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Characterization of TGFβ-specific CD4+T cells through the modulation of TGFβ expression in malignant myeloid cells

Cellular & Molecular Immunology volume 18, pages 2575–2577 (2021)Cite this article

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Transforming growth factor beta (TGFβ) is a pleiotropic cytokine with functions related to angiogenesis, tumor suppression, and immune tolerance [1]. TGFβ is involved in tumor immune escape and is a target for cancer immune therapy [1]. We have reported that healthy donors and cancer patients harbor CD8+ cytotoxic T lymphocytes (CTLs) that are specific to TGFβ-derived epitopes and that these CD8+ CTLs kill malignant myeloid cells in a TGFβ-dependent manner [2]. We also found that T cells specific to the TGFβ-derived epitope TGFβ141–160 (REAVPEPVLLSRAELRLLRL, or TGFβ-15) that were expanded from a patient with cancer contained both CD4+ and CD8+ TGFβ-15-specific T cells. The presence of CD4+ subsets, such as Th1 and Th9 cells, is important to antitumor immunity and has been shown to increase the efficacy of PD-L1/PD-1 immune checkpoint therapy [3, 4]. Hence, we set out to characterize CD4+ T cells that were specific to TGFβ-derived epitopes.

We have previously shown that stimulation of THP-1 cells with the cytokines IL-4 and TGFβ enhances their expression of TGFβ [2]. Hence, we investigated whether increased amounts of TGFβ produced by THP-1 cells could increase their recognition by TGFβ-15-specific CD4+ T cells. Importantly, the active form of the cytokine TGFβ does not include the TGFβ-15 epitope, which is found in the N-terminal latency-associated peptide of TGFβ. Accordingly, any increased recognition of THP-1 cells was not due to the processing and presentation of the TGFβ used for stimulation. Using western blotting, we confirmed that treatment of THP-1 cells for 72 h with 200 U/ml IL-4 and 5 ng/ml TGFβ increased the expression of TGFβ (Fig. 1C). THP-1 cells that were treated with these cytokines for 72 h were then used as target cells in an ICS experiment with an effector:target (E:T) ratio of 3:1. The results showed increased expression of TNF-α, IFN-γ, and CD107a in T cells that were stimulated with cytokine-treated THP-1 cells compared with unstimulated THP-1 cells (Fig. 1D, E). An increase in TGFβ expression by THP-1 cells was already evident after 48 h of stimulation with cytokines [2], and CD4+ T cells stimulated with THP-1 cells that were treated with cytokines for 48 h also responded with increased cytokine (data not shown). The experiment was repeated with 8 replicates using another TGFβ-15-specific CD4+ T cell culture. The number of replicates allowed us to perform statistical analysis. Again, we identified significant increases in cytokine release and CD107a expression in T cells stimulated with cytokine-treated THP-1 cells (Fig. 1F). Next, we asked whether the increased T cell activation was caused by increased HLA-II expression on cytokine-treated THP-1 cells. Using FACS analysis with Fc blocking and an appropriate isotype control, we demonstrated that this was not the case (Fig. 1G). In fact, cytokine-treated THP-1 cells expressed less HLA-II than untreated THP-1 cells.

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Acknowledgements

This project was supported by Danish Health Authority grant “Empowering Cancer Immunotherapy in Denmark” (grant number 4-1612-236/8), the Copenhagen University Hospital, Herlev, and Gentofte and through a research funding agreement between IO Biotech ApS and the National Center for Cancer Immune Therapy (CCIT-DK). We greatly appreciate the tremendous technical support from Merete Jonassen.

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  1. National Center for Cancer Immune Therapy, Department of Oncology, Copenhagen University Hospital, Herlev, Denmark

    Rasmus Erik Johansson Mortensen, Morten Orebo Holmström & Mads Hald Andersen

  2. Institute for Immunology and Microbiology, Copenhagen University, Copenhagen, Denmark

    Morten Orebo Holmström & Mads Hald Andersen

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  1. Rasmus Erik Johansson Mortensen
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  2. Morten Orebo Holmström
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Contributions

REJM performed experiments, interpreted the data, and wrote the manuscript; MOH performed experiments, interpreted the data, and wrote the manuscript; and MHA conceived the project, interpreted the data, and wrote the manuscript.

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Correspondence to Mads Hald Andersen.

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Competing interests

MHA has developed an invention based on the use of TGFβ for vaccinations. The rights of the invention have been transferred to Copenhagen University Hospital Herlev, according to the Danish Law of Public Inventions at Public Research Institutions. The capital region has licensed the rights to the company IO Biotech ApS. The patent application was filed by IO Biotech ApS. MHA is board member, consultant and shareholder in IO Biotech. The other authors declare no competing financial interests.

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Mortensen, R.E.J., Holmström, M.O. & Andersen, M.H. Characterization of TGFβ-specific CD4+T cells through the modulation of TGFβ expression in malignant myeloid cells. Cell Mol Immunol 18, 2575–2577 (2021). https://doi.org/10.1038/s41423-021-00770-0

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