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microRNAs are short noncoding RNAs that regulate protein expression posttranscriptionally. We previously showed that miR-155 promotes effector CD8+ T-cell responses. However, little is known about the regulation of miR-155 expression. Here, we report that antigen affinity and dose determine miR-155 expression in CD8+ T cells. In B16 tumors expressing a low-affinity antigen ligand, tumor-specific infiltrating CD8+ T cells showed variable miR-155 expression, whereby high miR-155 expression was associated with more cytokine-producing cells and tumor control. Moreover, anti–PD-1 treatment led to both increased miR-155 expression and tumor control by specific CD8+ T cells. In addition, miR-155 overexpression enhanced exhausted CD8+ T-cell persistence in the LCMV cl13 chronic viral infection model. In agreement with these observations in mouse models, miR-155 expression in human effector memory CD8+ T cells positively correlated with their frequencies in tumor-infiltrated lymph nodes of melanoma patients. Low miR-155 target gene signature in tumors was associated with prolonged overall survival in melanoma patients. Altogether, these results raise the possibility that high miR-155 expression in CD8+ tumor-infiltrating T cells may be a surrogate marker of the relative potency of in situ antigen-specific CD8+ T-cell responses.

Background: Immune checkpoint blockade remarkably provides durable clinical response in solid tumors and immunohistochemistry (IHC) measures are current companion diagnostics. Currently four (4) IHC companion diagnostics of Programed Death Ligand (PD-L1) are components of FDA approved clinical trials. The marked clinical responses seen in checkpoint inhibition outpace basic science advances. Our previous reports of interferon mediated viral response against dsRNA intermediates, namely in colon and bladder cancer, have examined the role of global demethylation (5-AZA) and combination therapies. Materials and Methods: Cell lines HCT116 (colon) and T24 (bladder) were treated with single and combination therapy of 5-AZA and Vitamin C were formalin fixed and processed for IHC staining for Ki67, MHL1 and markers of viral response: dsRNA markers (K1, J2), interferon activation (RIG I, MDA5, IFIT1, IFI27, IRF7, IRF9, and pSTAT1. Immune related markers included CD4, CD8, CD68, CD163 and PD-L1 (clone SP263). TMA sections were stained by automated immunostainers to include a subset of patient-matched whole sections. IHC staining documented for protein abundance (intensity 0-3) and cellular localization (nuclear, cytoplasmic, membranous) Results: Colon cell line experiments confirmed viral response activation with correlative staining of dsRNA antibodies with viral response markers RIG1, MDA5, IFIT1, IFI27, IRF7 and pSTAT1 with robust staining in formalin fixed paraffin embedded (FFPE) tissues with the exception of IFI27 and RIG1. Immune and macrophage markers CD8, CD4, CD68, and CD163 showed variable correlation with viral response markers and PD-L1 staining heterogeneous and most prevalent in non-small cell lung cancer (34%). Of 3 MMR deficient colon cancers, 2 stained positive for PD-L1. MDA5 staining was present in tumor and stroma but stronger peri-tumoral suggesting relation to tumor infiltrating lymphocytes. TMA cores and corresponding whole sections with co-expressed PD-L1 and MDA5 showed marked diminished CD8 staining as verified by nuclear quantitative algorithm. Summary: Here we evaluate the viral response phenotype via global demethylation in solid tumors with emphasis on colon and bladder cancer. lung and breast cancer. IHC methods in cell lines and FFPE clinical tissues provide a robust strategy to link viral and immune response in clinical tissue samples and provides glimpses of viral and immune pathways that might better elucidate tumor biology and thereby predict tumor response to drug regimens that include checkpoint inhibitors. Citation Format: Galen Hostetter, Liu Minmin, Marie Mustert, Bree Berghuis, Lisa Turner, Peter Jones, Steve Baylin, Scott Jewell, Lorenzo Sempere. Viral response markers in immune-competent solid tumors by immunohistochemistry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A39.

In the past few years, the field of cancer immunotherapy has made great progress and is finally starting to change the way cancer is treated. We are now learning that multiple negative checkpoint regulators (NCR) restrict the ability of T-cell responses to effectively attack tumors. Releasing these brakes through antibody blockade, first with anti-CTLA4 and now followed by anti-PD1 and anti-PDL1, has emerged as an exciting strategy for cancer treatment. More recently, a new NCR has surfaced called V-domain immunoglobulin (Ig)-containing suppressor of T-cell activation (VISTA). This NCR is predominantly expressed on hematopoietic cells, and in multiple murine cancer models is found at particularly high levels on myeloid cells that infiltrated the tumors. Preclinical studies with VISTA blockade have shown promising improvement in antitumor T-cell responses, leading to impeded tumor growth and improved survival. Clinical trials support combined anti-PD1 and anti-CTLA4 as safe and effective against late-stage melanoma. In the future, treatment may involve combination therapy to target the multiple cell types and stages at which NCRs, including VISTA, act during adaptive immune responses. Cancer Immunol Res; 2(6); 510–7. ©2014 AACR.