MiR-155 driven loss of ICOSL and SOCS1 in EBV+ gastric cancers renders \ abundant cytotoxic T cells ineffective, enabling immune evasion
Data files
Mar 30, 2026 version files 16.81 MB
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FIG._1_A_H_E.jpg
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FIG._1_B_H_E.jpg
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FIG._1_C_miR155.jpg
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FIG._1_D_EBER_12.jpg
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FIG._1_E_H_E.jpg
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FIG._1_F_H_E.jpg
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FIG._1_G_miR155.jpg
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FIG._1_H_EBER_12.jpg
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FIG._1_I__ICOSL.jpg
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FIG._1_J_CD8.jpg
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FIG._1_K_PDL1.jpg
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FIG._1_L_PD1.jpg
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FIG._1_M._ICOSL.jpg
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FIG._1_N_CD8.jpg
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FIG._1_O_PDL1.jpg
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FIG._1_P_PD1.jpg
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Fig._2__B_EBER_alone.jpg
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Fig._2__D_miR155___EBER___PDL1_merged.jpg
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Fig._2__E_CD8_alone.jpg
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Fig._2__G_CD8___ICOS_merged.jpg
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Fig._2_A_miR155_alone.jpg
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Fig._2_C_miR155___EBER_merged.jpg
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Fig._2_F_ICOS_alone.jpg
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Fig._2_H_CD8___ICOS_merged.jpg
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Fig._3_Panel_A.jpg
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Fig._3_Panel_B.jpg
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Fig._3_Panel_C.jpg
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Fig._3_Panel_D.jpg
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Fig._4__Panel_B_miR_155_EBV-.jpg
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Fig._4__Panel_C_miR_155_NL.jpg
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Fig._4__Panel_D_SOCS1_NL.jpg
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Fig._4__Panel_E_miR_155_EBV-.jpg
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Fig._4__Panel_F_SOCS1_EBV-.jpg
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Fig._4_Panel_A_miR_155_EBV_.jpg
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README.md
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Abstract
Poorly differentiated gastric carcinomas (PDGC) can be further subdivided into Epstein-Barr virus (EBV) positive (+) and EBV-negative (-) subtypes. EBV infection induces miR-155 expression. miR-155 targets ICOSL, a critical immune checkpoint. This study was done to compare the EBV+ and EBV- gastric cancers for their mechanisms of tumor rejection and how EBV-induced oncogenic miR-155 controls these events. For this, we interrogated PDGC samples for EBV status (by staining for EBV encoded factors), paralleled by CD8, PDL1, PD1, ICOSL, ICOS, MHC-I, SOCS1, and miR-155. We found that, despite the similar histological patterns of the PDGC cancer cells, EBV-driven induction of miR-155 suppresses SOCS1 expression, resulting in intense cytotoxic T-cell infiltration, but also loss of the other miR-155 target, ICOSL, making tumor cells invisible to the surrounding intense T cell infiltration. Conversely, EBV-negative tumors retain ICOSL/SOCS1 expression, but exhibit minimal T-cell infiltration, partly due to high SOCS1 expression, preventing immune-mediated clearance. Overall, our data indicates that SOCS1 expression, regulated by EBV-induced miR-155, along with ICOSL status determines whether tumors attract T cells and whether those T cells can effectively eradicate cancer cells.
Dataset DOI: 10.5061/dryad.gf1vhhn3q
Description of the data and file structure
these are images of different samples of tumors stained with a panel of different immune and oncogenic markers
Files and variables
File: FIG._1_A_H_E.jpg
Description: Fig 1A, is the H&E-stained image of an EBV-positive gastric cancer.
File: FIG._1_B_H_E.jpg
Description: Magnification (high resolution of the HE stained EBV-positive gastric cancer from Fig. 1A)
File: FIG._1_C_miR155.jpg
Description: miR-155 has been previously reported to be induced by EBV infection. This figure shows that EBV+ gastric cancers are as expected miR-155 positive. The tumor was analyzed by in-situ hybridization for miR-155. The signal for mir-155 positive is dark blue.
File: FIG._1_D_EBER_12.jpg
Description: The staining for EBV-expressed viral RNAs- known as EBER was done to confirm and prove that indeed the tumors we are studding are EBV-positive gastric cancers. The signal for EBR is dark brown.
File: FIG._1_G_miR155.jpg
Description: This figure shows that EBV-negative gastric cancers are miR-155 negative. The miR-155 expression was analyzed by in situ hybridization using an LNA probe that is complementary to miR-155 seed sequence. If mir-155 was expressed a dark blue signal was expected as in Figure 1C.
File: FIG._1_F_H_E.jpg
Description: H&E stain of a EBV-negative gastric cancer tumor slide, with patterns characteristic of gastric cancer. Fig. 1F is a magnification of Fig. 1E.
File: FIG._1_E_H_E.jpg
Description: H&E stain of a EBV-negative gastric cancer tumor slide.
File: FIG._1_J_CD8.jpg
Description: CD8 staining of EBV-positive gastric cancer showing heavy infiltration by CD8 positive ells (signal red).
File: FIG._1_I__ICOSL.jpg
Description: ICOSL staining of EBV-positive gastric cancer, showing in the same panel that tumor cells are ICOSL negative, while the non-tumor cells at the top right corner are ICOSL positive. Since the expression of ICOSL is controlled by miR-155 and we showed that EBV positive are miR-155 positive, this further confirms the involvement of ICOSL-miR-155 axes in EBV-positive gastric cancers.
File: FIG._1_K_PDL1.jpg
Description: PDL1 staining of EBV-positive gastric cancers showing that PDL1 is expressed (brown signal) in these tumors, however, the immunotherapy using anti-PDL1 nevertheless fails.
File: FIG._1_H_EBER_12.jpg
Description: Staining for EBV-expressed viral RNAs, known as EBER, in EBV-negative gastric cancers. The expected signal is brown color as in 1D.
File: FIG._1_L_PD1.jpg
Description: PD1 staining of an EBV-positive gastric cancer, showing that they are positive for this immune checkpoint (brown signal).
File: FIG._1_N_CD8.jpg
Description: CD8 staining of EBV-negative gastric cancer, showing that opposite to EBV-positive this tumor is cold and no CD8+ cells have infiltrated the tumor. Positive signal is expected to be red (as in Fig 1J).
File: FIG._1_P_PD1.jpg
Description: PD1 staining of an EBV-negative gastric cancer showing that there is lack of expression of this protein.
File: Fig._2_H_CD8___ICOS_merged.jpg
Description: CD8 (green) and ICOS (red) staining merged. When both proteins colocalize, they give a yellow signal, indicating that CD8+ cells infiltrating the EBV-positive gastric cancer are ICOS-positive.
File: Fig.4__Panel_H_SOCS1_EBV.jpg
Description: SOCS1 expression in an EBV-positive tumor showing that SOCS1 is not expressed. SOCS1 is a target of miR-155, and it is not expressed in EBV-positive tumors. a positive expression would have been marked by a brown signal as in Fig. 4D.
File: Fig._3_Panel_D.jpg
Description: MHCI expression in EBV negative cancer is lost. Expected signal is brown as in Fig 3C.
File: Fig._3_Panel_C.jpg
Description: MHCI is expressed at high levels in the normal gastric epithelia. Signal is brown.
File: Fig._2__B_EBER_alone.jpg
Description: EBER staining of a EBV-positive gastric tumor, showing that the cells express the EBV-encoded EBR (green color).
File: Fig._4__Panel_D_SOCS1_NL.jpg
Description: SOCS1 staining in the normal gastric epithelia. Signal is brown.
File: Fig._4__Panel_B_miR_155_EBV-.jpg
Description: miR-155 staining in an EBV negative tumor. If the tumor was expressing miR-155 the expected signal would have been dark blue as in panel 4A.
File: Fig._2__E_CD8_alone.jpg
Description: CD8 staining (green) alone in EBV-positive gastric cancers.
File: FIG._1_M._ICOSL.jpg
Description: ICOSL staining in EBV-negative gastric cancers, showing these tumors express ICOSL. Signal is brown.
File: Fig._2_C_miR155___EBER_merged.jpg
Description: miR-155 (blue color) and EBER (green color) staining merged, showing colocalization of EBER and miR-155 signals (Yellow). The image shows that most of the cells positive for EBER (EBV-positive) express miR-155 as well.
File: Fig._4__Panel_C_miR_155_NL.jpg
Description: miR-155 staining in a normal gastric epithelia. expected signal is dark blue. This is a serial section of the normal gastric epithelial shown in fig. 4D that was stained for SOCS1 and gave positive signal for this protein.
File: Fig._2_F_ICOS_alone.jpg
Description: ICOS staining (red) showing EBV positive gastric cancers are infiltrated by ICOS-positive T cells.
File: Fig._2_A_miR155_alone.jpg
Description: miR-155 staining (blue signal) in EBV-positive tumors. The image shows that EBV-positive gastric cancers have elevated levels of miR-155.
File: Fig._4__Panel_F_SOCS1_EBV-.jpg
Description: SOCS1 staining in an EBV negative tumor, showing strong expression (brown) of SOCS1. SOCS1 is a target of miR-155 and EBV negative tumors do not express miR-155, that can explain the high levels of SOCS1 expression. SOCs1 however is a suppressor of cytokine signaling, leading to suppression of CD8 cell infiltration.
File: FIG._1_O_PDL1.jpg
Description: PDL1 staining in EBV-negative gastric cancers where PDL1 is not expressed. If it was expressed it would have given a brown signal as in panel K of the same figure.
File: Fig._2__D_miR155___EBER___PDL1_merged.jpg
Description: Merging signals of miR-155 (blue), PDL1 (red), and EBER staining (green), showing that miR-155 and EBER colocalize with each other (expressed by the same cells), but not PDL1.
File: Fig._3_Panel_A.jpg
Description: H&E staining of an EBV-positive tumor that was used to test MHC-I expression in the Fig 3B.
File: Fig._3_Panel_B.jpg
Description: MHCI staining in an EBV-positive tumor showing this tumor had lost MHCI expression and was acting like the EBV-negative gastric cancer tumors (Fig. 3D). expected positive staining is brown color.
File: Fig.4_Panel_A_miR_155_EBV.jpg
Description: miR-155 stain in EBV tumor showing that EBV-positive gastric cancers express miR-155 at high levels. Positive signal for miR-155 is dark blue.
File: Fig.4_Panel_G_SOCS1_EBV.jpg
Description: SOCS1 stain in EBV positive tumor. Expression of SOCS1 gives brown signal after staining. Notice that the tumor section is negative for SOCS1, while the adjacent tissue -normal histopathology stains positive for SOCS1.
File: Fig._2__G_CD8___ICOS_merged.jpg
Description: Staining of CD8 and ICOS signals merged showing that ICOS is expressed by infiltrating CD8 T cells in EBV-positive gastric cancers.
File: Fig._4__Panel_E_miR_155_EBV-.jpg
Description: miR-155 staining in EBV negative tumors showing lack of expression of miR-155 in this tumor. expected color -blue if tumors expressed miR-155 as in Fig. 4A.
- Fig.4__Panel_H_SOCS1_EBV.jpg
- Fig.4_Panel_A_miR_155_EBV.jpg
- Fig.4_Panel_G_SOCS1_EBV.jpg
Code/software
NA
All work was done using immunohistochemistry (for all proteins studied) and in-situ hybridization (for EBV-encoded small RNAs (EBER-1/2) and miR-155) of mostly tumor tissue arrays.
all the work was done based on the methods described in very detail in
1- G. J. Nuovo. A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat. Protoc. 4:107–115 (2009).
2- G. J. Nuovo. In Situ Molecular Pathology and Co-Expression Analyses (Second Edition). Book. Academic Press, ISBN 9780128206539 (2020).