ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma
Dyke P. McEwen, Paramita Ray, Derek J. Nancarrow, Zhuwen Wang, Srimathi Kasturirangan, Saeed Abdullah, Ayushi Balan, Rishi Hoskeri, Dafydd Thomas, Theodore S. Lawrence, David G. Beer, Kiran H. Lagisetty, Dipankar Ray
Dyke P. McEwen, Paramita Ray, Derek J. Nancarrow, Zhuwen Wang, Srimathi Kasturirangan, Saeed Abdullah, Ayushi Balan, Rishi Hoskeri, Dafydd Thomas, Theodore S. Lawrence, David G. Beer, Kiran H. Lagisetty, Dipankar Ray
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Research Article Gastroenterology Immunology

ISG15/GRAIL1/CD3 axis influences survival of patients with esophageal adenocarcinoma

Abstract

Immunosuppression is a common feature of esophageal adenocarcinoma (EAC) and has been linked to poor overall survival (OS). We hypothesized that upstream factors might negatively influence CD3 levels and T cell activity, thus promoting immunosuppression and worse survival. We used clinical data and patient samples of those who progressed from Barrett’s to dysplasia to EAC, investigated gene (RNA-Seq) and protein (tissue microarray) expression, and performed cell biology studies to delineate a pathway impacting CD3 protein stability that might influence EAC outcome. We showed that the loss of both CD3-ε expression and CD3+ T cell number correlated with worse OS in EAC. The gene related to anergy in lymphocytes isoform 1 (GRAIL1), which is the prominent isoform in EACs, degraded (ε, γ, δ) CD3s and inactivated T cells. In contrast, isoform 2 (GRAIL2), which is reduced in EACs, stabilized CD3s. Further, GRAIL1-mediated CD3 degradation was facilitated by interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein. Consequently, the overexpression of a ligase-dead GRAIL1, ISG15 knockdown, or the overexpression of a conjugation-defective ISG15–leucine-arginine-glycine-glycine mutant could increase CD3 levels. Together, we identified an ISG15/GRAIL1/mutant p53 amplification loop negatively influencing CD3 levels and T cell activity, thus promoting immunosuppression in EAC.

Authors

Dyke P. McEwen, Paramita Ray, Derek J. Nancarrow, Zhuwen Wang, Srimathi Kasturirangan, Saeed Abdullah, Ayushi Balan, Rishi Hoskeri, Dafydd Thomas, Theodore S. Lawrence, David G. Beer, Kiran H. Lagisetty, Dipankar Ray

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Figure 4

GRAIL isoform 1 promotes CD3-ε degradation in a catalytic activity–dependent manner.

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GRAIL isoform 1 promotes CD3-ε degradation in a catalytic activity–depen...
(A and B) DDK-tagged CD3-ε was overexpressed in HEK193 cells in the presence or absence of V5-tagged GRAIL1. Twenty-four hours after transfection, cells were treated with cycloheximide (50 μg/mL); lysates were prepared at indicated time points and subjected to immunoblotting. To determine protein half-life, band intensities were measured using ImageJ (NIH) and normalized by considering 0-hour time point band intensity as “1” arbitrary unit (a.u.). (C and D) Similar experiments were performed using DDK-tagged CD3-ζ and half-life was calculated in the presence and absence of GRAIL1 overexpression. (E and F) This experiment was performed similarly to panel C, except to restore dimeric CD3-ζ, cells were treated with DSS (1 μg/mL) for the final 30 minutes prior to harvest. Half-lives were calculated for the CD3-ζ monomer and dimer as described above. (G) GFP-tagged CD3-δ was overexpressed either alone or in the presence of wild type (WT) GRAIL1 or its catalytically inactive C277A and C280A mutants. Twenty-four hours after transfection, cell lysates were prepared and subjected to immunoblotting as indicated. (H) Live cell image of CD3-δ-GFP–expressing cells either alone or in the presence of WT, C277A, and C280A GRAIL1 mutants. Scale bar: 50 mm.