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 6

ISG15 is required in GRAIL1-mediated CD3 degradation, and higher ISG15 expression is correlated with poor OS in EAC.

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ISG15 is required in GRAIL1-mediated CD3 degradation, and higher ISG15 e...
(A and B) CD3-ε and CD3-δ were overexpressed in the presence or absence of GRAIL1 in HeLa cells following transfection of either control or ISG15 siRNA. For panel A, cells were also treated with or without IFN-γ (10 ng/mL) for 6 hours, as indicated, prior to protein isolation. Cell lysates were subjected to immunoblotting using indicated antibodies. (C) GFP-tagged CD3-δ was transfected alone or along with a conjugation-deficient ISG15 LRAA mutant, as indicated. Twenty-four hours after transfection, cell lysates were prepared and subjected to immunoblotting. (D) Jurkat cells were treated with His-tagged ISG15 (250 ng/mL) for 24 hours, and cell lysates were subjected to immunoblotting. (E) PBMCs were treated with recombinant ISG15, and 24 hours after treatment cells were analyzed for surface CD3-ε expression using FACS. (F) PBMCs were treated with recombinant ISG15 for 15 minutes. Cells were then washed and subjected to immunofluorescence using anti-His antibody. Anti-CD3/CD28 beads used for T cell activation, showing nonspecific staining (*) in both vehicle control– and ISG15-treated samples. This was used for immunofluorescence intensity normalization, and His-ISG15 specific staining is shown by arrowhead (v). Scale bar, 10 μm. (G and H) ISG15 gene expression levels in EACs were compared with nondysplastic Barrett’s (NDBE) and dysplasia (DYS) based on previously published RNA-Seq data sets (57, 27). Box plots show the interquartile range, median (line), and minimum and maximum (whiskers). (I) Kaplan-Meier curves showing OS of patients with EAC expressing either negative (n = 6) or positive (n = 40) ISG15 expression (P = 0.09) based on log-rank Mantel-Cox test). (J) Representative EAC TMA cores showing numbers with no (scored 0) and different levels (scale of 1–3 as we defined) of ISG15 expression. Pictures were captured at 20× original magnification.