in 20 ␮M PP1 (22) (Biomol, Plymouth Meeting, PA)
at 37°C for 30 min and stained on ice in the presence of 20 ␮M PP1.
Flow cytometry
The mAbs anti-CD8-allophycocyanin (53.6.7), anti-CD4-allophycocyanin (RM4-5), anti-V␣2-PE (B20.1), and anti-CD3⑀-PE (145-2C11)
were obtained from BD PharMingen (San Diego, CA). CT-CD8a
(anti-CD8) was purchased from Caltag (Burlingame, CA). The anti-2C
clonotypic mAb 1B2 (23) was conjugated to PE using a Phycolink kit
from Prozyme (San Leandro, CA). Unconjugated polyclonal anti-IgG
was obtained from ICN Biomedicals (Costa Mesa, CA). FITCconjugated anti-IgG was purchased from Accurate Chemical and Scientific (Westbury, NY). PE-conjugated anti-IgG was obtained from
Serotec (Raleigh, NC). Lymph node (LN) cells and thymocytes were
isolated, and approximately 2 ⫻ 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. Paraformaldehyde 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 Biosciences, 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 CD8⫹ cells stained with
Kb/SIYR-PE, anti-CD8-allophycocyanin, or both Kb/SIYR-PE and antiCD8-allophycocyanin (Table I). Comparable FRET increases (gains in
the FL3 channel) were detected using compensated (115– 4 ⫽ 111
arbitrary mean fluorescence intensity units) or uncompensated (313–
188 ⫽ 124 arbitrary mean fluorescence intensity units) conditions.
Analytical gel filtration
Size exclusion gel filtration analysis was performed by the Mayo Protein
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
used as the buffer, and samples were run at room temperature. Samples of
Kb/SIYR tetramer and monomer were diluted to 200 ␮g/ml in PBS, and
100 ␮l of each sample was injected into the column. The relative protein
concentration was determined by measuring absorbance at 280 nm.
Staining Regimen
Uncompensated
MFIa
Compensated
MFI
Kb/SIYR
Kb/SIYR and anti-CD8 APC
Kb/SIYR
Anti-CD8-APC
Kb/SIYR and anti-CD8 APC
Anti-CD8-APC
Kb/SIYR and anti-CD8 APC
810
735
188
15
313
902
877
821
752
4
5
115
920
884
Signal
FL2
FL3
FL4
a
FL, Fluorescence; MFI, mean fluorescence intensity.
Results
Allophycocyanin is an efficient FRET acceptor in combination
with FITC or PE as FRET donors
Allophycocyanin is a widely available phycobiliprotein that is
maximally excited by light of 615– 655 nm (but not 488 nm), and
emits most efficiently at wavelengths above 650 nm (Fig. 1) (24).
FITC and PE both absorb light efficiently at 488 nm, and fluoresce
at wavelengths that overlap the excitation range for allophycocyanin. The large size of phycobiliproteins and the existence of multiple fluorescent moieties within each molecule make PE and allophycocyanin unsuitable for precise measurements of distance
using FRET. However, quantitation of distances between fluorophores is not necessary to effectively demonstrate that cell surface
molecules are or are not associating with one another. Thus, allophycocyanin paired with either FITC or PE is an ideal set of reagents to study cell surface interactions qualitatively by FRET.
Using a standard two-laser flow cytometry system, allophycocyanin fluorescence can be assessed both when directly excited
(aligned with the 635-nm laser) and when indirectly excited by
FRET (aligned with the 488-nm laser). Allophycocyanin fluoresces in a range similar to that of the dyes PerCP and Red613;
thus, allophycocyanin fluorescence due to FRET can be measured
using standard detectors normally assigned to PerCP or Red613
FIGURE 1. Excitation and Emission Spectra for FITC, PE, and allophycocyanin. a, Overlay of excitation and emission spectra of FITC (solid
line, excitation; dashed line, emission) and allophycocyanin (gray line,
excitation; dotted line, emission). Arrow, excitation at 488 nm. Wavelengths greater than 670 nm (detected by FL3 on FACSCalibur (BD Biosciences)) are shaded gray. FITC, but not allophycocyanin, is excited directly at 488 nm, whereas allophycocyanin, but not FITC, fluoresces at
wavelengths detected by FL3. b, Overlay of spectra for PE (excitation is
shown by a line, emission is shown by a dashed line) and allophycocyanin
(gray line, excitation; dotted line, emission). Only PE is excited at 488 nm,
and only allophycocyanin fluoresces in the FL3 range.
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K /SIYR and K /OVA monomers and tetramers were prepared as previously described (20). The expression vector for the production of Kb tetramers and monomers was generated by site-directed mutagenesis of Kb
cDNA and cloned into pET23 (Novagen, Madison, WI) for expression of
protein in Escherichia coli. The human ␤2-microglobulin construct was
described previously (21). SIYR (SIYRYYGL) and OVA (SIINFEKL)
peptides were generated at the Mayo Protein Core Facility.
Table I. Comparison of compensated vs uncompensated flow cytometry
readings
The Journal of Immunology
(670 long-pass filter; Fig. 1, shaded regions). Importantly, neither
FITC nor PE fluoresces appreciably at wavelengths ⬎670 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
670⫹ 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
expected to interact, such as CD4 and class I-restricted TCRs, no
FRET was observed despite the presence of both fluorochromes on
the same cell (Fig. 4, b and d, dotted and bold lines).
T cells that react against a particular peptide-MHC complex can be
effectively identified by their ability to bind to tetramers of soluble
MHC complexed with the reactive peptide (25). To demonstrate
that class I tetramers bind CD8 and recruit it to the TCR, we
stained LN cells from 2C and OT-1 TCR-transgenic mice (specific
for Kb/SIYR and Kb/OVA, respectively) with 53.6.7-allophycocyanin (a CD8-specific mAb) and PE-conjugated tetramers. With
both 2C and OT-1 CD8⫹ cells, incubation with the relevant tetramer stained the cells with PE and brought CD8 into close proximity with the TCR, as evidenced by allophycocyanin fluorescence
upon excitation at 488 nm (Fig. 3, shaded histograms). Tetramers
containing an irrelevant peptide did not label the CD8⫹ cells or
induce FRET, demonstrating that binding to the TCR is an essential step in the recruitment of CD8. To show that recruitment of
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
the 2C and OT-1 TCRs, respectively (23, 26). PE-conjugated 1B2
and anti-V␣2 bound effectively to their respective TCRs (Fig. 3, a
and c), but were unable to recruit CD8 and produced minimal
FRET (Fig. 3, b and d). The augmentation of the FRET signals
produced by the interaction between tetramers and anti-CD8 compared with those produced by anti-TCR Abs and anti-CD8 implies
that in the presence of tetramers, CD8 and the TCR are colocalized
and are not randomly distributed on the T cell membrane. The
inefficient induction of FRET by 1B2 and anti-V␣2 cannot be attributed to an intrinsic inability of these mAbs to interact with
anti-CD8-allophycocyanin, because FRET between these reagents
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
(light gray), Kb/OVA-PE tetramer (dark gray), or 1B2-PE (dotted line),
each followed by staining with 53.6.7-allophycocyanin (anti-CD8). An additional sample of 2C cells was incubated with both anti-CD8 reagents,
CT-CD8a and 53.6.7-allophycocyanin, then stained with Kb/SIYR-PE tetramer (thin line). All cells were analyzed for staining with tetramer-PE or
1B2-PE (a) and for FRET (b). 2C cells stained efficiently with Kb/SIYR
and 1B2 (a), but only exhibited FRET when bound by Kb/SIYR (b). Furthermore, when interactions between CD8 and class I molecules were
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
stained with 53.6.7-allophycocyanin along with Kb/SIYR-PE tetramer
(light gray), Kb/OVA-PE tetramer (dark gray), anti-V␣2 (B20.1) conjugated to PE (dashed line), or CT-CD8a and Kb/OVA-PE tetramer (bold
line). Again, cells were analyzed for both staining with tetramer-PE or
anti-V␣2-PE (c) and FRET (d). OT-1 cells stained efficiently with Kb/OVA
and anti-V␣2, but CT-CD8a abrogated binding of Kb/OVA (c). FRET
occurred when cells were stained with Kb/OVA, but not anti-V␣2 (d).
Because CT-CD8a blocked Kb/OVA binding, no FRET activity was
observed, as expected (d). Events were gated for CD8⫹ live cells.
can be efficiently induced by artificial cross-linking with polyclonal anti-IgG (data not shown). Therefore, the substantial levels
of FRET that occur upon tetramer binding are indicative of tetramer-dependent association of CD8 and the TCR.
The anti-CD8 mAb 53.6.7 enhances the ability of MHC tetramers to bind to T cells, but other anti-CD8 mAbs, including CTCD8a, impair tetramer binding (27). CT-CD8a and 53.6.7 bind to
distinct epitopes on CD8␣ and do not inhibit one another from
binding (data not shown). To determine whether recruitment of
CD8 to the TCR by MHC class I tetramers is dependent on binding
of CD8 by the MHC, we incubated 2C and OT-1 LN cells with
CT-CD8a and 53.6.7-allophycocyanin, followed by relevant tetramer. As has been reported previously, preincubation with CTCD8a abrogated binding of Kb/OVA to OT-1 CD8⫹ LN cells, but
only moderately reduced binding of Kb/SIYR to 2C CD8⫹ 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 combination with FITC or PE as FRET donors. OT-1 LN cells were stained with
allophycocyanin-labeled anti-CD8 mAb (53.6.7), washed, and incubated
with polyclonal anti-mouse IgG, either unconjugated (solid line), FITCconjugated (dotted line), or PE-conjugated (dashed line). Stained cells were
analyzed for FRET by detection at FL3. Both FITC and PE were efficient
FRET donors, as indicated by their ability to enhance the FL3 signal.
Events shown were gated for CD8⫹ (FL4 signal) live cells.
To demonstrate that MHC class I-mediated recruitment of coreceptor to the TCR is specific for CD8 and not CD4, we stained 2C
and OT-1 thymocytes with PE-conjugated Kb/SIYR or Kb/OVA
tetramers as well as allophycocyanin-conjugated anti-CD8 or antiCD4 mAbs. CD8⫹ thymocytes (mostly double-positive cells) that
were costained with 53.6.7-allophycocyanin and the relevant tetramer produced an enhanced FRET acceptor signal. However,
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MHC class I-peptide tetramers recruit CD8 to the TCR
823
824
CLASS I MOLECULES MEDIATE TCR/CD3⑀/CD8 INTERACTION
CD4⫹ thymocytes (again, mostly double-positive cells) labeled
with relevant tetramer and anti-CD4-allophycocyanin did not produce an acceptor signal despite the fact that the CD4-labeled thymocytes 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.
with CT-CD8a blocked Kb/SIYR-mediated FRET signaling (Fig.
5, b and d). Maximal cross-linking of anti-CD8 and anti-CD3⑀
using anti-IgG induced approximately the same intensity of FRET
TCR and CD8 receptors bind the same MHC ligand, assembling
TCR/CD3/CD8 complexes
FIGURE 4. MHC class I-induced FRET is observed only with the CD8
coreceptor. a and b, 2C thymocytes (predominantly CD4/CD8 doublepositive cells) were stained with Kb/SIYR-PE tetramer and anti-CD8
(53.6.7) conjugated to allophycocyanin (light gray), Kb/OVA-PE tetramer
and anti-CD8-allophycocyanin (dark gray), or Kb/SIYR-PE tetramer and
anti-CD4 (RM4 –5) conjugated to allophycocyanin (dotted line). Cells
were analyzed for tetramer staining (a) and FRET (b). Kb/SIYR tetramer
bound 2C thymocytes at comparable levels when costained with either
anti-CD4 or anti-CD8, but cells only exhibited FRET when anti-CD8 was
bound. Kb/OVA tetramer did not stain 2C thymocytes and did not induce
FRET (a and b). c and d, OT-1 thymocytes were stained with Kb/SIYR-PE
tetramer and anti-CD8-allophycocyanin (light gray), Kb/OVA-PE tetramer
and anti-CD8-allophycocyanin (dark gray), or Kb/OVA-PE tetramer and
anti-CD4-allophycocyanin (bold line). As in a and b, cells were analyzed
for tetramer staining (c) and FRET (d). Kb/OVA tetramer (but not Kb/SIYR
tetramer) stained the thymocytes in the presence of either anti-CD8 or
anti-CD4 (c), but only induced FRET when the cells were costained with
anti-CD8 (d). Events shown were gated for allophycocyanin⫹ (i.e., CD4⫹
or CD8⫹) live cells.
FIGURE 5. TCR and CD8 bind the same MHC ligand, assembling
TCR/CD3/CD8 complexes. a, 2C LN cells were labeled with antiCD3⑀-PE (145-2C11) and anti-CD8-allophycocyanin (53.6.7), washed, incubated with unlabeled Kb/SIYR (bold line) or Kb/OVA (light gray) tetramers, 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 antiCD3⑀-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 requirement 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
Kb/SIYR-mediated association and FRET (dark gray, d). e, Cells were
stained with anti-CD3⑀-PE and anti-CD8-allophycocyanin (53.6.7), then
incubated with FACS medium (dotted line) or anti-IgG (line). Cross-linking the PE- and allophycocyanin-linked Abs with anti-IgG produced FRET
with an efficiency similar to that of Kb/SIYR tetramers and monomers.
Because the magnitude of FRET induced by treatment of cells with MHC
monomers (c and d) was equivalent to levels induced by treatment with
MHC tetramers (a and b) and with anti-IgG (e), we conclude that the extent
of TCR/CD3/CD8 complex assembly by all three treatment schemes was
equivalent. f, Cells were incubated in the presence of 20 ␮M PP1 (light
gray) or medium (bold line) before and during staining, then stained with
anti-CD3⑀-PE and anti-CD8-allophycocyanin (53.6.7) and incubated with
Kb/SIYR monomers, as in c and d. The presence of PP1 at concentrations
sufficient to block Lck activity did not affect TCR/CD3/CD8 complex assembly. Events were gated for CD3⫹8⫹ live cells. g, Monomeric (gray) or
tetrameric (bold line) preparations of soluble Kb/SIYR were analyzed for
the presence of aggregates by analytical gel filtration. No aggregates were
found in the monomeric preparation, while the tetrameric preparation contained higher m.w. species.
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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 (antiCD8), 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
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 antiIgG polyclonal Abs, reagents expected to efficiently assemble
TCR/CD8 complexes. As a negative control, the 2C cells were
incubated with irrelevant (Kb/OVA) tetramers or monomers.
Kb/SIYR tetramers and monomers, but not Kb/OVA tetramers or
monomers, induced indirect allophycocyanin fluorescence on
CD3⫹8⫹ 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
The Journal of Immunology
Discussion
We have demonstrated the feasibility of detecting molecular associations on the surface of living T cells using the principal of
FRET. Surprisingly, the fluorochromes we used, PE and allophycocyanin, have not been used together in the literature to assess
molecular proximity despite the existence of ample commercial
sources and the convenient availability of standard detection systems to measure FRET using these fluorochromes. PE and allophycocyanin are an efficient donor-acceptor pair and are excellent
reagents to probe live cells for interactions between cell surface
proteins.
Using FRET, we have directly demonstrated that CD8 and the
TCR make intimate contact on the surface of T cells upon binding
to MHC class I molecules. Soluble MHC-peptide monomers alone
can induce this association. Even though bivalent Abs were used to
detect the relative positions of CD3⑀ and CD8, soluble monomeric
class I molecules only bind a single molecule each of TCR and
CD8. This implies that the interaction of CD8 with the TCR com-
plex is the result of binding of CD8 and TCR molecules to the
same MHC molecule.
Our experiments have shown FRET interactions between allophycocyanin-labeled anti-CD8 mAbs and both PE-labeled tetramers and PE-labeled anti-CD3⑀ mAbs. The PE-tagged reagents are
likely to position their fluors at different distances from the cell
membrane and from the allophycocyanin-tagged anti-CD8 mAb.
We do not know the precise location of either the PE or allophycocyanin fluors on the Abs or tetramers, nor do we know to what
extent these fluors can move about once the reagents are bound to
cell surface molecules on T cells. Thus, it is impossible to precisely state the relative positions of TCR, CD3⑀, and CD8 during
class I ligation based on our data. However, we can demonstrate
that class I-dependent interactions occur between the TCR and
CD8 as well as between CD3⑀ and CD8.
All our experiments used the anti-CD8 mAb 53.6.7 to detect
interactions between CD8 and CD3⑀ or the TCR. We used this
mAb because other available Abs against CD8, such as CT-CD8a,
block its ability to bind to class I. However, it is possible that
ligation of CD8 with 53.6.7 might alter the ability of CD8 to interact with either the TCR or class I. In fact, photoaffinity experiments show that addition of 53.6.7 augments the ability of class I
to bind to a CD8⫹ T cell (7). However, in our hands the presence
of 53.6.7 only minimally affects the affinity of tetramers for thymocytes (Fig. 4, a and c). Furthermore, even in the absence of
53.6.7, Kb/OVA tetramers require CD8 ligation to bind to OT-1
cells, while Kb/SIYR tetramer binding to 2C cells is augmented by
intact CD8-class I interactions (27). Thus, while 53.6.7 may affect
the interaction of CD8 with class I or the TCR, it is unlikely that
the observed class I-dependent CD8-TCR interactions require the
presence of 53.6.7.
Several reports indicate that a minor population of CD8 molecules on resting T cells interacts with the TCR or members of the
CD3 complex, as evidenced by immunoprecipitation (9, 10). Although we show that interactions between TCR and CD8 (Fig. 3)
and between CD3⑀ and CD8 (Fig. 5) are dramatically enhanced by
class I ligation, our results do not exclude a basal level of TCR/
CD8 association. In fact, while the anti-TCR reagents 1B2-PE and
V␣2-PE did not produce nearly as much FRET as did PE-conjugated tetramers, they did induce an FL3 signal distinguishable
from background (Fig. 3). This could be interpreted as indicative
of a low level interaction between the TCR and CD8. In fact, while
the dominant view in the literature is that CD8 is primarily not
associated with the TCR until Ag ligation, some have argued that
the majority of coreceptor molecules on resting T cell are associated with members of the CD3 complex (10). Under this model,
ligation of CD8 and the TCR by class I molecules would not cause
recruitment of CD8 to the TCR, but simply a rearrangement of the
TCR/CD3 complex, such that the orientations of the TCR and CD8
change relative to one another. Our data do not exclude either
model; however, they show that class I ligation is capable of mediating such a recruitment or rearrangement.
Since the interaction between the TCR and CD8 upon class I
binding occurs in cells poisoned with azide and in those with
blocked kinase function, no intracellular activation signals are required for this association. CD8 and the TCR each associate with
the same MHC molecule based on their combined molecular avidity for class I, leading to the assembly or rearrangement of the
TCR/CD3/CD8 complex. As FRET was observed between reagents targeting CD8 and CD3⑀, it is evident that complex assembly brings Lck into close proximity with CD3⑀, one of several
members of the CD3 complex shown to be dependent on Lck for
phosphorylation. Recruitment of CD8-lck to CD3⑀ would enhance
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as Kb/SIYR tetramers and monomers (Fig. 5e). FRET intensity is
a function of both the efficiency of formation of donor-acceptor
complexes and the degree of proximity between the complexed
donor and acceptor fluorochromes. Thus, although the absolute
intensity of FRET observed using anti-CD3⑀-PE as the donor
reagent (Fig. 5) is less than that induced by Kb/SIYR-PE (Fig. 3b),
we conclude that the efficiency of TCR/CD3/CD8 complex formation is the same in both cases, because monomer induced FRET
is as efficient as tetramer- and anti-IgG-induced FRET. To verify
that our Kb/SIYR preparation is indeed monomeric, we recharacterized our preparation by analytical gel filtration. No aggregates
were detected in the monomeric preparation, while our multimeric
preparation of Kb/SIYR did contain higher m.w. species as expected (Fig. 5g). Furthermore, washing of stained cells incubated
with monomers before fixation failed to produce an augmented
FRET signal (data not shown). Since tetramers have sufficient
avidity to remain bound during washing, it is implicit that our
preparation contains only low avidity monomers. Thus, a single
soluble MHC class I-peptide complex is able to bind to both CD8
and the TCR, thereby recruiting CD8 to the TCR/CD3 complex.
As mentioned previously, CD8 and the TCR can associate upon
Ab cross-linking in the absence of class I ligation (13, 28). Thome
et al. (13) coimmunoprecipitated CD8 with the TCR after activation of hybridoma cells with anti-CD3 Abs. As there is no MHC
ligand in the activation step in their experiment, the association of
CD8 with the TCR complex could not have been mediated by
simultaneous binding of the T cell surface coreceptors to the same
target molecule. Rather, some other explanation, such as redistribution of the molecules into the same membrane compartment,
followed by physical associations of the cytoplasmic components
of CD8-lck and the TCR complex must be responsible for the
observed coimmunoprecipitation of the two complexes.
Our experiments, which were conducted on ice and in an azidecontaining medium, imply that when the TCR is ligated by MHC,
it associates with CD8 in an ATP-independent fashion. To test
formally whether Src family kinase activity is required for MHCinduced TCR/CD8 interaction, we preincubated 2C LN cells with
the Src family kinase inhibitor PP1 before and during staining,
then stained the cells with anti-CD3⑀-PE and 53.6.7-allophycocyanin, followed by monomeric Kb/SIYR. The presence of PP1 at
levels that completely inhibit Src kinase activity in T and NK cells
had no impact upon the interaction between TCR and CD8 (Fig.
5f). This demonstrates that CD8 can be recruited by MHC to the
TCR complex independent of cellular activation or phosphorylation of receptor components.
825
826
CLASS I MOLECULES MEDIATE TCR/CD3⑀/CD8 INTERACTION
cellular activation by targeting CD3⑀ for phosphorylation and subsequent use as a docking molecule. Although we have demonstrated CD8-TCR interactions that are class I dependent and Lck
independent, others have shown that cross-linking of CD3 in the
absence of class I is able to mediate the formation of complexes
between the TCR and CD8 (13) or CD4 (18), and that these interactions depend on the intact coupling of Lck to the relevant
coreceptor. Our findings do not exclude a model in which TCR/
CD3 complexes interact with CD4 or CD8 in the absence of MHC
to modulate or propagate signals emanating from the TCR. However, we do demonstrate that simultaneous binding to class I is in
itself sufficient to cause the intimate association of CD8 with the
TCR and CD3⑀.
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