Two intermediate-avidity cytotoxic T lymphocyte clones with a disparity between functional avidity and MHC tetramer staining
Michael A. Derby,
Jian Wang1,,
David H. Margulies1, and
Jay A. Berzofsky
Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, and
1 Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA
Correspondence to:
J. A. Berzofsky
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Abstract
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The efficacy of cytotoxic T lymphocytes (CTL) has been shown to be highly dependent upon their functional avidity (the sensitivity of their cellular response to MHCpeptide complexes). To examine this relationship, we employed target cell lysis as a quantitative measure and established a set of four CTL clones that exhibited a range of functional avidities spanning more than three orders of magnitude. Within this set, clones displayed a linear correlation between functional avidity and the TCR down-regulation that occurred in response to increasing antigen density. Staining intensity of MHCpeptide tetramer, however, correlated only with the very highest and very lowest avidity clones; the two intermediate-avidity clones showed an inverse relationship between tetramer staining and functional avidity. Compensation for differences in surface levels of TCR improved the correlation, but failed to fully account for this discrepancy. Comparison of TCR signals generated by stimulation of CTL with substrate-bound soluble MHCpeptide or antigen-presenting cells suggested that internal TCR signaling efficiency accounts for at least a portion of the observed functional avidity and suggests the need for caution in directly relating tetramer staining to avidity.
Keywords: antigen binding, FACS, TCR, vaccinia, mouse
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Introduction
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The ability of cytotoxic T lymphocytes (CTL) to clear viral infections depends in great part upon the ability of TCR to specifically recognize peptide antigens presented by the MHC on antigen-presenting cells (APC). The advent of soluble, fluorescently labeled multivalent preparations of MHCpeptide oligomers, including MHCpeptide tetramers (1) and IgGMHCpeptide dimers (2), has provided powerful tools for a rapid and direct quantification of specific CTL by flow cytometry. Flow cytometric staining is interpreted to reflect a specific interaction between TCR and a particular MHCpeptide, an interpretation that has been confirmed by several independent approaches after initial reports of surprisingly high levels of tetramer-positive CTL following immunologic challenge (3). Additionally, the potential contributions of the CD8 co-receptor to the strength of tetramer staining have been characterized and can be eliminated by the choice of appropriate staining conditions (4). As a result, the intensity of tetramer staining has been accepted as a direct measure of the affinity of TCR for the MHCpeptide (58) and extrapolated to be an indication of the actual sensitivity of the CTL response to MHCpeptide antigen density (3). Regardless of the importance of the TCRMHCpeptide interaction, however, it remains only the initial step of a complex signaling process that terminates far down-stream in the CTL response. Therefore, we use the term functional avidity to refer to the overall sensitivity of the T cell response to antigen density, including not only the initial thermodynamic affinity of the TCRMHCpeptide interaction but also the subsequent efficiency with which that interaction is eventually realized as a cellular response.
While pursuing studies on high- and low-avidity CTL, we generated a series of >30 clones that varied in their functional avidity for peptide P18-I10 (I10), the principle H-2Dd CTL epitope in the V3 loop of HIV-1IIIB gp160. Four of those clones were selected for extended study based upon their functional avidity as defined by CTL lysis. TCR down-regulation reflects the amount of signaling through the TCR (912), and thus, as expected, TCR down-regulation showed a direct and linear relationship with functional avidity. We were surprised, however, to discover that staining with H-2Dd/I10 tetramer showed positive correlations only with the very highest and very lowest avidity clones of the four selected for extended study. Two intermediate-avidity clones demonstrated a striking inverse correlation with both functional avidity and TCR down-regulation. Since functional avidity measures the actual CTL response to MHCpeptide antigen density, it results from a complex summation of factors that reflect both the initial TCRMHCpeptide interactions (such as dwell time and concentration) as well as aspects of the signaling pathways downstream from TCR engagement. Further characterization of these two clones demonstrated that differences in surface concentrations of TCR could only partially account for the discrepancy between functional avidity and tetramer staining. As a result, we suggest that a portion of measured CTL functional avidity reflects intrinsic signaling efficiency.
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Methods
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Mice, cell lines and virus
BALB/c mice were purchased from the Frederick Cancer and Research Development Center (Frederick, MD). P815 target cells are derived from a DBA/2 mastocytoma. The E1B6 hybridoma, which recognizes the MCMV synthetic peptide in the context of H-2Ld, was the kind gift of L. F. Boyd (NIAID, Bethesda, MD). vPE16 is a recombinant vaccinia virus expressing the gp160 protein from HIV-1IIIB (13).
Peptides and proteins
The P18-I10 (I10) peptide (RGPGRAFVTI) represents the immunodominant CTL epitope in the V3 loop of HIV-1IIIB gp160 in mice of the H-2d haplotype (14,15) and was obtained from Peptide Technologies (Gaithersburg, MD). Soluble recombinant H-2Dd (
3) was isolated as previously described (16). An H-2Dd `motif' peptide (AGPARAAAL) and the H-2Ld synthetic peptide MCMV (YPHFMPTNL) were obtained from Bachem California (Torrance, CA).
H-2Dd tetramer
A cDNA construct encoding the extracellular domains of H-2Dd under control of the T7 promoter was engineered to include sequences encoding the BirA biotinylation signal (17) at the C-terminus of the protein and was provided by Dr K. Natarajan (NIAID, Bethesda, MD). H-2DdBirA was expressed in Escherichia coli, refolded in vitro in the presence of human ß2-microglobulin (ß2m) and either peptide I10 or `motif' peptide according to previously published methods (1,18), and then purified by size-exclusion chromatography on a Superdex-75 gel filtration column (Pharmacia Biotech, Piscataway, NJ). Following purification, MHCpeptide complexes were biotinylated using biotin ligase (Avidity, Denver, CO) at 25°C for 20 h. After removal of free biotin by dialysis against PBS, tetramers were produced by mixing the biotinylated H-2Dd/peptide complex with streptavidinR-phycoerythrin (PE) (Biosource International, Camarillo, CA) at a molar ratio of 8:1. Specificity of the H-2Dd/I10 tetramer was confirmed using the B4.2.3 T cell hybridoma (16). The H-2Dd/motif tetramer was tested against S167, a Ly49A-transfected CHO cell line (the gift of Dr W. Yokoyama, Washington University, St Louis, MO). A control H-2Ld/pMCMV tetramer was made by a similar procedure using an H-2LdBirA-encoding vector (provided by Dr J. Altman, Emory University, Atlanta, GA), human ß2m and synthetic peptide MCMV. It was tested against the E1B6 T cell hybridoma.
Antibodies
Fc Block, and fluorescently conjugated anti-CD3, anti-CD8 (53-6.7), anti-CD28, anti-TCR, anti-TCR-Vß6, anti-TCR-Vß7, anti-TCR-Vß8.3 and anti-TCR-Vß14 were purchased from PharMingen (San Diego, CA). Blocking anti-CD8 (CT-CD8a) was obtained from Caltag (Burlingame, CA). Specific anti-sera against a range of mouse TCR V
and TCR Vß subunits were the kind gifts of K. Hathcock (NCI, Bethesda, MD) and R. Hodes (NIA, Bethesda, MD). Fluorescent anti-rat, anti-rabbit and anti-mouse secondary antibodies were obtained from Sigma (St Louis, MO).
Generation of CTL lines and clones
CTL primary cultures were generated using BALB/c splenocytes from mice previously immunized with vPE16. Responding splenocytes (7.5x106) were co-cultured in a 24-well plate with 3.5x106 irradiated (3000 rad) stimulating splenocytes either previously pulsed for 2 h with 0.0001100 µM of I10 peptide or un-pulsed but in the presence of 0.000055 µM free peptide. Culture medium consisted of RPMI 1640 with HEPES buffer supplemented with L-glutamine, sodium pyruvate, non-essential amino acids, penicillin/ streptomycin, 50 µM ß-mercaptoethanol, 10% fetal bovine serum and 10% rat T-Stim (Collaborative Biomedical Products, Bedford, MA). CTL lines were established from primary cultures and were maintained by culturing 1x107 CTL in T75 flasks in the presence of 0.51x108 irradiated spleen cells in 12.5 ml of medium. CTL clones were established from CTL lines of various avidities by limiting dilution cloning. Individual clones were obtained from plates containing growth in fewer than eight of 96 wells, screened for uniformity of TCR Vß usage and maintained under conditions appropriate for their avidity.
Bis(acetoxymethyl)-2,2':6',2''-terpyridine-6,6''-dicarboxylate (BATDA)/europium CTL assays
Target cells (1x106) were either un-pulsed or pulsed with an appropriate concentration of peptide in culture medium containing 2 mM Probenecid for 2 h at 37°C. During the last 15 min of culture, 5 µl of BATDA was added to each tube of target cells. BATDA-loaded APC were washed 5 times in 12.5 ml of warm culture medium containing 2 mM Probenecid, with a 20 min rest between washes 4 and 5. The assay was initiated with the addition of CTL to the peptide-pulsed, BATDA-loaded and washed APC at a 12.5:1 E:T ratio, in culture medium that also contained 2 mM Probenecid. At either 3 or 4.5 h, 20 µl samples of supernatant were added to 200 µl of europium solution in a 96-well plate and shaken for 15 min. Free BATDA released into the supernatant by lysis of the target cells was then measured by the time-delayed fluorescence of BATDA-chelated europium in a Wallac Victor2 (Perkin-Elmer, Gaithersburg, MD). Results from means of triplicate samples were usually displayed as the percent specific target cell lysis, although occasionally results were normalized to the plateau of maximal fluorescence exhibited by each CTL line or clone in order to more clearly allow direct comparisons between lytic curves. Specific lysis was calculated as previously described (19). In those experiments where the avidities of CTL lines were directly compared, a peptide concentration was determined that resulted in a plateau of maximal lysis for all CTL lines and clones by 4.5 h, and the CTL lysis of target cells was normalized to 100% of maximal lysis at that antigen density. With I10 peptide, even low-avidity lines or clones showed maximal lysis of target cells pulsed with concentrations >5 µM I10 peptide. Maximal lysis usually varied between 70 and 90% of total APC.
Affinity and functional avidity
TCR affinity refers to the association constant of monomeric TCR for MHCpeptide in the absence of co-receptors and accessory molecules. In contrast, the functional avidity of CD8+ CTL refers to the actual sensitivity of the cellular response to MHCpeptide antigen density. In these experiments, it was defined as a value equal to the negative logarithm of the pulsed peptide concentration that resulted in 50% maximal target cell lysis.
Antigen presentation by P815 or substrate-bound soluble H-2Dd
For TCR down-regulation or proliferation assays, P815 target cells were pulsed for 2 h with various concentrations of I10 peptide in medium at 37°C and then washed 4 times with medium prior to use. Other assays utilized Nunc Immulon-IV or Maxisorb (PGC Scientific, Frederick, MA) 96-well plates or Delfia 12-well strips (Perkin-Elmer) coated with 0.25 µg/well of soluble H-2Dd and then pulsed overnight with 050 µM I10 peptide, as previously described (20). Wells were rinsed, blocked with complete medium for at least 2 h at 37°C and then washed 4 times with complete medium prior to addition of CTL.
Flow cytometry and tetramer staining
For flow cytometric analysis, solutions containing >2x105 CTL were washed and re-suspended in FACS buffer: Ca2+- and Mg2+-free Dulbecco's salt solution containing 0.2% BSA and 0.1% sodium azide. Cells were incubated at 4°C with the appropriate antibody in FACS buffer for 15 min and then washed. Where necessary, a secondary reagent was then added in FACS buffer for an additional 15 min at 4°C and the cells were again washed. Samples were analyzed using CellQuest software on a FACScan or FACStar (Becton Dickinson, Mountain View, CA). Background staining was assessed by staining with the secondary antibody following an isotypic control primary antibody. For tetramer staining, CTL were rested for >5 days, washed in FACS buffer, blocked for 20 min on ice with Fc Block and then stained with fluorescent anti-CD3, anti-CD8, anti-TCR or 24 µg MHC tetramer with blocking anti-CD8 (CT-CD8a) for 60 min in FACS buffer on ice. Some experiments examined tetramer staining for 60 min at 37°C. After washing once with cold FACS buffer, the cells were analyzed by flow cytometry. Fluorescence intensity was measured as the geometric mean.
Tetramer/TCR and controls for tetramer staining
CTL were stained as usual for tetramer and TCR levels. After subtracting the background staining of isotypic controls or un-conjugated streptavidinPE, the geometric mean of tetramer staining was divided by the geometric mean of TCR staining to produce the ratio of tetramer to TCR density. At very low levels of tetramer and/or TCR staining, small experimental variations in background staining increased the variation in this ratio, particularly for clone 6Aa which stained very poorly with tetramer (see Fig. 4A
). In part, this also reflects the difficulty in obtaining directly comparable negative controls for tetramer reagents. In this work, we used three controls: (i) un-conjugated streptavidinR-PE, (ii) an H-2Dd tetramer conjugate folded around a `motif' peptide designed to have little side-chain interaction with the TCR and presumably isolating those interactions unique to the TCRH-2Dd contact zone exclusive of the peptide, and (iii) an H-2Ld/pMCMV tetramer conjugate that should show no interaction with TCR specific for H-2Dd/I10 peptide.

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Fig. 4. (A) A correction for the level of TCR staining per cell can improve the relationship between tetramer staining and functional avidity. The levels of Dd/I10 tetramer staining were normalized by dividing by the levels of TCR fluorescence and then plotted against the functional avidity. Correcting for the TCR levels adjusts for both cell size and TCR density, and helps compensate for the high staining seen on the large MLd clonal cells. (B) The relationship between tetramer staining per TCR and functional avidity is linear only if the signaling efficiency is equivalent in all CTL (diagonal line). Functional avidity is the product of an interaction between TCR affinity and signaling efficiency: for any given functional avidity, the vertical arrows indicate that there is a range of tetramer/TCR staining that could result depending upon the signaling efficiency of the CTL. If the signaling efficiency is relatively low, CTL would fall above the diagonal, whereas if the signaling efficiency is relatively high, they would fall below. Thus, low signaling efficiency in CTL with `B' functional avidity would be compensated by high TCR affinity and result in high tetramer/TCR staining `b'. Correspondingly, high signaling efficiency in CTL with `C' functional avidity would be compensated by low TCR affinity and result in low tetramer/TCR staining `c.' As a result, the functional avidity of these two CTL (`B' `C') would be inversely related to their measured tetramer/TCR staining (`c' `b').
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Results
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CTL clones derived from high- and low-avidity lines retain their parental avidity
In earlier work, we described the generation of high- and low-avidity CTL lines by using APC presenting high antigen densities to select for CTL with low avidity and low antigen densities to produce CTL with high avidity (19). The functional avidity of CTL lines was previously defined operationally as inversely related to the MHCpeptide antigen density required to induce a specific CTL response. In the present work, we made new CTL lines using continuously present I10 peptide at concentrations ranging from 5.0 to 5x104 µM (Fig. 1A
), rather than peptide pulsed onto APC. Functional characteristics of the CTL were still assessed by peptide pulsed APC, however, and we more specifically defined functional avidity as the negative logarithm of the pulsed peptide concentration that resulted in 50% maximal target cell lysis. The range of CTL functional avidity obtained was comparable to the range previously obtained using APC pulsed with peptide, although the concentrations of peptide necessary to generate CTL lines of low or very low avidity differed. More than 30 clones were established from these lines (data not shown). As expected, CTL lines gave rise only to clones of similar functional avidity. Based on their lytic properties, four clones representing the entire range of functional avidity were selected for further characterization (Fig. 1B
).

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Fig. 1. CTL lines of varying functional avidity can be generated from memory splenocytes exposed to different concentrations of free peptide. (A) After 4 weeks selection against free I10 peptide concentrations ranging between 0.0005 and 5 µM, CTL lines already exhibit characteristic avidities when lytic activity is measured with peptide-pulsed P815 target cells. (B) Clones isolated from a panel of CTL lines of varying functional avidity retained the avidities expressed by their parental lines: (1) 6Aa, from a CTL line generated with 5 µM free peptide; (2) MLd, from a CTL line generated with 100 µM peptide-pulsed APC; (3) 1Da, from a CTL line generated with 0.005 µM free peptide; and (4) MHb, from a CTL line generated against 0.0001 µM peptide-pulsed APC. Note that free peptide is much more effective at inducing low-avidity CTL. Results are expressed as the percent of maximal specific target cell lysis in order to more easily compare the functional avidities of the CTL clones. (C and D) TCR signaling was measured by flow cytometry as the down-regulation of TCR at 3 h in response to antigen density. Both high- and low-avidity clones showed a similar range of functional avidity following stimulation either by APC presenting MHCpeptide (with an E:T ratio of 1:15) (C) or by isolated, substrate-bound soluble MHCpeptide without other accessory molecules (D). Note that in these two systems the similarity in effective peptide concentrations used to load the MHC is likely serendipitous.
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CTL exhibit similar avidities when tested against APC or H-2Dd
The CTLAPC interaction involves a number of co-stimulatory and adhesive molecules within and around the `immunological synapse' or `supramolecular activation cluster' (SMAC), several of which have been directly implicated in TCR signaling (21,22). We have previously demonstrated that co-stimulatory and adhesive molecules do not correlate with, and therefore are unlikely to explain, differences in apparent TCR avidity (19). Similarly, they cannot explain differences between the avidity of TCR transgenics (M. A. Alexander-Miller, pers. commun.). Moreover, as shown in Fig. 1
(C and D), and other data not shown, the TCR signal (as measured by TCR down-regulation) shows a comparable range of avidity regardless of whether the CTL clones were stimulated by peptide-pulsed APC expressing co-stimulatory and adhesive molecules or by purified, substrate-bound soluble H-2Dd/peptide. Consistent with previous reports, the high-avidity MHb clone initially shows TCR down-regulation at very low antigen densities comparable with those inducing cytolytic activity, while the very low-avidity 6Aa clone shows little or no down-regulation even at high antigen densities where it does have cytolytic activity (23). Furthermore, as shown in Fig. 2
(A), the TCR down-regulation at 2.5 h on clones exposed to substrate-bound H-2Dd/I10 pulsed with 5.0 µM I10 peptide exhibits a linear relationship with the functional avidity of the same clones determined by CTL lytic assay (correlation coefficient = 0.999). Not only does this provide evidence for the equivalence of the two methods, but also strongly suggests that accessory molecules on the APC are not required to produce this range of functional avidity using the strong peptide antigen I10, since they are completely absent from the H-2Dd/I10 substrate.

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Fig. 2. Functional avidity is directly correlated with TCR signal but not with TCR affinity. (A) TCR signal was measured as the percent down-regulation of TCR 2.5 h after exposure to substrate-bound H-2Dd pulsed with 5 µM I10. A typical result shows a direct correlation (R = 0.999) between TCR signal and functional CTL avidity (determined as the negative logarithm of the half-maximal lysis of peptide-pulsed P815 targets). (B) H-2Dd/I10 tetramer staining of clonal CTL was compared to their functional avidity and shows a dramatic reversal of expected staining for the two intermediate-avidity clones. The results from five experiments are averaged together for simplicity of presentation. (C) CTL clones were stained with PE-conjugated H-2Dd/I10 tetramer or control H-2Dd/motif or H-2Ld/MCMV tetramers and examined with flow cytometry. The intermediate-avidity clones show an inverse relationship between functional avidity and the amount of tetramer staining.
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Binding of H-2Dd tetramer is not necessarily indicative of CTL functional avidity
MHC tetramer binding has frequently been reported not only as an indication of TCR specificity, but also as a measure of T cell avidity (58). Since we were interested in determining whether TCR affinity would reflect the functional avidity of our CTL clones, we used PE-conjugated H-2Dd tetramers to examine their TCR affinity. As expected, the highest avidity clone, MHb, bound the highest amount of tetramer, while the lowest avidity clone, 6Aa, showed the lowest amount of specific tetramer staining, but the two intermediate clones showed a dramatic reversal in their tetramer staining compared to their functional avidity (Fig. 2B and C
). Control staining under the same conditions with H-2Dd/motif or H-2Ld/MCMV tetramers was very low, demonstrating a lack of non-specific TCR/tetramer interactions in this system. In other systems, staining with tetramers at 37°C has been reported to provide a more accurate indication of TCR affinity (24). In this case, however, all four clones showed nearly identical staining, at both 37 and 4°C (data not shown), and thus temperature had no apparent effect on the disparity between tetramer staining and functional avidity. As a result, all staining was conducted at 4°C to minimize extraneous effects due to clustering, etc.
TCR Vß usage is not correlated with functional avidity
Our laboratory originally reported that BALB/c CTL specific for H-2Dd/I10 utilize TCR Vß from a limited pool, primarily Vß8 and Vß14 (24). A more recent report described the usage of TCR Vß on CTL lines specifically selected for high and low avidity, and concluded that low-avidity lines might preferentially use Vß8 (19). In order to further characterize any relationship between TCR Vß and CTL functional avidity, we examined Vß usage in a series of CTL lines after they had undergone selection for 4 weeks with APC presenting various concentrations of free peptide. As shown in Table 1
, both high- and low-avidity CTL lines in this particular series used Vß8 as well as Vß7, but usage of Vß6 differed. If this series is considered in the context of all previous data from this laboratory (19,24), however, CTL avidity shows no correlation with Vß usage. For example, of the four clones characterized, both the lowest avidity clone (6Aa) and the highest avidity clone (MHb) used Vß8.3.
CD8 and TCR levels vary between clones but demonstrate no correlation with functional avidity
In order to assess the potential contribution of other factors to tetramer binding, staining was repeated in the presence of saturating levels of blocking anti-CD8 (clone CT-CD8a), while other aliquots from the same cell suspensions were stained for CD8, TCR and CD3 in the absence of blocking anti-CD8. There was little or no effect of anti-CD8 upon H-2Dd/I10 tetramer staining, and the intermediate clones continued to show an inverse relationship between tetramer staining and functional avidity (Fig. 3
). The levels of CD8 staining were very high in the very low-avidity clone 6Aa, but were roughly equivalent in the other clones. On the other hand, there appeared to be a substantially greater amount of TCR and CD3 present on clone MLd than on clone 1Da (Fig. 3
and data not shown).

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Fig. 3. Levels of TCR are much higher on the MLd clone than on the 1Da clone. Clones were stained with H-2Dd tetramer loaded either with I10 peptide or a non-specific `motif' peptide, or with H-2Ld tetramer loaded with a non-specific `motif' peptide, all in the presence of anti-CD8 to block non-specific CD8MHC interactions. Other cell aliquots were stained with labeled antibodies against TCR or CD8. Cells were then analyzed by flow cytometry. While the relative levels of CD8 staining were similar on all clones except the very low-avidity 6Aa clone, which showed background levels of tetramer staining, the amounts of TCR and CD3 on the surface of MLd were 8- to 12-fold higher than on the surface of 1Da.
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Correction for TCR levels only partially accounts for discrepancies between tetramer staining and functional avidity
Since we suspected that the observed difference in TCR levels might plausibly explain the increased tetramer binding to clone MLd (5), we corrected for the levels of TCR present on the cell surface and then re-examined tetramer staining on all four clones at 4°C. Even though the relationship between tetramer staining and functional avidity does improve, it is still obviously neither linear nor exponential (Fig. 4A
). Taken as a whole, these results indicate that, although tetramer staining cannot always be relied upon as a direct measure of functional CTL avidity, a correction for TCR per cell can improve its accuracy, consistent with the results of Crawford et al. (5) for class II MHC tetramers.
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Discussion
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Initially, studies examining tetramer binding related the intensity of staining to the specificity of CTL for the MHCpeptide employed in the tetramer (3). Furthermore, there is evidence suggesting that the intensity of tetramer staining reflects not only the affinity of the TCR, but also the functional avidity of the CTL; thus recent studies have used tetramer staining as a direct indication of CTL avidity (68). Following this rationale, we also attempted to relate the staining intensity of H-2Dd/I10streptavidin tetramers to CTL functional avidity. As expected, the high-avidity clone MHb stained very strongly with tetramer, while the very low-avidity clone 6Aa showed a level of staining only slightly above background. The two CTL clones of intermediate avidity, however, demonstrated staining intensities dramatically opposite their functional avidity. The MLd low-avidity clone stained more like the high-avidity clone MHb, yet demonstrated a functional avidity barely above that of the very low-avidity 6Aa clone. In contrast, the staining intensity of clone 1Da was barely above that of 6Aa, despite a functional avidity nearly as high as that of MHb (cf. Fig. 1B
and 2A with 2B
).
Accessory molecules are unlikely to account for this difference. An earlier description from this laboratory of high- and low-avidity CTL lines against H-2Dd/I10 showed no relationship between the surface levels of measured accessory molecules and the functional avidity of CTL lines (19). Additionally, that work demonstrated that use of anti-CD3 to bypass the TCRMHCpeptide interaction evoked a range of CTL avidity consistent with an additional mechanism beyond engagement of the TCR by MHCpeptide. In this work, we have more comprehensively demonstrated that such molecules have little functional impact since clones exhibited a nearly identical range of functional avidity (and the same maximal response) after stimulation with APC or with substrate-bound MHC that lacked all accessory molecules (Fig. 1C and D
, and data not shown). Although CD8 could potentially have had a modifying role, this appears unlikely since addition of blocking anti-CD8 antibodies had no effect upon tetramer staining. Additionally, I10 is a strong antigen (25,26), and CD4 and CD8 have previously been shown to have little effect on strong TCR signals (27). Correspondingly, it is equally difficult to ascribe the difference either to an altered binding of the TCR or to a modifying effect of CD8. Interestingly, the peptide concentrations used to load the APC and substrate-bound H-2Dd were within 10-fold of each other, and were thus strikingly similar, but this similarity is most likely fortuitous since functional densities of H-2Dd/I10 concentrated within the SMAC may differ from those densities achieved with soluble H-2Dd/I10 bound to plastic.
Crawford et al. (5) have previously reported that correcting the class II MHC tetramer fluorescence for TCR density (tetramer/TCR) allows a linear relationship between tetramer/TCR and TCR affinity. A similar correction for the clones in this report, although improving the correlation somewhat, failed to provide a comparably linear relationship with CTL functional avidity (Fig. 4A
). If TCR affinity were directly equivalent to CTL functional avidity, then a plot of avidity versus tetramer/TCR fluorescence should be linear. This is not always the case, since CTL exhibiting 3 log10 differences in avidity can be selected from transgenic splenocytes that display similar amounts of an identical TCR (M. A. Alexander-Miller, pers. commun.). On the other hand, if functional avidity is a product of TCR affinity, internal signaling efficiency and effector efficacy (i.e. the ability to effectively deliver perforin, granzymes, IFN-
, etc.), then tetramer/TCR fluorescence would vary with the affinity of the TCR, as shown diagrammatically by the vertical arrows in Fig. 4(B)
, but tetramer/TCR fluorescence might not be linear for any given set of CTL. Thus, referring to Fig. 4(B)
, if clones A, B, C and D consecutively increased in measured functional avidity, but clone B had a high-affinity TCR and C a low-affinity TCR, then their order of increasing tetramer/TCR fluorescence might actually be a, c, b and d. In this model, only CTL at the extremes of avidity would be unlikely to have combinations of affinity and signaling efficiency that could cause discrepancies between tetramer/TCR fluorescence and measured functional avidity.
While these two clones may not be typical of CTL in general, they are not unique. As mentioned above, transgenic CTL can exhibit 3 log10 differences in avidity (M. A. Alexander-Miller, pers. commun.) and, although Yee et al. (6) concluded that tetramer staining could be taken as a good indication of functional avidity based upon the majority of CTL, their results also indicated that tetramer staining of up to 20% of the CTL does not correlate with avidity, consistent with our findings. Furthermore, their comparisons of the tetramer staining in high- and low-avidity clones showed a 23 log10 difference in one case, but only a 2- to 3-fold difference in the other. Since TCR affinity (or the surface levels of TCR or accessory molecules) cannot fully account for functional CTL avidity, but avidity does closely correlate with TCR signaling as assessed by TCR down-regulation, then the discrepancy must reflect internal signaling efficiency. As a result, we suggest that tetramer staining should be corrected for levels of TCR in determinations of TCR affinity, but suggest caution when directly relating tetramer staining to functional avidity in populations of CTL where avidity of individual CTL cannot be ascertained.
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Acknowledgments
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We thank Dr K. Natarajan for providing the construct encoding the H-2DdBirA, and for guidance in the expression, refolding and purification of tetramers. We thank Dr J. Altman for providing the H-2LdBirA construct. We would also like to thank Dr P. Henkart and Dr M. A. Alexander-Miller for critical review of the manuscript and helpful discussions.
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Abbreviations
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ß2m ß2-microglobulin |
APC antigen-presenting cell |
BATDA bis(acetoxymethyl)-2,2':6',2''-terpyridine-6,6''-dicarboxylate |
SMAC supramolecular activation cluster |
tetramer/TCR MHC tetramer fluorescence divided by TCR density |
PE phycoerythrin |
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Notes
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Transmitting editor: I. Pecht
Received 9 December 2000,
accepted 5 March 2001.
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