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Checkpoint blockade immunotherapy enhances the frequency and effector function of murine tumor-infiltrating T cells but does not alter TCRβ diversity

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Abstract

Checkpoint blockade immunotherapy is now a first-line treatment option for patients with melanoma. Despite achieving objective responses in about half of patients, the exact immune mechanisms elicited and those required for therapeutic success have not been clearly identified. Insight into these mechanisms is key for improving outcomes in a broader range of cancer patients. We used a murine melanoma model to track responses by different subsets of tumor-infiltrating lymphocytes (TIL) during checkpoint blockade immunotherapy. Tumors from treated mice had increased frequencies of both CD4+ and CD8+ T cells, which also showed evidence of functional reinvigoration and elevated effector cytokine production after immunotherapy. We predicted that increased T cell numbers and function within tumors reflected either infiltration by new T cells or clonal expansion by a few high-affinity tumor-reactive T cells. To address this, we compared TIL diversity before and after immunotherapy by sequencing the complementarity determining region 3 (CDR3) of all T cell receptor beta (TCRβ) genes. While checkpoint blockade effectively slowed tumor progression and increased T cell frequencies, the diversity of intratumoral T cells remained stable. This was true when analyzing total T cells and when focusing on smaller subsets of effector CD4+ and CD8+ TIL as well as regulatory T cells. Our study suggests that checkpoint blockade immunotherapy does not broaden the T cell repertoire within murine melanoma tumors, but rather expands existing T cell populations and enhances effector capabilities.

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Abbreviations

ATCC:

American Type Culture Collection

BLAST:

Basic local alignment search tool

BLOSUM62:

BLOck substitution matrix 62

CDR3:

Complementarity determining region

DAB:

Diaminobenzidine

Eomes:

Eomesodermin

FIR:

Foxp3-IRES-mRFP

GzmB:

Granzyme B

ICB:

Immune checkpoint blockade

Iono:

Ionomycin

IRES:

Internal ribosome entry site

LAG-3:

Lymphocyte-activation gene-3

RFP:

Red fluorescent protein

T-bet:

T-box expressed in T cells

TbetZsG :

T-bet-ZsGreen

TH1:

T helper 1 cell

Treg:

T regulatory cell

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Acknowledgements

The authors thank Sherri Koehm and Joy Eslick (Saint Louis University) for technical assistance with flow cytometry and cell sorting.

Funding

This work was supported by a grant from the Alvin J. Siteman National Cancer Institute Comprehensive Cancer Center and The Foundation for Barnes-Jewish Hospital (Grant no. P30 CA091842) to Ryan M. Teague.

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Authors and Affiliations

  1. Molecular Microbiology and Immunology Department, Saint Louis University School of Medicine, 1100 South Grand Blvd, St. Louis, MO, 63104, USA

    Lindsey M. Kuehm, Kyle Wolf, John Zahour, Richard J. DiPaolo & Ryan M. Teague

  2. Alvin J. Siteman National Cancer Institute Comprehensive Cancer Center, St. Louis, MO, USA

    Richard J. DiPaolo & Ryan M. Teague

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  1. Lindsey M. Kuehm
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Contributions

LMK helped develop methodology, performed all experiments, analyzed and interpreted data, and helped write the manuscript. RMT was the study supervisor, designed the study, and helped write the manuscript. KW and RJD assisted in translating and interpreting the TCR sequencing results as well as writing the manuscript. JZ performed the immunohistochemistry staining and analysis.

Corresponding author

Correspondence to Ryan M. Teague.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All mice were maintained under specific pathogen-free conditions and used in accordance with protocols established by the Institutional Animal Care and Use Committee of the Department of Comparative Medicine, Saint Louis University School of Medicine. All work with mice was performed according to protocol #2437 approved by the Animal Care and Use Program at Saint Louis University (Office of Laboratory Animal Welfare Assurance number D16-00141) which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALACI).

Animal source

TbetZsG original breeder mice were obtained from Taconic Farms, and the FIR mice were a gift from Daniel Hawiger’s laboratory (Saint Louis University).

Cell line authentication

The B16-F0 cell line was obtained commercially from and authenticated by American Type Culture Collection (ATCC) (Cat# CRL-6322). The cell line was free of mycoplasma, and correct morphology was confirmed by microscopy.

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Kuehm, L.M., Wolf, K., Zahour, J. et al. Checkpoint blockade immunotherapy enhances the frequency and effector function of murine tumor-infiltrating T cells but does not alter TCRβ diversity. Cancer Immunol Immunother 68, 1095–1106 (2019). https://doi.org/10.1007/s00262-019-02346-4

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