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Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Little has been reported on CD8. A recent work analyzing the interaction between CD8 and TCR suggested that TCR and the CD8 coreceptor bind peptide-MHC independently and with distinct kinetics (16). However, CD8 binding to class I MHC-peptide complexes is known to be enhanced by signaling events. Moreover, activated CD8 binding to class I MHC mediated by the TCR results in activation and signaling (17, 18). The wealth of evidence suggests that similar to CD4, CD8 is recruited and participates in TCR-peptide-MHC interactions only after the TCR already has been stably bound to the MHC-peptide complex.
Recently, a study with transgenic mouse models revealed that CD8 plays a critical role in TCR binding and activation by peptide-MHC complex multimers (18). In this study, with mouse MHC class I tetramers and two well-defined class I-restricted TCR-transgenic systems (OT-1 and 2C), it was shown that CD8 was pivotal for both tetramer binding to TCR and the subsequent activation of T cell response mediated by the tetramers (18).
Finally, MHC-peptide tetramers have caused a revolution in molecular immunology by enabling for the first time the phenotypic analysis of T cell responses (19, 20, 21, 22, 23, 24). Several groups reported on the construction of MHC-peptide tetramers and their use to detect specific and in some cases very rare T cell populations (19, 20, 21, 22, 23, 24, 25). Recently, we also have constructed single-chain MHC (scMHC)4-peptide complexes in a bacterial expression system and generated functional scMHC-peptide tetramers (25). Staining with MHC-peptide tetramers is performed in the presence of anti-CD8 Abs (19, 20, 21, 22, 23, 24). This indicates that tetramer binding can occur in the presence of anti-CD8 Abs; however, the effect of anti-CD8 Abs on tetramer binding was not studied in detail in the staining experiments involving human T cells.
To determine the role of CD8 in the binding of MHC-peptide tetramers to TCR in a human T cell system, we analyzed the influence of anti-CD8 Abs on the binding of specific cancer-associated MHC-peptide tetramers to a CTL clone derived from PBMCs isolated from a melanoma patient who was treated with a cancer peptide vaccine (26). Importantly, we show that human CD8 has a pivotal role in tetramer binding to TCR, because anti-CD8 Abs completely blocked tetramer-specific binding to TCR.
Our results suggest that TCR binding to MHC-peptide tetramers is coreceptor dependent. Thus, CD8 is recruited and participates in TCR-peptide-MHC interactions before the tetramer has stably bound to the TCR. We have concluded that the use of anti-CD8 Abs in various experimental systems to characterize phenotypes and to isolate T cell populations can greatly influence the outcome of TCR binding to MHC-peptide ligands in their multimeric (tetramer) form.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R6C12 is a CTL clone derived from the PBMCs of a melanoma patient after immunization with the gp100-derived G9-209-2M peptide. This clone was kindly given by S. Rosenberg (Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD).
The R6C12 was cloned in several steps of limiting dilution from bulk cultures and was expanded to large numbers by a protocol using allogeneic irradiated PBMCs (26). Frozen cells were thawed in culture medium consisting of RPMI 1640 containing 20% AIM-V medium plus 10% heat-inactivated human serum, 1 mM HEPES, pH 7.0, 1 mM glutamine, 1 mM pyruvate, 120 IU/ml IL-2, and 10 IU/ml IL-4.
In vitro stimulation of cells was performed every 4–5 days with irradiated JY cells as APCs and the melanoma cell line FM3D, which expresses high levels of gp100 (27). JY and FM3D cells were grown in RPMI 1640 containing 10% FCS, 1 mM glutamine, and 1 mM pyruvate.
Peptides and MHC tetramers
The peptides G9-209-2M (IMDQVPFSV) and 154 (KTWGQYWQV), derived from gp100, and the peptide TAX (LLFGYPVYV), derived from human T cell leukemia virus (HTLV)-1, were synthesized by standard techniques by Genemed Synthesis (South San Francisco, CA) and were >95% pure.
Production of scMHC-peptide tetramers was performed as described
previously (25). Briefly, the plasmid encoding
single-chain 
2-microglobulin- HLA-A2 genes,
which are connected by a flexible linker, was transformed and
overexpressed in Escherichia coli BL21 cells. In this
construct, a sequence for site-specific biotinylation by the BirA
enzyme was engineered at the C terminus of the HLA-A2
gene. The overexpressed protein was purified from inclusion
bodies, denatured, reduced, and mixed with a 5- to 10-fold molar excess
of the appropriate peptide in a redox-shuffling refolding system for
24–48 h. Soluble monomeric MHC-peptide complexes were purified from
the refolding solution by fast protein liquid chromatography with
Q-Sepharose chromatography and subjected to biotinylation with BirA
(Avidity, Denver, CO). Biotinylation efficiency was determined
by an ELISA with avidin-conjugated peroxidase and biotinylated myelin
basic protein peptide as a control (Avidity). Biotinylation efficiency
was routinely estimated to be >80% by this method. The biotinylated
monomeric complexes were subsequently mixed with streptavidin-PE
(Jackson ImmunoResearch, West Grove, PA) at a 4:1 molar ratio.
Tetramers were purified and separated from monomers by size-exclusion
chromatography with a TSK3000 column. Multimers were tested for
specific binding over a range of doses by flow cytometry and were
typically used at a concentration of 20–40 µg/ml.
Abs and flow cytometry
CTLs (5 x 105) were stained in 30
µl of PBS with MHC-peptide tetramers (20–40 µg/ml) and
anti-CD8 Abs (10–40 µg/ml). In blocking experiments,
anti-CD8 Abs were added 15 min before tetramers, which then were
incubated for 45 min. In other experiments, anti-CD8 Abs and
tetramers were added simultaneously or tetramers were added first for
45 min followed by a 15-min incubation with anti-CD8 Abs. After
incubation, Abs and tetramers were washed with PBS and the cells were
analyzed with a FACStar flow cytometer (BD Biosciences, Mountain View,
CA). The following anti-CD8 Abs were used: clone 3B5 (mouse IgG2a,
; Caltag, Burlingame, CA); clone-DK-25 (mouse IgG1 ; Dako,
Glostrup, Denmark); and clone MCD8 (mouse IgG1, ; IqP).
Anti-CD3 Ab was obtained from clone UCHT1-9 (IgG1, ; Dako).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the effect and contribution of CD8 in the binding of
multivalent tetrameric MHC-peptide complexes to class I-restricted TCR
in a human system, we analyzed CTLs recognizing a tumor-associated Ag.
A CTL clone, R6C12, was derived from PBMCs from a melanoma patient
who was treated with the gp100-derived peptide vaccine G9-209-2M.
(26). Recently, we reported on the construction and
generation of human recombinant scMHC-peptide complexes and tetramers
made by in vitro refolding of E. coli-expressed
2-microglubulin and HLA-A2 fused into a single
gene by a flexible peptide linker (25). We have
synthesized scMHC tetramers with the gp100-derived G9-209-2M peptide
and other control peptides and have determined whether the interaction
of these multimers with R6C12 CD8+ T cells was
dependent on the specificity of the TCR. In vitro-stimulated R6C12 CTLs
were stained for 1 h at 37°C with the specific G9-209-2M
peptide-containing tetramers and with control scMHC tetramers
containing a different peptide derived from gp100, peptide 154, and
also the HTLV-1-derived peptide TAX. These binding assays revealed that
R6C12 bound only the specific G9-209-2M MHC tetramers and not the
control multimers containing peptides 154 and TAX (Fig. 1
). An average of 70–90%
staining of R6C12 CTLs was observed with the specific 209-containing
tetramers in comparison with a 2–4% background nonspecific staining
observed with the control nonspecific peptides 154 and TAX (Figs. 1 and 2 and Table I). As shown in Fig. 1E, the
specificity of staining with the cognate 209 peptide tetramers vs the
control nonspecific tetramers was similar when performed at 37°C or
4°C. These results correlate well with reports of functional assays
using these similar ligands on other CTL lines or PBMCs derived from
melanoma patients (19, 20, 21, 22, 23, 24, 25).
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Next, we tested a panel of Abs that recognize human CD8 to
determine their effect on the binding of scMHC-G9-209-2M peptide
tetramers to TCRs on R6C12 cells. We found that all three anti-CD8
Abs (3B5 (Caltag), DK-25 (Dako), MCD8 (IqP)) caused a total blockage of
scMHC-209 tetramer binding to R6C12 cells (Fig. 3 and Table I
). In these experiments, we
incubated the cells for 15 min with anti-CD8 Abs before staining
for 45–60 min with the scMHC tetramers. We performed the binding of
anti-CD8 Abs and tetramers at 37°C, as suggested previously
(28). Under these conditions, a blocking effect of >95%
was observed (Fig. 3 and Table I) when different anti-CD8 Abs at
various concentrations were used. Importantly, no staining of R6C12
cells with the nonspecific tetramers was observed regardless of the
anti-CD8 used (data not shown), which indicates that the
anti-CD8 Abs did not change the fine specificity of tetramer
binding. In control experiments, we used an anti-CD3 instead of an
anti-CD8 Ab, which had no influence on tetramer binding (Fig. 4C). Thus, the inhibition was
the direct result of binding the Ab to CD8.
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, B–D). Similar inhibition profiles of tetramer binding was
observed with three different anti-CD8 Abs directed against the
- or -chain of CD8 (data not shown).
The blocking effect of the anti-CD8 Abs was titratable and covered
a similar dose range (Fig. 3E and Table I). At an Ab
dilution of 1/20, corresponding to a concentration of 10–2.5 µg/ml
(depending on Ab used), maximal inhibition was achieved. A dilution of
1/1000 exhibited a minor inhibition effect. The blocking effect of CD8
Abs was similar when FITC-conjugated or unconjugated Abs were used
(data not shown).
The timing of Anti-CD8 Abs use in tetramer-CD8 double-staining experiments is critical to the inhibition effect
The binding of MHC-peptide tetramers to TCR is dynamic because the
half-life of the TCR-MHC-peptide complex is relatively short, and the
binding of the individual MHC-peptide complex units of the tetramer to
the TCR is expected to be dynamic rather than static. Each MHC-peptide
complex (here the scMHC-209 complex) dissociates and reassociates with
the TCR over time. In such a case, the timing of the addition of
anti-CD8 Abs in double-staining experiments may be very critical
for observing the blocking effect of the anti-CD8 Abs. Therefore,
to test this we compared the effects of anti-CD8 Abs on tetramer
binding to R6C12 cells under two conditions: (1) when
anti-CD8 Abs and scMHC-209 tetramers were added simultaneously
(Fig. 3D), and (2) when scMHC-209 tetramers
were allowed to bind first for 45 min and anti-CD8 was added to the
cells only later, after the tetramers had already stably bound to R6C12
TCRs (Fig. 2 and Table I).
As shown, when cells were incubated first with tetramers and only later with anti-CD8 Abs, no inhibition was observed, and multimer staining appeared to be as stable as staining with tetramers alone, without anti-CD8 Abs. Thus, although we expect that the affinity of the anti-CD8 Abs to CD8 is much higher than that of the MHC-peptide complex to TCR, the high avidity of the tetramers enables them to bind strongly enough to overcome the blocking effect of the anti-CD8 Abs in the final stage of the experiment when the Abs are added to the assay.
When tetramers and anti-CD8 Abs are added simultaneously, a
complete blocking effect on tetramer binding was observed (Fig. 3D) that was indistinguishable from the blocking effect
observed when anti-CD8 Abs and tetramers were added sequentially
(first anti-CD8 Abs followed by tetramers, Fig. 3C).
Blockage of tetramer binding to TCR by CD8 Abs is not mediated by receptor down-regulation
We postulated that the staining of CTLs at 37°C could allow T
cell biological effects such as CD8 and/or TCR internalization. To
control these activities and to rule out the possibility that the
blockage of tetramer binding to TCR by anti-CD8 Abs is mediated by
increased internalization of TCRs, we incubated with anti-CD8 Abs
and stained with tetramers only at 4°C in the presence of 3 mM azide
to prevent Ab-induced internalization and TCR down-regulation, as
previously reported (29). The binding and specificity of
multimer binding at 4°C to R6C12 was maintained and was similar or in
several cases even somewhat improved than that obtained with staining
at 37°C (Fig. 2, A and C). Seventy percent
staining with the specific 209-containing tetramer was observed under
both conditions, whereas the nonspecific tetramers (containing peptides
154 and TAX) did not stain the R6C12 cells at both temperatures (data
not shown). However, the blocking effect of the anti-CD8 Abs on the
binding of the cognate tetramers to the R6C12 cells was identical under
both conditions: all showed a significant 70–90% blockage of tetramer
binding at 4°C as well as at 37°C (Fig. 2, B and
D). The inhibitory effect of the anti-CD8 Abs here was
somewhat better at 4°C (96% inhibition, Fig. 3D),
probably due to the improved binding of anti-CD8 Abs at 4°C.
However, as shown in Fig. 3, B–D, total inhibition of
>95% of the tetramer binding by CD8 Abs was also observed at 37°C.
Thus, the data indicate that the CD8 effects on tetramer binding
are not dependent on T cell activation and are not caused by TCR-CD8
internalization.
Anti-CD8-mediated blockage of tetramer binding is not a result of steric interference
Finally, we tested the effect of tetramers on the binding of
anti-CD8 Abs to rule out the possibility that the
anti-CD8-mediated blockage of tetramer binding is the result of a
steric interference. The binding of anti-CD8 Abs was not affected
by the presence of tetramers, and in fact was identical with the
binding of anti-CD8 Abs to R6C12 cells in the absence of tetramers
(Fig. 4A). Moreover, there was no influence on the sequence
of incubation, because similar staining with anti-CD8 was observed
when anti-CD8 Ab was added first followed by the addition of
tetramers, or that tetramers were allowed to bind before incubating
with anti-CD8 (Fig. 4A). As a control, we measured
the influence of tetramers on the binding of CD3 Abs and showed that
the tetramers compete with the binding of anti-CD3 Abs (Fig. 4B). Thus, the specific scMHC-209 tetramers blocked the
binding of anti-CD3 Abs; however, when anti-CD3 Abs were added
first before the addition of the 209-specific tetramers, neither
inhibition in anti-CD3 binding (Fig. 5A) nor inhibition of tetramer
binding was observed (Fig. 4C).
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A, can be accounted for by tetramer-induced TCR
internalization and down-regulation because in a similar experiment
performed at 4°C, the tetramer-mediated blockade of anti-CD3
binding was significantly diminished (Fig. 5B). It was reported previously that the competition of class I MHC-peptide tetrameric complexes with anti-CD3 provides evidence of the specificity of peptide binding to the TCR complex (30).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent study contradicts these findings. With two well-defined transgenic mouse systems, it showed that at least in the mouse system, CD8 plays a critical and sometimes even an obligatory role in Ag-specific TCR binding and T cell activation (18). The results in these mouse systems contradict the view that the multimeric (tetramer) form of MHC-peptide ligands bind directly and solely to the TCR, but rather, highlight a pivotal contribution of CD8 for this association.
Our results in the human system presented here come to the same
conclusion, namely that CD8 interaction with MHC-peptide tetramers is a
crucial and pivotal step in the binding events occurring between the
TCR and MHC-peptide complexes. Anti-CD8 Abs were able to completely
block the specific binding of scMHC-peptide tetramers to TCR. We used
three different anti-CD8 Abs directed to the - and -chain of
CD8 and found that all of them blocked tetramer binding to TCR with
similar dose response curves. Interestingly, in their study of the
mouse system, Daniels and Jameson (18) found that one
anti-CD8 Ab of the four tested enhanced TCR association by cognate
(but not noncognate) MHC-peptide tetramer (18). The other
three Abs tested were all inhibitory and blocked MHC-peptide tetramer
binding to the transgenic TCR.
Our results have important implications for understanding the early molecular events occurring during TCR-peptide-MHC interaction and the consequences of these interactions on T cell activation and signaling. They show that CD8 is recruited and participates directly in TCR-peptide-MHC interactions before the MHC-peptide complex has stably bound to the TCR. However, they also shed new light on the use of peptide-MHC tetramers for the phenotypic characterization of various T cell responses. The ability to produce large quantities of MHC-peptide tetramers has caused a revolution in T cell biology, allowing the detection of Ag-specific T cell populations (sometimes very rare) by using the increased avidity of the tetrameric form to compensate for the very low affinity of the TCR for MHC-peptide complexes (19, 20, 21, 22, 23, 24). Numerous reports have been published in recent years that show the ability of MHC-peptide tetramers to bind to Ag-specific TCRs. These T cell populations were analyzed under various pathological conditions such as after viral infections, cancer, and autoimmune diseases. All of these studies analyzing class I MHC-peptide tetramer binding used a double-staining procedure so that the ability to stain CD8+ T cells by the MHC-peptide tetramers was tested (19, 20, 21, 22, 23, 24).
However, the role of the CD8 coreceptor and its influence on tetramer binding was not determined. There are no studies for the human system reporting blocking effects of anti-CD8 Abs on the binding of MHC-peptide tetramers to CTLs under various pathological or normal conditions. Thus, our results suggest that the use of anti-CD8 Abs in flow cytometry analysis of T cell populations can drastically influence TCR binding to MHC-peptide tetramers. The order of incubation events with the reagents (anti-CD8 Ab and tetramers) used in these staining experiments is critical when an inhibitory Ab is used. Therefore, the influence of the commonly used anti-human CD8 Abs must be determined. The blocking effects shown here, with three commonly used Abs, raise a cautionary note as to the current strategies that are being used to stain with MHC-peptide tetramers and to detect low frequencies of T cell populations in various experimental systems and under different pathological conditions.
Our results may reflect interesting biological phenomena on differences in CD8 dependence of different T cells, thus causing the conflicting results in the literature on the role of CD8 in MHC-peptide-TCR interactions. Alternatively, as described above, the results represent differences in experimental design of Ab dose and/or order of Ab and tetramer addition. To reach a well-defined conclusion, more experiments should be performed with CTL clones or lines as well as heterogeneous CTL populations from PBMCs of individuals under various pathological or normal conditions.
Several studies also demonstrated the ability to differentiate between T cell subsets that have relative high or low affinity to MHC-peptide tetramers (18, 24). These subpopulations were designated tetramerhigh and tetramerlow populations, or in other cases, high-avidity and low-avidity populations. Interestingly, in the 2C TCR-transgenic mice, there is a natural population of T cells that are positive for the 2C TCR yet are negative for CD8 (18). These cells are functional, as they can respond to TCR ligand but only at high doses. The results suggest that the CD8- population is perhaps coreceptor-independent and that it may differ in tetramer binding compared with the CD8+ population. Analysis of tetramer binding by these two populations revealed that all 2C cells stained specifically with the specific tetramer in the absence of CD8 Ab, but the staining profile was bimodal, with a tetramerhigh and a tetramerlow population (18). The percentage of tetramerlow and tetramerhigh populations was correlated with the percentage of CD8- and CD8+ cells, respectively. The use of inhibitory anti-CD8 Abs caused the disappearance of the tetramerhigh population, this group of cells becoming tetramerlow. When using another melanoma-specific CTL line that recognizes MHC-peptide tetramers containing the melanoma-derived MART-1 peptide 27–35, we have indications that a similar situation exists in a human system. These in vitro-stimulated cells contain two distinct populations that are CD8- or CD8+. The specific MHC-peptide tetramers bind to the CD8- cells and stain this T cell population with a pattern similar to the tetramerlow population observed by Daniels and Jameson and others (18, 24). Interestingly, the staining of this tetramerlow population was not affected by the various anti-CD8 Abs that were used in this study (C. J. Cohen, G. Denkberg, and Y. Retire, manuscript in preparation). Thus, we postulate that CD8 engagement and involvement in binding by MHC-peptide complexes and multimers to TCR enables the formation and detection of the tetramerhigh T cell population.
The staining intensities of the tetramerlow T cell population are much weaker because of the absence of CD8 interaction with the TCR-peptide-MHC complex. Thus, anti-CD8 Abs will not exert any effect on the staining of the tetramerlow population by MHC-peptide tetramers. However, the tetramerhigh T cell population is CD8-dependent and thus, it is significantly influenced by the use of inhibitory CD8 Abs. With CD8-dependent binding, the inherent affinity of the tetramers is insufficient to allow multimeric ligand binding if CD8 participation is blocked.
It is possible that in many experimental systems where MHC-peptide tetramers have been used in combination with anti-CD8 Abs to analyze and isolate tetramer-specific T cell populations, the experimental procedures caused an undesirable preference for staining the tetramerlow population only, because of the blocking effects of the anti-CD8 Abs. Consequently, an important subpopulation of the tetramerhigh cells was not detected because of the presence of the inhibitory anti-CD8 Ab in the staining assays. These findings are supported by results showing that CD8 has a role in enhancing TCR-MHC-peptide interactions, including in the transgenic 2C TCR mouse model (18).
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Acknowledgments |
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Footnotes |
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2 G.D. and C.J.C. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haita 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il
4 Address correspondence and reprint requests to Dr. Yoram Reiter, Faculty of Biology, Technion-Israel Institute of Technology, Technion City, Room 333, Haifa 32000, Israel. E-mail address: reiter{at}tx.technion.ac.il
5 Abbreviations used in this paper: scMHC, single-chain MHC; HTLV, human T cell leukemia virus.
Received for publication January 30, 2001. Accepted for publication April 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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bind peptide-MHC independently and with distinct kinetics. Immunity 10:219.[Medline]
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C. J. Cohen, O. Sarig, Y. Yamano, U. Tomaru, S. Jacobson, and Y. Reiter Direct Phenotypic Analysis of Human MHC Class I Antigen Presentation: Visualization, Quantitation, and In Situ Detection of Human Viral Epitopes Using Peptide-Specific, MHC-Restricted Human Recombinant Antibodies J. Immunol., April 15, 2003; 170(8): 4349 - 4361. [Abstract] [Full Text] [PDF]
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P. Guillaume, D. F. Legler, N. Boucheron, M.-A. Doucey, J.-C. Cerottini, and I. F. Luescher Soluble Major Histocompatibility Complex-Peptide Octamers with Impaired CD8 Binding Selectively Induce Fas-dependent Apoptosis J. Biol. Chem., February 7, 2003; 278(7): 4500 - 4509. [Abstract] [Full Text] [PDF]
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C. J. Cohen, N. Hoffmann, M. Farago, H. R. Hoogenboom, L. Eisenbach, and Y. Reiter Direct Detection and Quantitation of a Distinct T-Cell Epitope Derived from Tumor-specific Epithelial Cell-associated Mucin Using Human Recombinant Antibodies Endowed with the Antigen-specific, Major Histocompatibility Complex-restricted Specificity of T Cells Cancer Res., October 15, 2002; 62(20): 5835 - 5844. [Abstract] [Full Text] [PDF]
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G. Denkberg, E. Klechevsky, and Y. Reiter Modification of a Tumor-Derived Peptide at an HLA-A2 Anchor Residue Can Alter the Conformation of the MHC-Peptide Complex: Probing with TCR-Like Recombinant Antibodies J. Immunol., October 15, 2002; 169(8): 4399 - 4407. [Abstract] [Full Text] [PDF]
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