Abstract
Background/Aim: Adoptive cell therapy using antigen-specific T cells is a promising treatment modality for cancer patients. Various methods to isolate specific T cells and identify corresponding T cell receptor (TCR) sequences are known. This study aimed to identify antigen-specific TCR from T cells isolated using carboxyfluorescein succinimidyl ester (CFSE), which marks proliferating activated T cells. Materials and Methods: CFSE stained healthy donor peripheral blood mononuclear cells (PBMCs) were treated with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) peptides for seven days. Then, proliferating T cells with decreased CFSE staining were isolated and single cell VDJ sequencing was performed on isolated T cells to identify antigen-specific TCRs. Results: As antigen-specific TCR candidates, ten TCR clones were selected for the CMV antigen and five for the EBV antigen. The reactivity of ten CMV TCR-transduced T cells and one EBV TCR-transduced T cell toward T2 cells pulsed with CMV or EBV peptide was confirmed via NFAT-luciferase, IFN-γ ELISA, and cytotoxicity assays. Conclusion: Identification of antigen-specific TCRs with CFSE staining is a valid method for the development of effective immunotherapy. The identified CMV- or EBV-specific TCRs can be used for adoptive cell therapy to treat cancer.
In recent times, several immunotherapeutic strategies harnessing the immune system’s specificity to target and eliminate tumors have garnered significant interest. Despite the success of immune checkpoint inhibitor (ICI) therapies, such as anti-cytotoxic T lymphocyte antigen 4 (CTLA-4) or anti-programmed death-1 (PD-1)/PD-L1 inhibitors, only 15-30% of patients with solid tumors benefit from ICIs (1). ICIs operate by alleviating immune suppression induced by immune checkpoint molecules on cytotoxic T cells, thereby fostering immune activation (2, 3). However, the lack of anticancer T cells in the tumor microenvironment (TME) is one of the resistance mechanisms toward ICIs (4, 5). Anticancer T cells can be cultured and used as adoptive cell therapy (ACT). ACT includes T cells cultured from peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs), and engineered T cells, such as chimeric antigen receptor T cells (CAR-T) and T cell receptor engineered T cells (TCR-T). CAR-T consists of three domains: single-chain variable fragment (scFv), immunoreceptor tyrosine-based activation motif (ITAM), and CD3, which can recognize antigens and activate T cells. However, CAR-T is limited to recognizing only cell surface membrane proteins, which constitute approximately 1% of the total proteins expressed in cells (6). CAR-T therapy has demonstrated clinical success in hematological malignancies, with several products receiving approval for integration into daily medical practice. However, its effectiveness in solid tumors has been limited (7). TCR-T has a TCR that recognizes specific major histocompatibility complex (MHC)-peptide antigen complex. A TCR consists of an alpha chain and a beta chain. Targets for TCR-T are antigens derived from intracellular and surface proteins, representing a strength of TCR-T over CAR-T, given that most antigens are intracellular (6).
Antigens are recognized by the amino acid sequence of complementarity-determining region 3 (CDR3) in the alpha and beta chains. CDR3 is determined through specific recombination of variable (V), diversity (D), and joining (J) gene segments, and reacts with various antigens through numerous recombinations (8, 9). It is important to analyze CDR3, which is a site that specifically binds antigens. DNA sequencing technology-based platforms include the 454/Roche platform based on pyrosequencing technology, Illumina/Solexa platform, and Ion Torrent/Life Technologies based on Sanger-sequencing technology (10-12). The advancement of next-generation sequencing (NGS) technology has enabled the analysis of TCR sequences. In addition, the development of single-cell analysis has rendered analysis of alpha and beta pairs of TCR sequences possible (11, 13). With the development of single-cell analysis technology, immune repertoire research is becoming crucial because it is possible to increase the level of repertoire analysis by accurately identifying and analyzing pairs rather than analyzing a group of cells (13).
Tumor antigens consist of tumor-associated antigens (TAAs) and tumor-specific antigens (neoantigens). NY-ESO-1 and Epstein-Barr Virus (EBV) are well-known TAAs (14, 15). Viral antigens can elicit high-affinity TCR responses. The development of some solid tumors is directly attributed to viral infections. For instance, nasopharyngeal carcinoma is caused by EBV infection. It is important to identify and selectively classify specific T cells recognizing tumor antigens (13). There are several methods for identifying antigen-specific T cells, including sorting of T cells using MHC-tetramer or identification based on expression of markers, such as 4-1BB and PD-1 (16-19). In this study, we identified cytomegalovirus (CMV) or EBV-specific TCR through carboxyfluorescein diacetate succinimidyl ester (CFSE) staining method. Proliferated cells, which show decreased CFSE staining intensity, were isolated as antigen-responsive T cells. Subsequently, dominant clones of the cells were selected, followed by validation of antigen-specific TCR.
Materials and Methods
Human samples. PBMCs were isolated from whole blood obtained from healthy donors. Informed consent was obtained from the donors (IRB#2017-0784). Separation of blood cells was performed using density centrifugation (Ficoll Paque, Cytiva, Marlborough, MA, USA). After isolation, healthy donor PBMCs were immediately used or stored in a liquid nitrogen tank.
Staining of PBMCs with CFSE dye and stimulation with antigen peptide. The isolated PBMCs were treated with 0.5 μM of CFSE (Thermo Fisher Scientific, Waltham, MA, USA). Stained PBMCs (1 e6 cells/tube) were stimulated with CMV peptide (NLVPMVATV, 1 μg/ml) or EBV peptides (EBV-1 LTAGFLIFL, EBV-2 CLGGLLTMV, EBV-3 FLYALALLI, EBV-4 TYGPVFMCL, and EBV-5 RYCCYYCLTL, 1 μg/ml) and cultured for 7 days to determine T cell proliferation.
Sorting analysis. CFSE-stained PBMCs treated with antigens were harvested after 7 days and washed with FACS buffer. After Fc blocking with Human Tru-Stain FcX (Fc Receptor Blocking Solution, Biolegend, San Diego, CA, USA), cells were stained with APC/Cy7 anti-human CD3 antibody and PerCP/Cy5.5 anti-human CD8 antibody (Biolegend, San Diego, CA, USA). After 20 min, PBMCs were washed and suspended in DAPI mixed Sorting buffer (1% FBS in DPBS). FACS (The BD FACSCanto™ II Flow Cytometer; BD, San Jose, CA, USA) was conducted to analyze the samples. For identification antigen-specific TCR, CD8+ and CD8− T cells with decreased CFSE fluorescence were sorted and collected in CTL medium (Thermo Fisher Scientific). Data analysis was conducted using the FlowJo.7.6.5 software (Tree Star, Ashland, OR, USA).
Single-cell VDJ library construction and sequencing. Sorted T cells were used for the preparation of a single-cell library. Single-cell libraries were prepared from CMV or EBV-specific T cells according to the manufacturer’s protocol of Chromium Next GEM Single Cell 5′ v2 Reagent Kits (10× Genomics, Pleasanton, CA, USA). The sorted cells and kit reagents were mixed with gel beads containing barcoded oligonucleotides (unique molecular identifier, UMIs) and oligo dTs (used for reverse transcription of polyadenylated RNAs) to generate a single gel bead-in-emulsion (GEMs). The barcoded cDNAs in each GEM were pooled for PCR amplification, and adapter and sample indices were added. Single-cell libraries of gene expression and VDJ were sequenced on the Illumina Novaseq 6000 sequencing platform (Illumina, San Diego, CA, USA) with paired-end 100 bp and 150 bp reads, respectively.
Analysis of sequence data. Sequencing data generated by single-cell platforms, was analyzed using the 10X Genomics platform, such as Cellranger multi or Cellranger vdj. Gene expression data and TCR data were aligned with GRCh38-2020-A and refdata-cellranger-vdj-GRCh38-alts-ensembl-5.0.0, respectively. Filtered gene expression data matrix as an initial Cellranger output was imported using the Read10X function of the Seurat R package. nCount_RNA (number of UMIs), nFeature_RNA [number of detected genes (features)], percent_mito (frequency of reads that map to mitochondrial genes), and percent_ribo (frequency of reads that map to ribosomal genes) were quantified in each sample using summary and PercentageFeatureSet functions. The scRepertoire R package was used for the TCR clonotype analysis. The contig list per each cell barcode was generated using the combineTCR function. A unique clonotype was defined as demonstrating the same V(D)J and CDR3 nucleotide sequences of TCR alpha/beta chains. The total count or relative frequency of unique clonotypes was quantified in each sample. In the case of EBV-specific T cells, clonotypic information of a T cell subset was preferentially attached to our Seurat object using the combined expression function, and the relative proportion of clonotypes was calculated in each group.
Searching in TCR database. Antigen-specificity of the top 10 dominant clones in each sample was confirmed using VDJmatch tools (20) with the latest version VDJdb (21). VDJmatch was carried out with input data containing V, D, and J genes, CDR3 nucleotide sequences, and CDR3 amino acid sequences and ran separately for TRA and TRB genes (e.g., -R TRB). Matching against the Immune epitope database (IEDB) (22) was performed using the TCRMatch tools (23) as an input data for only CDR3beta amino acid sequences.
Generation of antigen-specific TCR-T construct and production of lentivirus particles. To generate CMV or EBV-specific TCRs, analyzed TCRs were synthesized by the BIONICS company (Seoul, Republic of Korea) and cloned into a lentiviral vector (FUGW-IRES-GFP plasmid). To distinguish from endogenous TCR, a constant region of mouse TCRα/β was used.
Lipofectamine, lentiviral packaging plasmids (pMDL-prre, pRSV-rev and pMD2.G) and the TCR plasmid were added to the culture media of Lenti-X 293T cells and incubated for two days. The supernatant of Lenti-X 293T cell was obtained, filtered, and concentrated with the Lenti-X concentrator (Takara, Shiga, Japan) at 4°C for two days. To confirm the titer of the prepared lentiviral particles, we measured the titer of p24 using an ELISA method with a plate coated with anti-HIV p24 capture antibody according to the manufacturer’s (Takara) instructions.
Generation of TCR-T. For Jurkat [TCR knock-out (KO)]-NFAT-Luc cell line transduction, 1e6 cells were treated with TCR lentiviral particles and protamine, followed by centrifugation (800×g/90 min/32°C). For PBMC transduction, PBMCs were stimulated with TransAct (100 μl/2e7 cells, Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-2 (20 IU/ml, Boehringer Ingelheim, Biberach, Germany) for two days. Activated PBMCs (2e6 cells) were treated with TCR lentiviral particles and protamine, followed by centrifugation (800×g/90 min/32°C). The TCR expression in transduced cells was confirmed via FACS Canto II-based flow cytometry analysis using an anti-mouse TCRβ constant antibody (Biolegend).
Co-culture of T cells and target cells. T2 cells or T2-Luc cells were pulsed with the antigen peptide corresponding to each TCR (100 μg/ml) and β2m (3 μg/ml; BD), followed by incubation at room temperature for 2 h. For T cell activation assay, Jurkat (TCR KO)-NFAT-Luc cells and T2 cells were seeded at a ratio of 10:1 in a white 96 well plate and incubated for 24 h. For IFN- γ ELISA assay, primary T cells and T2 cells were seeded at a ratio of 4:1 in a 96-well plate and incubated for 24 h. For cytotoxicity assay, primary T cells and T2-Luc cells were seeded at ratios of 30:1, 10:1, 3:1, and 1:1 in a white 96-well plate and incubated for 24 h.
Luciferase assay was performed using Bright-Glo™ Luciferase Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The luminescence was measured via a microplate reader (Glomax, Promega). IFN- γ ELISA assay was performed using a human IFN- γ ELISA kit (Koma Biotech, Seoul, Republic of Korea) to measure the concentration of INF- γ in the supernatant.
Results
Isolation of antigen-specific T cells. CFSE-stained PBMCs (PBMC 21002, PBMC21003, and PBMC21010) were treated with CMV or EBV peptides. To obtain antigen-specific T cells, we sorted cells based on CFSE staining and CD8 positivity. Proliferation of CMV- or EBV-stimulated T cells was observed at 10% and 2-3% on day seven, respectively (Figure 1). The number of sorted cells in response to CMV and EBV was 3.4e4 and 2.6e5 for PBMC21002, 2.9e4 and 4.8e3 for PBMC21003, and 9.5e5 and 1.6e6 for PBMC21010, respectively (Table I). All isolated cells were used for TCR sequencing.
Sorting of antigen-specific T cells from healthy donor PBMC21002, PBMC21003, and 21010 on day 7. A) Proliferating cells after activation with cytomegalovirus (CMV) peptide or Epstein-Barr virus (EBV) peptide mix were assessed by carboxyfluorescein succinimidyl ester (CFSE) staining analysis and sorted from PBMC 21002,21003, and 21010. B) Flow cytometry analysis in CD3+ T cells assessed via CFSE staining.
Number of sorted carboxyfluorescein succinimidyl ester (CFSE)-negative cells reacted with cytomegalovirus (CMV) peptide or Epstein-Barr virus (EBV) peptides mix.
CMV-specific TCR sequences. Based on the single-cell gene expression profile, the frequency of CD3+ T cells in total cells was 81.4% (2,297/2,822), 92.6% (1,160/1,253), and 91.0% (3,846/4,225) in PBMC21002, 21003, and 21010, respectively. Among these, 92% (2,113/2,297), 97.5% (1,131/1,160), and 97.2% (3,739/3,846) were identified as CD3E+CD4−CD8+ (Table II). CD8+ T cells were analyzed using the output of the CellRanger vdj.
Single cell count of cytomegalovirus (CMV) activated T cells in PBMC21002, 21003, and 21010.
The top 10 clonotypes were also present in >80% of all healthy donors (Figure 2). Antigen specificity of the top 10 clonotypes in each sample was analyzed using searching tools (i.e., VDJtools and VDJMatch) in the database (VDJDB and IEDB) with information on known TCRs. TCRα or TCRβ were compared using the TCR database that includes TCRs specific for the various antigens. Several clonotypes (top 2 and top 6 in PBMC21002, top 4 and top 5 in PBMC21003, and top 2 in PBMC21010) were identified as TCRs reacting to HLA-A*02:01 restricted CMV pp65 antigen (NLVPMVATV). The top 1 clonotype in PBMC21002 was identified as TCRs reacting to HLA-A*02:01 restricted to influenza A antigen (GILGFVFTL). Based on the frequency and TCR database findings, we selected clones having >10% frequency and clones reacting to CMV pp65 antigen in each case as a CMV-specific TCR candidate (Table III).
Frequency of the top 10 T cell receptor (TCR) clones. Cytomegalovirus (CMV) or Epstein-Barr virus (EBV) TCR repertoires were sorted in order of frequency (%) and the ratio of the top 10 clones is shown.
Selected cytomegalovirus (CMV)-specific T cell receptor (TCR) candidates.
EBV-specific TCR sequences. In EBV-specific T cells, the frequency of CD3+ T cells in total cells was 89.3% (3,679/4,118), 93.1% (283/304), and 94.1% (5,020/5,332) in PBMC21002, 21003, and 21010, respectively. Of these 64.9% (2,388/3,679), 64.7% (183/283), and 74.7% (3,748/5,020) were CD3E+CD4−CD8+ cells, respectively (Table IV).
Cell count of Epstein-Barr virus (EBV)-specific T cells according to the presence/absence of CD3E, CD4, and CD8 gene expression in PBMC21002, 21003, and 21010.
Within the CD3E+CD4-CD8+ group, identified dominant clones comprised a substantial fraction ranging from 30% to 80% of the group repertoire (Figure 2). The data of TCRα or TCRβ of the top 10 clonotypes in each sample were also compared to the information in the TCR database including data on TCRs specific to various antigens. Several clonotypes (top 4 and top 9 in PBMC21002 and top 8 in PBMC21010) were identified as TCRs reacting to HLA-A*02:01 restricted to CMV pp65 antigen (NLVPMVATV). The top 2, top 4, and top 9 clonotypes in PBMC21002 were identified as TCRs reacting to HLA-A*02:01 restricted to influenza A antigen (GILGFVFTL). Because there were no confirmed EBV-reactive TCRs, we selected clones having >10% frequency in each case as an EBV-specific TCR candidate (Table V). A total of 5 clones (EBV TCR 1~5) were selected as EBV-specific TCR candidates. Among the candidates, EBV TCR 2 was identical to CMV TCR 1, and EBV TCR 4 was identical to CMV TCR 5.
Selected Epstein-Barr virus (EBV)-specific T cell receptor (TCR) candidates.
Validation of CMV-specific TCR-T. Ten plasmids with CMV-specific TCR candidates were transduced into TCR-KO Jurkat-NFAT-Luc cell line using lentiviral particles, and the expression level of CMV TCR was confirmed using FACS based on the antibody against mouse TCRβ (mTCRβ) constant region on days 1 and 7. On day 1, CMV TCRs 4 and 6 showed a low mTCRβ+ cell rate of 48.3% and 57.2%, respectively, compared to other CMV TCRs. However, on day 7, all 10 CMV-specific TCRs showed a mTCRβ+ cell rate of over 90% (Figure 3A). In the Jurkat-NFAT-Luc cell line in which the expression of CMV-specific TCR candidates was confirmed, the level of luminescence expression (TCR activation by antigen) was examined after 24 h using T2 cells pulsed with CMV or influenza A peptide. Among the 10 CMV-specific TCR candidates, all candidates showed reactivity with CMV peptide. TCRs 1, 5, 9, and 10 showed higher reactivity compared to other TCRs (Figure 3B).
Responsiveness of cytomegalovirus (CMV) T cell receptors (TCRs) to CMV antigen. A) Efficiency of the transduction of selected CMV-TCRs into the Jurkat NFAT-Luc cell line on days 1 and 7. B) Reaction between transduced Jurkat NFAT-Luc cell line and CMV peptide-pulsed T2 cells on day 7. The reactivity of the Jurkat NFAT-Luc cell line in which CMV-specific TCR was transduced by pulsing CMV peptide into the T2 cell line was confirmed by luciferase assay. C) Transduction efficiency of the selected CMV-specific TCR into healthy donor peripheral blood mononuclear cells (PBMCs) on days 7 and 14. D) Analysis of the reaction between CMV TCR-transduced T cells and T2 cells pulsed with CMV peptide by IFN-γ ELISA. E) Cytotoxicity of CMV TCR-transduced T cells against T2 cells pulsed with CMV peptide.
Next, 10 types of CMV-specific TCR candidates were transduced using lentiviral particles into healthy donor PBMCs, and expression was confirmed through mTCRβ expression on days 7 and 14 (Figure 3C). On day 14, TCR 4 and 6 showed a mTCRβ+ cell rate of less than 5%, but TCR 10 showed a rate of more than 30%. TCRs 1, 2, 3, 5, 7, and 9 showed approximately 10% expression. IFN-γ secretion level was confirmed using ELISA assay after 24 h of co-culture of TCR-transduced T cells and T2 cells pulsed with CMV or influenza A peptide. All CMV TCRs reacted specifically to the CMV antigen, of which TCR 8 secreted the lowest concentration of IFN-γ at 230 pg/ml), and TCR 10 secreted the highest concentration of IFN-γ at 650 pg/ml (Figure 3D). The killing efficacy of CMV TCR was measured using a cytotoxicity assay after 24 h of co-culture of T2 cells pulsed with CMV peptides and TCR-transduced T cells at ratios of 1:1, 3:1, 10:1, and 30:1. CMV TCR 1, 3, 5, 7, 8, 9, and 10 showed a killing efficiency of over 60% at a ratio of 30:1 (Figure 3E).
Validation of EBV-specific TCR-T. Five EBV-specific TCR candidates were transduced in the TCR-KO Jurkat NFAT-Luc cell line through lentiviral particles. The expression level of EBV TCR was measured by FACS using mTCRβ (Figure 4A). On day 7, expression levels of all five types were over 90%. Jurkat NFAT-Luc cells expressing EBV-specific TCR candidates were reacted with T2 cells pulsed with EBV peptides, and then luminescence was measured. Since the EBV-specific TCR candidates are likely to bind to CMV in the bioinformatics analysis, the CMV peptide was additionally pulsed to react. Only EBV TCR 3 specifically reacted with EBV peptides, and EBV TCR 2 and 4 had no response to EBV peptides but showed a response to CMV peptides. Since EBV TCR 2 and 4 were identical to the clones of CMV TCR 1 and 5, respectively, they responded to the CMV peptide (Figure 4B). As a positive group, the EBV-6 peptide, which differs from EBV peptide 3 in only one amino acid sequence and whose reactivity has already been confirmed in other studies, was also tested (Figure 4C) (24, 25). Almost no difference was observed in luminescence reactivity between EBV mix and EBV-6. Since the reactivity was tested using all 5 types of EBV peptides that are expected to react, we checked the reactivity of the EBV TCR 3 with 5 types of EBV peptides to determine which peptides showed a reaction. Of the five EBV peptides, only EBV peptide 3 (FLYALALLI) was confirmed to react with EBV TCR 3, and little reaction with other peptides (LTAGFLIFL, CLGGLLTMC, TYGPVFMCL, and RYCCYYCLTL) was observed (Figure 4D).
Responsiveness of Epstein-Barr virus (EBV) T cell receptors (TCRs) to EBV antigens. A) Expression efficiency of EBV TCR in Jurkat NFAT-Luc cell line on day 7. B) Luminescence analysis of Jurkat NFAT-Luc cells, which express five EBV-specific TCRs, and T2 cells exposed to EBV mix or cytomegalovirus (CMV) peptides. C) Luminescence analysis of Jurkat NFAT-Luc cells, expressing five EBV-specific TCRs, and T2 cells exposed to EBV mix or EBV-6. D) Investigation of the reactions of Jurkat NFAT-Luc cells expressing each EBV peptide and EBV TCR 3. E) Transduction efficiency of EBV-specific TCR into healthy donor peripheral blood mononuclear cells (PBMCs). The expression of mTCRβ was analyzed using flow cytometry on days 7 and 14. F) Analysis of the reaction between EBV TCR-transduced T cells and T2 cells pulsed with EBV peptides. G) Cytotoxicity of EBV TCR-transduced T cells against T2 cells pulsed with EBV peptide.
To confirm whether EBV-specific TCR candidates are applicable to PBMCs, we transduced all five candidates into healthy PBMCs and tested their reactivity. The expression of EBV TCR in CD3+ T cells on day 14 was the lowest in EBV TCR 5 at 8.8% and the highest in EBV TCR 1 at 43.1%. The expression was 25.1%, 15.9%, and 15.9% in EBV TCR 2, 3, and 4, respectively (Figure 4E). Based on the level of IFN-γ observed after co-culture with T2 cells after pulsing EBV peptide, we inferred that only EBV peptide 3 reacted with EBV TCR 3. In addition, EBV peptide 6 (FLYALALLL), which represents a sequence with a change in one amino acid in the sequence of EBV peptide 3, was also pulsed to confirm the reactivity. The response was not significantly different from that of EBV peptide 3 (Figure 4F). Killing efficacy of EBV TCR 3 against EBV peptide-pulsed T2 cells was observed to be 85%~99% at ratios of 10:1 and 30:1 (Figure 4G).
Discussion
Immunotherapy involving the analysis of TCR in neoantigen-specific T cells has emerged as a promising approach (15, 26-28). Various separation methods using markers, such as 4-1BB and PD-1, are under investigation for isolating antigen-specific T cells (29-32). In addition to neoantigens, isolating antigen-specific T cells using antigens that are well known tumor-derived antigens, such as EBV and CMV, has been studied with various methods (33, 34).
Among the separation methods, tetramers have already been widely used and studied, but specific tetramers are necessary for each experiment. One of the disadvantages of tetramers is the high cost of synthesizing each specific antigen and MHC type. Therefore, other methods have been developed (16, 35, 36). Hong et al. compared three methods (CD137, IFN-γ, or tetramer) to isolate CD8+ T cells activated by CMV and analyzed the TCR repertoires (37). A comparative analysis of the TCR repertoire using the three methods suggested that the tetramer separation method and the CD137 separation method exhibited the highest degree of similarity (37). However, another study proposed that TCR analysis using CFSE is more sensitive and can secure more clonotypes than the CD137 separation method (38). Therefore, we established a method using CFSE, which has advantages in terms of cost and labor, to isolate T cells that respond to antigen.
A VDJ library was produced from sorted cells through 10X GENOMICS, and the clones identified by each analysis were arranged in order of frequency. The top 10 clones were searched in the TCR database including VDJDB and IEDB to determine whether they were known TCRs. As antigen specific TCR candidates, clones with a frequency of more than 10% that were identified as known TCRs for CMV/EBV antigen were selected.
To validate each of the CMV and EBV-specific TCR candidates selected in this way, we first transduced the Jurkat (TCR KO)-NFAT-Luc cell line and PBMCz to confirm the expression level. Both the transduced Jurkat (TCR KO)-NFAT-Luc cells and PBMCs were evaluated for reactivity through the antigen-presenting T2 cells, which were pulsed with CMV or EBV peptides. The reaction was confirmed in all 10 types of CMV-specific TCR candidates and EBV TCR 3. Because TCR expression was much better observed in the cell line and the reactivity patterns in the cell line and PBMCs were similar, the reactivity of TCR can be sufficiently confirmed in the cell line.
Upon searching the TCR database, there was a potential association with a response to influenza A. However, not all TCR candidates exhibited a response, indicating that the CMV TCR 1 to 10 and EBV TCR 3 are potentially specific to CMV or EBV. Upon analyzing the clones for EBV-specific TCR, two out of the five clones were found to be reactive to the CMV peptide. Among the five EBV antigens, the antigen with a known binding HLA type was EBV-3. Since the verification of the EBV TCR candidates was conducted only for HLA-A*02:01, there is a possibility that antigen reactivity of other EBV TCR candidates may be observed for other HLA types.
We analyzed antigen-specific T cells from healthy donor PBMCs and identified 10 CMV-specific TCRs and 1 EBV-specific TCR. CMV-specific TCRs can be used in cell therapy to treat glioblastoma multiforme and prevent CMV infection following hematopoietic stem cell transplantation (HSCT) or other organ transplantation (39, 40). EBV-specific TCR can also be used for the treatment of nasopharyngeal carcinoma and post-transplant lymphoproliferative disorder in solid organ and bone marrow transplant recipients (41). Clinical research on cell therapy using CMV or EBV TCR is also being conducted for this disease, but no candidates have yet been approved for treatment. Therefore, the CMV- or EBV-specific TCRs we identified can be used to develop treatments for various diseases, including cancer. Moreover, the utility of the method in this study may extend to other situations, given the challenges associated with isolating antigen-specific T cells. For example, this TCR sequence-based method can be employed to identify TCR candidates targeting tumor neoantigens, which can subsequently be used in adoptive cell therapy. In conclusion, we developed a potentially valuable tool for the development of a cell therapy for diverse immunological challenges.
Footnotes
Authors’ Contributions
All Authors made substantial contributions to the conceptualization and design of this study; the acquisition, interpretation, and validation of the data; drafting and critical revision of the manuscript; and final approval of the version to be published.
Funding
This study was supported by the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea (A20223741), Basic Science Research Programs through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning, Korea (NRF-2021R1F1A1063946), and Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (RS-2022-DD124076, Korea).
Conflicts of Interest
K.A.K., J.J., W.S.B., B.H., and Y.A.K. were employees of NeogenTC during the conduct of this study. H.J.L. is a founder of NeogenTC.
- Received December 30, 2023.
- Revision received January 17, 2024.
- Accepted January 18, 2024.
- Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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