T cell expressed miR-155 promotes antitumor immunity

MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional knockout (KO) mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote interferon gamma (IFNγ) production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage (TAM) expression of IFNγ-inducible genes. We also found that immune checkpoint–blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte–associated protein 4 (CTLA-4) restored antitumor immunity in miR155 T cell–conditional KO (TCKO) mice. We noted that these ICB antibodies rescued the levels of IFNγ expressing T cells, expression of multiple activation and effector genes expressed by tumorinfiltrating CD8 and CD4 T cells, and TAM activation. Moreover, the ICB approach partially restored expression of several de-repressed miR155 targets in tumor-infiltrating, miR-155 deficient CD8 T cells, suggesting that miR-155 and the ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight miR-155’s multifaceted role in T cells in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies. MicroRNA are a class of non-coding RNAs that function in immune cells by modulating gene expression post-transcriptionally via binding to the 3’ untranslated region (3’ UTR) of their target mRNA in a sequence specific manner to repress the expression of these targets(1). MicroRNA-155 (miR-155) is a key regulator of the immune response in contexts ranging from autoimmunity to infection to immunization(2-4). miR-155 has more recently emerged as a regulator of tumor immunity due to its influence on the anti-tumor activity of a variety of immune cells including T cells, NK cells and myeloid populations(5-7). We previously reported a defect in the accumulation of interferon gamma (IFNγ) expressing CD4 and CD8 T cells in tumors from mice genetically deficient in miR155, and this correlated with increased growth of syngeneic B16f10 melanomas(8), an observation also made by an independent laboratory(9). Other http://www.jbc.org/cgi/doi/10.1074/jbc.M117.808121 The latest version is at JBC Papers in Press. Published on September 14, 2017 as Manuscript M117.808121 Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 8, 2017 hp://w w w .jb.org/ D ow nladed from T cell expressed miR-155 promotes antitumor immunity 2 studies found that overexpression of miR-155 in OVA specific CD8 T cells resulted in a robust antigen specific immune response to B16 melanoma tumors that expressed OVA(5) and that miR-155 overexpression promoted the proliferation and effector function of CD8 T cells in response to anti-tumor γc cytokines(10). A regulatory role for miR-155 in Tregs was also reported in the setting of tumor immunity(11). Altogether, these studies provide evidence that miR-155 functions in various T cell types as they respond to solid tumors. miR-155 is highly upregulated in macrophages responding to LPS and other inflammatory stimuli(12). It is also expressed in tumor-associated macrophages (TAMs)(7) and is thought to be important in the polarization process of macrophages and monocytes to a proinflammatory anti-tumor phenotype(13). Ex-vivo knockdown and overexpression of miR-155 in TAMs demonstrated that miR-155 expression in these cells promotes a pro-inflammatory M1 phenotype(14). This work, along with evidence showing that MMTV-PyMT mice develop spontaneous breast cancer at a higher rate when miR-155 is knocked down using a lentivirusdelivered inhibitory sponge in TAM populations(7), suggests that miR-155 expression within the macrophage compartment inhibits tumor growth by creating a pro-inflammatory tumor micro-environment. Additionally, there is evidence that miR-155 also regulates myeloid derived suppressor cell (MDSC) responses in tumor bearing mice(9,15). Thus, in addition to T cells, miR-155 also appears to play important biological functions within the myeloid compartment during tumor immunity. Despite this important progress, several unanswered questions about the role of miR-155 during antitumor immunity remain. The cell intrinsic roles of miR-155 during T and myeloid cell responses to solid tumors have not been examined using miR-155 conditional knockout mice that do not require manipulations such as bone marrow reconstitution or adoptive transfers. Further, a potential role for miR-155 in regulating crosstalk between T cells and TAM populations within the tumor microenvironment has not been explored, nor has it been determined whether or not defective antitumor responses by miR-155 T cells can be reversed. In the current study, we employed miR155 conditional knockout mice to test T cell and macrophage specific roles of miR-155 in response to a syngeneic B16f10 melanoma tumor. We found that miR-155 expression within the T cell compartment is required to promote optimal antitumor CD4 and CD8 T cell responses and reduce tumor growth. Additionally, miR-155 expression by T cells promoted the activation of TAMs through the induction of IFNγ inducible genes, while its expression by LysM TAMs was not required for this response to occur. We also discovered that ICB therapy largely rescues antitumor immune responses in miR-155 TCKO mice, and does so by restoring the levels of IFNγ expressing T cells, TAM activation, and expression of several T cell activation and effector genes. Additionally, ICB also reduced expression of several miR-155 target genes that were derepressed in T cells lacking miR-155. This indicates that miR-155 and ICB reagents regulate overlapping pathways. Our findings clearly demonstrate that T cell expressed miR-155 plays a significant role in promoting the endogenous, multicellular immune response against solid tumors, and that evaluation and/or augmentation of its expression may be a clinically relevant tool for immunotherapy. RESULTS T cell-specific deletion of miR-155 reduces the levels of intra-tumor IFNγ-expressing T cells and promotes the growth of B16-f10 tumors––To assess the role of miR-155 expression within T cells following a solid tumor challenge, we injected syngeneic B16f10 melanoma cells into miR-155 TCKO mice in which miR-155 was conditionally deleted in CD4 and CD8 T cells via CD4-Cre(3). During the development of T cells in the thymus, all CD4 and CD8 T cells undergo a double positive CD4CD8 stage in they will express Cre under the control of CD4 and thus delete floxed genes in cells that will become either CD4 or CD8 T cells. At day 12 post injection, miR-155 TCKO mice exhibited modestly increased tumor sizes compared to 155 controls as measured by diameter (Fig. 1A) and weight (Fig. 1B) of the transplanted tumors. These experiments were also performed using CD4Cre controls and we obtained similar results (data not shown). We previously observed increased tumor by gest on Sptem er 8, 2017 hp://w w w .jb.org/ D ow nladed from T cell expressed miR-155 promotes antitumor immunity 3 growth in whole body miR-155 deficient mice along with a defect in the numbers of intra-tumor IFNγ expressing CD4 and CD8 T cells(8). Therefore, we assayed the production of this cytokine by tumor infiltrating T cell populations from miR-155 TCKO mice and controls. We found an overall decrease in the percentage of CD45 immune cells within tumors of these knockout mice, accompanied by a further reduction in the percentage of IFNγ cells within the CD45 compartment (Fig. 1C,D). We also noted a trending decrease in the percentage of CD8 T cells and a significant decrease in CD8IFNγ T cells (Fig. 1E,F) within the tumor microenvironment when miR-155 was specifically lacking from T cells. Additional analysis of the T cell compartments within the tumors also revealed a significant decrease in the percentages of CD4 T cells and CD4IFNγ T cells in our miR-155 TCKO mice compared to controls (Fig. 1G,H). Reduced activation of TAMs in miR-155 TCKO tumor bearing mice––To further assess the effects of macrophage intrinsic versus extrinsic miR-155 expression on TAM phenotypes within the tumor microenvironment we sorted macrophages from B16f10 tumors growing in miR-155 TCKO, miR-155 macrophage conditional knockout mice (MCKO), and control mice on day twelve post-tumor administration based on their expression of CD45, CD11b and F4/80 (Fig. 2A). Deletion of miR-155 from miR155 floxed macrophages by LysM-Cre was verified by qPCR analysis of mature miR-155 transcript levels in bone marrow derived macrophages (Fig. S1A). TAMs sorted from miR155 TCKO mice had a marked decrease in mRNA levels encoding several IFNγ inducible genes (Fig. 2B), consistent with reduced percentages of tumor infiltrating, IFNγ producing T cells in these mice, while TAMs from miR-155 MCKO mice expressed near Wt levels of these genes. This particular gene subset was also confirmed to be IFNγ inducible, as their expression was blocked in IFNγR macrophages isolated from B16f10 tumors (Fig. 2C). Further, TAMs from miR-155 TCKO mice also had defective surface expression of MHCI and CD40 as determined by FACS, while miR-155 MCKO mice expressed Wt levels of these cell surface proteins (Fig. 2D,E,F,G and data not shown). Also of note, miR-155 MCKO mice challenged with B16f10 tumors showed no difference in tumor growth or in the percentages of IFNγ producing T cells in the tumor microenvironment (Fig. S1B,C,D). Additionally, tumor growth in miR-155 MCKO mice given B16f10-OVA tumors was similar to control mice (Fig. S1E). T