RESULTS
We selected three epitope-specific populations of CD8+T cells from two individuals (CL-MCRL and CL-3089) with acutely resolving HCV infection for this study. Both individuals were selected from a cohort of high-risk injecting drug users followed prospectively for recent HCV infection19,20. CL-MCRL experienced a brief period of acute HCV infection (Figure 1A) that was bound by negative HCV RNA PCR tests at 80 days before and 115 days after the estimated date of infection21. Similarly, we identified a single time point for individual CL-3089 at 180 days post-infection (DPI) with a positive HCV RNA PCR test. Two peripheral HCV-specific CD8+ T cell populations were identified from CL-MCRL targeting the HLA-B*07:02 GPRLGVRAT (GPR) and HLA-A*01:01 ATDALMTGF (ATD) epitopes whilst a single population targeting the HLA-B*07:02 GPRLGVRAT epitope was identified from CL-3089 (Table 1).
We stained for epitope-specific populations using MHC-I Dextramers and flow cytometry (Figure 1B) and tracked the stable persistence of the GPR-specific population from CL-MCRL over at least two years (Figure 1C). We applied TCR reconstruction from single-cell RNA-sequencing using the VDJPuzzle pipeline22 and found that the GPR-specific population from CL-MCRL consisted almost completely of a monoclonal population of CD8+ T cells expressing an identical paired CDR3αβ clonotype across all four sampled time points (Figure 1D). More specifically, this monoclonal clonotype (TCRα: CAVRATGQNFVF, TCRβ: CASSQAPPGQGVDIQYF) accounted for 171/174 of all reconstructed paired CDR3αβ clonotypes from the GPR-specific population from CL-MCRL. By comparison, the ATD-specific and GPR-specific CD8+ T cell populations from individuals CL-MCRL and CL-3089, respectively, were highly diverse and polyclonal (Supplementary Figure 1). Notably, we did not observe the monoclonal GPR clonotype from CL-MCRL in the repertoire derived from CL-3089. Taken together, these observations point to the expansion of a naturally-occurring monoclonal population following natural HCV infection.
We next explored whether the recruitment and expansion of a monoclonal population may have been driven by the affinity characteristics of the monoclonal TCR for its cognate peptide target. To this end, we employed the use of reversible MHC Streptamers14 to measure the dissociation time of the TCR-pMHC interaction and determine its koff affinity (Figure 1E). Briefly, reversible Streptamers were formed from conjugating fluorescently labelled pMHC monomers with a Strep-Tactin backbone via Strep-tag binding sites, and the addition of biotin dissociates the backbone by competitive binding and leaves monomeric TCR-pMHC complexes. The dissociation speed of monomeric TCR-pMHC complexes can subsequently be measured and corresponds to the koff rate. We applied this assay directly to cryopreserved peripheral blood mononuclear cells (PBMC) from CL-MCRL by first co-staining with reversible Streptamers and non-reversible Dextramers for the GPR-epitope (Figure 1F). Following the addition of biotin, we observed rapid loss of the backbone signal (half-life: 26 seconds) and gradual loss of pMHC signal (Figure 1G). We periodically paused acquisition to preserve sample during the extended dissociation between the monoclonal TCR population GPR-epitope presented on pMHC complexes, and after analysis determined the dissociation to have a half-life of 794 seconds and a koff constant of 1.3 x 10-3 (95% confidence interval: 1.5 x 10-3 to 1.1 x 10-3).
We were unable to directly measure the koff affinities of the polyclonal populations from CL-MCRL and CL-3089 because of the breadth of unique TCR arrangements, and thus we applied an in vitro colony expansion approach to generate pure, monoclonal populations of epitope-specific CD8+ cells from single-cell sorted cells. Single clones from the GPR- and ATD-specific populations from CL-MCRL and CL-3089 respectively, were identified by staining with non-reversible Dextramers, isolated by fluorescence-activated cell sorting, and expanded by stimulation with phytohemagglutinin (PHA) and IL-2, in the presence of gamma irradiated feeder cells for 4 weeks (Table 1). We also applied this approach to the monoclonal GPR-specific population from CL-MCRL and successful expansions typically yielded up to one million CD8+ T cells. We next repeated the dissociation assay using these colony expanded cells by first re-identifying epitope-specific cells by non-reversible multimer staining (Figure 2A and Methods) followed by measurement of reversible Streptamer dissociation comprising of the backbone (Figure 2B) and pMHC complex (Figure 2C).
Notably, the dissociation half-life varied in duration between colonies, epitope specificity, and individual origin. The strongest koff affinity was recorded from CL-MCRL GPR-specific colonies (half-lives 1039 to 1321 seconds), followed by CL-3089 GPR-specific colonies (half-lives 247 to 1191 seconds), and the weakest koff affinities were recorded from CL-MCRL ATD-specific colonies (half-lives 22 to 196 seconds) (Figure 2D). More specifically, the koff constants for the CL-MCRL ATD-specific colonies ranged from 3.54 x 10-3 to 3.14 x 10-2 (Table 2), the koff constants for the CL-MCRL GPR-specific colonies ranged from 5.25 x 10-4 to 6.67 x 10-4 (Table 3), and the koff constants for the CL-3089 GPR-specific colonies ranged from 5.82 x 10-4 to 2.81 x 10‑3 (Table 3). Intriguingly for one pair of colonies (F4 and F4-2) derived from the polyclonal GPR-specific population, their dissociation half-lives were within the range of the monoclonal clonotype (Table 3) which suggested that it may be possible to identify similarly high affinity TCRs within polyclonal populations.
Our sorting strategy for colony expansion resulted in the random selection of clonotypes and thus we performed targeted TCR sequencing to link clonotype identity with measured koff affinities. Total RNA was extracted from a subset of samples and the CDR3 regions from TCRα and TCRβ chain transcripts were enriched by nested PCR followed by Sanger sequencing. All ATD-specific colonies had distinct clonotypes (Table 2), which was consistent with the polyclonal nature of the original repertoire (Supplementary Figure 1). Notably, around half of all clonotypes from the colony expansions had also been observed in the scRNA-seq dataset (Tables 2 and 3).
Finally, we considered whether, at the population level, transcriptional phenotypes may be associated with differences in the distribution of koff affinities. We first performed a differential gene expression analysis between the GPR- and ATD-specific populations from the early time points (<120 days post-infection) in CL-MCRL and identified enrichment for effector-associated signatures (NKG7 , GZMH , GZMA ) in the former and a memory-associated signature (IL7R ) in the latter (Figure 3A). Next, we compared the GPR-specific monoclonal population from CL-MCRL and the polyclonal one from CL-3089, but identified differences primarily related to TCR gene usage and mitochondrial gene expression which may reflect inter-individual differences (Figure 3B). Rather, the GPR population from CL-3089 resembled an intermediate population between the ATD- and GPR-specific populations when characterised by expression for the phenotypic markers NKG7 , GZMH , and IL7R(Figure 3C). We further applied gene set enrichment analysis using Hallmark pathways from the Molecular Signatures database which confirmed that the GPR-specific population exhibited an effector-polarised phenotype with evidence for elevated cell cycling and cellular metabolism pathways in its enriched genes (Figure 3D and E). Taken together, our findings suggest an association between affinity and transcriptional phenotype at the population level whereby higher affinity responses can also be observed in combination with highly active effector phenotypes.