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Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer

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Abstract

Immune checkpoint inhibitors (ICIs) have shown superior clinical responses and significantly prolong overall survival (OS) for many types of cancer. However, some patients exhibit long-term OS, whereas others do not respond to ICI therapy at all. To develop more effective and long-lasting ICI therapy, understanding the host immune response to tumors and the development of biomarkers are imperative. In this study, we established an MC38 immunological memory mouse model by administering an anti-PD-L1 antibody and evaluating the detailed characteristics of the immune microenvironment including the T cell receptor (TCR) repertoire. In addition, we found that the memory mouse can be established by surgical resection of residual tumor following anti-PD-L1 antibody treatment with a success rate of > 40%. In this model, specific depletion of CD8 T cells revealed that they were responsible for the rejection of reinoculated MC38 cells. Analysis of the tumor microenvironment (TME) of memory mice using RNA-seq and flow cytometry revealed that memory mice had a quick and robust immune response to MC38 cells compared with naïve mice. A TCR repertoire analysis indicated that T cells with a specific TCR repertoire were expanded in the TME, systemically distributed, and preserved in the host for a long time period. We also identified shared TCR clonotypes between serially resected tumors in patients with colorectal cancer (CRC). Our results suggest that memory T cells are widely preserved in patients with CRC, and the MC38 memory model is potentially useful for the analysis of systemic memory T-cell behavior.

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Data availability

Bulk RNA-seq and TCR sequencing data presented in this work will be submitted through the Sequencing Read Archive. The remaining data generated and/or analyzed during the current study are available within the article and its Supplementary Data files or are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank Dr. Ai Takemoto, Ms. Junko Sugihara, Ms. Saki Matsumoto, and Ms. Satoko Baba for their help in technical assistance for the in vitro and in vivo study.

Funding

This study was supported in part by MEXT/JSPS KAKENHI grant number JP22K18383 (to R. Katayama), JP20K07399 (to S. Sakata) and the grant from the AMED grant number JP21ck0106472h0003, JP23ama221201h0002, JP23ama221210h0002, and JP23ck0106795h0001 (to R. Katayama), JP22ck0106543h0003 (to K. Kiyotani) and the grants from the Princess Takamatsu Cancer Research Fund, the Cell Science Research Foundation, and Chugai Foundation for Innovative Drug Discovery Science (to R. Katayama), and the grants from the Nippon Foundation and Takeda Science Foundation.

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Authors and Affiliations

  1. Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-Ku, Tokyo, 135-8550, Japan

    Mizuki Haraguchi, Ray Sagawa, Satoshi Takagi & Ryohei Katayama

  2. Department of Respiratory Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan

    Mizuki Haraguchi & Kazuhisa Takahashi

  3. Immunopharmacogenomics Group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan

    Kazuma Kiyotani & Tomohiro Tate

  4. Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Tomohiro Tate & Satoshi Nagayama

  5. Pathology Project for Molecular Targets, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan

    Seiji Sakata & Kengo Takeuchi

  6. Department of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan

    Satoshi Nagayama

  7. Department of Surgery, Uji-Tokushukai Medical Center, Kyoto, Japan

    Satoshi Nagayama

  8. Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan

    Kengo Takeuchi

  9. Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan

    Ryohei Katayama

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  1. Mizuki Haraguchi
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  2. Kazuma Kiyotani
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  3. Tomohiro Tate
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Contributions

MH, KK, KT, and RK designed the research. MH, KK, TT, SS, RS, SN, KT, and RK conducted the experiments and collected the data. MH, KK, TT, SS, ST, SN, KT, and RK analyzed the data. MH and RK wrote the initial draft of the manuscript. All authors provided critical review of the manuscript.

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Correspondence to Ryohei Katayama.

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Conflict of interest

R. Katayama received research funding from Chugai, TAKEDA, Toppan Printing outside the submitted work. K. Kiyotani is a scientific adviser of Cancer Precision Medicine, Inc. Other authors have declared that no conflict of interest exists.

Ethics approval and consent to participate

Informed consent was obtained from all individual participants included in the study. All experiments involving CRC patients were performed in accordance with approved protocols by the Institutional Review Board of Japanese Foundation for Cancer Research (No. 2018-GA-1021). All mice studies were conducted in line with the protocols approved by the Committee for the Use and Care of Experimental Animals of the Japanese Foundation for Cancer Research (No. 10-01-20). The authors affirm that human research participants provided informed consent for publication of this study.

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262_2023_3473_MOESM1_ESM.pdf

Supplementary file. Fig. S1 MC38 escape host immune attack using the PD-1/PD-L1 interaction. (A) MC38 cells were subcutaneously injected into C57BL/6 mice. The tumor volumes were measured and shown as the mean ± SEM at each time point. (B) The percentage of CD8a + T cells in CD45 + cells in MC38 tumors and the expression of PD-1 were measured using flow cytometry at each time point. Day 3, 6, 9 and 12, n = 3 per time point. (C)The expression of TNF, IFN, GzmB, and Ki67 in CD8 + TILs was measured using flow cytometry at each time point. Day 3, 6, 9 and 12, n = 3 per time point. (D) MC38 PD-L1+ / + and MC38 PD-L1− / − cells were stimulated overnight with 50 ng/ml IFN to induce PD-L1 expression and PD-L1 expression was measured using flow cytometry. (E) Genomic deletion around the PD-L1 locus was confirmed by Sanger sequencing of the PCR products around the sgRNA targeting region using genomic DNA from MC38 PD-L1− / −. (F) The changes in tumor volume are plotted individually and correspond to Fig. 1A. (G) The changes in tumor volume are plotted individually and correspond to Fig. 1B. Error bars represent the mean ± SEM (A, B, C). Fig. S2 MHC expression in MC38 B2M− / − cells. (A) Genomic deletion around the B2M locus was confirmed by Sanger sequencing of the PCR products around the sgRNA targeting region using genomic DNA from MC38 B2M− / − cells. (B)The expression of pan-MHC class I on the surface of MC38 B2M+ / + and MC38 B2M− / − cells was measured using flow cytometry. Fig. S3 Acquired immune responses are increased in TME of memory mice and FTY720 did not abrogate tumor rejection of memory mice. (A) Related GO terms of genes specifically decreased in the TME of the memory mice (upper). Related GO terms of genes specifically increased in the TME of the memory mice (lower). (B) The frequency of Treg cells in the TME was measured using flow cytometry (naïve n = 3, memory n = 3). (C) The frequency of Gr1 + CD11b + cells in the TME was measured using flow cytometry (naïve n = 3, memory n = 3). (D) The expression of PD-1 on CD8 + T cells in the TME was measured using flow cytometry. (E) The expression of PD-L1 on the CD45 − cells in the TME was measured using flow cytometry. (F) The confirmed memory mice and naïve mice were administrated 75 μg/mouse of FTY720 (n = 5) or vehicle (EtOH, n = 5) 3 times a week before MC38 reinjection. The frequency of CD8 + T cells (left) or CD4 + T cells (right) in PBMCs was evaluated using flow cytometry. (G) MC38 cells were subcutaneously injected into memory mice on day 0. FTY720 injection was started on day 1 following tumor injection. Tumor volume was measured and plotted individually (FTY720 n = 9, EtOH n = 5, naïve n = 3). (H) MC38 cells were subcutaneously injected into memory mice on days 0 and 25. FTY720 injection was started before tumor injection (FTY720 n = 5, EtOH n = 5, naïve n = 3). Tumor volume was measured and plotted individually. Error bars represent the mean ± SEM (B, C, F). Data were analyzed by a two-tailed unpaired Student’s t test (B, C, F). Fig. S4 Neoadjuvant ICI treatment induces immunological memory against tumors. (A) The average number of the clonotypes contained in the right and left side tumors (naïve n = 4, memory n = 3). (B) MC38 cells were subcutaneously injected into C57BL/6 mice. The tumor-bearing mice were administrated 100 µg/mouse anti-PD-L1 antibody intraperitoneally twice a week for a total of five times, and then, the residual tumors were surgically resected. The change in tumor volume is plotted individually. (C) The change in the proportion of the shared among the resected primary tumor as well as the first and second reinjected tumors in the other memory mice corresponding to Fig. 6D. Error bars represent the mean ± SEM (A). Data were analyzed by a two-tailed unpaired Student’s t test (A). Fig. S5 Clinical course of the CRC patient. The clinical course of the CRC patient (C053) is shown. Briefly, the patient was a 71-year-old with Stage IVa colorectal cancer. Her tumor contained a KRAS G12V mutation, BRAF WT, and MSI-L/MSS. The patient was treated with primary tumor resection followed by chemotherapy. Five months later, she had an operation to resect the liver metastasis and received additional chemotherapy; however, new metastases appeared in the liver and lung sequentially and they were resected. The change in CT imaging for each resected tumor is shown. Fig. S6 Representative IHC images of C053. Representative IHC images (CD8, PD-1, GzmB) for Tumor (A), LS1-3 (B), LS4 (C), Lung (D). B2M staining of the Lung tumor is shown (E). Scale bar: 300 μm (A–D), 200 μm (E). Fig. S7 The shared TCR repertoires are observed in other patients. The resected tumor was prepared, and TCR sequencing was performed by next-generation sequencing. Only TCRs with a proportion of ≥ 0.1% were selected and presented. The change in the proportion of the shared TCR clonotype is shown. (A) C124, diagnosed as a colorectal neuroendocrine tumor. The patient recurred with liver metastasis after resection of the primary tumor. Each tumor was analyzed using TCR repertoire sequencing. (B) C105, diagnosed as colorectal cancer with lung metastasis. Each tumor was analyzed using TCR repertoire sequencing. (C) C152, diagnosed as colorectal cancer with liver metastasis. Each tumor was analyzed using TCR repertoire sequencing. Fig. S8 TILs presented in LS4 respond against other resected tumors. TILs from each resected tumor were cocultured with individual cancer cells, and specific immune responses were evaluated using an IFNγ ELISpot assay. PMA/Ionomycin was used as a positive control. The number in the upper right corner of the well represents the number of counted spots. Fig. S9 Expression of tissue resident memory T-cell markers in the skin of memory mouse. (A) The scheme of the experiment is shown. MC38 cells were subcutaneously injected into memory and naïve mouse at right side on day 0, and skin at the left side of mouse was surgically resected on days 3 after euthanized the mice. (B,C) CD8 + T cells were stained with the fluorescent antibody to detect CD45/CD3/CD4 or CD8/CD69/CD103 with live-dead detecting reagent and analyzed with multi-color flow cytometry. CD69 expression in CD8 + T-cell in the mouse skin was shown in histogram (B), and CD69 with CD103 expression was shown in dot plot (C)

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Haraguchi, M., Kiyotani, K., Tate, T. et al. Spatiotemporal commonality of the TCR repertoire in a T-cell memory murine model and in metastatic human colorectal cancer. Cancer Immunol Immunother 72, 2971–2989 (2023). https://doi.org/10.1007/s00262-023-03473-9

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