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Hepatology Communications
Volume 5, Issue 6 p. 1106-1119
Original Article
Open Access
NEXT GENERATION SEQUENCING-BASED IDENTIFICATION OF T-CELL RECEPTORS FOR
IMMUNOTHERAPY AGAINST HEPATOCELLULAR CARCINOMA
Yipeng Ma,
Yipeng Ma
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
These authors contributed equally to this work.
Search for more papers by this author
Jiayu Ou,
Jiayu Ou
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
These authors contributed equally to this work.
Search for more papers by this author
Tong Lin,
Tong Lin
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
Search for more papers by this author
Lei Chen,
Lei Chen
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
Search for more papers by this author
Junhui Chen,
Junhui Chen
Intervention and Cell Therapy Center, Peking University Shenzhen Hospital,
Shenzhen, China
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Mingjun Wang,
Corresponding Author
Mingjun Wang
* mingjunw@szinno.com
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO:
Mingjun Wang, M.D., Ph.D.
Shenzhen Institute for Innovation and Translational Medicine
Shenzhen International Biological Valley-Life Science Industrial Park
Dapeng New District, Shenzhen, China
E-mail: mingjunw@szinno.com
Tel.: +86 755 84200365
Search for more papers by this author
Yipeng Ma,
Yipeng Ma
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
These authors contributed equally to this work.
Search for more papers by this author
Jiayu Ou,
Jiayu Ou
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
These authors contributed equally to this work.
Search for more papers by this author
Tong Lin,
Tong Lin
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
Search for more papers by this author
Lei Chen,
Lei Chen
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
Search for more papers by this author
Junhui Chen,
Junhui Chen
Intervention and Cell Therapy Center, Peking University Shenzhen Hospital,
Shenzhen, China
Search for more papers by this author
Mingjun Wang,
Corresponding Author
Mingjun Wang
* mingjunw@szinno.com
Department of Research and Development, Shenzhen Institute for Innovation and
Translational Medicine, Shenzhen International Biological Valley-Life Science
Industrial Park, Shenzhen, China
ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO:
Mingjun Wang, M.D., Ph.D.
Shenzhen Institute for Innovation and Translational Medicine
Shenzhen International Biological Valley-Life Science Industrial Park
Dapeng New District, Shenzhen, China
E-mail: mingjunw@szinno.com
Tel.: +86 755 84200365
Search for more papers by this author
First published: 28 February 2021
https://doi.org/10.1002/hep4.1697
Supported by the Shenzhen Basic Research Program (grant numbers
JCYJ20170412102821202 to M.W., JCYJ20180507182902330 to L.C.,
JCYJ20190809115811354 to M.W.), Shenzhen Peacock Plan (grant number
KQTD20130416114522736 to M.W.), Special Funds for Dapeng New District Industry
Development (grant numbers KJYF202001-13 to Y.M., KY20150116 to M.W.,
KJYF202001-12 to M.W., KY20160309 to M.W., KY20170203 to M.W., KY20180105 to
M.W., KY20180214 to L.C., PT201901-12 to M.W.), by Shenzhen Sanming Project of
Medicine (grant number SZSM201612071 to J.C.), and by National Human Genetic
Resources Platform of China (grant number YCZYPT (2018)03-1 to J.C.
Potential conflict of interest: Nothing to report.
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ABSTRACT
Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains a global
health concern, and HBV proteins may be ideal targets for T cell-based
immunotherapy for HCC. There is a need for fast and efficient identification of
HBV-specific T cell receptors (TCRs) for the development of TCR-transduced T
(TCR-T) cell-based immunotherapy. Two widely employed TCR identification
approaches, T cell clonal expansion and single-cell sequencing, involve a TCR
singularization process for the direct identification of Vα and Vβ pairs of TCR
chains. Clonal expansion of T cells is well known to have tedious time and
effort requirements due to the use of T cell cultures, whereas single-cell
sequencing is limited by the requirements of cell sorting and the preparation of
a single-cell immune-transcriptome library as well as the massive cost of the
whole procedure. Here, we present a next-generation sequencing (NGS)-based
HBV-specific TCR identification that does not require the TCR singularization
process. Conclusion: Two pairing strategies, ranking-based strategy and α–β
chain mixture-based strategy, have proved to be useful for NGS-based TCR
identification, particularly for polyclonal T cells purified by a peptide-major
histocompatibility complex (pMHC) multimer-based approach. Functional evaluation
confirmed the specificity and avidity of two identified HBV-specific TCRs, which
may potentially be used to produce TCR-T cells to treat patients with
HBV-related HCC.
ABBREVIATIONS
ALT alanine aminotransferase CD cluster of differentiation CDR3 complementarity
determining region 3 DMSO dimethyl sulfoxide EC50 50% effective concentration
ELISA ezyme-linked immunosorbent assay ELISpot enzyme-linked immune absorbent
spot FCM flow cytometry HBc hepatitis B virus core protein HBV hepatitis B virus
HCC hepatocellular carcinoma HLA human leukocyte antigen IFN-γ interferon-gamma
IL interleukin MHC major histocompatibility complex mTRBC mouse T cell receptor
beta chain constant region NGS next-generation sequencing PBMC peripheral blood
mononuclear cell REP rapid expansion protocol TCR T cell receptor TCR-T T cell
receptor-transduced T TRAV T cell receptor alpha chain variable region TRBV T
cell receptor beta chain variable region
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer
and causes more than 700,000 deaths annually.(1) At least 50% of HCC is
hepatitis B virus (HBV)-related disease.(2) HBV infection, cirrhosis, and
hepatoma are typical characteristics of HBV-related HCC, especially in Asia.(3)
Currently, the treatments for HCC mainly include surgery, antiviral drugs, and
targeted therapies. The response rate is low, and the overall efficacy is still
limited.(4, 5) Immunotherapy has increasingly become a trend in tumor treatment
because of its proven efficacy and clear targeting. T cell receptor
(TCR)-transduced T (TCR-T) cell therapy has shown promising efficacy in the
clinical treatment of malignant cancers, such as the treatment of metastatic
synovial cell sarcoma, melanoma, and non-small cell lung cancer by the TCR-T
cell targeting the cancer germline antigen New York esophageal squamous cell
carcinoma 1 (NY-ESO-1) as well as the treatment of human papillomavirus
(HPV)-positive epithelial cancers by a TCR-T cell targeting HPV-derived
antigen.(6, 7) HBV antigens, which are expressed in cancer cells of HBV-related
HCC and HBV-infected hepatocytes but not in uninfected liver cells and other
organ tissues, may be ideal targets for T cell-based immunotherapy.(8, 9)
Currently, the populations covered by reported HBV-specific TCRs are still
limited, and there is a need for fast identification of HBV-specific TCRs. Once
polyclonal-reactive T cells have been isolated by a peptide-major
histocompatibility complex (pMHC) multimer-based approach, limiting dilution is
commonly employed to obtain T cell clones for subsequent TCR sequencing.(10)
This procedure can reduce the complexity of obtained antigen-specific TCRs and
is essentially a TCR singularization process that simplifies the pairing of Vα
and Vβ chains. However, this strategy is also known to have tedious requirements
of time and effort due to the use of T cell cultures and cloning.(11) Recently,
great advances have been made in the field of single-cell sequencing, which has
proven to be efficient in TCR identification by sorting and directly cloning
single Vα–Vβ pairs from a T cell.(11, 12) However, single-cell sequencing is
currently limited by the requirements of cell sorting and the preparation of a
single-cell immune-transcriptome library as well as the massive cost of the
whole procedure.
Bulk sequencing approaches, such as next-generation sequencing (NGS), have been
widely employed for TCR repertoire studies, but they are not considered to be
applicable to the identification of TCR pairs.(13) Although TCR β chains are
normally unique for a T cell, the major concern with NGS is that approximately
30% of T cells in peripheral blood mononuclear cells (PBMCs) have the potential
to produce two distinct TCR α chain messenger RNAs (mRNAs) because of a
different allelic exclusion mechanism that influences the frequency and ranking
of TCR α chains in NGS.(14) Despite such limitations, NGS-based pairing has been
successfully employed for the identification of neoantigen-targeted TCRs.(15)
Furthermore, the peptide-MHC multimer-based purification process can greatly
reduce the complexity of the obtained TCR chains in NGS, which makes an α–β
chain mixture-based pairing strategy possible. Here, we present a study of
NGS-based fast identification of HBV-specific TCRs that may potentially be used
to produce TCR-T cells to treat patients with HBV-related HCC.
MATERIALS AND METHODS
T CELL STIMULATION, ISOLATION, AND RAPID EXPANSION
PBMCs from human leukocyte antigen A (HLA-A)*02-positive (+) donors with chronic
HBV infection were isolated through a standard Ficoll gradient. Informed consent
in writing was obtained from each patient. PBMCs were stimulated by 10 μM of HBV
core18-27I peptide (FLPSDFFPSI; Sangon Biotech, Shanghai, China) for 10 days in
X-VIVO 15 (#04-418QCN; Lonza) plus 10% human AB serum, 1% GlutMAX supplement
(#35050061; Gibco), and penicillin-streptomycin (#15140122; Gibco). We added
50 IU/mL interleukin (IL)-2 (#200-02; PEPROTECH), 10 ng/mL IL-7 (#200-07,
PEPROTECH), and IL-15 (#C016; Novoprotein) on day 1 and changed this solution
every 2 days. T cells stained with R-phycoerythrin (R-PE)-labeled Pro5
HLA-A*02:01/FLPSDFFPSI pentamer (#F283-2A-G; Proimmune) were isolated by anti-PE
MicroBeads (#130-105-639; Miltenyi Biotec) according to the manufacturer’s
protocol, followed by a rapid expansion protocol.
T CELL REPERTOIRE PREPARATION, HIGH-THROUGHPUT SEQUENCING, AND DATA ANALYSIS
T cells after rapid expansion were collected, and RNA isolation, T cell
repertoire preparation, high-throughput sequencing, and data analysis were
performed by GENEWIZ (Guangzhou, China). Briefly, total RNA was extracted from
the T cell sample using Trizol (#15596018; Invitrogen) according to the user
manual. We performed 5’ RACE with the SMARTer RACE complementary DNA (cDNA)
Amplification Kit (634859; Clontech); total RNA input was 1 μg. TCR α chain
variable region (TRAV) and β chain variable region (TRBV) NGS libraries were
made by using NEBNext Ultra DNA Library Prep Kit for Illumina. After quality
control on a Bioanalyzer High Sensitivity DNA chip (Agilent), libraries were
sequenced on the Illumina Miseq 2 × 300 platform.
FLOW CYTOMETRY
T cells were analyzed by flow cytometry (FCM), as described in the Supporting
Methods.
RETROVIRAL TRANSDUCTION
TCR chains were codon optimized and synthesized by Sangon Biotech, fused by a
furin plus P2A element when needed, substituted with murine constant domains, as
described, and cloned into the retroviral vector MSGV1 (#107227; Addgene). We
used 293T cells for transfection of the retrovirus plasmids TCR plasmid, VSV-G,
and Gag-Pol by Lipofectamine 2000 (#11668019; Invitrogen) according to the
manufacturer’s protocol. Retrovirus supernatant was centrifuged at 2000g for
2 hours in nontissue culture plates precoated with 20 μg/mL RetroNectin (#T100A;
Takara). The retrovirus supernatant was then removed, and prestimulated PBMCs
were added and cultured overnight in a 5% CO2 incubator at 37°C. When the α–β
chain mixture-based pairing strategy was employed, retrovirus supernatant of TCR
α chains and β chains were generated individually and mixed at a volume ratio of
1:1 for cotransfection of T cells.
ENZYME-LINKED IMMUNE ABSORBENT SPOT ASSAY
Ezyme-linked immunosorbent assay (ELISA) and enzyme-linked immune absorbent spot
(ELISpot) assay were performed as described in the Supporting Methods.
VΔ2+ T CELL CULTURE AND TCR TRANSDUCTION
Healthy donor PBMCs were selectively activated to culture Vδ2+ T cells by adding
1 μmol/L zoledronic acid (#SML0223; Sigma-Aldrich) and transduced as described
in the Supporting Methods. For the cytotoxicity assay, cells were stained with
anti-human TCR Vδ2-BV605 (clone B6; #331430; Biolegend) and sorted by
fluorescence-activated cell sorting (FACS) using BD FACS Aria II before resting.
CYTOTOXICITY ASSAY
In vitro cytotoxicity of TCR-T cells was evaluated by lactate dehydrogenase
(LDH) release of target cells according to the manufacturer’s protocol (#C0017;
Beyotime), as described in the Supporting Methods.
RESULTS
Nearly 96% of chronic HBV infections are caused by five HBV genotypes (A-E),
although genotype C is the most common.(16) HBV core 18-27V (FLPSDFFPSV) is a
proven epitope derived from HBV genotypes A, D, and E, which are the dominant
genotypes in Africa, Europe, and Western Asia, respectively. The hepatitis B
core (HBc) proteins of HBV genotypes B and C contain a slightly different
epitope in the same region (FLPSDFFPSI), 18-27I in this study.(17) As HBV
genotypes B and C are endemic in Eastern Asia, we attempted to focus on
searching for TCRs targeting core proteins derived from these genotypes.
Furthermore, considering that V27 epitope-specific cytotoxic T lymphocytes may
not cross-react with the I27 epitope and may be inhibited by the simultaneous
presentation of V27 and I27 epitopes,(17, 18) an HBV core18-27I pentamer was
used to isolate HBV-specific T cells from PBMCs of patients who were A*02+/HBV+.
A proportion of pentamer-positive T cells was identified from 1 patient with a
chronic HBV infection (Fig. 1A). Pentamer-stained T cells were then isolated by
magnetic beads and expanded by a rapid expansion protocol (REP) (Fig. 1A). The
expanded T cells were purified by pentamer staining again for TCR sequencing.
Instead of limiting dilution or single-cell sequencing, NGS was employed for TCR
identification. The top five abundant TCR Vα chains accounted for 99.6% of all
the presented Vα chains, which in order were TRAV12-2, TRAV14/DV4, TRAV12-2,
TRAV8-3, and TRAV38-2/DV8 (Fig. 1B). Similarly, the top five abundant TCR Vβ
chains accounted for 99.9% of all the presented Vβ chains, which in order were
TRBV27, TRBV4-1, TRBV9, TRBV19, and TRBV29-1. The ranking of the α
complementarity determining region 3 (αCDR3) and βCDR3 of these TCR chains was
correlated with the ranking of the corresponding Vα and Vβ chains (Fig. 1C).
FIG. 1
Open in figure viewerPowerPoint
Isolation of polyclonal HBV-specific T cells and NGS. (A) HBV-specific T cells
were isolated using a peptide-MHC tetramer and expanded by an REP, after which
the expanded T cells were purified by pentamer staining again for NGS. (B)
Frequency of the top five abundant TCR Vα and Vβ genes. (C) Frequency of the top
five abundant αCDR3s and βCDR3s. Abbreviations: PE, phycoerythrin; SSC, side
scatter.
Theoretically, an equimolar endogenous expression of TCR α and β chains would
lead to an equal abundance of Vα and Vβ chains in NGS when all T cells express a
unique TCR. Therefore, a ranking-based pairing strategy was employed. Given that
the purity of pentamer staining-based isolation is approximately 90%, TRAV12-2
and TRBV27 as well as TRAV14/DV4 and TRBV4-1, which account for approximately
90% of Vα and Vβ chains, were paired directly based on their abundances and
ranking in NGS (Fig. 2A) and were constructed into one retroviral vector with
respective modified mouse constant regions (Fig. 2B).(19) We termed the TRAV12-2
and TRBV27 pair TCR1 and the TRAV14/DV4 and TRBV4-1 pair TCR2. Most cluster of
differentiation (CD)8+ T cells transduced with both TCRs (TCR1-T and TCR2-T
cells) can be stained simultaneously by the HBV core18-27I pentamer and
anti-mouse β chain antibody (Fig. 2C), further confirming the reliability of the
pairings of Vα and Vβ chains based on NGS abundances and rankings.
FIG. 2
Open in figure viewerPowerPoint
Ranking-based pairing strategy for TCR identification. (A) TRAV12-2 and TRBV27
as well as TRAV14/DV4 and TRBV4-1, which account for approximately 90% of Vα and
Vβ chains, were paired directly based on their abundances and ranking in NGS.
(B) Schematic representation of TCR α and β chains cloned into one transgene
cassette. Constant regions were replaced by corresponding modified mouse TCR
constant chains (mTRAC and mTRBC) and were fused by a furin-P2A element. (C) FCM
analysis of CD8+ T cells transduced with both TCRs (TCR1-T and TCR2-T cells) and
stained simultaneously by the HBV core18-27I pentamer and anti-mouse β chain
antibody. Abbreviation: LTR, long-terminal repeat.
At the same time, an α–β chain mixture-based pairing strategy was also employed
for the identification of possible TCR pairs. First, the top four Vβ chains,
which account for 99.7% of all the sequenced TCR β chains, were chosen.
Considering that the frequency of pentamer-positive T cells in NGS-sequenced
polyclonal T cells is approximately 90%, these top four Vβ chains should be able
to cover all possible functional TCRs. When dual α chain T cells are present in
pentamer-purified polyclonal T cells, their influence on the overall abundance
of Vα chains can be difficult to predict. However, given that the two α chains
from the same T cell will most likely result in equal abundance in NGS, its
influence can be easily accommodated by including extra Vα chains in
ranking-based selection of Vα chains. When the frequency of dual receptor T
cells is approximately 30%,(14) the possible number (according to the Poisson
probability distribution) of dual α chain T cells (0-4) that correspond to the
four selected Vβ chains and their possibilities (p) are p(0) = 0.3, p(1) = 0.36,
p(2) = 0.22, p(3) = 0.09, and p(4) = 0.03. Therefore, selection of the top six
Vα chains can cover approximately 90% of all possibilities. These chains were
inserted into an MSGV vector. All 24 possible α–β pairs were tested by
cotransfecting T cells with 24 different mixtures of Vα virus and Vβ virus. Only
two engineered T cells showed specific recognition of the core 18-27I epitope
(Fig. 3A,B). Further, FCM analysis showed that activation markers of T cells,
such as 4-1BB, tumor necrosis factor receptor superfamily member 4 (OX40), and
programmed death 1 receptor (PD-1), were also up-regulated (Fig. 3C,D). The two
transfected Vα and Vβ pairs were TRAV12-2 and TRBV27 and TRAV14/DV4 and TRBV4-1,
the same TCR pairs identified by the ranking-based pairing strategy.
FIG. 3
Open in figure viewerPowerPoint
An α–β chain mixture-based pairing strategy for TCR identification. (A,B) Top
four Vβ and top six Vα retroviruses were generated separately and mixed for
subsequent transfection of T cells. T cells transferred with 24 different α–β
chain mixtures were tested by ELISA for the secretion of IFN-γ. TRAV12-2 and
TRAV14/DV4 showed specific recognition of core 18-27I epitope when paired with
TRBV27*01 and TRBV4-1, respectively. (C,D) FCM analysis of the activation
markers (4-1BB, OX40 and PD-1) of CD4 + and CD8 + T cells. Abbreviations: OX40,
tumor necrosis factor receptor superfamily, member 4; PD-1, programmed death 1
receptor.
Both TCRs recognized the core 18-27I and core 18-27V epitopes presented on
HLA-A*02:01 molecules (Fig. 4A). Neither showed recognition of the polymerase
575-583 epitope. Similar results were obtained in the ELISpot assay (Fig. 4B);
many more TCR1-T cells showed recognition of both the core 18-27I and core
18-27V epitopes than TCR2-T cells, whereas neither responded to T2 cells
(Fig. 4B). In addition, both TCR-T cells also recognized intrinsically processed
epitopes (core 18-27V) by HepG2.2.15 (Fig. 4C). Furthermore, this recognition
was blocked by the HLA-I blocking antibody (Fig. 4C).
FIG. 4
Open in figure viewerPowerPoint
Functional evaluation of T cells transduced with TCR1 and TCR2. (A) T2 cells
loaded with different epitopes or DMSO control were incubated with TCR1-T and
TCR2-T cells overnight. The supernatant was tested by ELISA for the amount of
IFN-γ. (B) T2 cells loaded with different epitopes or DMSO control were
incubated with TCR1-T and TCR2-T cells overnight in an anti-IFN-γ
antibody-coated ELISpot plate for the detection of IFN-γ secreting T cells. (C)
HepG2 cells and HepG2.2.15 cells were incubated with TCR1-T and TCR2-T cells
overnight with or without the addition of anti-HLA-A/B/C antibody (clone W6/32).
Supernatants were taken for the quantification of IFN-γ by ELISA. Data in (A,B)
represent mean ± SD.
Potential antitumor function was evaluated by the LDH detection assay. We first
generated a HepG2 cell line that stably integrated the HBc coding sequence
(18-27I) derived from HBV genotypes B and C. Both TCR1-T cells and TCR2-T cells
specifically lysed HepG2-HBc cells in a dose-dependent manner (Fig. 5A) but did
not lyse wild-type HepG2 cells (Fig. 5B). As shown by intracellular staining
(Fig. 5C,D), only T cells transduced with TCRs (mouse TRB constant region
positive [mTRBC+]) specifically recognized HepG2-HBc cells and did not respond
to HepG2 cells.
FIG. 5
Open in figure viewerPowerPoint
Recognition of intrinsically processed core 18-27I epitope. (A) HepG2 stably
transferred with HBV core antigens derived from genotypes B and C (HepG2-HBc) or
(B) HepG2 wild type were incubated with TCR1-T and TCR2-T cells. The specific
lysis of target cells was measured by LDH assay. Data represent mean ± SD. (C,D)
FCM analysis of intracellular IFN-γ of both TCR1-T and TCR2-T cells after
incubating overnight with (C) HepG2-HBc or (D) HepG2. Abbreviations: Con-T,
control T cells; E:T, effector to target cell ratio.
The functional avidity of the two TCRs was further evaluated by a peptide
dilution assay. The 50% effective concentration (EC50) values of TCR1-T and
TCR2-T cells for the core 18-27I epitope were 1.263 × 10−6 mol/L and
2.218 × 10−6 mol/L, respectively, (Fig. 6A,B). For the core 18-27V epitope, the
EC50 values were 7.711 × 10−11 mol/L and 3.145 × 10−10 mol/L, respectively.
Neither showed recognition of the polymerase 575-583 epitope. Furthermore, the
recognition of core 18-27 epitopes on the most frequent HLA-A*02 subtypes in
China was also evaluated. Based on the common and well-documented alleles in
Chinese, the top 10 most frequent HLA-A*02 subtypes (HLA-A*02:01, A*02:07,
A*02:03, A*02:05, A*02:06, A*02:09, A*02:10, A*02:11, A*02:48, and A*02:53N)
were constructed and transduced into Cos-7 cells. Both TCRs recognized their
cognate peptides derived from the HBV genotype or A/D/E (core 18-27V) or B/C
(core 18-27I) not only when presented by HLA-A*02:01 but also when presented by
02:05, 02:06, 02:07, 02:09, 02:10, and 02:11 (Fig. 6C,D).
FIG. 6
Open in figure viewerPowerPoint
Functional affinity and recognition of different HLA-A*02 subtypes. (A,B) T2
cells loaded with a serial dilution of different epitopes were incubated with
(A) TCR1-T and (B) TCR2-T cells overnight. Supernatants were taken for
quantification of IFN-γ by ELISA. (C,D) Cos-7 cells transduced with different
A*02 subtypes were loaded with 18-27I and 18-27V epitopes and then incubated
with (C) TCR1-T and (D) TCR2-T cells overnight for the detection of IFN-γ in the
supernatant by ELISA. Data represent mean ± SD. Abbreviations: HBV Pol, HBV
polymerase; WT, wild type.
The use of mouse constant regions can reduce the mispairing of TCR chains(19)
but may lead to immunologic rejection when used in vivo. Therefore, the
functions of the two paired HBV-specific TCRs were further verified by
transduction to γδ-T cells, which can also greatly reduce potential mispairings
of TCR chains because of the employment of different constant chains.(20)
Furthermore, it is known that a subgroup of Vδ2+ T cells expresses its
coreceptor CD8 through an α–α homodimer instead of an α–β heterodimer at a
relatively low level of constant expression; this makes them an ideal tool for
the functional evaluation of the dependence TCRs on coreceptors. Vδ2+ T cells
transduced with both TCRs (Supporting Fig. S1) specifically recognized the core
18-27V epitope loaded on T2 cells (Fig. 7A,B). In contrast, nontransduced Vδ2+ T
cells did not show any recognition of target cells. This specific recognition
was further confirmed by intracellular interferon-γ (IFN-γ) staining
(Fig. 7B,C). Moreover, the proportion of IFN-γ-positive Vδ2+ T cells was much
higher than the proportion of Vδ2+ CD8+ T cells, which suggests that the TCR
functioned without coreceptors. The recognition of intrinsically processed
epitope was further verified. Vδ2+ T cells transduced with TCR1 lysed HepG2.2.15
cells dose dependently, while the nontransduced Vδ2+ control T cells only showed
moderate cytotoxicity against HepG2.2.15 (Fig. 7D). This is not surprising as
Vδ2+ T cells is known for non-TCR-mediated activity, such as natural killer
group 2 member D (NKG2D)/ligand interaction-triggered cytotoxicity.(21, 22)
FIG. 7
Open in figure viewerPowerPoint
Functional evaluation of Vδ2 T cells transduced with TCR1 and TCR2. (A) Vδ2+ T
cells transduced with both TCRs were incubated overnight with T2 cells loaded
with core 18-27V epitopes or DMSO. Supernatants were taken for the
quantification of IFN-γ by ELISA. (B) An illustration of the FCM analysis of
intracellular IFN-γ of Vδ2+ T cells after overnight incubation with T2 cells
loaded with core 18-27V or DMSO. (C) Statistics summary of the proportion of
intracellular IFN-γ positive Vδ2+ T cells after overnight incubation with T2
cells loaded with core 18-27V or DMSO. (D) HepG2.2.15 cells were incubated
overnight with Vδ2+ T cells transduced with TCR1 or the nontransduced control T
cells. Supernatants were taken for cytotoxicity assay. Data in (A,C,D) represent
mean ± SD. Abbreviations: E:T, effector to target cell ratio; SSC, side scatter.
DISCUSSION
In this study, polyclonal T cells specific to the HLA-A*02-restricted 18-27
epitope of the HBV core antigen were purified by a peptide-MHC multimer-based
approach and TCRs were sequenced by NGS, a bulk sequencing approach. Without the
typically involved TCR singularization processes, such as limiting dilution for
single-cell expansion or single-cell sequencing, the Vα and Vβ chains were
paired based solely on NGS. We suggest that NGS-based pairing strategies
(ranking-based and α–β chain mixture-based strategies) are feasible for the fast
and efficient identification of HBV-specific TCRs.
The abundance of Vα chains showed a correlation with the abundance of the
corresponding αCDR3s in NGS, suggesting that there are no shared αCDR3s by
several different Vα chains or one shared Vα chain by different αCDR3s, which is
also true for Vβ chains and βCDR3s. Nevertheless, as mentioned earlier, a
proportion of T cells may encode two mRNAs of two different TCR α chains.
Dual-receptor T cells may present TCRs at a lower quantity on the cell surface
than single-receptor T cells, which may lead to less clonal expansion when
encountering repeated antigen stimulation in a chronic virus infection, such as
HBV.(23) This means that the outgrowth of single-receptor virus-reactive T cells
may account for a much larger proportion than 70%. Equimolar expression of
endogenous TCR α and β chains would theoretically lead to equal abundances of Vα
and Vβ chains in NGS, but there could always be some deviations considering that
the Vα and Vβ chains are normally cloned separately by different primer sets and
that two different NGS libraries are generated. However, the ranking of the TCR
chains presented in their own library should not be affected by such different
manipulations. Therefore, it is also important to correlate the ranking of
different Vα and Vβ chains when processing the pairing. Among the two identified
TCR pairs, the rankings of Vα chains (TRAV12-2 and TRAV14/DV4) are the same as
the corresponding Vβ chains (TRBV27 and TRBV4-1), whereas their abundances
showed differences of 17% and 9%, respectively. Pentamer staining confirmed that
both Vα and Vβ chains paired correctly when introduced on the surface of T
cells.
Interestingly, the α–β chain mixture-based pairing strategy identified the same
two TCR pairs as the ranking-based pairing, which suggests that the NGS
frequency-based selection of Vβ chains and dual-receptor T cell frequency-based
selection of Vα chains can cover most functional T cells. Furthermore, the
results of the α–β chain mixture-based pairing strategy suggest that the two
identified TCRs are paired endogenously, although they are not obtained from
single cells (single-cell sequencing or limiting dilution for single-cell
expansion). As demonstrated previously,(24) consistent and repeated antigenic
stimulation in chronic infection continues to narrow the TCR repertoire, which
results in the vast majority of antigen-specific responses being clonal.
Additionally, both our in vitro stimulation and the REP processes may have
contributed to the narrowing of the HBV-specific TCR repertoire, given that not
all T cells are expandable under the same conditions.(25, 26) Our results
highlight the potential value of the α–β chain mixture-based pairing strategy
for fast TCR identification when there is a limited number (e.g., <10) of Vα and
Vβ chains presented, which is normally true for peptide-MHC multimer-purified
polyclonal virus-specific T cells from donors with a chronic virus
infection.(10, 27) Certainly, this strategy is not suitable for TCR
identification from T cell mixtures that do not undergo peptide-MHC multimer
purification because neither the potential functional Vβ chains nor the
corresponding Vα chains can be selected.(11) A reasonable concern about this
strategy is that the virus mixtures we used here led to a limited cotransfection
ratio, which was approximately 2% in our case, as detected by mTRBC in FCM
analysis. The reasons for this result are multiple. One is that the titer of
viruses available for transfection is limited when they are generated in 24-well
plate wells for feasible manipulation. Another is that the nonpaired transferred
chains are internalized quickly. Either way, the high sensitivity of functional
assays, such as ELISA and ELISpot, can accommodate such a low frequency of
functional T cells.(28, 29) Moreover, the plasmid-based TCR-introducing approach
can employ much simpler manipulation processes (e.g., mixture of plasmids and
electroporation for transposon-based gene transfer) than virus-based approaches,
which further increases the applicability of this strategy.(30)
The specificity of both TCRs was verified by the recognition of HBV core 18-27I
and HBV core 18-27V epitopes but not nonrelevant HBV polymerase 585-593 epitopes
in ELISA. Although no significant differences in functional avidity were
detected between the two TCRs by ELISA, the ELISpot assay clearly showed a much
higher functional avidity of TCR1 for both core 18-27V and 18-27I epitopes. This
result may be explained by the higher sensitivity of the ELISpot assay and
highlights its potential value in the comparison of the functional avidity of
different TCRs. Additionally, the function of the two paired TCRs was further
confirmed by showing recognition of the endogenously presented epitope (core
18-27V) by HepG2.2.15. The blocking of recognition by anti-HLA-I blocking
antibody suggests that this recognition is HLA-I molecule restricted. The
peptide dilution assay also revealed a much higher functional avidity of both
TCRs for the core 18-27V epitope in contrast to the core 18-27I epitope. This
result may be attributed to the stronger binding affinity between the core
18-27V epitope and HLA-A*02 subtypes and also to the fact that while the 27I
reduces the binding of the epitope to HLA-A*02, it does not alter TCR
interaction.(17, 31) It is suggested that binding stability between the epitope
and HLA molecule is a better indication of immunogenicity than affinity,
considering that the epitope should be able to bind to the MHC molecule and
remain bound for long enough to be presented to and recognized by T cells to
elicit an immune response.(32, 33) As described,(34) the comparatively lower
binding stability of the 18-27I epitope with A*02:03 may partially explain the
inactivity of both TCRs. Similar low-binding stability of the 18-27I epitope
with A*02:48 was predicted by using NetMHCstabpan version1.0 (data not
shown).(35) This suggests that other epitopes from HBV genotypes B/C are
required when performing TCR identification for these two types of HLAs (A*02:03
and A*02:48). Furthermore, the activity of both TCRs for multiple HLA-A2
subtypes makes them potentially applicable to a wide range of populations.
Both TCRs showed a similar pattern for dose-response curves toward two epitopes,
but TCR1 (TRAV12-2 and TRBV27) was more potent with a lower EC50 value. The
ELISpot assay further confirmed the higher functional avidity, as previously
described. Interestingly, the prevalent expression of TRAV12-2 was identified in
different TCRs targeting HLA-A*02-restricted epitopes derived from viruses as
well as cancer antigens.(10, 36-38) Structural analysis revealed an unusual
“α-centric” TCR binding mode in which the invariable CDR1α loop, not the CDR3
loops, dominated the interaction with the HLA-A*02 molecule. This finding
suggests that TCRs expressing the TRAV12-2 could have an intrinsic advantage
when binding to cognate antigens restricted by HLA-A*02 due to the innate
recognition of residues on the MHC surface.(36) This advantage may explain why
the two V chains of TCR1 are mostly abundant in NGS. A higher functional avidity
may lead to the outgrowth of TCR1-T cells in vivo following repeated antigenic
stimulation and when stimulated in vitro using the core 18-27I peptide. Given
that as the abundance of TCRs increases so does their chances of being covered
using NGS-based pairing strategies, this positive association between the
abundances of TCR chains and their functional avidity lends further credence to
the potential value of an MHC multimer-aided NGS-based strategy for the fast
identification of TCRs with superior functional avidity. Additionally, the NGS
abundance of TCR chains can be valuable information in the choice of TCRs with
variable affinities, such as an intrinsic higher abundance; hence, the higher
avidity of TCR1 supports its potentially better efficacy for TCR-T cell
therapy.(39) Although the strategy based on the abundance of TCR reads is shown
to work in a single patient and for a single epitope in the present study, it
has been successfully and repeatedly applied in our laboratory to identify
functional TCRs specific to epitopes derived from other types of viruses,
including Epstein-Barr virus and cytomegalovirus (unpublished data). In
addition, it would be interesting to determine whether single-cell analysis is
able to verify the pair of TCR α and TCR β detected by calculating the abundance
of TCR reads in bulk sequencing. We will address this issue in future studies.
The possible mispairing of endogenous and introduced TCR chains that do not
experience negative selection in the thymus may potentially lead to autoimmune
reactions; therefore, Vα and Vβ chains were expressed from one construct with
the modified mouse TCR constant domains by adding an extra disulfide bond. As
reported,(19) this strategy can reduce the chance of mispairings. However, this
strategy may lead to allergic reactions when used in vivo and therefore is not
widely employed in clinical TCR-T cell therapies. An alternative approach to
avoid mispairing would be transducing TCRs to γδ-T cells, such as Vδ2+ T cells,
which employ different TCR constant regions from αβ T cells.(20) The superior
functional avidity of both TCRs was further verified by the activities of Vδ2+ T
cells without the expression of coreceptors. In addition to directly acting on
target cells, Vδ2+ T cells can also function as antigen-presenting cells
following activation for the recruitment of reactive CD4 and CD8 T cells to
bridge innate and adaptive immunity.(22, 40) Furthermore, Vδ2+ T cells, as the
first-line defenders of infections, can defend against infection by secreting a
unique panel of antiviral cytokines following activation.(41, 42) For example,
Vδ2+ T cells have been found to be capable of uniquely secreting the dendritic
cell-inducing cytokine granulocyte-macrophage colony-stimulating factor
following activation,(41) which has been proven to eliminate hepatitis B surface
antigen-positive hepatocytes.(43) This is the first study to confirm the
activities of HBV-specific TCRs when introduced into γδ-T cells, which have the
potential to contribute uniquely to the treatment of HBV-related diseases,
including HCC.
A reasonable concern about HBV-specific TCR-T cell therapy is the potential
liver toxicity induced by the lysis of HBV-infected yet functional liver cells.
However, as demonstrated in bone marrow transplantation, the transfer of
HBV-specific immune cells into patients with chronic HBV can lead to HBV
clearance while causing limited and acceptable liver toxicity.(44-46) In a
humanized mouse model, the data suggest that when a minor proportion of liver
cells is infected by HBV, the adoptive transfer of TCR-T cells only leads to
moderate toxicity.(47) In addition, antiviral therapies, including nucleotide
analogs as well as upcoming RNA interference therapies, can greatly reduce the
antigen presence in normal liver cells, which can potentially be combined with
TCR-T cell therapies for patients with HCC.(48) Furthermore, TCR-T cell therapy
has already been shown to be a safe approach for the treatment of metastatic HCC
in patients with a liver transplant.(9) Transient introduction of HBV-specific
TCRs has also been proven to be a safe approach to avoid robust and consistent T
cell reactions.(49, 50)
In summary, we present an example of fast and efficient NGS-based identification
of HBV-specific TCRs. Functional evaluation confirmed their specificity and
avidity, which means that they may be able to be used to generate TCR-T cells to
treat patients with HBV-related HCC.
SUPPORTING INFORMATION
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Author names in bold designate shared co-first authorship.
Volume5, Issue6
June 2021
Pages 1106-1119
* FIGURES
* REFERENCES
* RELATED
* INFORMATION
RECOMMENDED
* Identification of α‐fetoprotein‐specific T‐cell receptors for hepatocellular
carcinoma immunotherapy
Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Xiaotao Jiang, Qi Li, Heping Liu,
Lei Huang, Juan Wu, Esteban Celis, Todd Merchen, Edward Kruse, Yukai He,
Hepatology
* Identification of a hepatitis C virus–reactive T cell receptor that does not
require CD8 for target cell recognition
Glenda G. Callender, Hugo R. Rosen, Jeffrey J. Roszkowski, Gretchen E. Lyons,
Mingli Li, Tamson Moore, Natasha Brasic, Mark D. McKee, Michael I. Nishimura,
Hepatology
* High‐throughput T‐cell receptor sequencing across chronic liver diseases
reveals distinct disease‐associated repertoires
Evaggelia Liaskou, Eva Kristine Klemsdal Henriksen, Kristian Holm, Fatemeh
Kaveh, David Hamm, Janine Fear, Marte K. Viken, Johannes Roksund Hov, Espen
Melum, Harlan Robins, Johanna Olweus, Tom H. Karlsen, Gideon M. Hirschfield,
Hepatology
* High frequency of the MAGE‐1 gene expression in hepatocellular carcinoma
N Yamashita, H Ishibashi M.D., K Hayashida, J Kudo, K Takenaka, K Itoh, Y
Niho,
Hepatology
* Immunosuppressive Drug‐Resistant Armored T‐Cell Receptor T Cells for Immune
Therapy of HCC in Liver Transplant Patients
Morteza Hafezi, Meiyin Lin, Adeline Chia, Alicia Chua, Zi Zong Ho, Royce Fam,
Damien Tan, Joey Aw, Andrea Pavesi, Thinesh Lee Krishnamoorthy, Wan Cheng
Chow, Wenjie Chen, Qi Zhang, Lu-En Wai, Sarene Koh, Anthony T. Tan, Antonio
Bertoletti,
Hepatology
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