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Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity
Published on: December 7, 2019
TGFβ limits proximal CD8+ TCR signaling via PTPN22 following strong and moderate agonism
Andrew J Gunderson1,2, Kelley Jordan1, Tomoko Yamazaki1
1Earle A. Chiles Research Institute, Providence Cancer Institute, Portland, OR, United States.
Abstract:
Transforming growth factor beta (TGFβ) is an immunosuppressive cytokine that is overexpressed in tumor microenvironments. We have shown that CD8+ T cells with genetic ablation of the TGFβ type I receptor, Alk5 (CD8ΔALK5), were more sensitive to αCD3 stimulation resulting in enhanced proliferation and cytokine production. Based on these data, we hypothesized that TGFβ impaired T-cell receptor (TCR) signaling. We tested in vitro cytotoxicity of wild-type (WT) and CD8ΔALK5 OT-I T cells against murine oral carcinoma models transduced with ovalbumin altered peptide ligands (APLs) of differing affinities and found that loss of TGFβ renders CD8+ T cells more cytotoxic, but with diminishing effect at lower TCR agonism. TGFβ limits proximal TCR signaling intensity and duration, mediated by an interaction between the TGFβ type II receptor, PTPN22, and Zap70 that requires the Alk5 receptor. Downstream TCR signal integration is impaired by TGFβ following high and moderate, but not low TCR agonism. In vitro and in vivo models of chronic antigen stimulation demonstrate that TGFβ promotes both stem-like differentiation and terminal exhaustion, with loss of the more cytotoxic transitory exhausted population. Tumors of mixed APL clonality were implanted into Rag-/- animals followed by adoptive cell transfer of WT or CD8ΔALK5 OT-I T cells and monitored for clonal outgrowth. CD8ΔALK5 OT-I T cells were better able to control tumor clones with moderate TCR agonism compared to WT OT-I T cells. Targeting TGFβ signaling is one approach to enhance TCR signaling following strong or moderate agonism, alter differentiation toward more cytotoxic transitory exhaustion, and reduce terminal exhaustion, to improve antitumor immunity.
Insights
Transforming growth factor beta (TGFβ) suppresses T-cell receptor (TCR) signaling, hindering anti-tumor immunity. Ablating TGFβ signaling in CD8+ T cells enhances their cytotoxicity and promotes a more effective anti-tumor response.
Area of Science:
- Immunology
- Cancer Biology
- Molecular Signaling
Background:
- Transforming growth factor beta (TGFβ) is an immunosuppressive cytokine often overexpressed in tumor microenvironments.
- TGFβ's role in impairing T-cell receptor (TCR) signaling and its impact on anti-tumor immunity requires further elucidation.
Purpose of the Study:
- To investigate the hypothesis that TGFβ impairs T-cell receptor (TCR) signaling.
- To determine the effect of TGFβ ablation on CD8+ T cell function and anti-tumor cytotoxicity.
Main Methods:
- Utilized genetic ablation of the TGFβ type I receptor (Alk5) in CD8+ T cells (CD8ΔALK5).
- Assessed in vitro cytotoxicity of wild-type (WT) and CD8ΔALK5 OT-I T cells against murine oral carcinoma models with varying ovalbumin altered peptide ligand (APL) affinities.
- Examined proximal TCR signaling events and downstream signal integration.
- Modeled chronic antigen stimulation in vitro and in vivo, including adoptive cell transfer studies in Rag-/- mice.
Main Results:
- Loss of TGFβ signaling renders CD8+ T cells more cytotoxic, particularly against moderate TCR agonism.
- TGFβ limits proximal TCR signaling intensity and duration via an Alk5-dependent interaction involving the TGFβ type II receptor, PTPN22, and Zap70.
- TGFβ impairs downstream TCR signal integration under high and moderate, but not low, TCR agonism.
- In chronic stimulation models, TGFβ promotes stem-like differentiation and terminal exhaustion, reducing the cytotoxic transitory exhausted population.
- CD8ΔALK5 OT-I T cells demonstrated superior control over tumors with moderate TCR agonism compared to WT OT-I T cells.
Conclusions:
- TGFβ signaling critically limits CD8+ T cell anti-tumor immunity by impairing TCR signaling and promoting exhaustion.
- Targeting TGFβ signaling can enhance TCR signaling, modulate T cell differentiation towards a more cytotoxic phenotype, and reduce terminal exhaustion, thereby improving anti-tumor responses.
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