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 7

Mutant p53 influences ISG15 secretion.

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Mutant p53 influences ISG15 secretion. (A) Wild-type (WT) p53–containing...
(A) Wild-type (WT) p53–containing nondysplastic CpA cells were either left untransfected or transfected with DDK-tagged WT or different p53 mutants (C135S, R175H, R213Q, R248Q, R273H) as indicated. Forty-eight hours after transfection, cell culture supernatants were collected, concentrated (see Methods), and subjected to immunoblotting to quantify secreted ISG15 (s-ISG15) levels. Cellular ISG15 (c-ISG15) levels were determined using immunoblotting. Panels also show the levels of p53 expression, and Hsc70 was used as loading control. (B and C) OE33 cells were transfected using control, TP53, or 2 different GRAIL1 siRNAs, and 48 hours after transfection, supernatant and cell lysates were prepared and immunoblotted as above. In the indicated lanes, cells were also treated with IFN-γ (10 ng/mL) for the last 6 hours prior to harvest. (D) OE33 cells were treated with DMSO or different concentrations (3 and 5 μM) of simvastatin. Forty-eight hours after treatment, samples (secreted and cellular) were prepared as above and immunoblotted.