AUTHOR CONTRIBUTIONS E.A. conceived and designed the experiments, collected the data, performed and interpreted the analyses, and wrote the manuscript. D.M.L and A.P.M planned experiments, and collected and analyzed data. I.K. conceived of and designed the hmMHC algorithm and performed analyses using it, and wrote the methodological description found in this manuscript. M.D. generated the KP9025 sarcoma cell line. A.M.L provided technical assistance and helped plan experiments using MHC class II tetramers. W.M. and C.F.L. planned, performed and analyzed mass spectrometry experiments. E.E. assisted with bioinformatics analyses. A.N.V. assisted with the generation of the CD4 + T cell hybridomas, and helped design and perform experiments using them. D.R. designed, collected, and analyzed data for experiments involving multi-color flow cytometry. J.P.W. provided technical support for MHC class I tetramer staining. M.M.G assisted in experiment planning. R.F.V.M. collected and analyzed data for experiments involving multi-color flow cytometry. C.D.A., K.C.F.S. and J.M.W. provided technical assistance throughout the study. A.C. collected data. K.W.W. provided mITGB1-MHC class II monomers and provided assistance in experimental design. T.J. provided support in experimental design and data analysis regarding the KP9025 sarcoma line. M.N.A. conceived and designed the hmMHC algorithm and provided bioinformatics support. E.R.U. provided assistance in experimental design. R.D.S. conceived experiments, interpreted data, and wrote the manuscript. All authors contributed to manuscript revision. R.D.S. is a cofounder, scientific advisory board member, stockholder, and royalty recipient of Jounce Therapeutics and Neon Therapeutics and is a scientific advisory board member for A2 Biotherapeutics, BioLegend, Codiak Biosciences, Constellation Pharmaceuticals, NGM Biopharmaceuticals and Sensei Biotherapeutics. K.W.W. serves on the scientific advisory board of Tscan Therapeutics and Nextechinvest and receives sponsored research funding from Bristol-Myers Squibb and Novartis; these activities are not related to the findings described in this publication. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. Dr. Jacks's laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and Calico, but this funding did not support the research described in this manuscript. DATA AVAILABILITY Nucleotide variant calls generated from cDNA capture sequencing of the T3 and KP9025 sarcoma lines and used in the prediction of antigens shown in Figure 1a, Extended Data Figure 3a-b, and Extended Data Figure 6b are available within the article as Supplem...

SUMMARY While current immune checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor infiltrating cells by single cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Originally identified in studies of cellular resistance to viral infection, interferon (IFN)-γ is now known to represent a distinct member of the IFN family and plays critical roles not only in orchestrating both innate and adaptive immune responses against viruses, bacteria, and tumors, but also in promoting pathologic inflammatory processes. IFN-γ production is largely restricted to T lymphocytes and natural killer (NK) cells and can ultimately lead to the generation of a polarized immune response composed of T helper (Th)1 CD4 T cells and CD8 cytolytic T cells. In contrast, the temporally distinct elaboration of IFN-γ in progressively growing tumors also promotes a state of adaptive resistance caused by the up-regulation of inhibitory molecules, such as programmed-death ligand 1 (PD-L1) on tumor cell targets, and additional host cells within the tumor microenvironment. This review focuses on the diverse positive and negative roles of IFN-γ in immune cell activation and differentiation leading to protective immune responses, as well as the paradoxical effects of IFN-γ within the tumor microenvironment that determine the ultimate fate of that tumor in a cancer-bearing individual.

Neoplastic cells rely on the tumor microenvironment (TME) for survival and progression factors. Indeed, senescent and cancer-associated fibroblasts (CAFs) express factors that promote tumorigenesis that are collectively referred to as the senescence-associated secretory phenotype (SASP). Despite their importance in tumorigenesis, the mechanisms that control TME-derived factor expression remain poorly understood. Here we address a key unanswered question, how the SASP is sustained in senescent fibroblasts and CAFs. We find that the mitogen-activated protein kinase p38 (p38MAPK) controls AUF1 occupancy on SASP mRNAs and thus controls their stability. The importance of this regulatory mechanism is underscored by our findings that stromal-specific p38MAPK inhibition abrogates the tumor-promoting activities of CAFs and senescent fibroblasts. Our data suggest that targeting SASP mRNA stability through inhibition of p38MAPK will significantly aid the development of clinical strategies to target the TME.

Age is a major risk factor for the development of cancer. Senescent fibroblasts, which accumulate with age, secrete pro-tumorigenic factors collectively referred to as the senescence-associated secretory phenotype (SASP). Here we examined the molecular mechanisms that control SASP activation, focusing on the known SASP factor osteopontin (OPN). We found that expression of the canonical SASP members IL6 and IL8, but not OPN, were dependent upon a persistent DNA damage response (DDR) as evidenced by ATM and NF-κB activation. Treatment with several histone deacetylase (HDAC) inhibitors robustly activated SASP in the absence of DNA breaks, suggesting that DDR-dependent SASP activation occurs in response to chromatin remodeling rather than physical breaks in DNA. In the setting of histone deacetylase inhibition, IL6 and IL8 expression remained dependent upon ATM and NF-κB, while OPN expression remained independent of these factors. Further analysis revealed that HDAC1 was sufficient to induce OPN expression, which is interesting given that loss of HDAC1 expression correlates with increased OPN expression within the stromal compartment of invasive breast cancers. Importantly, fibroblasts treated with HDAC inhibitors promoted tumor growth in vivo. Our findings therefore indicate that HDAC modulation plays an important role in stromal cell activation, with important implications for the use of HDAC inhibitors in the treatment of cancer.

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Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.