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.

The tetra-primer amplification refractory mutation system-polymerase chain (ARMS-PCR) reaction is a simple and economical method to genotype single-nucleotide polymorphisms (SNPs). It uses four primers in a single PCR and is followed just by gel electrophoresis. However, the optimization step can be very hardworking and time-consuming. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. Two SNPs that provided different amplification conditions were selected. DNA extraction methods, annealing temperatures, PCR cycles protocols, reagents, and primers concentration were also analyzed. The use of tetra-primer ARMS-PCR could be impaired for SNPs in DNA regions rich in cytosine and guanine and for samples with DNA not purified. The melting temperature was considered the factor of greater interference. However, small changes in the reagents concentration significantly affect the PCR, especially MgCl2. Balancing the inner primers band is also a key step. So, in order to balance the inner primers band, intensity is important to observe which one has the weakest band and promote its band by increasing its concentration. The use of tetra-primer ARMS-PCR attends the expectations of modern genomic research and allows the study of SNPs in a fast, reliable, and low-cost way.

During the last decades, the pleiotropic antitumor functions exerted by type I interferons (IFNs) have become universally acknowledged, especially their role in mediating interactions between the tumor and the immune system. Indeed, type I IFNs are now appreciated as a critical component of dendritic cell (DC) driven T cell responses to cancer. Here we focus on IFN-α and IFN-β, and their antitumor effects, impact on immune responses and their use as therapeutic agents. IFN-α/β share many properties, including activation of the JAK-STAT signaling pathway and induction of a variety of cellular phenotypes. For example, type I IFNs drive not only the high maturation status of DCs, but also have a direct impact in cytotoxic T lymphocytes, NK cell activation, induction of tumor cell death and inhibition of angiogenesis. A variety of stimuli, including some standard cancer treatments, promote the expression of endogenous IFN-α/β, which then participates as a fundamental component of immunogenic cell death. Systemic treatment with recombinant protein has been used for the treatment of melanoma. The induction of endogenous IFN-α/β has been tested, including stimulation through pattern recognition receptors. Gene therapies involving IFN-α/β have also been described. Thus, harnessing type I IFNs as an effective tool for cancer therapy continues to be studied.

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