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Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks., Competing Interests: Declaration of interests K.P., M.H., J.H.C., V.K.K., A.J.A., O.R.-R., A. Regev., and N.H. are co-inventors on US Patent Application No. 16/995,425 relating to methods for predicting outcomes and treating colorectal cancer as described in the manuscript. A.J.A. is a Consultant for Oncorus, Arrakis Therapeutics, and Merck and receives research funding from Mirati Therapeutics, Deerfield, and Novo Ventures. R.B.C. receives consulting/speaking fees from Abbvie, Amgen, Array Biopharma/Pfizer, Asana Biosciences, Astex Pharmaceuticals, AstraZeneca, Avidity Biosciences, BMS, C4 Therapeutics, Chugai, Elicio, Fog Pharma, Fount Therapeutics/Kinnate Biopharma, Genentech, Guardant Health, Ipsen, LOXO, Merrimack, Mirati Therapeutics, Natera, N-of-one/QIAGEN, Novartis, nRichDx, Revolution Medicines, Roche, Roivant, Shionogi, Shire, Spectrum Pharmaceuticals, Symphogen, Tango Therapeutics, Taiho, Warp Drive Bio, and Zikani Therapeutics; holds equity in Avidity Biosciences, C4 Therapeutics, Fount Therapeutics/Kinnate Biopharma, nRichDx, and Revolution Medicines; and has received research funding from Asana, AstraZeneca, Lilly, and Sanofi. V.K.K. consults for Pfizer, GSK, Tizona Therapeutics, Celsius Therapeutics, Bicara Therapeutics, Compass Therapeutics, Biocon, and Syngene. G.M.B. has sponsored research agreements with Palleon Pharmaceuticals, Olink Proteomics, and Takeda Oncology; served on SABs for Novartis and Nektar Therapeutics; and received honoraria from Novartis. A.C.A. is a paid consultant for iTeos Therapeutics, and is an SAB member for Tizona Therapeutics, Compass Therapeutics, Zumutor Biologics, and ImmuneOncia, which have interests in cancer immunotherapy. A.C.A.’s interests were reviewed and managed by the BWH and Partners Healthcare in accordance with their conflict of interest policies. M.G. receives research funding from BMS, Merck, and Servier. J.W.R., C.A.F., and M.L.H. are employees of and stockholders for NanoString Technologies Inc. D.R.Z. is a former employee of NanoString Technologies Inc. B.I. is a consultant for Merck and Volastra Therapeutic. R.B. is an UptoDate Author. A. Rotem is an equity holder in Celsius Therapeutics and NucleAI. K.N. has research funding from Janssen, Revolution Medicines, Evergrande Group, Pharmavite; advisory board: Seattle Genetics, BiomX; consulting: X-Biotix Therapeutics; research funding: BMS, Merck, and Servier. B.E.J. is on the SAB for Checkpoint Therapeutics. O.R.-R. is a named inventor on patents and patent applications filed by the Broad Institute in single-cell genomics. From October 2020, O.R.-R. is an employee of Genentech. A. Regev. is a founder of and equity holder in Celsius Therapeutics, an equity holder in Immunitas Therapeutics, and was an SAB member for Thermo Fisher Scientific, Syros Pharmaceuticals, and Neogene Therapeutics until August 1, 2020. From August 1, 2020, A. Regev. is an employee of Genentech. A. Regev. is a named inventor on several patents and patent applications filed by the Broad Institute in single-cell and spatial genomics. N.H. holds equity in BioNTech and is an advisor for Related Sciences/Danger Bio., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

FoxP3 + T regulatory (Treg) cells are central elements of immunologic tolerance. They are abundant in many tumors, where they restrict potentially favorable antitumor responses. We used a three-pronged strategy to identify genes related to the presence and function of Tregs in the tumor microenvironment. Gene expression profiles were generated from tumor-infiltrating Tregs (TITRs) of both human and mouse tumors and were compared with those of Tregs of lymphoid organs or normal tissues from the same individuals. A computational deconvolution of whole-tumor datasets from the Cancer Genome Atlas (TCGA) was performed to identify transcripts specifically associated with Tregs across thousands of tumors from different stages and locations. We identified a set of TITR-differential transcripts with striking reproducibility between tumor types in mice, between mice and humans, and between different human patients spanning tumor stages. Many of the TITR-preferential transcripts were shared with "tissue Tregs" residing in nonlymphoid tissues, but a tumor-preferential segment could be identified. Many of these TITR signature transcripts were confirmed by mining of TCGA datasets, which also brought forth transcript modules likely representing the parenchymal attraction of, or response to, tumor Tregs. Importantly, the TITR signature included several genes encoding effective targets of tumor immunotherapy. A number of other targets were validated by CRISPR-based gene inactivation in mouse Tregs. These results confirm the validity of the signature, generating a wealth of leads for understanding the role of Tregs in tumor progression and identifying potential targets for cancer immunotherapy., Competing Interests: Conflict of interest statement: C.B. co-authored a consortium position paper with Miriam Merad in 2017 that stems from the Human Cell Atlas, a large consortium to which they both belong; they did not collaborate directly on the paper. C.B. is a principal investigator in a bridge funding for a consortium (Immune Cell Atlas), of which Dr. Merad is the Program Director; Dr. Merad’s review of this paper was concluded before this award.

Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics., Competing Interests: The authors declare no conflict of interest.

T lymphocyte activation by antigen conditions adaptive immune responses and immunopathologies, but we know little about its variation in humans and its genetic or environmental roots. We analyzed gene expression in CD4(+) T cells during unbiased activation or in T helper 17 (T(H)17) conditions from 348 healthy participants representing European, Asian, and African ancestries. We observed interindividual variability, most marked for cytokine transcripts, with clear biases on the basis of ancestry, and following patterns more complex than simple T(H)1/2/17 partitions. We identified 39 genetic loci specifically associated in cis with activated gene expression. We further fine-mapped and validated a single-base variant that modulates YY1 binding and the activity of an enhancer element controlling the autoimmune-associated IL2RA gene, affecting its activity in activated but not regulatory T cells. Thus, interindividual variability affects the fundamental immunologic process of T helper activation, with important connections to autoimmune disease., (Copyright © 2014, American Association for the Advancement of Science.)

To extend our understanding of the genetic basis of human immune function and dysfunction, we performed an expression quantitative trait locus (eQTL) study of purified CD4(+) T cells and monocytes, representing adaptive and innate immunity, in a multi-ethnic cohort of 461 healthy individuals. Context-specific cis- and trans-eQTLs were identified, and cross-population mapping allowed, in some cases, putative functional assignment of candidate causal regulatory variants for disease-associated loci. We note an over-representation of T cell-specific eQTLs among susceptibility alleles for autoimmune diseases and of monocyte-specific eQTLs among Alzheimer's and Parkinson's disease variants. This polarization implicates specific immune cell types in these diseases and points to the need to identify the cell-autonomous effects of disease susceptibility variants.

FOXP3(+) regulatory T (Treg) cells enforce immune self-tolerance and homeostasis, and variation in some aspects of Treg function may contribute to human autoimmune diseases. Here, we analyzed population-level Treg variability by performing genome-wide expression profiling of CD4(+) Treg and conventional CD4(+) T (Tconv) cells from 168 donors, healthy or with established type-1 diabetes (T1D) or type-2 diabetes (T2D), in relation to genetic and immunologic screening. There was a range of variability in Treg signature transcripts, some almost invariant, others more variable, with more extensive variability for genes that control effector function (ENTPD1, FCRL1) than for lineage-specification factors like FOXP3 or IKZF2. Network analysis of Treg signature genes identified coregulated clusters that respond similarly to genetic and environmental variation in Treg and Tconv cells, denoting qualitative differences in otherwise shared regulatory circuits whereas other clusters are coregulated in Treg, but not Tconv, cells, suggesting Treg-specific regulation of genes like CTLA4 or DUSP4. Dense genotyping identified 110 local genetic variants (cis-expression quantitative trait loci), some of which are specifically active in Treg, but not Tconv, cells. The Treg signature became sharper with age and with increasing body-mass index, suggesting a tuning of Treg function with repertoire selection and/or chronic inflammation. Some Treg signature transcripts correlated with FOXP3 mRNA and/or protein, suggesting transcriptional or posttranslational regulatory relationships. Although no single transcript showed significant association to diabetes, overall expression of the Treg signature was subtly perturbed in T1D, but not T2D, patients.