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\begin{document}

\title{Discovery of a monoclonal, high-affinity CD8+ T cell clone following
natural hepatitis C virus infection}



\author[1]{Curtis Cai}%
\author[2]{Elizabeth Keoshkerian}%
\author[3]{Kristof Wing}%
\author[4]{Jerome Samir}%
\author[3]{Manuel Effenberger}%
\author[5]{Kilian Schober}%
\author[6]{Rowena Bull}%
\author[1]{Andrew Lloyd}%
\author[7]{Dirk Busch}%
\author[1]{Fabio Luciani}%
\affil[1]{UNSW}%
\affil[2]{The Kirby Institute}%
\affil[3]{Technical University of Munich}%
\affil[4]{UNSW Sydney}%
\affil[5]{Friedrich-Alexander-Universität Erlangen-Nürnberg}%
\affil[6]{University of New South Wales School of Medical Sciences}%
\affil[7]{Technische Universität München}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
CD8+ T cells recognising their cognate antigen are typically recruited
as a polyclonal population consisting of multiple clonotypes with
varying T-cell receptor (TCR) affinity to the target peptide-MHC (pMHC)
complex. Advances in single-cell sequencing have increased accessibility
towards identifying TCRs with matched antigens. Here we present the
discovery of a monoclonal CD8+ T cell population with specificity for a
hepatitis C virus (HCV)-derived HLA class I epitope (HLA-B*07:02
GPRLGVRAT) which was isolated directly ex vivo from an individual with
an episode of acutely resolved HCV infection. This population was absent
prior to infection and underwent expansion and stable maintenance for at
least two years after infection as measured by HLA-multimer staining.
Furthermore, the monoclonal clonotype was characterised by an unusually
long dissociation time (half-life = 794 seconds and koff = 5.73\selectlanguage{ngerman}×10-4)
for its target antigen when compared to previously published results. A
comparison with related populations of HCV-specific populations derived
from the same individual and a second individual suggest that
high-affinity TCR-pMHC interactions may be inherent to epitope identity
and shape the phenotype of responses which has implications for rational
TCR selection and design in the age of personalised immunotherapies.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{INTRODUCTION}

The diversity and functionality of CD8\textsuperscript{+} T cells
recruited during an epitope-specific response is thought to arise
through the contribution of antigen density\textsuperscript{1}, antigen
sensitivity\textsuperscript{2}, TCR gene biases\textsuperscript{3}, and
other factors, but the precise contributions are incompletely
understood. There are further constraints which shape the diversity of
the final repertoire based on structural features at key conserved
residues\textsuperscript{4} and consideration of a T-cell clone's
pre-existing frequency in the na\selectlanguage{ngerman}ïve
repertoire\textsuperscript{5,6}although this may be overcome by repeated
antigen re-exposure through vaccination\textsuperscript{7}. Typically,
these factors conspire to generate epitope-specific responses consisting
of a range of unique TCR arrangements (clonotypes)\textsuperscript{8},
with a distinct affinity for their cognate peptide in the context of MHC
(pMHC). Notably however, epitope-specific populations have been observed
to comprise few or single unique clonotypes during infection with human
immunodeficiency virus\textsuperscript{9} and \emph{Mycobacterium
tuberculosis} \textsuperscript{10}. Such monoclonal populations may have
arisen through a selective advantage intrinsically conferred by its
specific TCR clonotype.

The affinity of the TCR-pMHC interaction may be measured by both its
association (k\textsubscript{on}) and dissociation
(k\textsubscript{off}) constants, which collectively contribute to its
host cell's activation potency\textsuperscript{11}. One such method for
the measurement of the k\textsubscript{off} rate utilises dissociable
HLA multimers, so-called MHC Streptamers, which allow the real-time
tracking of dissociation of monomeric TCR-pMHC complexes by flow
cytometry\textsuperscript{12-14}. Notably, CD8\textsuperscript{+} T cell
clonotypes with lower k\textsubscript{off} constants (or practically,
high-affinity clones with extended dissociation times) have improved
protective capacity when used for adoptive
transfer\textsuperscript{13}and high affinity neoantigen-specific clones
are more effective at slowing tumor growth\textsuperscript{15}. By
contrast, low-affinity clonotypes are preferentially expanded long-term
during chronic cytomegalovirus infection\textsuperscript{16}, may be
better suited for providing responses optimised against antigens with
relatively higher abundance\textsuperscript{17}, and provide the
flexibility for recognising and responding to mutated
epitopes\textsuperscript{18}. Taken together, these studies provide
evidence that the affinity of TCR-pMHC interactions likely influence the
functional capacity of epitope-specific populations.

In this study, we characterised the phenotype and affinity of a
naturally-derived monoclonal CD8\textsuperscript{+} T cell population
arising from an individual with acutely resolving infection that we had
reported on previously\textsuperscript{19}. We examined three hepatitis
C virus (HCV)-specific CD8\textsuperscript{+} T cell populations from
two individuals and profiled each population's respective repertoire
diversity, phenotypic characteristics, and dissociation affinities at
the single-cell level. The k\textsubscript{off} rate-derived affinity of
the monoclonal TCR clonotype was the strongest out of all tested
sequences, although clonotypes from a polyclonal population targeting
the same epitope also displayed surprisingly strong affinities. Notably,
analysis of the transcriptional signatures from each population revealed
associations between epitope specificity, affinity and molecular
phenotype. Our findings contribute to an improved understanding of the
heterogeneity of TCR repertoires generated by viral infections and offer
insights into how affinity may shape the phenotype and expansion of T
cells at the epitope-specific level.

\textbf{RESULTS}

We selected three epitope-specific populations of
CD8\textsuperscript{+}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 infection\textsuperscript{19,20}. 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 infection\textsuperscript{21}. 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\textsuperscript{+} T cell populations were identified
from CL-MCRL targeting the HLA-B*07:02 \emph{GPRLGVRAT} (GPR) and
HLA-A*01:01 \emph{ATDALMTGF} (ATD) epitopes whilst a single population
targeting the HLA-B*07:02 \emph{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 pipeline\textsuperscript{22} and found that the
GPR-specific population from CL-MCRL consisted almost completely of a
monoclonal population of CD8\textsuperscript{+} T cells expressing an
identical paired CDR3\selectlanguage{greek}αβ \selectlanguage{english}clonotype across all four sampled time points
(Figure 1D). More specifically, this monoclonal clonotype (TCR\selectlanguage{greek}α:
\selectlanguage{english}CAVRATGQNFVF, TCR\selectlanguage{greek}β: \selectlanguage{english}CASSQAPPGQGVDIQYF) accounted for 171/174 of all
reconstructed paired CDR3\selectlanguage{greek}αβ \selectlanguage{english}clonotypes from the GPR-specific population
from CL-MCRL. By comparison, the ATD-specific and GPR-specific
CD8\textsuperscript{+} 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 Streptamers\textsuperscript{14} to measure the
dissociation time of the TCR-pMHC interaction and determine its
k\textsubscript{off} 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 k\textsubscript{off} 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 k\textsubscript{off} constant of
1.3 x 10\textsuperscript{-3} (95\% confidence interval: 1.5 x
10\textsuperscript{-3} to 1.1 x 10\textsuperscript{-3}).

We were unable to directly measure the k\textsubscript{off} affinities
of the polyclonal populations from CL-MCRL and CL-3089 because of the
breadth of unique TCR arrangements, and thus we applied an \emph{in
vitro} colony expansion approach to generate pure, monoclonal
populations of epitope-specific CD8\textsuperscript{+} 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\textsuperscript{+} 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
k\textsubscript{off} 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
k\textsubscript{off} affinities were recorded from CL-MCRL ATD-specific
colonies (half-lives 22 to 196 seconds) (Figure 2D). More specifically,
the k\textsubscript{off} constants for the CL-MCRL ATD-specific colonies
ranged from 3.54 x 10\textsuperscript{-3} to 3.14 x
10\textsuperscript{-2} (Table 2), the k\textsubscript{off} constants for
the CL-MCRL GPR-specific colonies ranged from 5.25 x
10\textsuperscript{-4} to 6.67 x 10\textsuperscript{-4} (Table 3), and
the k\textsubscript{off} constants for the CL-3089 GPR-specific colonies
ranged from 5.82 x 10\textsuperscript{-4} to 2.81 x
10\textsuperscript{-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 k\textsubscript{off} affinities.
Total RNA was extracted from a subset of samples and the CDR3 regions
from TCR\selectlanguage{greek}α \selectlanguage{english}and TCR\selectlanguage{greek}β \selectlanguage{english}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
k\textsubscript{off} affinities. We first performed a differential gene
expression analysis between the GPR- and ATD-specific populations from
the early time points (\textless{}120 days post-infection) in CL-MCRL
and identified enrichment for effector-associated signatures
(\emph{NKG7} , \emph{GZMH} , \emph{GZMA} ) in the former and a
memory-associated signature (\emph{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 \emph{NKG7} , \emph{GZMH} , and
\emph{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.

\textbf{DISCUSSION}

During a typical CD8\textsuperscript{+} T cell immune response, a range
of clonotypes are recruited, each with their own distinct affinity for
the target epitope. In this study we identified an HCV-specific
clonotype from an individual with acutely resolved infection that was
monoclonal expanded and exhibited a strong affinity for the
HLA-B*07:02\emph{GPR} epitope, with a dissociation rate significantly
smaller than the ones of previously reported
TCRs\textsuperscript{13,14}. Intriguingly, a polyclonal repertoire from
a second individual that also generated a response against the same
epitope also exhibited similar dissociation rates, suggesting that
affinity alone may not be a sufficient factor regulating whether
monoclonal or polyclonal populations arise.

Our findings provide evidence that high-affinity TCRs may be more common
than previously observed, or particular epitopes may bias the
recruitment of higher affinity receptors. Previous studies of TCR
dissociations using the same assay have reported half-lives typically of
up to 200 seconds \textsuperscript{13,14,23}, which was exceeded by all
GPR-specific clonotypes in this study.

The affinity between a TCR and its target pMHC is particularly
meaningful for dictating the activation potency of
CD8\textsuperscript{+} T cell clones\textsuperscript{24} which may have
contributed to the accelerated differentiation and expansion of the
monoclonal GPR-specific clonotype at the expense of recruitment of
additional clonotypes. It is plausible that the range of affinities
measured from our colony expansions may have been biased to favour
subsets such as central memory T cells which have greater capacity for
clonal expansion \emph{in vitro} by cytokine
stimulation\textsuperscript{25} or high affinity clonotypes which are
more responsive to memory recall responses \textsuperscript{26,27}.

The role of affinity in determining T cell fate remains unclear, with
contrasting reports on the role of high affinity in driving memory T
cell fate\textsuperscript{28-30}. High-affinity clones were found
dominating during the acute phase of infection, while under chronic
conditions, low-affinity repertoires may be favoured through mechanisms
such as mutational escape epitopes upon viral evolution or
re-infection\textsuperscript{18}. Our new study is consistent with a
model that high-affinity memory cells can be detected in the acute phase
of viral infection, and these are phenotypically and transcriptionally
distinct from effector cells that contribute to IFN-?
production\textsuperscript{19}. In this study we studied high-risk
behaviour individuals who inject drugs, thus it is likely that
continuous exposure to HCV may contribute to the formation of memory
populations, and therefore to future protective
immunity\textsuperscript{31,32}. Nevertheless, more studies are needed
to dissect the role of affinity in determining the fate of a cytotoxic T
cell response\textsuperscript{33} and whether the affinity profile of
memory populations continue to be shaped by future antigen encounter.

Our findings will benefit the growing field of personalized
immunotherapies involving the rational TCR design. Notably, rational
engineering of the monoclonal receptor offers the possibility to further
enhance its affinity, for example to synthesize specific high-affinity
TCRs against neoantigen targets\textsuperscript{34}.

\textbf{CONCLUSION}

In summary, this study expanded the known range of
k\textsubscript{off}rates and provided a collection of TCR sequences
which may have application for comparative studies between high and low
affinity receptors.

\textbf{METHODS}

Study subjects

Two individuals with acutely resolving HCV infection (CL-MCRL and
CL-3089) were sourced from the HITS cohort (reference) for single-cell
RNA-sequencing (scRNA-seq). For each population, cells from a single
timepoint following resolution of infection were selected for sorting
cells for single-cell colony expansion.

Single-cell RNA-sequencing

scRNA-seq was performed as described previously\textsuperscript{19}.
Briefly HCV-specific CD8\textsuperscript{+} T cell populations were
isolated by HLA-I Dextramer staining (HLA-A*01:01 \emph{ATDALMTGF} or
HLA-B*07:02 \emph{GPRLGVRAT} ) (Immudex) and sorting with flow cytometry
(BD FACSAria III) for plate-based Smart-Seq2
scRNA-seq\textsuperscript{35,36}. Sequencing was performed with 150bp
paired end sequencing on Illumina NovaSeq 6000 and NextSeq 500
platforms. Full length TCR sequences were reconstructed using the
VDJPuzzle package with default parameters \textsuperscript{22}.

Reversible and non-reversible multimer preparation

Reversible MHC Streptamers were prepared by incubating 1\selectlanguage{greek}μ\selectlanguage{english}g of the
relevant pMHC monomer conjugated to AF488 (HLA-B*07:02
\emph{GPRLGVRAT}or HLA-A*01:01 \emph{ATDALMTGF} ) (laboratory synthesis)
with 1\selectlanguage{greek}μ\selectlanguage{english}g Strep-Tactin-APC (IBA Lifescience, PN 6-5010-001) in 25 \selectlanguage{greek}μ\selectlanguage{english}l of
FACS buffer (PBS/1\% BSA). Non-reversible multimers were similarly
prepared by incubating 1\selectlanguage{greek}μ\selectlanguage{english}g of the relevant pMHC monomer conjugated to
biotin (laboratory synthesis) with 1 \selectlanguage{greek}μ\selectlanguage{english}g of Streptavidin-PE (BioLegend,
PN 405245). Incubations were performed on ice in the dark for 45
minutes. Streptamers and multimers were used within 6 hours of
preparation.

K\textsubscript{off}dissociation assay using PBMC

The dissociation rate for the GPR-specific population from CL-MCRL was
measured directly from cryopreserved PBMC. Cryopreserved cells were
thawed and stained first with the reversible Streptamer for 20 minutes
followed by surface antibody staining: CD8-eFluor450 (OKT-8) and
CD19-PE-Cy5 (HIB19) for an additional 20 minutes without washing. Cells
were then washed twice and stained with a HLA-B*07:02
\emph{GPRLGVRAT}Dextramer (Immudex, Copenhagen, Denmark) for 20 minutes.
Two final washes were performed, and cells were resuspended with one
drop of propidium iodide (Molecular Probes) and 100 \selectlanguage{greek}μ\selectlanguage{english}l of FACS buffer.
Data was acquired on a LSR Fortessa x20 (BD Biosciences) in a cooled
tube holder. After a brief period of acquisition, a 1X biotin solution
(Sigma-Aldrich) was added to dissociate the reversible Streptamer.
Additional biotin solution was added as required and the tube containing
cells was periodically placed on ice without sample acquisition to
preserve sample availability during the dissociation process. The time
between subsequent acquisitions was embedded in raw FCS files and used
to determine the time since initial acquisition.

Single-cell colony expansion

Total PBMC were thawed and stained first with the relevant Dextramer
conjugated to PE (Immudex), followed by Fixable yellow viability stain
(Invitrogen) and finally a panel of surface antibodies: CD3-BV480
(UCHT1), CD8-APC-R700 (RPA-T8), CD19-PE-Cy5 (HIB19). Single HCV-specific
CD8\textsuperscript{+} T cells (lymphocytes/singlets/live
cells/CD3\textsuperscript{+}/CD19\textsuperscript{-}/CD8\textsuperscript{+}/Dextramer\textsuperscript{+})
were sorted into separate wells of a 384-well flat bottom plate
containing expansion media (CTS OpTmizer T Cell Expansion serum free
media) (Gibco) supplemented with L-glutamine (2mM) (Sigma-Aldrich),
penicillin/streptomycin (1X) (Sigma-Aldrich), IL-2 (500IU/ml) (STEMCELL
Technologies), PHA-L (1\selectlanguage{greek}μ\selectlanguage{english}g/\selectlanguage{greek}μ\selectlanguage{english}l) (Sigma-Aldrich) and containing
1\selectlanguage{ngerman}×10\textsuperscript{6} gamma irradiated feeder PBMC cells from
unrelated blood donors. Sorting was performed with single cell precision
on a FACS Aria III flow cytometer (BD) and plates were incubated in a
37°C, 5\% CO\textsubscript{2} incubator.

On day 7 after sorting, 50\selectlanguage{greek}μ\selectlanguage{english}l of supplementing media containing CTS
OpTmizer T Cell Expansion serum free media with L-glutamine (2 mM),
penicillin/streptomycin (1X~concentration), and IL-2 (500 IU/ml) was
added to each well. On every subsequent 3-4 days, 50 \selectlanguage{greek}μ\selectlanguage{english}l of existing
media was removed and replaced with an equal volume of supplementing
media. If cell colonies reached 100\% confluency, they were transferred
to 96-well flat bottom plates (typically after three weeks) in
supplementing media containing PHA-L (1\selectlanguage{greek}μ\selectlanguage{english}g/\selectlanguage{greek}μ\selectlanguage{english}l). Colonies continued to be
monitored and supplemented every 3-4 days and after an additional two
weeks or when 100\% confluency was reached, they were cryopreserved in
two identical aliquots containing 50\% media, 40\% FBS, and 10\% DMSO.

K\textsubscript{off} dissociation assay using colony expansions

Single-cell expansions were retrieved from cryopreservation and stained
first with the reversible Streptamer for 20 minutes followed by surface
antibody staining: CD8-eFluor450 (OKT-8) and CD19-PE-Cy5 (HIB19) for an
additional 20 minutes without washing. Cells were then washed twice and
stained with the non-reversible multimer for 20 minutes. Two final
washes were performed, and cells were resuspended with one drop of
propidium iodide (molecular probes) and 100 \selectlanguage{greek}μ\selectlanguage{english}l of FACS buffer.

Data acquisition by flow cytometry for dissociation assay

Stained cells were transferred to 5 mL round-bottom polystyrene tubes
which had been pierced with a syringe to allow delivery of a biotin
solution during acquisition. Cells were kept cool at 4\selectlanguage{ngerman}°C or on ice at
all points during preparation and acquisition. Data were acquired on an
LSR Fortessa x20 (BD Biosciences). At the beginning of data acquisition,
events were recorded for up to 60 seconds followed by rapid injection of
1.5 mL PBC containing 1X biotin (Sigma-Aldrich). Samples were
continuously acquired until loss of MHC monomer signal was observed and
additional biotin solution was added as required.

Calculation of dissociation and k\textsubscript{off} constants

Compensated fluorescence and time values from the
lymphocyte/singlet/live/CD8+/non-reversible multimer subpopulation were
exported from raw FCS files using FlowJo (v10.6.1, BD) and loaded into
Prism (v7.04, GraphPad). Time values (in seconds), MHC-AF488, and
Strep-Tactin-APC fluorescence values were used as input for non-linear
regression under `XY analyses' to compute the half-life values for the
associated dissociation curve. k\textsubscript{off} values were
calculated by taking the reciprocal of the computed half-life in seconds
and upper and lower 95\% confidence intervals for half-lives were also
reported.

Targeted TCR amplification of colony expansions

Total RNA was extracted from an aliquot of cells from selected
single-cell colony expansions using an RNeasy micro kit (Qiagen) and
stored at -20°C. RNA was reverse transcribed using the SuperScript IV
Reverse Transcriptase kit (Invitrogen) according to manufacturer's
recommendations. First, template RNA was incubated with a poly dT primer
(AAGCAGTGGTATCAACGCAGAGTACT\textsubscript{30}VN) and nucleotides for 5
minutes at 65°C then immediately placed on ice. Next, the mix containing
reverse transcriptase and buffer was added and the mixture was incubated
at 50°C for 15 minutes followed by 80°C for 10 minutes.

Two rounds of nested PCRs were performed to enrich for TCR-associated
transcripts using previously published primer pools\textsuperscript{37}.
All reactions used identical cycling conditions consisting of an initial
denaturation (95°C for one minute) followed by 35 cycles of: 95°C for 20
seconds, 52°C for 20 seconds, 72°C for 45 seconds, followed by a final
extension at 72°C for 4 minutes. For the first round PCR, alpha and beta
chain transcripts were amplified in a single reaction consisting of PCR
buffer (1X), dNTPs (0.2 mM), TRAC external primer (0.4 \selectlanguage{greek}μ\selectlanguage{english}M), TRBC
external primer (0.4 \selectlanguage{greek}μ\selectlanguage{english}M), TRAV external primer pool (0.1 \selectlanguage{greek}μ\selectlanguage{english}M), TRBV
external primer pool (0.1 \selectlanguage{greek}μ\selectlanguage{english}M), Taq DNA polymerase (Qiagen), and 5ul of
reverse transcribed template. For the second round nested PCR, 2.5 \selectlanguage{greek}μ\selectlanguage{english}l of
first round product was added to a total reaction mix of 25 \selectlanguage{greek}μ\selectlanguage{english}l
containing PCR buffer (1X), dNTPs (0.2mM) and Taq DNA polymerase
(Qiagen). Separate reactions were prepared for the amplification of
alpha and beta chain transcripts containing either TRAC internal primer
(0.4 \selectlanguage{greek}μ\selectlanguage{english}M) and TRAV internal primer pool (0.1 \selectlanguage{greek}μ\selectlanguage{english}M), or TRBC internal primer
(0.4 \selectlanguage{greek}μ\selectlanguage{english}M) and TRBV internal primer pool (0.1 \selectlanguage{greek}μ\selectlanguage{english}M).

Amplification products were validated by gel electrophoresis and
purified using ExoSAP-IT Express (Applied Biosystems). Samples were
submitted for Sanger sequencing at the UNSW Ramaciotti Centre for
Genomics using the relevant internal constant region primer.
Electropherograms after sequencing were manually inspected for clear
signals. Nucleotide sequences were used to query the online IMGT/V-QUEST
tool and obtain TCR gene usage and CDR3 amino acid sequences (available
at http://www.imgt.org/IMGT\_vquest/input).

\textbf{ACKNOWLEDGEMENTS}

We thank the study participants for their generous donation of samples.
The HITS-p investigators include Andrew Lloyd, Lisa Maher, Kate Dolan,
Paul Haber, William Rawlinson, Carla Treloar and Gregory Dore.~ This
research was supported from National Health and Medical Research Council
of Australia (NHMRC) Project Nos. APP1121643, 1027551, 1060199,
Partnership No. 1016351, and Programme Nos. 510488 and 1053206. The
HITS-c cohort was supported by the UNSW Hepatitis C Vaccine Initiative
and NHMRC Project Grant No. 630483. F.L., A.R.L. and R.A.B. are
supported by NHMRC Research Fellowships (Numbers: 1128416, 1041897 and
1084706). F.L., A.R.L. and R.A.B. are supported by NHMRC Research
Fellowships (Numbers: 1128416, 1041897 and 1084706). C.C. and J.S. are
supported by Australian Government Research Training Program (RTP)
Scholarships. D.H.B. was supported by the Deutsche
Forschungsgemeinschaft (DFG, SFB-TRR338/1-452881907-A01). K.S. is
supported by the German Federal Ministry of Education and Research
(BMBF, projects 01KI2013). The authors acknowledge the facilities and
technical support from the staff at the UNSW Flow Cytometry core
facility located within the Mark Wainwright Analytical Centre.
Sequencing was performed by staff at the Ramaciotti Centre for Genomics,
UNSW. Analyses of single-cell sequencing data were performed with
resources allocated from the National Computational Infrastructure,
Australia.

\textbf{AUTHOR CONTRIBUTIONS}

\textbf{Curtis Cai} : Data curation, formal analysis; investigation;
methodology; writing -- original draft. \textbf{Jerome Samir} : Data
curation; formal analysis. \textbf{Elizabeth Keoshkerian} :
Investigation; methodology. \textbf{Kristof Wing} : Formal analysis;
investigation; methodology. \textbf{Manuel Effenberger} :
Investigation.\textbf{Kilian Schober} : Investigation. \textbf{Rowena A
Bull} : Investigation. \textbf{Andrew R Lloyd} : Investigation.
\textbf{Dirk H Busch} : Conceptualization; investigation. \textbf{Fabio
Luciani} : Conceptualization; data curation; formal analysis; funding
acquisition; investigation; methodology; project administration;
supervision; writing -- review and editing.

\textbf{CONFLICT OF INTEREST}

All the other authors declare no conflict of interest related to this
work.

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\textbf{FIGURES}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. A monoclonal, high affinity CD8\textsuperscript{+} T
cell population generated following natural HCV infection.}

A. Longitudinal measurements of viral load and IFN-\selectlanguage{greek}γ \selectlanguage{english}ELISpot responses
to two HCV-derived CD8\textsuperscript{+} T cell epitopes in CL-MCRL.
IU: international units. SFU: spot-forming units. B. Representative flow
cytometry gating for Dextramer staining of HCV-specific
CD8\textsuperscript{+} T cells. C. Frequency of cells specific for the
GPR-epitope measured by Dextramer staining in CL-MCRL. D. Stacked bar
plot showing the diversity of GPR-specific clonotypes with paired CDR3\selectlanguage{greek}α
\selectlanguage{english}and CDR3\selectlanguage{greek}β \selectlanguage{english}amino acid sequences at time points from CL-MCRL. E. Schematic
of the k\textsubscript{off} dissociation assay using reversible
Streptamers. pMHC: peptide-MHC. F. Flow cytometry gating for the
identification of the GPR-specific population for the koff dissociation
assay. G. Dot plot of staining intensity of the reversible Streptamer
backbone and pMHC components over time. The dotted red line indicates
the time of addition of biotin. The green curve shows the non-linear
regression. The half-life of the curve was calculated and numbers in
brackets represent 95\% confidence intervals. Cells were placed on ice
during intervening periods.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Comparative HCV-specific clonotypes from polyclonal
populations are lower affinity than the monoclonal clonotype.}

A. Representative flow cytometry gating for HCV-specific
CD8\textsuperscript{+} T cell colonies stained with non-reversible
multimers. B. Representative dots plots of fluorescence intensity from
the reversible Streptamer backbone during the
k\textsubscript{off}dissociation assay of single cell colony expansions.
C. Representative dots plots of fluorescence intensity from the
reversible Streptamer pMHC during the k\textsubscript{off} dissociation
assay of single cell colony expansions D. Box and whiskers plots of
dissociation half-life times of all measured clonotypes. Whiskers
represent 95\% confidence intervals from non-linear regression. Pairs of
solid colored symbols show cells derived from the same colony expansion
but cryopreserved and dissociations measured independently.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. The monoclonal GPR-specific response is characterised
by effector signatures.}

A. Volcano plot of differentially expressed genes between GPR- and
ATD-specific populations from CL-MCRL. Dashed line indicates p-value
\textless{} 0.05. Genes highlighted in red indicate adjusted p-values
corrected by Bonferroni correction \textless{} 0.05. B. Volcano plot of
differentially expressed genes between GPR-specific populations from
CL-MCRL and CL-3089 calculated using Mann-Whitney U test. Dashed line
indicates p-value \textless{} 0.05. Genes highlighted in red indicate
adjusted p-values corrected by Bonferroni correction \textless{} 0.05.
C. Violin plots showing the transcriptional expression level of
effector-associated markers. D. GSEA summary of differentially expressed
genes between GPR- and ATD-specific populations from CL-MCRL, using the
set of Hallmark pathways from the Human MSigDB collections. E. GSEA plot
showing selected enriched pathways between GPR- and ATD-specific
populations from CL-MCRL. NES: normalised enrichment score, pval:
p-value, padj: adjusted p-value. Statistical significance was determined
by Mann-Whitney U test (A and B) and Broad GSEA test (D and E). n.s. not
significant, **** p \textless{} 0.0001\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Supplementary figure 1. Polyclonal repertoires of comparative
HCV-specific CD8+ T cell populations.}

A. Pie charts of the distribution of ATD-specific clonotypes (paired
CDR3\selectlanguage{greek}α \selectlanguage{english}and CDR3\selectlanguage{greek}β \selectlanguage{english}amino acid) from CL-MCRL recovered by single-cell
RNA-sequencing. B. Pie charts of the distribution of GPR-specific
clonotypes (paired CDR3\selectlanguage{greek}α \selectlanguage{english}and CDR3\selectlanguage{greek}β \selectlanguage{english}amino acid) from CL-3089 recovered by
single-cell RNA-sequencing. DPI: days post-infection. Colored segments
represents a unique expanded clonotypes and grey segments represent all
singleton clonotypes.

\textbf{TABLES}

\textbf{Table 1} . Subjects details.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Subject} & \textbf{Gender} & \textbf{Age at infection} &
\textbf{Infecting virus genotype} & \textbf{Epitope} & \textbf{Protein
source (amino acid position)} & \textbf{Timepoint selected for colony
expansion}\tabularnewline
\midrule
\endhead
CL-3089 & M & 26 & 1b & HLA-B*0702 GPRLGVRAT & CORE (41-49) & 357
DPI\tabularnewline
CL-MCRL & F & 25 & 1a & HLA-B*0702 GPRLGVRAT & CORE (41-49) & 255
DPI\tabularnewline
CL-MCRL & F & 25 & 1a & HLA-A*0101 ATDALMTGF & NS3 (1436-1444) & 731
DPI\tabularnewline
\bottomrule
\end{longtable}

DPI: days post-infection

\textbf{Table 2:} Single cell colonies and affinity for T-cell receptors
specific for HLA-A*0101 ATDALMTGF.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\textbf{Subject} & \textbf{Colony} & \textbf{Half-life (sec)} &
\textbf{95\% CI (sec)} & \textbf{k\textsubscript{off} constant} &
\textbf{95 \% CI k\textsubscript{off}} & \textbf{V gene} & \textbf{D
gene} & \textbf{J gene} & \selectlanguage{greek}\textbf{CDR3α aa sequence}\selectlanguage{english} & \selectlanguage{greek}\textbf{CDR3β aa
sequence}\selectlanguage{english} & \textbf{Observed in scRNA-seq dataset?}\tabularnewline
\midrule
\endhead
CL-MCRL & CL-D1/D2-4 & 195.8 & 163-237.2 & 3.54E-03 & 2.92E-03 to
4.25E-03 & & & & - & - & -\tabularnewline
CL-MCRL & CL-C1-2 & 52.41 & 44.5-61.9 & 1.32E-02 & 1.12E-02 to 1.56E-02
& TRAV8-4*03; TRBV6-6*02 & TRBD1*01 & TRAJ20*01; TRBJ1-5*01 &
CAVTTGDYKLSF & CASSYEGAGNQPQHF & CDR3\selectlanguage{greek}α \selectlanguage{english}and CDR3\selectlanguage{greek}β \selectlanguage{english}observed
separately\tabularnewline
CL-MCRL & CL-C1-6 & 56.33 & 47.8-66.5 & 1.23E-02 & 1.04E-02 to 1.45E-02
& TRAV21*01; TRBV7-2*01 & TRBD2*01 & TRAJ9*01; TRBJ2-5*01 &
CAVRPTGGFKTIF & CASSLVGQLDEQYF & CDR3\selectlanguage{greek}αβ \selectlanguage{english}match\tabularnewline
CL-MCRL & CL-D1/D2-38 & 195.6 & 177.7-216.4 & 3.54E-03 & 3.20E-03 to
3.90E-03 & TRAV38-2/ DV8*01 & & TRAJ42*01 & CAYFSLYGGSQGNLIF & - & CDR3\selectlanguage{greek}α
\selectlanguage{english}match\tabularnewline
CL-MCRL & CL-A1/A2-16 & 66.41 & 18.6-555.3 & 1.04E-02 & 1.25E-03 to
3.73E-02 & TRAV3*01 & & TRAJ4*01 & CASGSGGNSKLIF & - & Not
observed\tabularnewline
CL-MCRL & CL-C1-7 & 22.07 & 13.6-37.9 & 3.14E-02 & 1.83E-02 to 5.10E-02
& TRAV8-3*01; TRBV2*01 & TRBD1*01 & TRAJ13*01; TRBJ2-1*01 & CAVSMGYQKVTF
& CASSRDPSYNEQFF & Not observed\tabularnewline
CL-MCRL & CL-B1-12 & 67.12 & 33.4-138.3 & 1.03E-02 & 5.01E-03 to
2.08E-02 & TRAV8-4*07 & & TRAJ20*01 & CAVSLGAYKLIF & - & Not
observed\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3} . Single cell colonies and affinity for T-cell
receptors specific for HLA-B*0702 GPRLGVRAT.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllll@{}}
\toprule
\textbf{Subject} & \textbf{Colony} & \textbf{Half-life (sec)} &
\textbf{95\% CI (sec)} & \textbf{k\textsubscript{off} constant} &
\textbf{95 \% CI k\textsubscript{off}} & \textbf{V gene} & \textbf{D
gene} & \textbf{J gene} & \selectlanguage{greek}\textbf{CDR3α aa sequence}\selectlanguage{english} & \selectlanguage{greek}\textbf{CDR3β aa
sequence}\selectlanguage{english} & \textbf{Was clonotype observed in scRNA-seq
dataset?}\tabularnewline
\midrule
\endhead
CL-MCRL & CL-P1-1A* & 1311 & 1299-1323 & 5.29E-04 & 5.24E-04 to 5.34E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P1-1B* & 1194 & 1174-1215 & 5.81E-04 & 5.70E-04 to 5.90E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P1-6B & 1228 & 1207-1249 & 5.64E-04 & 5.55E-04 to 5.74E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P2-1B & 1104 & 1089-1120 & 6.28E-04 & 6.19E-04 to 6.36E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P2-5B & 1321 & 1285-1360 & 5.25E-04 & 5.10E-04 to 5.39E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P2-7B & 1039 & 985-1097 & 6.67E-04 & 6.32E-04 to 7.04E-04 &
& & & & &\tabularnewline
CL-MCRL & CL-P2-9B & 1180 & 1160-1201 & 5.87E-04 & 5.77E-04 to 5.98E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P1-11B & 1319 & 1294-1346 & 5.26E-04 & 5.15E-04 to 5.36E-04
& & & & & &\tabularnewline
CL-MCRL & CL-P1-21 & 1263 & 1247-1279 & 5.49E-04 & 5.42E-04 to 5.56E-04
& & & & & &\tabularnewline
CL-MCRL & Monoclonal from PBMC & 794 & 679-941 & 8.73E-04 & 7.37E-04 to
1.02E-03 & TRAV3*01 TRAJ26*01 TRBV4-3*01 & TRBD1*01 & TRBJ2-4*01 &
CAVRAYGQNFVF & CASSQAPPGQGVDIQYF & N/A\tabularnewline
CL-3089 & D7 & 790 & 681-934 & 8.77E-04 & 7.42E-04 to 1.02E-03 &
TRAV1-2*01 & & TRAJ6*01 & CAVLSSGGSYIPTF & - & CDR3\selectlanguage{greek}α
\selectlanguage{english}match\tabularnewline
CL-3089 & E6* & 333 & 295-378 & 2.08E-03 & 1.83E-03 to 2.35E-03 &
TRAV3*01; TRBV27*01 & & TRAJ8*01; TRBJ1-1*01 & CAVRGPMNTGFQKLVF &
CASSTQENTEAFL & CDR3\selectlanguage{greek}α \selectlanguage{english}match, identical gene usage for \selectlanguage{greek}αβ
\selectlanguage{english}chains\tabularnewline
CL-3089 & E6-2* & 247 & 212-291 & 2.81E-03 & 2.38E-03 to 3.27E-03 & As
above & & As above & As above & As above & As above\tabularnewline
CL-3089 & F3 & 891 & 803-998 & 7.78E-04 & 6.95E-04 to 8.63E-04 &
TRAV8-4*07; TRBV9*01 & TRBD1*01 & TRAJ6*01; TRBJ2-3*01 & CAARRETGGSYILTF
& CASSVARDRASTDTQYF & Not observed\tabularnewline
CL-3089 & F4* & 926 & 789-1111 & 7.49E-04 & 6.24E-04 to 8.79E-04 &
TRAV1-2*01; TRBV28*01 & TRBD1*01 & TRAJ6*01; TRBJ2-7*01 & CAVLSSGGSYIPTF
& CASSKFRDSSYEQYF & CDR3\selectlanguage{greek}αβ \selectlanguage{english}match\tabularnewline
CL-3089 & F4-2* & 1191 & 1065-1345 & 5.82E-04 & 5.15E-04 to 6.51E-04 &
As above & As above & As above & As above & As above & As
above\tabularnewline
CL-3089 & CL10-060417 & 563 & 356-1197 & 1.23E-03 & 5.79E-04 to 1.95E-03
& & & & & &\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

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