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Esophageal cancer cell-derived small extracellular vesicles decrease circulating Tfh/Tfr via sEV-PDL1 to promote immunosuppression

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Abstract

Esophageal cancer (EC) is a deadly malignancy. Small extracellular vesicles (sEVs) with programmed death ligand 1 (sEV-PDL1) induce immune escape to promote tumor progression. Furthermore, the imbalance between circulating follicular helper T (Tfh) and circulating follicular regulatory T (Tfr) cells is related to the progression of many malignant tumors. However, the role of the EC-derived sEV-PDL1 in circulating Tfh/Tfr is unknown. Circulating Tfh and Tfr cells were detected by flow cytometry. sEVs were isolated through differential centrifugation and cultured for cell expansion assays. Naïve CD4+ T cells were isolated, stimulated, and cultured with sEVs to evaluate the frequencies, phenotypes, and functions of Tfh and Tfr cells. The proportion of circulating Tfh in patients with EC was lower than that in healthy donors (HDs), whereas that of circulating Tfr was higher. The EC group showed significantly lower circulating Tfh/Tfr and a higher level of sEV-PDL1 than HDs. Notably, sEV-PDL1 was negatively correlated with circulating Tfh/Tfr in the EC group. In vitro assays, sEV-PDL1 inhibited Tfh expansion, enhanced the cytotoxic T lymphocyte-associated antigen 4+ (CTLA4+) Tfh cell percentage, decreased the levels of interleukin (IL)-21 and interferon-γ, and increased IL-10. sEV-PDL1 promoted the expansion and immunosuppressive functions of circulating Tfr; the increased percentages of CTLA4+ Tfr and inducible T cell co-stimulator+ Tfr were accompanied with high IL-10. However, applying an anti-PDL1 antibody significantly reversed this. Our results suggest a novel mechanism of sEV-PDL1-mediated immunosuppression in EC. Inhibiting sEV-PDL1 to restore circulating Tfh/Tfr balance provides a novel therapeutic approach for EC.

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Data availability

Data are available upon reasonable request.

Abbreviations

Ae:

Abdominal esophagus

ATCC:

American Type Culture Collection

CCK-8:

Cell counting Kit-8

CTLA4:

Cytotoxic T lymphocyte-associated antigen 4

CXCR5:

The chemokine receptor 5

EC:

Esophageal cancer

Eca109-PDL1kd :

PDL1 knocked down Eca109 cells

Eca109-PDL1nc :

PDL1 negative controlled Eca109 cells

FMO:

The fluorescent minus one method

HD:

Healthy donor

ICIs:

Immune checkpoint inhibitors

ICOS:

Inducible T cell co-stimulator

Lt:

Lower thoracic esophagus

Mt:

Middle thoracic esophagus

NTA:

Nanoparticle tracking analysis

PBMC:

Peripheral blood mononuclear cell

PD1:

Programmed death receptor 1

PDL1:

Programmed death ligand 1

qPCR:

Quantitative PCR

sEVs:

Small extracellular vesicles

sEV-PDL1:

PDL1 of the small extracellular vesicle

Tfr:

Follicular regulatory T cell

Tfh:

Follicular helper T cell

TNM:

Tumor size (T), lymph node infiltration (N), metastasis (M)

Ut:

Upper thoracic esophagus

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Acknowledgements

We sincerely thank all the people who have provided helpful support.

Funding

This work was supported by the Basic Research Cooperation Project of Beijing, Tianjin, Hebei from the Natural Science Foundation of Hebei (H2020206649), Tianjin (20JCZXJC00070), and Beijing (J200018) and the key Research and Development Program of Hebei Province (21377704D).

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Authors and Affiliations

  1. Hebei Medical University, Shijiazhuang, 050011, Hebei, China

    Zijie Li, Tingting Lv, Xiaokuan Zhang, Yuying Qi & Zhiyu Wang

  2. Department of Immuno-Oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, 050010, Hebei, China

    Yuehua Zhang & Lu Chen

  3. Department of Internal Medicine, Henan Cancer Hospital Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, 450008, Henan, China

    He Hao

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  1. Zijie Li
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Contributions

ZW and ZL conducted conceptualization of the study and designed the experiment. ZL performed major Imageflow experiments and mass cytometry experiments and analysis. HH and YZ interpreted and analyzed the data. LC, XZ, and TL assisted with the experiments. YQ provided advice with the experiments. ZL wrote, reviewed, and edited the manuscript. ZW is responsible for the overall content as guarantor.

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Correspondence to Zhiyu Wang.

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No potential conflict of interest was reported by the authors.

Ethics approval and consent to participate

This study involves human participants. Participants gave informed consent to participate in the study before taking part. This study was approved by the Fourth Hospital of Hebei Medical University Ethics Committee (2020ky241).

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Li, Z., Zhang, Y., Hao, H. et al. Esophageal cancer cell-derived small extracellular vesicles decrease circulating Tfh/Tfr via sEV-PDL1 to promote immunosuppression. Cancer Immunol Immunother 72, 4249–4259 (2023). https://doi.org/10.1007/s00262-023-03561-w

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