Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 23;20(1):241.
doi: 10.1186/s12967-022-03444-1.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy

Kazumi Okamura  1 , Satoshi Nagayama  2 , Tomohiro Tate  3 , Hiu Ting Chan  3 , Kazuma Kiyotani  3 , Yusuke Nakamura  3
Affiliations

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy

Kazumi Okamura et al. J Transl Med. .

Abstract

Background: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy.

Methods: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs).

Results: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells.

Conclusion: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.

Keywords: Adoptive T cell therapy; Colorectal cancer; Tumor-draining lymph nodes; Tumor-infiltrating lymphocytes.

PubMed Disclaimer

Conflict of interest statement

Kazuma Kiyotani reported advisory roles to Cancer Precision Medicine Inc, Japan. Yusuke Nakamura is a stockholder and an advisor of OncoTherapy Science, Inc, Japan.

Figures

Fig. 1
Fig. 1
Screening and expansion of tumor-reactive T cells in non-metastatic lymph nodes. a Lymphocytes (1 × 105/well) after making cell suspension were stimulated by autologous tumor cells (1 × 105/well) and IFNγ secretions were detected by ELISPOT assay as shown Pre. Lymphocytes co-culture with autologous tumor-cells for 2 weeks were rested without tumor-cells for 2 days. Subsequently, lymphocytes (1 × 105/well) were stimulated by autologous tumor-cells (1 × 105/well) and IFNγ secretions were detected by ELISPOT assay as shown Post. Samples of which spot number (lymphocytes + tumor cells) is over 15 and more 1.5 folds than the negative control (only lymphocytes) were considered as positive. Experiments were conducted in duplicate. b Absolute live cell numbers were counted before and 2 weeks after co-culture with autologous tumor-cells. A part of lymphocytes was stained with anti-CD3, CD4, CD8α, CD56 and CD19 antibody and their expressions were evaluated by flow cytometry. The number indicates CD8+ T cells after co-culture with autologous tumor cells from 1 × 106 total lymphocytes
Fig. 2
Fig. 2
Inhibitory T cell expansion induced induction failure of cytotoxic CD8 T cells. a Lymphocytes co-culture with autologous tumor-cells for 2 weeks were rested without tumor cells for 2 days. Subsequently, lymphocytes (1 × 105/well) were stimulated by autologous tumor-cells (1 × 105/well) for 24 h and IFNγ secretions were detected by ELISPOT assay. Only lymphocytes samples were negative control. Experiments were conducted in duplicate. b Evaluation of KRAS mutation status on codon G12/G13 by ddPCR by using gDNA of tumor tissues and lymph nodes of C177. c Lymphocytes were stained with anti-TCRαβ, CD4, CD8α, TIM-3, PD-1, CTLA4 and LAG3 antibodies. TIM-3, PD-1, CTLA4 and LAG3 expression on CD4 or CD8 T cells were evaluated by flow cytometry respectively. The number indicates the proportion and mean fluorescence intensity (MFI) of each population in live cells
Fig. 3
Fig. 3
Shared clonotypes with TILs were expanded by co-culture with tumor cells. a TCRβ clonotypes of lymphocytes before, 1 and 2 weeks after co-cultured with tumor-cells were compared with those of 4 TIL fragments which were recognized over 0.1% in at least 1 TIL fragment of C165. The number indicates the % of the frequency of shared clonotypes detected in each lymph node. b TCR clonotypes of lymphocytes before and after co-cultured with tumor-cells were compared with those of TILs which occupied over 0.01% in total T cells of C207. The number indicates the % of the frequency of shared clonotypes detected in each lymph node. c Lymphocytes of non-metastatic lymph nodes of C207 were stained with anti-TCRαβ, CD4, CD8α and PD-1 antibodies. CD4 and CD8 T cells were sorted by PD1 expression pattern. TCRβ clonotypes of TILs were compared with each population in each lymph nodes. The number indicates the frequency of shared clones. d TCRβ clonotypes of lymphocytes that occupied more than 0.1% of CD8 T cells in each lymph nodes were compared between lymph nodes by PD1 expression pattern. The number indicates the % of the frequency of shared clonotypes between each sample
Fig. 4
Fig. 4
Tumor reactivities depend on shared TCR clonotypes between lymph nodes. a, b TCRβ clonotypes of lymphocytes that occupied more than 0.1% of T cells in lymph node were compared between each lymph node before and after co-culture with tumor-cells. The number indicates the % of the frequency of shared clonotypes in each lymph node. C177 (a) and C215 (b). Rectum and sigmoid colon show the condition with respective tumor cell lines for co-culture and control without tumor cell lines for co-culture period
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Watanabe T, et al. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2016 for the treatment of colorectal cancer. Int J Clin Oncol. 2018;23:1–34. doi: 10.1007/s10147-017-1101-6. - DOI - PMC - PubMed
    1. Hashiguchi Y, et al. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines 2019 for the treatment of colorectal cancer. Int J Clin Oncol. 2020;25:1–42. doi: 10.1007/s10147-019-01485-z. - DOI - PMC - PubMed
    1. Lichtenstern CR, Ngu RK, Shalapour S, Karin M. Immunotherapy inflammation and colorectal cancer. Cells. 2020 doi: 10.3390/cells9030618. - DOI - PMC - PubMed
    1. Le DT, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med. 2015;372:2509–2520. doi: 10.1056/NEJMoa1500596. - DOI - PMC - PubMed
    1. Xiang B, Snook AE, Magee MS, Waldman SA. Colorectal cancer immunotherapy. Discov Med. 2013;15:301–308. - PMC - PubMed

Publication types

Substances