Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR

Thomas M. Lawson, Stephen Man, Eddy C. Y. Wang, Sheila Williams, Nicholas Amos, Geraldine M. Gillespie1, Paul A. Moss2 and Leszek K. Borysiewicz

Department of Medicine, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK
1 Institute of Molecular Medicine, Oxford OX3 9DU, UK
2 Department of Haematology, University of Birmingham, Birmingham B15 2TH, UK

Correspondence to: T. M. Lawson


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have shown that the dominance of CD8+ T cells expressing TCR Vß17 in the adult HLA-A*0201-restricted influenza A/M158–66-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vß17+ cells, subdominant M158–66-specific clones expressing non-Vß17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M158–66 complex could influence functional properties, M158–66-specific clones expressing subdominant (non-Vß17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vß17+) TCR. The Vß17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vß gene segments. All Vß17+ CTL clones tested bound HLA-A*0201/M158–66 tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vß17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vß17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vß17+ CTL. The dominance of Vß17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M158–66 complex.

Keywords: cytotoxic T lymphocyte, infectious immunity virus, human, repertoire development, TCR


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) response to influenza A is dominated by M158–66-specific CD8+ T cells which express Vß17+ TCR (1,2). Such TCR dominance may reflect a trimming of the available TCR repertoire against an antigen (2,3) or might be explained by maturation of the response with selection of a narrow range of high-affinity TCR after repeated antigen exposure (46). In the preceding paper in this issue we showed that TCR usage during in vitro primary HLA-A*0201-restricted M158–66-specific responses is diverse and TCR Vß17 dominance is acquired following the first exposure to influenza A. This observation suggests that thymic selection is not an over-riding factor in shaping the TCR repertoire selected by influenza A in HLA-A*0201 subjects. Having demonstrated that oligoclonal TCR Vß17 usage in the M158–66-specific CD8+ T cell response is acquired, it becomes important to determine the fate of the `non-Vß17+' M158–66-specific clones identified during the primary in vitro cord blood response. Do they persist, but remain subdominant? Previous studies have failed to identify subdominant M158–66-specific clones in HLA-A*0201+ adult subjects. It is possible that this resulted from the limitations of the in vitro conditions used (1,2,7,8)

In this study we removed Vß17+ cells from peripheral blood mononuclear cells (PBMC) prior to in vitro stimulation with influenza A and generated subdominant M158–66-specific clones in all adult individuals studied. The isolation of M158–66-specific clones expressing different TCR enabled a comparison of the functional properties at a clonal level. We compared phenotype, peptide sensitivity, CD8 dependence and HLA-A*0201/M158–66 tetramer binding of these CTL clones to allow comparison of the relative avidity of different M158–66-specific clones for cognate antigen. Tetramers are particularly valuable reagents for this type of comparative study as their intensity of binding to and the rate of dissociation from T cells has been used to estimate the affinity and kinetics of the specific TCR–MHC–peptide interaction (6,9).


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of Vß17+ and `non-Vß17+' M158–66-specific CTL lines and clones
HLA-A*0201-restricted influenza A/M158–66-specific CTL lines and clones were generated as previously described (2). Briefly, freshly isolated PBMC from an HLA-A*0201 adult volunteer were depleted of either Vß17+ cells or control Vß22+ cells by immunomagnetic depletion. Depletion was typically >97%. The PBMC depleted of Vß17+ or Vß22+ cells were infected with 200 HAU of influenza A (AX/31). After 1 h incubation at 37°C, cells were cultured in RPMI-10(AB) [RPMI 1640 (Sigma, St Louis, MO), 10% human AB serum, 2 mM L-glutamine, 1 mM sodium pyruvate and 25 mM HEPES buffer] for 7 days. On day 7 and weekly thereafter, the cells were harvested, counted and replated in 24-well plates at 2x106 cells/well in RPMI-10(AB) containing 10 IU/ml IL-2 (Boehringer Mannheim, Mannheim, Germany). Cultures were re-stimulated with irradiated autologous M158–66-pulsed (10–6 M) PBMC at a responder:stimulator ratio of 1:1. On day 21 cultures were tested for influenza A and M158–66-specificity by 51Cr-release assay and lines were confirmed to be Vß17+ and `non-Vß17+' respectively by two-colour immunofluorescence analysed by flow cytometry. If M158–66-specificity was not detected from Vß17 depleted samples, the procedure was repeated using a re-stimulation protocol designed to induce `primary' in vitro peptide-specific CTL responses (11).

CTL clones were generated from Vß17+ and `non-Vß17+' CTL lines by limiting dilution as described (2), and stimulated weekly using irradiated allogeneic PBMC, phytohemagglutinin (1 µg/ml; Sigma), lymphocult-T (1 IU/well; Biotest, Dreieich, Germany), rhIL-2 (20 IU/ml; Boehringer Mannheim) and IL-7 (5 ng/ml; Genzyme, Cambridge, MA). Clones were assayed for M158–66 specificity after 21 days.

Cytotoxicity assays
Standard 51Cr-release assays were performed using 51Cr-labelled HLA-A*0201+ CIRA2 targets (2000/well). Targets were pulsed with various concentrations of M158–66 peptide (10–6 to 10–13 M) or infected with 200 HAU influenza A (AX/31). Control targets included CIRA2 cells in the absence of peptide and CIRA2 cells pulsed with the HLA-A*0201-binding Plasmodium cp36 peptide (36). For peptide sensitivity assays an E:T ratio of 5:1 was used for each clone. For CD8 blocking experiments, the CTL clones were incubated with varying concentrations of anti-CD8 antibody (OKT8) for 30 min prior to adding the targets. A control isotype-matched CD4 blocking antibody (OKT4) was used in each experiment.

Staining and analysis of CD8+T cells
Cells were analysed by flow cytometry using the FACScan (Becton Dickinson, La Jolla, CA). Live cells were gated based on FSC/SSC profiles. The TCR Vß gene segment usage by the various clones generated was determined by two-colour immunofluorescence using a panel of FITC-conjugated anti-TCR Vß mAb (TCR Workshop, San Francisco, 1995) and anti-CD8–phycoerythrin (Becton Dickinson). TCR {alpha}ß and CD8 expression levels were determined using FITC-conjugated anti-TCR{alpha}ß and phycoerythrin-conjugated anti-CD8 (both from Becton Dickinson). The staining procedure was as previously described (2).

Tetramer binding
The soluble tetrameric HLA-A*0201/M158–66 complex conjugated to streptavidin–TriColor used in this study has previously been described (8). In brief, cells were incubated for 1 h at 4°C, with soluble tetrameric HLA-A*0201/M158–66 complexes conjugated to streptavidin–TriColor (0.5 mg/ml), before washing twice with PBS and fixing in 2% paraformaldehyde. Single-colour fluorescence intensity was measured by flow cytometry using the FACScan (Becton Dickinson).

Cytokine production
CTL clones were incubated with CIRA2 cells (2000/well) which had been pulsed with M158–66 peptide (10–6 M), a control Plasmodium cp36 peptide (10–6 M) or no peptide at an effector to stimulator ratio of 5:1 for 6 h. IFN-{gamma}, tumour necrosis factor-{alpha} (TNF-{alpha}) and IL-4 production by the clones was measured in the supernatant using standard ELISA kits (Genzyme). IFN-{gamma} production as a function of the concentration of M158–66 peptide used to pulse target cells was also determined.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TCR Vß gene segment usage of HLA-A*0201-restricted M158–66 peptide-specific clones
CTL clones recognizing M158–66 peptide are largely Vß17+ and previous reports have suggested that the HLA-A*0201-restricted M158–66-specific CTL response is highly dependent on the Vß17+ population (1,7,8). To investigate whether subdominant, `non-Vß17+' M158–66-specific CTL clones exist, we depleted Vß17+ cells from fresh PBMC prior to in vitro re-stimulation. `Non-Vß17+' M158–66-specific CTL lines were successfully generated from six of six HLA-A*0201 individuals (Table 1Go). For three of the subjects (MJ, MA and LM), `non-Vß17+' M158–66-specific CTL lines were successfully generated using the `standard' re-stimulation protocol (10). However, in the other three subjects (LF, MR and JM), `non-Vß17+' M158–66 CTL could only be generated by using a re-stimulation protocol designed to generate in vitro primary responses (11) (Table 1Go).


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Table 1. Cytotoxicity and TCR Vß gene segment usage of M158–66-specific CTL lines generated from adult PBMC depleted of either Vß17+ or Vß22+ cells prior to in vitro stimulation
 
The TCR usage of these CTL lines was shown to be heterogeneous and different for each individual (Table 1Go). No significant TCR Vß expansion was detected for two of the CTL lines (MA and JM). M158–66-specific clones expressing Vß13.6, Vß23 and Vß8.1 were derived from one of these CTL lines (MR). These Vß cell populations comprised 1.5, 1 and 4% respectively of the CD8 population of freshly isolated PBMC from this individual. Untreated PBMC from the same individual resulted in a CTL line containing >90% CD8+Vß17+ cells and several Vß17+, but no `non-Vß17+' clones, were generated. Therefore, although Vß17+ CTL dominate the HLA-A*0201-restricted influenza A/M158–66-specific CTL response, subdominant CTL expressing other TCR specific for the same MHC–peptide complex persist and can be expanded in the absence of the dominant Vß17+ CTL population during in vitro culture.

Peptide sensitivity of HLA-A*0201-restricted M158–66-specific CTL clones
Efficiency of target cell lysis by CTL is dependent on the TCR affinity for and the half-life of the interaction of the TCR with the MHC–peptide complex (6,12). Therefore, the functional cytotoxic activity of both Vß17+ and non-Vß17+ M158–66-specific clones was compared. In the presence of high concentrations of exogenous M158–66 peptide (10–6 M), all clones produced similar levels of target cell lysis at equivalent E:T ratios (Fig. 1Go). Similar levels of lysis were also seen at equivalent E:T ratios using targets infected with influenza A virus (Fig. 1Go) confirming that clones expressing either the dominant Vß17+ TCR or non-dominant TCR were able to recognize the M158–66 epitope after endogenous processing and presentation. Cytotoxicity was then compared using reducing concentrations of M158–66 peptide and a fixed E:T ratio of 5:1 for each clone. Three TCR Vß17+ clones were compared with similar results (data not shown) and one selected for further study. Peptide sensitivity assays were performed in parallel on the Vß17+ clone and the three clones expressing `non-Vß17+' TCR, using the same peptide-pulsed CIRA2 target cells. The TCR Vß17+ clone lysed target cells at concentrations of M158–66 peptide >3 log10 fold lower than the `non-Vß17+' clones (Fig. 2Go), suggesting that the Vß17+ TCR have a greater affinity for HLA/A*0201/M158–66 than other M158–66-specific TCR.



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Fig. 1. Cytotoxic activity of M158–66-specific clones expressing different TCR. Influenza A/M158–66-specific clones expressing different TCR produce similar levels of lysis at equivalent E:T ratios in the presence of excess exogenous M158–66 peptide and were also able to recognize endogenously processed peptide. 51Cr-release assays were performed using CIRA2 targets (2000/well) pulsed with M158–66 peptide (10–6 M) or infected with influenza A virus (200 HAU). Lysis of targets in the absence of peptide or pulsed with a control A2-binding Plasmodium cp36 peptide was <3% for each clone. Assays were performed in triplicate and the SD of the mean is shown in each case.

 


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Fig. 2. Peptide sensitivity of M158–66-specific clones expressing different TCR. The M158–66-specific clone expressing Vß17 recognized targets pulsed with lower M158–66 concentrations than the other M158–66-specific clones. CIRA2 targets (2000/well) were pulsed with reducing concentrations of peptide and the same batch of targets was used for each clone. A fixed E:T ratio of 5:1 was used for each clone. Assays were performed in triplicate and the SD of the mean was <5% in each case.

 
Binding of soluble HLA-A*0201/M158–66 tetramers
Given the reported relation between tetramer binding, TCR affinity and functional response observed following T cell activation (6,9,12,13), we assessed the binding of soluble HLA-A*0201/M158–66 tetramers to M158–66-specific CTL clones, and compared this to the differences observed in the cytotoxicity assays. Equivalent tetramer binding to two Vß17+ clones was confirmed in this study (data not shown), and similar binding of the same reagent to TCR Vß17+ M158–66-specific CTL lines and clones has been demonstrated in other studies (8,14). The TCR Vß17+ and the TCR Vß8.1+ clones bound tetramer with equal fluorescence intensity, but interestingly the Vß13.6+ and the Vß23+ clones did not bind tetramer, despite evidence of M158–66-specific cytotoxicity (Fig. 3Go). The concentration of tetramer used (0.5 mg/ml), produced saturation binding to Vß17+ clones and the Vß8.1+ clone, but even greater tetramer concentrations (1 mg/ml) did not result in any detectable binding to the Vß13.6+ and the Vß23+ clones (data not shown). The differences observed were independent of TCR expression; surface TCR {alpha}ß expression was least for the Vß17+ clone and identical for the three `non-Vß17+' clones (Fig. 4Go). The most likely explanation for the observed differences in tetramer binding was that it reflected variation in the affinity of the individual TCR for the HLA-A*0201/M158–66 complex.



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Fig. 3. Level of HLA-A*0201/M158–66 tetramer binding to M158–66-specific clones. Cells were incubated with the tetramer at 4°C for 1 h before being washed twice and fixed in 2% paraformaldehyde. Tetramer binding to each clone is compared to a negative control (dotted line) and the Vß17+ clone (shaded). Tetramer binding to the `non-Vß17+' clones is represented by a bold continuous line. Tetramer staining was performed in parallel to the assays measuring IFN-{gamma} production and cytotoxic activity of the clones.

 


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Fig. 4. The M158–66-specific clones express similar levels of TCR {alpha}ß. Cell-surface expression of TCR{alpha}ß was determined using TCR {alpha}ß–FITC followed by immunofluorescence analysis by FACS. This was performed on the same day as tetramer staining and determination of M158–66-specific cytotoxicity and IFN-{gamma} production. The Vß8.1+, Vß13.6+ and Vß23+ clones exhibited virtually identical levels of TCR {alpha}ß expression which were marginally greater than that of the Vß17+ clone (shaded).

 
Recently non-specific binding of tetramers through interaction with CD8 has been reported (15). It was therefore important to exclude such an interaction for the Vß8.1 clone, the only `non-Vß17' clone to bind tetramer. The Vß8.1 clone and, as a control, the Vß17+ clone were stained with anti-CD8 (OKT8) to block CD8 binding sites prior to staining with tetramer. No significant decrease in tetramer binding to either clone was observed after CD8 staining (data not shown), suggesting that non-specific interaction with CD8 is unlikely to be the explanation for the observed differences in tetramer staining.

CD8 dependence
The importance of the CD8 co-receptor in the activation of CTL and the stabilization of the TCR–MHC–peptide interaction has been described in a number of studies (1618). CD8 is of particular importance when the interaction between the TCR and its MHC–peptide complex is of very low affinity and characterized by a rapid `off-rate' (12,18). The CD8 dependence of the various M158–66-specific CTL clones was compared by estimating the susceptibility of the clones to inhibition by anti-CD8 mAb (OKT8) to determine whether there was a difference between TCR Vß17+ and `non-Vß17+' clones. These experiments were not performed on the TCR Vß13.6+ clone because of insufficient cell numbers. Both the TCR Vß17+ clone and the TCR Vß23+ clone were CD8 independent, whereas the Vß8.1+ clone was highly susceptible to inhibition by anti-CD8 mAb (Fig. 5Go). Thus the clones could not be separated into TCR Vß17+ and `non-Vß17+' groups based on their individual CD8 dependence. Furthermore, CD8 dependence did not correlate with TCR affinity inferred from peptide–dose response experiments (Fig. 2Go).



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Fig. 5. CD8 dependence of M158–66-specific clones expressing different TCR determined by susceptibility to inhibition by anti-CD8 mAb. CTL were incubated with various concentrations of OKT8 ({blacksquare}) or as a control, equivalent concentrations of OKT4 (•) for 30 min before adding CIRA2 targets (2000/well) pulsed with M158–66 peptide (10–6 M). Lysis of targets in the absence of M158–66 peptide as <5%. E:T ratios of 5:1 were used for each clone. Assays were performed in triplicate wells and the mean percent specific lysis ± SD is plotted. The Vß13.6 clone was not examined because of insufficient cell numbers.

 
Cell-surface phenotypes and cytokine production
Cytotoxicity is not the only effector function of CD8+ T cells; IFN-{gamma} and TNF-{alpha} release is also important (19). In the presence of M158–66 peptide concentrations shown to produce maximal cytotoxic activity (10–6 M), the M158–66-specific clones expressing TCR incorporating the Vß17, Vß23 and Vß13.6 gene segments produced similar quantities of IFN-{gamma} (Fig. 6AGo). However, the Vß8.1+ clone did not produce IFN-{gamma} at peptide concentrations shown to produce maximal cytolytic activity (10–6 M) (Fig. 6AGo). The production of IFN-{gamma} by antigen-specific CTL has previously been shown to require higher peptide concentrations than those required for cytotoxicity (20). These findings were confirmed for the three clones that produced IFN-{gamma}, by performing cytotoxicity assays and measuring IFN-{gamma} production in parallel (Figs 2 and 6BGoGo). Interestingly, however, the differences in peptide sensitivity between the Vß17+ and `non-Vß17+' clones observed in cytotoxicity assays were not seen for IFN-{gamma} release (Fig. 6BGo). All IFN-{gamma}-producing clones were able to produce TNF-{alpha}, but the Vß8.1+ clone also failed to release this cytokine (data not shown). None of the clones released IL-1 or IL-4 (data not shown).



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Fig. 6. (A) IFN-{gamma} production by Vß17+, Vß23+, Vß8.1+ and Vß13.6+ M158–66-specific clones. Stimulator cells were CIRA2 cells pulsed with M158–66 peptide (10–6 M). Control targets were either pulsed with the HLA-A*0201-binding Plasmodium cp36 peptide (10–6 M) or used in the absence of peptide. Stimulator cells were used at 2000/well and a fixed E:T ratio of 5:1 for each clone. IFN-{gamma} was measured by ELISA after a 6-h incubation. Columns represent the mean of triplicate wells ± SD. Cytotoxicity assays were performed in parallel to the above experiments using the same stimulator cells as targets and all clones produced >70% specific lysis under these conditions (data not shown). (B) Effect of peptide concentration on IFN-{gamma} release by M158–66-specific clones expressing different TCR. IFN-{gamma} release was measured by ELISA after a 6-h incubation of effector cells with CIRA2 stimulator cells (2000/well) pulsed with reducing concentrations of M158–66 peptide. Each assay was performed in triplicate wells using an effector:stimulator ratio of 5:1. The SD was <10% for each result.

 
The Vß8.1+ CTL clone was functionally distinct from the other clones in that it did not produce IFN-{gamma} and was CD8 dependent. One possibility is that this CTL clone might belong to a CD8 T cell subset (21). The expression levels of cell-surface markers CD11a, CD11b, CD27 and CD28 on the Vß8.1+ clone were determined by flow cytometry and compared to those on Vß17+, Vß23+and Vß13.6+ clones. The Vß8.1+ clone expressed high levels of CD11b, CD27 and CD28 (Fig. 7Go). In contrast the Vß17+, Vß13.6+ and Vß23+ clones were C11b, CD27 and CD28 (Fig. 7Go).



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Fig. 7. Overlay FACS histograms comparing CD11a, CD11b, CD27, CD28 and CD38 expression in the TCR Vß8 and TCR Vß13.6 clones. The TCR Vß8 clone (shaded area) showed increased surface expression of (C) CD27 (45%) and (D) CD28 (62%) compared to no expression in the other CTL clones, represented here by the TCR Vß13.6 clone (bold black line). There were no significant differences in (A) CD11a, (B) CD11b and (E) CD38 expression. Clones were stained with anti-CD8–TriColor, the appropriate anti-TCR Vß–FITC and a range of phycoerythrin-conjugated mAb to the different cell-surface molecules. Analysis was carried out using standard lymphocyte gates on FSC/SSC, followed by gating on populations positive for CD8 and the appropriate TCR Vß chain.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have compared the functional characteristics of dominant (Vß17+ TCR) and subdominant CTL (`non-Vß17+ `TCR) clones recognizing the same HLA-A*0201/M158–66 complex. Generation of non-Vß17+ CTL clones in vitro required the prior depletion of Vß17+ T cells from PBMC, suggesting that Vß17+ cells have a competitive advantage in vitro. In three donors it was not possible to generate non-Vß17+ CTL clones unless dendritic cell-enriched stimulator cells were used for re-stimulation. This suggests either an extremely low frequency of non-Vß17+ M1 specific CTL or that these CTL required additional co-stimulatory signals provided by dendritic cells.

We have shown that the TCR Vß17+ clones require far lower concentrations of cognate peptide than the `non-Vß17+' clones to mediate cytotoxicity, although all CTL clones were able to lyse influenza A-infected target cells expressing endogenously processed antigen. This suggests that the Vß17+ TCR have a greater affinity for the HLA-A*0201/M158–66 complex than non-Vß17+ TCR (6,12,13,22,23). This is supported by the observed binding of saturating concentrations of tetrameric complexes of HLA-A*0201/M158–66. All Vß17+ clones bound this tetramer at high levels, while two out of three of the `non-Vß17+' clones were unable to bind tetramer despite clear evidence of peptide-specific cytotoxicity and IFN-{gamma} production. Furthermore all tetramer staining was carried out at 4°C, under conditions shown to be optimal for detecting low-affinity cross-reactive TCR (24). Since levels of TCR expression of Vß17+ and `non-Vß17+' clones was equivalent, it is likely that the tetramer binding reflected the affinity of the TCR for the HLA-A*0201/M158–66 complex. However, the Vß8.1+ clone which has a low affinity based on peptide titration (Fig. 2Go) was able to bind tetramer. This raises the possibility that tetramers, as currently configured are not the optimal structure for TCR recognition compared with the MHC class I–peptide complex on a target cell surface. Therefore, some caution must be exercised when assigning absolute TCR affinity and dissociation constants using these reagents.

However, this does not negate their value in relative and comparative studies (6). The frequency of these non-tetramer binding CD8+ CTL clones in the HLA-A*0201/M158–66 response and other antigen-specific responses remains unknown. The detection of such CTL among subdominant CTL suggests that the use of tetramer reagents in isolation may fail to detect a subset of antigen-specific T cells.

Anti-CD8 mAb have been used to block CTL–target interactions. These have been particularly effective in blocking responses of `primary' CTL which have low avidity for target cells expressing the appropriate MHC–peptide complex. It is more difficult or impossible to block `secondary' CTL derived by repeated antigen challenge in vivo or in vitro. In this study, two `low-affinity' T cell clones as defined by peptide sensitivity and tetramer binding could not be blocked using anti-CD8 antibodies. This was also the case for Vß17+ clones, so no correlation could be drawn between operationally defined TCR affinity and TCR dependence.

In this study, higher CTL avidity for HLA-A*0201/M158–66 correlated with expression of TCR Vß17. However, we did not observe any functional advantage of Vß17+ clones in terms of IFN-{gamma} production, as both Vß17+ and `non-Vß17+' CTL (apart from the Vß8.1+ clone) produced similar amounts of IFN-{gamma}. This suggests that cytotoxicity may be the more important parameter for functional selection of CD8+ T cells in this system.

The Vß8.1+ CTL clone was remarkable in several respects. Firstly, despite having a lower affinity for the HLA-A*0201/M158–66 complex as defined by peptide sensitivity, this clone was able to bind tetramer to the same level as Vß17+ CTL clones. These results appear to be contradictory and do not necessarily imply that the Vß8.1+ TCR has the same affinity for the HLA-A*0201/M158–66 complex as the Vß17+ TCR as saturating doses of tetramer were used in this study. It is also possible that TCR with similar affinities for the same MHC–peptide ligand may differ with respect to their off-rate. This may be a more accurate indicator of the functional outcome following TCR activation by specific MHC–peptide ligand than TCR affinity (22,2527). Determination of the kinetics of interaction of the Vß8.1+ TCR with the HLA-A*0201/M158–66 complex might therefore explain the apparent discrepancy between tetramer binding and peptide sensitivity in cytotoxicity assays.

The Vß8.1 clone was the only clone whose cytotoxicity could be inhibited by anti-CD8 mAb, but was also the only `non-Vß17+' M158–66-specific clone able to bind tetramer. Without detailed topological study of the interaction between the complete TCR {alpha}ß complex and the HLA-A*0201/M158–66 complex, it is difficult to explain this result. We confirmed that the tetramer staining of this clone was not through non-specific interaction with CD8. In addition the Vß8.1+clone was the only CTL clone unable to produce detectable IFN-{gamma}:. The identification of a CD8+ CTL clone which exhibits cytotoxicity, but fails to produce IFN-{gamma} has implications for in vitro assays used in the ex vivo detection of CD8+ CTL by the ELISPOT technique (7). Although more sensitive than traditional cytotoxicity assays (7), in isolation it may fail to detect some antigen-specific cells. However, further studies are required to determine if this failure of IFN-{gamma} release by the Vß8.1+ clone is a consequence of long-term in vitro propagation.

The apparently contradictory results obtained with the Vß8.1+ CTL illustrate the importance of studying TCR interactions with MHC–peptide complexes, free of other cell–cell interactions. Most studies to date have examined the relationship between TCR affinity for MHC–peptide ligand and functional responses by introducing minor changes in the peptide (9,12). These studies have focused on a single TCR in each case. In this study we have identified four functionally heterogeneous clones with the same peptide specificity but different TCR. This provides an opportunity for comparative studies on the affinity and kinetics of TCR– MHC–peptide interaction against the same pathogen-derived MHC–peptide complex. It will therefore be important to compare the affinity and kinetics of interaction with the HLA-A*0201/M158–66 complex for these different TCR by surface plasmon resonance, thus removing the influence of other cell–cell interactions.

The respective in vivo roles of the dominant Vß17+ CTL and subdominant `non-Vß17+' CTL in recovery from influenza infection is unknown. The selective expansion of CD8+ CTL expressing TCR with the highest affinity for the HLA-A*0201/M158–66 complex may allow more rapid destruction of influenza-infected cells. If this is the case then why do subdominant `non-Vß17+' CTL persist in the peripheral blood? We have shown that these subdominant CTL can be expanded in vitro in the absence of the dominant Vß17+ CTL. Such a phenomenon has been shown to occur in vivo for HLA-B8-restricted Epstein–Barr virus-specific CTL responses (28). Studies in mouse strains with a deficiency of T cells bearing particular TCR Vß genes have shown that peptide-specific T cell responses characterized by restricted TCR usage can be either crippled (29,30), reduced (3133) or unaffected (34). It remains to be determined whether this is dependent on the nature of the antigen or the experimental model used. Nevertheless, the persistence of subdominant CTL clones with heterogeneous TCR may provide a mechanism to cope with pathogen mutation.

In summary, we have demonstrated that although CTL expressing TCR incorporating the Vß17 gene segment dominate the HLA-A*0201-restricted influenza A/M158–66-specific CTL response in man, a subdominant repertoire of CTL with heterogeneous Vß gene segment usage exists, which can substitute for the dominant population in its absence in vitro. We identify an in vitro functional advantage of Vß17+ clones over other clones in that they were able to mediate cytolysis at lower concentrations of peptide. We have also shown that two-thirds of the `non-Vß17+' clones generated failed to bind tetrameric HLA-A*0201/M158–66 molecules, thus identifying a potential limitation of these reagents particularly when used to study the antigen-selected TCR repertoire (35). These studies also document the considerable functional heterogeneity of human CD8+ CTL, even when specific for a single MHC–peptide complex and where oligoclonality of TCR usage is established. Thus, although the use of ELISPOT and tetramer assays have greatly increased the sensitivity of detection of CD8+ CTL (36), it is clear that these techniques will still fail to detect some functional antigen-specific CD8+ CTL in vitro.


    Acknowledgments
 
This study was supported by an MRC ROPA award. T. M. L. is an ARC Clinical Lecturer in Rheumatology. S. M. is a Royal Society University Research Fellow.


    Abbreviations
 
CTL cytotoxic T lymphocyte
PBMC peripheral blood mononuclear cell
TNF tumour necrosis factor

    Notes
 
Transmitting editor: E. Simpson

Received 24 July 2001, accepted 30 July 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Moss, P. A., Moots, R. J., Rosenberg, W. M., Rowland-Jones, S. J., Bodmer, H. C., McMichael, A. J. and Bell, J. I. 1991. Extensive conservation of alpha and beta chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl Acad. Sci. USA 88:8987.[Abstract]
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