InVivoMAb anti-mouse IFNγ

The innate immune system protects cells against viral pathogens in part through the autocrine and paracrine actions of interferons (IFN)-alpha/beta (type I), -gamma (type II), and -lambda (type III). The transcription factor interferon regulatory factor (IRF)-1 has a demonstrated role in shaping innate and adaptive antiviral immunity by inducing the expression of IFN stimulated genes (ISGs) and mediating signals downstream of IFN-gamma. Although ectopic expression experiments have suggested an inhibitory function of IRF-1 against infection of alphaviruses in cell culture, its role in vivo remains unknown. Here, we infected Irf1(-/-) mice with two distantly related arthritogenic alphaviruses, chikungunya (CHIKV) and Ross River (RRV), and assessed the early antiviral functions of IRF-1 prior to induction of adaptive B and T cell responses. IRF-1 expression limited CHIKV-induced foot swelling in joint-associated tissues and prevented dissemination of CHIKV and RRV at early time points. Virological and histological analysis revealed greater infection of muscle tissues in Irf1(-/-) compared to wild-type mice. The antiviral actions of IRF-1 appeared independent of the induction of type I IFN or effects of type II and III IFNs but were associated with altered local pro-inflammatory cytokine and chemokine responses and differential infiltration of myeloid cell subsets. Collectively, our in vivo experiments suggest that IRF-1 restricts CHIKV and RRV infection in stromal cells, especially muscle cells, and this controls local inflammation and joint-associated swelling.IMPORTANCE Interferon regulatory factor (IRF)-1 is a transcription factor that regulates the expression of a broad range of antiviral host defense genes. In this study, using Irf1(-/-) mice, we investigated the role of IRF-1 in modulating pathogenesis of two related arthritogenic alphaviruses, chikungunya and Ross River viruses. Our studies show that IRF-1 controlled alphavirus replication and swelling in joint-associated tissues within days of infection. Detailed histopathological and virological analyses revealed that IRF-1 preferentially restricted CHIKV infection in cells of non-hematopoietic lineage, including muscle cells. The antiviral actions of IRF-1 resulted in decreased local inflammatory responses in joint associated tissues, which prevented immunopathology.

Antibody blockade of programmed death-1 (PD-1) or its ligand, PD-L1, has led to unprecedented therapeutic responses in certain tumor-bearing individuals, but PD-L1 expression’s prognostic value in stratifying cancer patients for such treatment remains unclear. Reports conflict on the significance of correlations between PD-L1 on tumor cells and positive clinical outcomes to PD-1/PD-L1 blockade. We investigated this issue using genomically related, clonal subsets from the same methylcholanthrene-induced sarcoma: a highly immunogenic subset that is spontaneously eliminated in vivo by adaptive immunity and a less immunogenic subset that forms tumors in immunocompetent mice, but is sensitive to PD-1/PD-L1 blockade therapy. Using CRISPR/Cas9-induced loss-of-function approaches and overexpression gain-of-function techniques, we confirmed that PD-L1 on tumor cells is key to promoting tumor escape. In addition, the capacity of PD-L1 to suppress antitumor responses was inversely proportional to tumor cell antigenicity. PD-L1 expression on host cells, particularly tumor-associated macrophages (TAM), was also important for tumor immune escape. We demonstrated that induction of PD-L1 on tumor cells was IFNgamma-dependent and transient, but PD-L1 induction on TAMs was of greater magnitude, only partially IFNgamma dependent, and was stable over time. Thus, PD-L1 expression on either tumor cells or host immune cells could lead to tumor escape from immune control, indicating that total PD-L1 expression in the immediate tumor microenvironment may represent a more accurate biomarker for predicting response to PD-1/PD-L1 blockade therapy, compared with monitoring PD-L1 expression on tumor cells alone. Cancer Immunol Res; 5(2); 106-17. (c)2017 AACR.

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2(+) tumors, some in excess of 150 mm(2), in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNgamma. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478-90. (c)2016 AACR.

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-gamma production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-gamma and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-gamma but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-gamma. Stimulation of human Caco-2BBe cells with IFN-gamma caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-gamma promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.