Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function

Yuto Tan; Naoko Kumagai-Takei; Yurika Shimizu; Akira Yamasaki; Mari Hara-Yamamoto; Shigeru Mitani; Tatsuo Ito

doi:10.1371/journal.pone.0331468

. 2025 Sep 8;20(9):e0331468. doi: 10.1371/journal.pone.0331468

Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function

Yuto Tan ¹, Naoko Kumagai-Takei ^2,^*, Yurika Shimizu ², Akira Yamasaki ², Mari Hara-Yamamoto ³, Shigeru Mitani ¹, Tatsuo Ito ²

Editor: Xianmin Zhu⁴

PMCID: PMC12416725 PMID: 40920775

Abstract

T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient’s body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8⁺T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.

Introduction

Programmed Cell Death 1 (PD-1) is a type I transmembrane protein preferentially expressed on immune cells including T, B, and NK cells. Its ligand, Programmed Cell Death 1 Ligand 1 (PD-L1), is expressed in various cell types including cancer cells. PD-L1 is also expressed on antigen-presenting cells and is a member of the B7 family. B7 family members regulate immune responses by delivering co-stimulatory or co-inhibitory signals [1].

Upon interaction with PD-L1, the receptor PD-1 potently interferes with T cell receptor (TCR) signaling through intracellular molecular mechanisms. The protein structure of PD-1 consists of an extracellular immunoglobulin variable region (IgV)-like domain, a transmembrane region, and a cytoplasmic domain that contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and an immunoreceptor tyrosine-based switch motif (ITSM) [2]. Interference with PD-1 signaling by immune checkpoint inhibitors enhances T cell function by enhancing signal transduction from the TCR signalosome [3].

PD-1 plays an important role in controlling T cell responses. PD-1 expression is induced at the cell surface of T cells following activation. Constitutive PD-1 expression by tumor-specific T cells is known to be associated with the expression of additional inhibitory receptors, leading to impaired T cell function and tumor escape, upon ligation to its ligand PD-L1 expressed by tumor cells or immune infiltrating cells within tumor microenvironments [4]. In the antigen-specific T cell response, Zap70 is phosphorylated following binding to the T cell receptor. However, this phosphorylation is counteracted by the interaction of PD-1 with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation [5,6]. Our research hypothesis holds that depletion of lymphocytes through this PD-1-mediated mechanism may lead to decreased efficacy in the treatment for cancer patients.

In recent years, T-cell therapies have proven to be a promising treatment option for cancer patients, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, current challenges include insufficient activation of T cells or poor persistence in the patient’s body, which can lead to incomplete elimination of cancer cells and recurrence. As an alternative method, the permanent removal of PD-1 using genome editing (e.g., via CRISPR/Cas9) is considered, although knockout of PD-1 in therapeutic T cells carries high risks such as the development of tumor cells associated with genotoxicity [7]. In an effort to avoid these risks, methods involving temporary inhibition of PD-1 expression have been investigated. In this study, we extracted an RNA region to target PD-1 pre-mRNA and temporarily inhibit PD-1 expression using our CRISPR/dCas13 system. We also examined the effect of targeting the Exon2 splice cis-element on lymphocyte function, focusing particularly on cytokine production.

Materials and methods

Cell culture of the human CD8⁺ T cell line

The human CD8⁺ T cell line EBT-8 was a gift from Prof. H. Asada [8] and was maintained in GIT medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 80 U/ml recombinant human IL-2 (KYOWA Pharmaceutical Industry Co., Ltd., Osaka, Japan), 100 µg/ml streptomycin, and 100 U/ml penicillin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan). EBT-8 was established simply by continuously culturing mononuclear cells obtained from a patient with leukemia, which means that EBT-8 was not established as a specific T cell clone for a specific antigen. Cells were placed in 25 cm² tissue culture flasks in portrait style in a volume of 10 ml, and incubated at 37°C in a humidified atmosphere of 5% carbon dioxide in air.

Guide RNA (gRNA) design

gRNA design was performed as described by Bandaru et al [9]. Briefly, a total of 18 different gRNAs were designed with SnapGene Viewer 4.1.9. Prior to oligosynthesis, BbsI restriction sequence was added at the 5′end of the designed gRNAs. All the gRNA oligonucleotides were procured from Integrated DNA Technologies, Inc. (IDT) (Coralville, IA). Details of the gRNAs designed for the study are provided in supplementary form (Supporting Information-1 (gRNAs for PD-1)).

The gRNA oligonucleotides were subcloned into the backbone vector (pLKO5.U6.crRNA.tRFP) expressing RFP following restriction enzyme treatment with BsmbI.

Transient transfection assays

dCas13 plasmid vector and 18 different gRNA expression vectors with RFP were prepared. Transformation was performed using E. coli strain DH5-alpha competent cells. Bacterial cells were harvested and pelleted by centrifugation after preparation of large overnight culture. Plasmid DNA was extracted from this bacterial culture using the NucleoBond Xtra Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) according to the manufacturer’s instructions. To deliver vectors into EBT-8 cells, electroporation was performed using NEPA21 Type II (Nepa Gene Co., Ltd. Chiba, Japan).

Cell isolation and DNA extraction

Following the staining of EBT-8 cells with anti-human PD-1-APC antibody (BioLegend, Inc., San Diego, CA), PD-1 positive and negative RFP⁺ cells were isolated by flow cytometry with FACS Aria III (Becton Dickinson, Franklin Lakes, NJ). PD-1 positive and negative RFP⁺ cell DNA was isolated using Genomic DNA from Tissue (Macherey-Nagel GmbH & Co. KG, Düren, Germany).

Detection of gRNAs

gRNA regions were amplified by PCR using DNA extracted from cells as templates. The sequences of the gRNAs were identified by next-generation sequencing (Azenta Life Sciences, Burlington, MA). A new screening system was developed and used to identify the gRNAs detected in each RFP⁺ cell population (PD-1 positive and negative cells) and to calculate the number of gRNA reads.

Assay for expression levels of cell surface molecules

EBT-8 cells were stained with PD-1-PE antibody (BioLegend, Inc.) to examine the expression levels of PD-1. The percentage of cells positive for mean fluorescence intensity was analyzed using a FACSCalibur^TM (Becton Dickinson) flow cytometer.

Multiplex cytokine/chemokine analysis

One day following electroporation with or without dCas13 plasmid vector and one type of gRNA vector, cells were stimulated with bead-bound antibodies. Monoclonal antibody CD3 (Beckman Coulter, Inc., Brea, CA) was incubated with anti-mouse IgG-coated beads (Spherotech, Inc., Lake Forest, IL) at room temperature for 30 min. After washing the beads with PBS, 2.0 × 10⁴ cells were incubated with 2.0 × 10⁴ beads in GIT medium supplemented with 80 U/ml recombinant human IL-2, 100 µg/ml streptomycin and 100 U/ml penicillin in 96-well round-bottomed plates. After the plates were incubated at 37°C for 48 h in a humidified atmosphere of 5% CO2, culture supernatants were collected and assayed for cytokines and chemokines. The assay was performed using a human cytokine/chemokine magnetic bead panel kit (EMD Millipore Corporation, Billerica, MA) according to the manufacturer’s instructions. Data was collected using a Luminex-200 Instrument System (Luminex, Austin, TX).

Statistical analysis

Significant differences were determined using Student’s t-test and are indicated by asterisks (*P < 0.05, **P < 0.01).

Results

Expression levels of PD-1 in a human CD8⁺ T cell line, EBT-8 cells, and gRNA design

The EBT-8 cell line was established from a large granular lymphocyte leukemia of T cell origin and shows surface expression of CD2, CD3, CD8, HLA-DR, and T cell receptor alpha/beta, which are characteristic of cytotoxic T lymphocytes [8]. In this study, we examined the expression of PD-1, which is not yet known in cell lines. Flow cytometry revealed that PD-1 is expressed on the surface of EBT-8 cells (Fig 1A). PD-1 acts by down-regulating the immune system [10], and Exon2 contains the binding domain for PD-L1/PD-L2, which are PD-1 ligands. We hypothesized that disrupting the interaction of the Exon2 splice cis-trans element on PD-1 pre-mRNA might lead to lymphocytes exerting their inherent functions. In an effort to regulate RNA splicing using our CRISPR/dCas13 system, it was important to identify an RNA sequence that can guide dCas13 to a specific region of the pre-mRNA. Therefore, in this study, we designed 18 different guide RNAs that target Exon2 splice cis-elements in pre-mRNA of PD-1 (Fig 1B). The guide RNAs were designed based on the following three considerations. Some of the gRNAs needed for CRISPR/dCas13 were designed to bind to GU and AG sequences. gRNAs were also designed to target splicing factor binding sites on the PD-1 pre-mRNA. It is better to avoid designing gRNAs that recognize RNA sequences where the protospacer flanking site (PFS) contains a G at the 3’-end [9].

Screening of gRNAs using the CRISPR/dCas13 system

If the gRNA sequence that regulates the RNA splicing can be identified using dCas13 and the 18 gRNAs shown in Fig 1B above, dCas13 with the identified gRNA will bind to that region and inhibit the splicing of Exon 2. In other words, Exon 2 will not be translated and the extracellular domain of the PD-1 receptor will be absent. To conduct screening of gRNAs using the CRISPR/dCas13 system, gRNA-transfected cells in PD-1-negative cells or PD-1-positive cells were sorted (Fig 2A). The distribution of the different gRNAs was highly heterogeneous (Fig 2B). For instance, in PD-1 negative cells, guide1 was highly abundant. These results indicated that guide1 targeted the Exon2 splice cis-element that regulates the splicing of PD-1 pre-mRNA.

Fig 2 — A) Dot plots with gRNA and PD-1 parameters were used to define gRNA(+) cells in **(a)** PD-1-negative and **(b)** PD-1-positive populations. b) Next generation sequencing was then performed to determine the number of each gRNA. The number of reads for each gRNA was converted to number of reads per million mapped reads. The converted number of each gRNA region is shown as the ratio of gRNA-introduced PD-1-negative cells to PD-1-positive cells.

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of interferon-γ (IFN-γ), TNF-α, IL-6, and CXCL10

Porter and coworkers [11] reported delayed onset of cytokine secretion with vigorous in vivo chimeric antigen receptor T-cell expansion and prominent antileukemia activity, demonstrating substantial and sustained effector functions of CART19 cells. Therefore, in this study, we also examined the effect of targeting the Exon2 splice cis-element on the production of cytokines, which are indicators of anti-tumor activity. The amount of IFN-γ was significantly lower in RNA-targeted cells than in non-targeted cells (Fig 3A). The amount of IFN-γ in the culture supernatant of targeted cells was approximately 1230 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8⁺ T cells retain the ability to produce IFN-γ following stimulation (Fig 3A). TNF-a is known to promote the differentiation of cytotoxic CD8⁺ T cells and eliminate tumors [12,13]. The amount of TNF-α was also significantly lower in RNA-targeted cells than in non-targeted cells (Fig 3B). The amount of TNF-α in the culture supernatant of targeted cells was approximately 570 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8⁺ T cells retain the ability to produce TNF-α following stimulation (Fig 3B). IL-6 also promotes the differentiation of cytotoxic CD8⁺ T cells [14]. The amount of IL-6 was also significantly lower in RNA-targeted cells than in non-targeted cells (Fig 3C). The amount of IL-6 in the culture supernatant of targeted cells was approximately 20 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8⁺ T cells retain the ability to produce IL-6 following stimulation (Fig 3C). CXCL10 is a critical chemokine that attracts T cells into the tumor microenvironment [15]. Although the amount of CXCL10 was significantly higher in RNA-targeted cells than in non-targeted cells, the amount of CXCL10 in the culture supernatant of non-targeted and targeted cells was found to be comparable (Fig 3D). The result indicated that targeted CD8⁺ T cells retain the ability to produce CXCL10 following stimulation (Fig 3D).

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of G-CSF, GM-CSF, IL-5, IL-8, and fractalkine

Granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), which promote neutrophil survival and activation, are known to induce adaptive antitumor immune responses and regression of established tumors based on neutrophil–T-cell interactions [16,17]. The amounts of G-CSF and GM-CSF were significantly lower in RNA-targeted cells than in non-targeted cells (Fig 4A, 4B). The amount of GM-CSF in the culture supernatant of targeted cells was approximately 3920 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8⁺ T cells retain the ability to produce GM-CSF following stimulation. It is known that IL-5 recruits eosinophils, which then activate CD8⁺ T cells [18]. The IL-8–responsive CD8 T-cell subset was enriched in perforin, granzyme B, and IFN-γ, and had high cytotoxic potential [19]. There were no significant differences in the production of IL-5 and IL-8 among the RNA-targeted and non-targeted cells (Fig 4C, 4D). The amount of IL-5 and IL-8 in the culture supernatant of targeted cells was approximately 2480 pg/ml and 2740 pg/ml, respectively. The result indicated that targeted CD8⁺ T cells retain the ability to produce IL-5 and IL-8 following stimulation. Fractalkine is a chemokine involved in the migration of cytotoxic T lymphocytes [20]. There were no significant differences in the production of fractalkine among the targeted and non-targeted cells (Fig 4E).

Fig 4 — Cytokine and chemokine levels were determined using a Luminex-based multiplex assay. The concentration (pg/ml) of G-CSF **(A)**, GM-CSF **(B)**, IL-5 **(C)**, IL-8 **(D)** and fractalkine **(E)** is indicated in the case of control (non-targeting) and cells targeting the cis-element of Exon2 (targeting).

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of IL-4, IL-10, and IL-13

IL-4 is known to promote eosinophil expansion or migration [21]. The amount of IL-4 was significantly lower in RNA-targeted cells than in non-targeted cells (Fig 5A). The amount of IL-4 in the culture supernatant of targeted cells was approximately 1140 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8⁺ T cells retain the ability to produce IL-4 following stimulation. Classically, IL-10 is known to inhibit T cell responses. On the other hand, IL-10 enhances CD8⁺ T cell proliferation, cytotoxic activity, and IFN-γ production [22]. IL-13 is also known to promote eosinophil expansion or migration. Eosinophils are expected to be of use as cellular biomarkers and effector cells in cancer therapy following ICI, especially with anti-CTLA4 and anti-PD-1 antibodies [21]. There were no significant differences in the production of IL-10 and IL-13 among the targeted and non-targeted cells (Fig 5B, 5C). The amount of IL-13 in the culture supernatant of targeted cells was approximately 10570 pg/ml (Fig 5C). The result indicated that targeted CD8⁺ T cells retain the ability to produce IL-13 following stimulation.

Fig 5 — Cytokine levels were determined using a Luminex-based multiplex assay. The concentration (pg/ml) of IL-4 **(A)**, IL-10 **(B)** and IL-13 **(C)** is indicated in the case of control (non-targeting) and cells targeting the cis-element of Exon2 (targeting).

Discussion

The present study investigated guide1 RNA targeting of the Exon2 splice cis-element in pre-mRNA of PD-1 using 18 different guide RNAs. It is important that the guide RNA was extracted by screening in proliferating T cells using the CRISPR/dCas13 system. While targeting the Exon2 splice element in pre-mRNA of PD-1, cytokine secretion capacity was maintained in RNA-targeted CD8⁺T cells. For some cytokines, cytokine production levels were lower in RNA-targeted cells compared to non-targeted cells, which may imply that the RNA editing technology itself has some effect on the state of T cells.

The unique CRISPR/dCas13 technology used in this study allows for the biology-based search of drug targets on RNA. This makes it possible to take an approach that selectively inhibits only specific interactions with RNA involving various molecules without degrading the RNA [23]. In future, we would like to apply the RNA targeting method obtained in this study to lymphocytes derived from ascites of cancer patients. By targeting PD-1 pre-mRNA, it will be possible to prevent lymphocytes from expressing the extracellular domain of PD-1. Ascites of cancer patients is often discarded with symptomatic treatment, but contains valuable lymphocytes from cancer patients. Therefore, we intend to extract these lymphocytes from ascites scheduled for disposal, target the pre-mRNA of lymphocytes, and then returning them to the cancer patient in an effort to contribute towards the promotion of anti-tumor activity. To do this, it is necessary to assess tumor clearance capacity by conducting in vitro cell-killing assays or in vivo murine tumor model experiments, which has not yet been examined in present study.

Previous studies have been reported on exon 3 of PD-1 pre-mRNA in cancer cells [24], but our study differs in that we target Exon2 of PD-1 pre-mRNA in CD8⁺ T cells. PD-1 is known to be expressed in various cell types, so existing immunotherapy with anti-PD-1 antibodies may have unexpected effects depending on the cells. We used CD8⁺T cell lines in this study. If only the patient’s lymphocytes are targeted, as in this study, PD-1 expression on lymphocytes could be suppressed, it might lead to enhanced antitumor activity. Further validation experiments using primary human T cells are needed.

Supporting information

S1 Table. A list of the guide RNA sequences.

(PDF)

pone.0331468.s001.pdf^{(97.6KB, pdf)}

S2 Table. Data the values used to build graphs.

(PDF)

pone.0331468.s002.pdf^{(169KB, pdf)}

Acknowledgments

We thank Dr. H. Asada for generously providing the EBT-8 cells.

Data Availability

All relevant data are within the manuscript and its Supporting Information file.

Funding Statement

N.K.-T. In full Naoko Kumagai-Takei This work was supported by JSPS KAKENHI (JP22K10497) and Research Project Grants (R03B039, R04B042, R05B043, R06B029) from Kawasaki Medical School.

References

1.Greaves P, Gribben JG. The role of B7 family molecules in hematologic malignancy. Blood. 2013;121(5):734–44. doi: 10.1182/blood-2012-10-385591 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Gao M, Shi J, Xiao X, Yao Y, Chen X, Wang B, et al. PD-1 regulation in immune homeostasis and immunotherapy. Cancer Lett. 2024;588:216726. doi: 10.1016/j.canlet.2024.216726 [DOI] [PubMed] [Google Scholar]
3.Arasanz H, Gato-Cañas M, Zuazo M, Ibañez-Vea M, Breckpot K, Kochan G, et al. PD1 signal transduction pathways in T cells. Oncotarget. 2017;8(31):51936–45. doi: 10.18632/oncotarget.17232 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Simon S, Labarriere N. PD-1 expression on tumor-specific T cells: friend or foe for immunotherapy?. Oncoimmunol. 2017;7(1):e1364828. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Okazaki T, Chikuma S, Iwai Y, Fagarasan S, Honjo T. A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat Immunol. 2013;14(12):1212–8. doi: 10.1038/ni.2762 [DOI] [PubMed] [Google Scholar]
6.Buchbinder EI, Desai A. CTLA-4 and PD-1 pathways: similarities, differences, and implications of their inhibition. Am J Clin Oncol. 2016;39(1):98–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Balke-Want H, Keerthi V, Cadinanos-Garai A, Fowler C, Gkitsas N, Brown AK, et al. Non-viral chimeric antigen receptor (CAR) T cells going viral. Immunooncol Technol. 2023;18:100375. doi: 10.1016/j.iotech.2023.100375 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Asada H, Okada N, Hashimoto K, Yamanishi K, Sairenji T, Hirota S, et al. Establishment and characterization of the T-cell line, EBT-8 latently infected with Epstein-Barr virus from large granular lymphocyte leukemia. Leukemia. 1994;8(8):1415–23. [PubMed] [Google Scholar]
9.Bandaru S, Tsuji MH, Shimizu Y, Usami K, Lee S, Takei NK, et al. Structure-based design of gRNA for Cas13. Sci Rep. 2020;10(1):11610. doi: 10.1038/s41598-020-68459-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Javed SA, Najmi A, Ahsan W, Zoghebi K. Targeting PD-1/PD-L-1 immune checkpoint inhibition for cancer immunotherapy: success and challenges. Front Immunol. 2024;15:1383456. doi: 10.3389/fimmu.2024.1383456 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725–33. doi: 10.1056/NEJMoa1103849 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol. 2000;18:275–308. doi: 10.1146/annurev.immunol.18.1.275 [DOI] [PubMed] [Google Scholar]
13.Koh C-H, Lee S, Kwak M, Kim B-S, Chung Y. CD8 T-cell subsets: heterogeneity, functions, and therapeutic potential. Exp Mol Med. 2023;55(11):2287–99. doi: 10.1038/s12276-023-01105-x [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Jiang Z, Liao R, Lv J, Li S, Zheng D, Qin L, et al. IL-6 trans-signaling promotes the expansion and anti-tumor activity of CAR T cells. Leukemia. 2021;35(5):1380–91. doi: 10.1038/s41375-020-01085-1 [DOI] [PubMed] [Google Scholar]
15.Dong M, Lu L, Xu H, Ruan Z. DC-derived CXCL10 promotes CTL activation to suppress ovarian cancer. Transl Res. 2024;272:126–39. doi: 10.1016/j.trsl.2024.05.013 [DOI] [PubMed] [Google Scholar]
16.Dranoff G. GM-CSF-based cancer vaccines. Immunol Rev. 2002;188:147–54. [DOI] [PubMed] [Google Scholar]
17.Stoppacciaro A, Melani C, Parenza M, Mastracchio A, Bassi C, Baroni C, et al. Regression of an established tumor genetically modified to release granulocyte colony-stimulating factor requires granulocyte-T cell cooperation and T cell-produced interferon gamma. J Exp Med. 1993;178(1):151–61. doi: 10.1084/jem.178.1.151 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Blomberg OS, Spagnuolo L, Garner H, Voorwerk L, Isaeva OI, van Dyk E, et al. IL-5-producing CD4+ T cells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer. Cancer Cell. 2023;41(1): 106–23. [DOI] [PubMed] [Google Scholar]
19.Hess C, Means TK, Autissier P, Woodberry T, Altfeld M, Addo MM, et al. IL-8 responsiveness defines a subset of CD8 T cells poised to kill. Blood. 2004;104(12):3463–71. doi: 10.1182/blood-2004-03-1067 [DOI] [PubMed] [Google Scholar]
20.Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T. Fractalkine in vascular biology: from basic research to clinical disease. Arterioscler Thromb Vasc Biol. 2004;24(1):34–40. doi: 10.1161/01.ATV.0000095360.62479.1F [DOI] [PubMed] [Google Scholar]
21.Grisaru-Tal S, Rothenberg ME, Munitz A. Eosinophil-lymphocyte interactions in the tumor microenvironment and cancer immunotherapy. Nat Immunol. 2022;23(9):1309–16. doi: 10.1038/s41590-022-01291-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Saraiva M, Vieira P, O’Garra A. Biology and therapeutic potential of interleukin-10. J Exp Med. 2020;217(1):e20190418. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Shimizu Y, Bandaru S, Hara M, Young S, Sano T, Usami K, et al. An RNA-immunoprecipitation via CRISPR/dCas13 reveals an interaction between the SARS-CoV-2 5’UTR RNA and the process of human lipid metabolism. Scient Rep. 2023;13(1):10413. doi: 10.1038/s41598-023-36812-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Wang X, Yan L, Guo J, Jia R. An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3. Int Immunopharmacol. 2024;126:111280. doi: 10.1016/j.intimp.2023.111280 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0331468.r001

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: This study addresses key limitations of current T-cell therapies—particularly Chimeric Antigen Receptor T-cell (CAR-T) therapy—including insufficient T-cell activation, poor in vivo persistence leading to incomplete tumor cell clearance and recurrence risks, and genotoxicity associated with conventional genome editing. It proposes an RNA editing strategy based on CRISPR/dCas13. Following PD-1 knockout, CD8⁺ T cells retain the ability to secrete critical cytokines (e.g., IFN-γ, TNF-α, GM-CSF) and proliferate.

However, significant problems exist in the article, as follows:

1. Invalidity of cross-system comparisons:

The methodology of directly contrasting cytokine concentrations in in vitro culture supernatants with serum data from other studies is unreasonable. Culture supernatants reflect transient local secretion, whereas serum is the result of systemic metabolic equilibrium. Culture supernatants cause cytokine accumulation due to lacking clearance mechanisms, while serum undergoes dynamic metabolic regulation. Absolute concentration comparisons are meaningless due to differences in kits, antibody affinity, and calibrator standards across studies.

2. Lack of evidence for functional inference:

Data indicate that PD-1-knockout cells retain in vitro secretory capacity but at levels far lower than non-target cells, demonstrating the knockout itself has substantially compromised T-cell status. Moreover, the authors did not assess tumor clearance capacity by conducting in vitro cell-killing assays or in vivo murine tumor model experiments. Therefore, cytokine secretion capacity cannot be equated with its actual effect within the in vivo tumor microenvironment (TME).

3. PD-1 mRNA knockout was conducted only in two T-cell lines in this study, not verified in primary T cells, resulting in lack of persuasiveness."

In summary, while the proposed CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity, the study's conclusions regarding preserved T-cell function are undermined by flawed comparative analyses, insufficient functional validation, and the absence of primary T-cell data. Translationally relevant claims require rigorous validation in biologically appropriate models and assays.

**********

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PLoS One. 2025 Sep 8;20(9):e0331468. doi: 10.1371/journal.pone.0331468.r002

Author response to Decision Letter 1

9 Aug 2025

Response to Reviewer

We are grateful for the critical comments and useful suggestions, which helped us to improve our manuscript PONE-D-25-31015, entitled “Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.” As indicated in the following responses, we have taken all comments and suggestions into account and have improved the manuscript with five figures and one Supplementary Information.

Reviewer Comments to Author

However, significant problems exist in the article, as follows:

Comment 1:

1. Invalidity of cross-system comparisons:

Response1:

In accordance with the reviewer’s comment, we agreed that comparisons with absolute cytokine levels from other studies, including serum measurement results from other studies, were not valid, so we performed statistical processing on the data obtained from these experiments (Fig. 3-Fig. 5) and interpreted the results.

In Abstract, we replaced the sentence “We extracted the RNA region of PD-1 pre-mRNA in the present study. By targeting this region of RNA, expression of the extracellular domain of PD-1 was specifically blocked while maintaining cytokine production and cell proliferation in CD8+ T cells.” with the sentence “We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-�, TNF-�, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells.”.

See Abstract (p.3)

In Materials and Methods, we added about Statistical Analysis with the sentences “Significant differences were determined using Student's t-test and are indicated by asterisks (*P < 0.05, **P < 0.01).”

See Materials and Methods (p.9)

In the Results, about Figure 3, the following words and sentences were deleted:

We deleted the word “Figure 3A” from the sentence “Therefore, in this study, we also examined the effect of targeting the Exon2 splice cis-element on the production of cytokines, which are indicators of anti-tumor activity.”. We deleted the sentences “The amount of IFN-� exceeded the levels reported in a previous study (11).”, and “Although serum and bone marrow tumor necrosis factor α levels remained unchanged following CART19-cell infusion (11), the amount of TNF-� in the culture supernatant of targeted cells exceeded the levels reported with CART19-cell infusion (Figure 3B).”. We deleted the words “to previous reported levels in CART-cell fusion” from the sentence “The amount of CXCL10 in the culture supernatant of non-targeted and targeted cells was found to be comparable to previous reported levels in CART-cell fusion (Figure 3D).”

See Results (p.12-13)

In Results, about Figure 3, the following sentences were added:

We add the sentences “The amount of IFN-� was significantly lower in RNA-targeted cells than in non-targeted cells (Figure 3A).” and “The amount of TNF-� was also significantly lower in RNA-targeted cells than in non-targeted cells (Figure 3B). The amount of TNF-� in the culture supernatant of targeted cells was approximately 570 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8+ T cells retain the ability to produce TNF-� following stimulation (Figure 3B).”, and “The amount of IL-6 was also significantly lower in RNA-targeted cells than in non-targeted cells (Figure 3C).” and “Although the amount of CXCL10 was significantly higher in RNA-targeted cells than in non-targeted cells, the”.

See Results (p.12-13)

In the Results, about Figure 4, the following words and sentences were deleted:

We deleted the sentences “The amount of G-CSF in the culture supernatant of non-targeted and targeted cells was found to be comparable to previous reported levels in the serum of healthy volunteers (Figure 4A) (18). The amount of GM-CSF in the culture supernatant of non-targeted and targeted cells was greater than in the serum of healthy volunteers (Figure 4B) (19).　The result indicated that targeted CD8+ T cells retain the ability to produce GM-CSF following stimulation.”. We deleted the sentence “The amount of IL-5 and IL-8 in culture supernatants of non-targeted and targeted cells was greater than in the serum of healthy volunteers (Figure 4CD), as previously reported (22).”. We deleted the sentence “The amount of fractalkine in the culture supernatant of non-targeted and targeted cells was found to be comparable to previous reported levels in the serum of healthy volunteers (Figure 4E) (24).”.

See Results (p. 14-15)

In Results, about Figure 4, the following sentences were added:

About Fig. 4, we add the sentences “The amounts of G-CSF and GM-CSF were significantly lower in RNA-targeted cells than in non-targeted cells (Figure 4AB). The amount of GM-CSF in the culture supernatant of targeted cells was approximately 3920 pg/ml.” We added the words “Although less than in non-targeted cells” before the sentences “the result indicated that targeted CD8+ T cells retain the ability to produce GM-CSF following stimulation.”. We added the sentences “There were no significant differences in the production of IL-5 and IL-8 among the RNA-targeted and non-targeted cells (Figure 4CD). The amount of IL-5 and IL-8 in the culture supernatant of targeted cells was approximately 2480 pg/ml and 2740 pg/ml, respectively.” And “There were no significant differences in the production of Fractalkine among the targeted and non-targeted cells (Figure 4E).”.

See Results (p.14-15)

In Results, about Figure 5, the following sentences were deleted:

About Fig. 5, we deleted the sentences “The amount of IL-4 in culture supernatants of non-targeted and targeted cells was greater than in the serum of healthy volunteers, as previously reported (26) (Figure5A).”, and “The amount of IL-10 in culture supernatants of non-targeted and targeted cells was found to be comparable to previous reported levels in the serum of healthy volunteers (Figure 5B) (28). The amount of IL-13 in culture supernatants of non-targeted and targeted cells was greater than in the serum of healthy volunteers, as previous reported (22) (Figure5C).”.

See Results (p.16)

In Results, about Figure 5, the following sentences were added:

About Fig. 5, we added the sentences “The amount of IL-4 was significantly lower in RNA-targeted cells than in non-targeted cells (Figure 5A). The amount of IL-4 in the culture supernatant of targeted cells was approximately 1140 pg/ml. Although less than in non-targeted cells, the result indicated that targeted CD8+ T cells retain the ability to produce IL-4 following stimulation.” and “There were no significant differences in the production of IL-10 and IL-13 among the targeted and non-targeted cells (Figure 5BC). The amount of IL-13 in the culture supernatant of targeted cells was approximately 10570 pg/ml (Figure 5C). The result indicated that targeted CD8+ T cells retain the ability to produce IL-13 following stimulation.”.

See Results (p.16)

In Discussion, we replaced the sentences “cytokine production was sufficient and T cell function was not regulated. This means that the guide1 RNA extracted using the CRISPR/dCas13 system specifically binds to the pre-mRNA region of PD-1 and does not affect other functions, such as cytokine production in immune cells.” with the sentences “cytokine secretion capacity was maintained in RNA-targeted CD8+T cells. For some cytokines, cytokine production levels were lower in RNA-targeted cells compared to non-targeted cells, which may imply that the RNA editing technology itself has some effect on the state of T cells.”.

See Discussion (p.18)

In References, we removed 6 references describing serum cytokine levels.

References (p.24-25)

In Figures, we have reflected the results of statistical processing in the figures 3-5.

See Figures 3-5

Comment 2:

2. Lack of evidence for functional inference:

Response2:

In accordance with the reviewer’s comment, we agree that the knockout itself may be affecting the state of T cells. So, we added the sentences “For some cytokines, cytokine production levels were lower in RNA-targeted cells compared to non-targeted cells, which may imply that the RNA editing technology itself has some effect on the state of T cells.” in Discussion.

As pointed out, we have not evaluated the tumor elimination potential in in vitro cell killing assays or in vivo mouse tumor model experiments. Nevertheless, we apologize for the misleading statement that equates cytokine secretion potential with actual effects in the in vivo tumor microenvironment (TME). In Abstract, we replaced the sentence “In this study, we overcame the aforementioned problems by extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13.” with the sentence “By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future.”. In Introduction, we replaced the sentence “In this study, we overcame the aforementioned problems by extracting the splice cis-element of PD-1 pre-mRNA using biology based on CRISPR/dCas13. Expression of the extracellular domain of PD-1 was specifically blocked while maintaining cytokine production and cell proliferation. It is expected that the use of RNA editing technology that targets only mRNA maturation should provide safe novel T cell therapy in the absence of genotoxicity.” with “We also examined the effect of targeting the Exon2 splice cis-element on lymphocyte function, focusing particularly on cytokine production.”. In Discussion, we added the sentences “To do this, it is necessary to assess tumor clearance capacity by conducting in vitro cell-killing assays or in vivo murine tumor model experiments, which has not yet been examined in present study.”.

See Abstract (p. 3), Introduction (p.5), Discussion (p.18-19).

Comment3:

3. PD-1 mRNA knockout was conducted only in two T-cell lines in this study, not verified in primary T cells, resulting in lack of persuasiveness."

Response3:

As pointed out, in this study, PD-1 mRNA knockout was performed only on two T cell lines. We are in the preparation stage to begin further research using primary T cells, but due to confidentiality concerns regarding our collaborative research, we only wish to include experiments using cell lines in this study. Therefore, in Abstract, we deleted the sentences “These results suggested that the use of the RNA editing technology maintains the safety of novel T cell therapy without genotoxicity by only targeting mRNA maturation.”. Instead, in Discussion, we added the sentences “we used CD8+T cell lines in this study. If only the patient's lymphocytes are targeted, as in this study, PD-1 expression on lymphocytes could be suppressed, it might lead to enhanced antitumor activity. Further validation experiments using primary human T cells are needed.”.

See Abstract (p. 3), Discussion (p. 19)

Comment4:

Response4:

In accordance with the comments, we agree that rigorous validation in biologically relevant models and assays is required to make claims of translational significance. While we focused on cytokine secretion and cell proliferation, we understand that future claims of translational significance require experiments using those appropriate models, assays, and primary T cells. Therefore, we have revised our manuscript as shown in Responses 1-3 above.

Attachment

Submitted filename: Response to Reviewers.pdf

pone.0331468.s004.pdf^{(166.1KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0331468.r003

Decision Letter 1

Xianmin Zhu

18 Aug 2025

<p>Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.

PONE-D-25-31015R1

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: The authors have addressed all my concerns. Now this manuscript is ready to be published in the journal of PlosOne.

**********

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Reviewer #1: Yes: Haopeng Wang

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PLoS One. doi: 10.1371/journal.pone.0331468.r004

Acceptance letter

Xianmin Zhu

PONE-D-25-31015R1

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. A list of the guide RNA sequences.

(PDF)

pone.0331468.s001.pdf^{(97.6KB, pdf)}

S2 Table. Data the values used to build graphs.

(PDF)

pone.0331468.s002.pdf^{(169KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.pdf

pone.0331468.s004.pdf^{(166.1KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information file.

[pone.0331468.ref001] 1.Greaves P, Gribben JG. The role of B7 family molecules in hematologic malignancy. Blood. 2013;121(5):734–44. doi: 10.1182/blood-2012-10-385591 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref002] 2.Gao M, Shi J, Xiao X, Yao Y, Chen X, Wang B, et al. PD-1 regulation in immune homeostasis and immunotherapy. Cancer Lett. 2024;588:216726. doi: 10.1016/j.canlet.2024.216726 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref003] 3.Arasanz H, Gato-Cañas M, Zuazo M, Ibañez-Vea M, Breckpot K, Kochan G, et al. PD1 signal transduction pathways in T cells. Oncotarget. 2017;8(31):51936–45. doi: 10.18632/oncotarget.17232 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref004] 4.Simon S, Labarriere N. PD-1 expression on tumor-specific T cells: friend or foe for immunotherapy?. Oncoimmunol. 2017;7(1):e1364828. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref005] 5.Okazaki T, Chikuma S, Iwai Y, Fagarasan S, Honjo T. A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application. Nat Immunol. 2013;14(12):1212–8. doi: 10.1038/ni.2762 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref006] 6.Buchbinder EI, Desai A. CTLA-4 and PD-1 pathways: similarities, differences, and implications of their inhibition. Am J Clin Oncol. 2016;39(1):98–106. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref007] 7.Balke-Want H, Keerthi V, Cadinanos-Garai A, Fowler C, Gkitsas N, Brown AK, et al. Non-viral chimeric antigen receptor (CAR) T cells going viral. Immunooncol Technol. 2023;18:100375. doi: 10.1016/j.iotech.2023.100375 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref008] 8.Asada H, Okada N, Hashimoto K, Yamanishi K, Sairenji T, Hirota S, et al. Establishment and characterization of the T-cell line, EBT-8 latently infected with Epstein-Barr virus from large granular lymphocyte leukemia. Leukemia. 1994;8(8):1415–23. [PubMed] [Google Scholar]

[pone.0331468.ref009] 9.Bandaru S, Tsuji MH, Shimizu Y, Usami K, Lee S, Takei NK, et al. Structure-based design of gRNA for Cas13. Sci Rep. 2020;10(1):11610. doi: 10.1038/s41598-020-68459-4 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref010] 10.Javed SA, Najmi A, Ahsan W, Zoghebi K. Targeting PD-1/PD-L-1 immune checkpoint inhibition for cancer immunotherapy: success and challenges. Front Immunol. 2024;15:1383456. doi: 10.3389/fimmu.2024.1383456 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref011] 11.Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 2011;365(8):725–33. doi: 10.1056/NEJMoa1103849 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref012] 12.Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in resistance to infection. Annu Rev Immunol. 2000;18:275–308. doi: 10.1146/annurev.immunol.18.1.275 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref013] 13.Koh C-H, Lee S, Kwak M, Kim B-S, Chung Y. CD8 T-cell subsets: heterogeneity, functions, and therapeutic potential. Exp Mol Med. 2023;55(11):2287–99. doi: 10.1038/s12276-023-01105-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref014] 14.Jiang Z, Liao R, Lv J, Li S, Zheng D, Qin L, et al. IL-6 trans-signaling promotes the expansion and anti-tumor activity of CAR T cells. Leukemia. 2021;35(5):1380–91. doi: 10.1038/s41375-020-01085-1 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref015] 15.Dong M, Lu L, Xu H, Ruan Z. DC-derived CXCL10 promotes CTL activation to suppress ovarian cancer. Transl Res. 2024;272:126–39. doi: 10.1016/j.trsl.2024.05.013 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref016] 16.Dranoff G. GM-CSF-based cancer vaccines. Immunol Rev. 2002;188:147–54. [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref017] 17.Stoppacciaro A, Melani C, Parenza M, Mastracchio A, Bassi C, Baroni C, et al. Regression of an established tumor genetically modified to release granulocyte colony-stimulating factor requires granulocyte-T cell cooperation and T cell-produced interferon gamma. J Exp Med. 1993;178(1):151–61. doi: 10.1084/jem.178.1.151 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref018] 18.Blomberg OS, Spagnuolo L, Garner H, Voorwerk L, Isaeva OI, van Dyk E, et al. IL-5-producing CD4+ T cells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer. Cancer Cell. 2023;41(1): 106–23. [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref019] 19.Hess C, Means TK, Autissier P, Woodberry T, Altfeld M, Addo MM, et al. IL-8 responsiveness defines a subset of CD8 T cells poised to kill. Blood. 2004;104(12):3463–71. doi: 10.1182/blood-2004-03-1067 [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref020] 20.Umehara H, Bloom ET, Okazaki T, Nagano Y, Yoshie O, Imai T. Fractalkine in vascular biology: from basic research to clinical disease. Arterioscler Thromb Vasc Biol. 2004;24(1):34–40. doi: 10.1161/01.ATV.0000095360.62479.1F [DOI] [PubMed] [Google Scholar]

[pone.0331468.ref021] 21.Grisaru-Tal S, Rothenberg ME, Munitz A. Eosinophil-lymphocyte interactions in the tumor microenvironment and cancer immunotherapy. Nat Immunol. 2022;23(9):1309–16. doi: 10.1038/s41590-022-01291-2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref022] 22.Saraiva M, Vieira P, O’Garra A. Biology and therapeutic potential of interleukin-10. J Exp Med. 2020;217(1):e20190418. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref023] 23.Shimizu Y, Bandaru S, Hara M, Young S, Sano T, Usami K, et al. An RNA-immunoprecipitation via CRISPR/dCas13 reveals an interaction between the SARS-CoV-2 5’UTR RNA and the process of human lipid metabolism. Scient Rep. 2023;13(1):10413. doi: 10.1038/s41598-023-36812-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0331468.ref024] 24.Wang X, Yan L, Guo J, Jia R. An anti-PD-1 antisense oligonucleotide promotes the expression of soluble PD-1 by blocking the interaction between SRSF3 and an exonic splicing enhancer of PD-1 exon 3. Int Immunopharmacol. 2024;126:111280. doi: 10.1016/j.intimp.2023.111280 [DOI] [PubMed] [Google Scholar]

PERMALINK

Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function

Yuto Tan

Naoko Kumagai-Takei

Yurika Shimizu

Akira Yamasaki

Mari Hara-Yamamoto

Shigeru Mitani

Tatsuo Ito

Roles

Abstract

Introduction

Materials and methods

Cell culture of the human CD8+ T cell line

Guide RNA (gRNA) design

Transient transfection assays

Cell isolation and DNA extraction

Detection of gRNAs

Assay for expression levels of cell surface molecules

Multiplex cytokine/chemokine analysis

Statistical analysis

Results

Expression levels of PD-1 in a human CD8+ T cell line, EBT-8 cells, and gRNA design

Fig 1. gRNAs targeting the Exon2 splice cis-element in pre-mRNA of PD-1.

Screening of gRNAs using the CRISPR/dCas13 system

Fig 2. Cell sorting of gRNA(+) cells.

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of interferon-γ (IFN-γ), TNF-α, IL-6, and CXCL10

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of G-CSF, GM-CSF, IL-5, IL-8, and fractalkine

Fig 4. Verification of the maintenance of cytokine and chemokine production ability involved in various immune responses in CD8+ T cells targeting the cis-element.

Effect of targeting the Exon2 splice cis-element in PD-1 on the production of IL-4, IL-10, and IL-13

Fig 5. Verification of the maintenance of cytokine production ability involved in various anti-immune responses in CD8+ T cells targeting the cis-element.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Xianmin Zhu

Roles

Author response to Decision Letter 1

Decision Letter 1

Xianmin Zhu

Roles

Acceptance letter

Xianmin Zhu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Cell culture of the human CD8⁺ T cell line

Expression levels of PD-1 in a human CD8⁺ T cell line, EBT-8 cells, and gRNA design

Fig 4. Verification of the maintenance of cytokine and chemokine production ability involved in various immune responses in CD8⁺ T cells targeting the cis-element.

Fig 5. Verification of the maintenance of cytokine production ability involved in various anti-immune responses in CD8⁺ T cells targeting the cis-element.