Multimeric soluble peptide-MHC class I complexes bind to T cell receptors (TCR) on the surface of CD8 cytotoxic T lymphocytes (CTL) with sufficient avidity to enable the labelling of CTL according to their antigen specificity . Visualization of antigen-specific CD8+T lymphocytes with fluorogen-conjugated peptide-MHC class I multimers enables both the physical quantification and the phenotypic characterization of CTL responses directly ex vivo, and these properties have led to the widespread adoption of this technique throughout the field of experimental immunology. However, it is well documented that TCR can exhibit a degree of promiscuity in terms of ligand recognition . We recently demonstrated that the multimerization of peptide-MHC class I can allow binding to TCR with affinities that are too low to generate ligand-induced physiological responses, and result in crossreactive staining of HIV-specific CTL lines and clones by unrecognized natural variant antigens . These results suggest that crossreactivity between peptide-MHC class I multimers and low avidity CTL could be relevant to the interpretation of direct ex-vivo staining. The possibility of such cross-staining is likely to be considerably greater when using peptide-MHC class I multimers to stain CTL specific for epitopes derived from antigenically variable pathogens such as HIV-1 . Here we extend our previous study with the demonstration that the cross-staining of CTL populations with peptide-MHC class I multimers can complicate analyses performed directly ex vivo.
In a detailed analysis of virus-specific T cell responses during primary HIV-1 infection, we defined a substantial population of CD8 T lymphocytes in donor SC21 that stained with phycoerythrin-conjugated tetramers of human leukocyte antigen (HLA) B7, folded around the HIV-1 Nef epitope RPMTYKAAL (residues 75–83; reference strain HIV-IIIB) (Fig. 1a) . However, the tetramer-positive population consisted of a smear of CD8+T lymphocytes with different staining intensities. This suggested either that a spectrum of TCR expression densities was present within the tetramer-positive population of CD8+T lymphocytes, or that the RPMTYKAAL-specific response was polyclonal and comprised TCR with variable affinities for the peptide-HLA B7 ligand. Consistent with the former possibility, extensive staining of the population with a monoclonal antibody panel recognizing individual Vβ TCR chain determinants revealed that more than 95% of the antigen-specific CD8+T lymphocytes expressed Vβ14 TCR at three separate time-points (Fig. 1a and data not shown). Similar antigen-specific oligoclonal expansions within the CD8+T lymphocyte population have been reported previously in acute primary HIV-1 infection, and appear to typify the early cellular immune response to virus infection . Furthermore, there was a large discrepancy between the number of antigen-specific CD8+T cells determined physically with the RPMTYKAAL-HLA B7 tetramer compared with functional measurements based on IFN-γ release in ELISpot assays. These incongruities prompted the sequencing of plasma virus from donor SC21, which revealed that 39/40 Nef clones isolated from two separate time-points encoded the variant epitope RPMTYKGAL (data not shown). We therefore performed both peptide-MHC class I tetramer staining and ELISpot analysis using the autologous virus epitope sequence. Although the total number of antigen-specific CD8 T lymphocytes determined with the RPMTYKGAL-HLA B7 tetramer was similar in magnitude to that measured with the RPMTYKAAL-HLA B7 tetramer, staining with tetramer folded around the autologous epitope improved both the intensity and uniformity of staining (Fig. 1a and b). In contrast to staining with the RPMTYKGAL-HLA B7 tetramer, staining with the non-autologous tetramer was abrogated at lower concentrations (Fig. 1b); this observation is consistent with the generation of a high affinity response to the autologous viral epitope, and indicates that the naturally dominant Vβ14 TCR had a lower affinity for the RPMTYKAAL-HLA B7 antigen. In addition, IFN-γ ELISpot analysis demonstrated that the autologous peptide epitope activated a substantially higher proportion of CD8+T lymphocytes, which were stimulated to produce spots that were larger both in size and intensity, compared with the RPMTYKAAL sequence (Fig. 1c). Quantitative differences between the epitopes were also seen in direct ex-vivo chromium release lysis assays (data not shown). The ‘wild-type’ sequence RPMTYKAAL therefore acted as a poor or weak agonist for SC21 CTL.
These findings demonstrate that crossreactivity can complicate direct ex-vivo peptide-MHC class I multimer-based analyses of CTL populations responding to pathogens with variable antigenicity. Marked discrepancies between physical and functional measurements of antigen-specific CTL, together with non-uniform staining of low intensity in oligoclonal CD8+T lymphocyte populations, should alert investigators to the possibility that individual CTL responses are directed against variant epitopes.
The authors would like to thank Anele Waters and the donor SC21 for excellent collaboration.
David A. Priceb*
Sara J. Dawsona
Philippa J. Easterbrookc
Rodney E. Phillipsa
Andrew K. Sewella
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