CONCLUSION
In summary, this study expanded the known range of koffrates and provided a collection of TCR sequences which may have
application for comparative studies between high and low affinity
receptors.
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 previously19.
Briefly HCV-specific CD8+ T cell populations were
isolated by HLA-I Dextramer staining (HLA-A*01:01 ATDALMTGF or
HLA-B*07:02 GPRLGVRAT ) (Immudex) and sorting with flow cytometry
(BD FACSAria III) for plate-based Smart-Seq2 scRNA-seq35,36. 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 22.
Reversible and non-reversible multimer preparation
Reversible MHC Streptamers were prepared by incubating 1μg of the
relevant pMHC monomer conjugated to AF488 (HLA-B*07:02 GPRLGVRATor HLA-A*01:01 ATDALMTGF ) (laboratory synthesis) with 1μg
Strep-Tactin-APC (IBA Lifescience, PN 6-5010-001) in 25 μl of FACS
buffer (PBS/1% BSA). Non-reversible multimers were similarly prepared
by incubating 1μg of the relevant pMHC monomer conjugated to biotin
(laboratory synthesis) with 1 μg 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.
Koffdissociation 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 GPRLGVRATDextramer (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 μl 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+ T cells (lymphocytes/singlets/live
cells/CD3+/CD19‒/CD8+/Dextramer+)
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 (1μg/μl) (Sigma‑Aldrich) and containing
1×106 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% CO2 incubator.
On day 7 after sorting, 50μl 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 μl 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 (1μg/μl). 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.
Koff 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 μl 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°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 koff 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. koff 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
(AAGCAGTGGTATCAACGCAGAGTACT30VN) 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 pools37.
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 μM), TRBC
external primer (0.4 μM), TRAV external primer pool (0.1 μM), TRBV
external primer pool (0.1 μM), Taq DNA polymerase (Qiagen), and 5ul of
reverse transcribed template. For the second round nested PCR, 2.5 μl of
first round product was added to a total reaction mix of 25 μl
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 μM) and TRAV internal primer pool (0.1 μM), or TRBC internal primer
(0.4 μM) and TRBV internal primer pool (0.1 μM).
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).
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.