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T cell alloreactivity against minor histocompatibility antigens (mHAgs)-polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms-is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D-R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D-R pairs demonstrated that total and organ-specific mHAg load could independently predict the occurrence of acute GvHD and chronic pulmonary GvHD, respectively, and defined promising GvL targets, confirmed in a validation cohort of 58 D-R pairs, for the prevention or treatment of post-transplant disease recurrence., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens and found an association with beneficial response to PD-1 blockade. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcome. This hub is distinct from mature tertiary lymphoid structures and is enriched for stem-like TCF7 + PD-1 + CD8 + T cells, activated CCR7 + LAMP3 + dendritic cells and CCL19 + fibroblasts as well as chemokines that organize these cells. Within the stem-immunity hub, we find preferential interactions between CXCL10 + macrophages and TCF7 - CD8 + T cells as well as between mature regulatory dendritic cells and TCF7 + CD4 + and regulatory T cells. These results provide a picture of the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Full-length RNA-sequencing methods using long-read technologies can capture complete transcript isoforms, but their throughput is limited. We introduce multiplexed arrays isoform sequencing (MAS-ISO-seq), a technique for programmably concatenating complementary DNAs (cDNAs) into molecules optimal for long-read sequencing, increasing the throughput >15-fold to nearly 40 million cDNA reads per run on the Sequel IIe sequencer. When applied to single-cell RNA sequencing of tumor-infiltrating T cells, MAS-ISO-seq demonstrated a 12- to 32-fold increase in the discovery of differentially spliced genes., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Targeted synthetic vaccines have the potential to transform our response to viral outbreaks, yet the design of these vaccines requires a comprehensive knowledge of viral immunogens. Here, we report severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides that are naturally processed and loaded onto human leukocyte antigen-II (HLA-II) complexes in infected cells. We identify over 500 unique viral peptides from canonical proteins as well as from overlapping internal open reading frames. Most HLA-II peptides colocalize with known CD4 + T cell epitopes in coronavirus disease 2019 patients, including 2 reported immunodominant regions in the SARS-CoV-2 membrane protein. Overall, our analyses show that HLA-I and HLA-II pathways target distinct viral proteins, with the structural proteins accounting for most of the HLA-II peptidome and nonstructural and noncanonical proteins accounting for the majority of the HLA-I peptidome. These findings highlight the need for a vaccine design that incorporates multiple viral elements harboring CD4 + and CD8 + T cell epitopes to maximize vaccine effectiveness., Competing Interests: Declaration of interests S.W.-G., D.-Y.C., S.S., K.R.C., N.H., S.A.C., J.G.A., M.S., and P.C.S. are named co-inventors on a patent application related to this work, filed by The Broad Institute, that is being made available in accordance with the COVID-19 technology licensing framework to maximize access to university innovations. N.H. is a founder of Neon Therapeutics (now BioNTech US), was a member of its scientific advisory board, and holds shares. N.H. is also an advisor for IFM Therapeutics. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer, and PrognomIQ. J.G.A. is a past employee of Neon Therapeutics (now BioNTech US). P.C.S. is a cofounder of and consultant to Sherlock Biosciences and Delve Biosciences and a board member of Danaher Corporation and holds equity in the companies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC., Competing Interests: Declaration of interests M.J.S., C.L.S., W.M.G., and K.N. receive funding from Janssen. C.L.S., M.G., and K.N. receive funding from Bristol Myers Squibb. W.M.G. receives funding from Tempus and Pavmed technologies; is a board member for Freenome, Guardant Health, and SEngine; and consults for DiaCarta. A.J.A. receives funding from Mirati Therapeutics, Deerfield, and Novo Ventures and consults for Oncorus, Arrakis Therapeutics, and Merck. G.M.B. receives funding from Palleon Pharmaceuticals, Olink Proteomics, and Takeda Oncology and is a board member for Novartis and Nektar Therapeutics. A.C.A. is a board member for Tizona Therapeutics, Compass Therapeutics, Zumutor Biologics, and ImmuneOncia and consults for iTeos Therapeutics. M.G. and K.N. receive funding from Merck and Servier. K.N. receives funding from Revolution Medicines, Evergrande Group, Pharmavite, and Merck; is a board member for Seattle Genetics and BiomX; and consults for X-Biotix Therapeutics. A. Regev is a founder of and equity holder in Celsius Therapeutics and holds equity in Immunitas Therapeutics. O.R.-R. and A. Regev are employees of Genentech. N.H. holds equity in BioNTech and consults for Related Sciences/Danger Bio. All other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development., Competing Interests: Declaration of interests S.W.-G., S.K., S.S., K.R.C, N.H., S.A.C., J.G.A., M.S., and P.C.S. are named co-inventors on a patent application related to immunogenic compositions of this manuscript filed by The Broad Institute that is being made available in accordance with the COVID-19 technology licensing framework to maximize access to university innovations. D.L.-E., C.T., Y.W., M.R.D., W.A.D., and D.C.P. are employees and stockholders of Repertoire Immune Medicines. N.B. is an extramural member of the Parker Institute for Cancer Immunotherapy; receives research funds from Regeneron, Harbor Biomedical, DC Prime, and Dragonfly Therapeutics; and is on the advisory boards of Neon Therapeutics, Novartis, Avidea, Boehringer Ingelheim, Rome Therapeutics, Roswell Park Comprehensive Cancer Center, BreakBio, Carisma Therapeutics, CureVac, Genotwin, BioNTech, Gilead Therapeutics, Tempest Therapeutics, and the Cancer Research Institute. A.S. is a consultant for Gritstone, Flow Pharma, CellCarta, OxfordImmunotech, Immunoscape, and Avalia. La Jolla Institute for Immunology has filed for patent protection for various aspects of T cell epitope and vaccine design work. D.B.K. has previously advised Neon Therapeutics and has received consulting fees from Neon Therapeutics. D.B.K. owns equity in AduroBiotech, Agenus Inc., Armata Pharmaceuticals, Breakbio Corp., Biomarin Pharmaceutical Inc., Bristol Myers Squibb Com., Celldex Therapeutics Inc., Editas Medicine Inc., Exelixis Inc., Gilead Sciences Inc., IMV Inc., Lexicon Pharmaceuticals Inc., Moderna Inc., and Regeneron Pharmaceuticals. D.B.K. receives SARS-CoV-2 research support from BeiGene for a project unrelated to this publication. N.H. is a founder of Neon Therapeutics, Inc. (now BioNTech US), was a member of its scientific advisory board, and holds shares. N.H. is also an advisor for IFM Therapeutics. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, and Seer and a scientific advisor to Pfizer and Biogen. J.G.A. is a past employee and shareholder of Neon Therapeutics, Inc. (now BioNTech US). P.C.S. is a co-founder and shareholder of Sherlock Biosciences and a non-executive board member and shareholder of Danaher Corporation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

Birdshot Uveitis (BU) is a blinding inflammatory eye condition that only affects HLA-A29-positive individuals. Genetic association studies linked ERAP2 with BU, an aminopeptidase which trims peptides before their presentation by HLA class I at the cell surface, which suggests that ERAP2-dependent peptide presentation by HLA-A29 drives the pathogenesis of BU. However, it remains poorly understood whether the effects of ERAP2 on the HLA-A29 peptidome are distinct from its effect on other HLA allotypes. To address this, we focused on the effects of ERAP2 on the immunopeptidome in patient-derived antigen presenting cells. Using complementary HLA-A29-based and pan-class I immunopurifications, isotope-labeled naturally processed and presented HLA-bound peptides were sequenced by mass spectrometry. We show that the effects of ERAP2 on the N-terminus of ligands of HLA-A29 are shared across endogenous HLA allotypes, but discover and replicate that one peptide motif generated in the presence of ERAP2 is specifically bound by HLA-A29. This motif can be found in the amino acid sequence of putative autoantigens. We further show evidence for internal sequence specificity for ERAP2 imprinted in the immunopeptidome. These results reveal that ERAP2 can generate an HLA-A29-specific antigen repertoire, which supports that antigen presentation is a key disease pathway in BU., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Venema, Hiddingh, de Boer, Claas, Mulder, den Hollander, Stratikos, Sarkizova, van der Veken, Janssen, van Veelen and Kuiper.)

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies., Competing Interests: Conflict of interest S. A. C. is a member of the scientific advisory boards of Kymera, PTM Biolabs, and Seer and a scientific advisor to Pfizer and Biogen. J. G. A. is a past employee and shareholder of Neon Therapeutics, Inc (now BioNTech US). S. K., S. S., K. R. C., S. A. C., and J. G. A. are named co-inventors on patent application(s) related to HLA peptide motif technology that is related to topics in this article filed by The Broad Institute., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens., Competing Interests: Conflict of interest D. A. B. reported nonfinancial support from Bristol-Myers Squibb, honoraria from LM Education/Exchange Services, and personal fees from Octane Global, Defined Health, Dedham Group, Adept Field Solutions, Slingshot Insights, Blueprint Partnerships, Charles River Associates, Trinity Group, and Insight Strategy, outside the submitted work. P. A. O. has received research funding from and has advised Neon Therapeutics, Bristol-Meyers Squibb, Merck, CytomX, Pfizer, Novartis, Celldex, Amgen, Array, AstraZeneca/MedImmune, Armo BioSciences, and Roche/Genentech, outside the submitted work. D. B. K. has previously advised Neon Therapeutics and has received consulting fees from Neon Therapeutics. D. B. K. owns equity in Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Bristol Myers Squibb, Celldex Therapeutics, Chinook Therapeutics, Editas Medicine, Exelixis, Gilead Sciences, IMV, Lexicon Pharmaceuticals, Moderna, and Regeneron Pharmaceuticals. BeiGene, a Chinese biotech company, supports unrelated research at TIGL. C. J. W. holds equity in BioNTech, Inc and receives research funding from Pharmacyclics, Inc. S. A. C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, and Seer and an ad hoc scientific advisor to Pfizer and Biogen. All the other authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor-associated macrophages (TAM) are regulators of extracellular matrix (ECM) remodeling and metastatic progression, the main cause of cancer-associated death. We found that disabled homolog 2 mitogen-responsive phosphoprotein (DAB2) is highly expressed in tumor-infiltrating TAMs and that its genetic ablation significantly impairs lung metastasis formation. DAB2-expressing TAMs, mainly localized along the tumor-invasive front, participate in integrin recycling, ECM remodeling, and directional migration in a tridimensional matrix. DAB2 + macrophages escort the invasive dissemination of cancer cells by a mechanosensing pathway requiring the transcription factor YAP. In human lobular breast and gastric carcinomas, DAB2 + TAMs correlated with a poor clinical outcome, identifying DAB2 as potential prognostic biomarker for stratification of patients with cancer. DAB2 is therefore central for the prometastatic activity of TAMs. SIGNIFICANCE: DAB2 expression in macrophages is essential for metastasis formation but not primary tumor growth. Mechanosensing cues, activating the complex YAP-TAZ, regulate DAB2 in macrophages, which in turn controls integrin recycling and ECM remodeling in 3-D tissue matrix. The presence of DAB2 + TAMs in patients with cancer correlates with worse prognosis. This article is highlighted in the In This Issue feature, p. 1611 ., (©2020 American Association for Cancer Research.)

Massively parallel single-cell and single-nucleus RNA sequencing has opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so is the need for computational pipelines for scaled analysis. Here we developed Cumulus-a cloud-based framework for analyzing large-scale single-cell and single-nucleus RNA sequencing datasets. Cumulus combines the power of cloud computing with improvements in algorithm and implementation to achieve high scalability, low cost, user-friendliness and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

Background and Aims: CD14+ mononuclear phagocytes [MNPs] and T cells infiltrate colon in ulcerative colitis [UC]. Here we investigated how CD14+ MNPs and the cytokines they produce shape the colonic effector T cell profile., Methods: Colonic or mesenteric lymph node [mLNs] CD4+ T cells isolated from UC or Crohn's disease [CD] patients were stimulated with cytokines or autologous CD14+ MNPs. Cytokine expression was assessed by intracytoplasmic staining and multiplex ELISA. Unsupervised phenotypic multicolour analysis of colonic CD14+ MNPs was performed using the FlowSOM algorithm., Results: Among CD14+CD64+HLA-DR+SIRPα + MNPs, only the pro-inflammatory cytokine-producing CD163- subpopulation accumulated in inflamed UC colon and promoted mucosal IL-1β-dependent Th17, Th17/Th1, Th17/Th22 but not Th1 responses. Unsupervised phenotypic analysis of CD14+CD64+ MNPs segregated CD163- monocyte-like cells and CD163+ macrophages. Unexpectedly, IL-12, IL-1β and CD163-, but not CD163+, cells induced IL-8 expression in colonic CD4+ T cells, which co-expressed IFN-γ and/or IL-17 in UC and not CD. The CD163- monocyte-like cells increased the frequency of IL-8+IL-17+/-IFN-γ +/- T cells through IL-1β and IL-12. Finally, colonic IL-8+ T cells co-expressing GM-CSF, TNF-α and IL-6 were detected ex vivo and, promoted by IL-12 in the mucosa and mLNs in UC only., Conclusions: Our findings established a link between monocyte-like CD163- MNPs, IL-12, IL-1β and the detection of colonic memory IL-8-producing CD4+ T cells, which might all contribute to the pathogenesis of UC., (Copyright © 2019 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Inflammatory bowel diseases are associated with dysregulated immune responses in the intestinal tissue. Four molecularly identified macrophage subsets control immune homeostasis in healthy gut. However, the specific roles and transcriptomic profiles of the phenotypically heterogeneous CD14 + macrophage-like population in inflamed gut remain to be investigated in Crohn's disease (CD). Here we identified two phenotypically, morphologically and functionally distinct colonic HLADR + SIRPα + CD14 + subpopulations that were further characterized using single-cell RNA-sequencing (scRNAseq) in CD. Frequencies of CD64 hi CD163 -/dim cells selectively augmented in inflamed colon and correlated with endoscopic score of disease severity. IL-1β and IL-23-producing CD64 hi CD163 -/dim cells predominated over TNF-α-producing CD64 hi CD163 hi cells in lesions. Purified "inflammatory monocyte-like" CD163 - , but not "macrophage-like" CD163 hi cells, through IL-1β, promoted Th17/Th1 but not Th1 responses in tissue memory CD4 + T cells. Unsupervised scRNAseq analysis that captures the entire HLADR + SIRPα + population revealed six clusters, two of which were enriched in either CD163 - or CD163 hi cells, and best defined by TREM1/FCAR/FCN1/IL1RN or CD209/MERTK/MRCI/CD163L1 genes, respectively. Selected newly identified discriminating markers were used beyond CD163 to isolate cells that shared pro-Th17/Th1 function with CD163 - cells. In conclusion, a molecularly distinct pro-inflammatory CD14 + subpopulation accumulates in inflamed colon, drives intestinal inflammatory T-cell responses, and thus, might contribute to CD disease severity.

Given the many cell types and molecular components of the human immune system, along with vast variations across individuals, how should we go about developing causal and predictive explanations of immunity? A central strategy in human studies is to leverage natural variation to find relationships among variables, including DNA variants, epigenetic states, immune phenotypes, clinical descriptors, and others. Here, we focus on how natural variation is used to find patterns, infer principles, and develop predictive models for two areas: (a) immune cell activation-how single-cell profiling boosts our ability to discover immune cell types and states-and (b) antigen presentation and recognition-how models can be generated to predict presentation of antigens on MHC molecules and their detection by T cell receptors. These are two examples of a shift in how we find the drivers and targets of immunity, especially in the human system in the context of health and disease.

After entering tissues, monocytes differentiate into cells that share functional features with either macrophages or dendritic cells (DCs). How monocyte fate is directed toward monocyte-derived macrophages (mo-Macs) or monocyte-derived DCs (mo-DCs) and which transcription factors control these differentiation pathways remains unknown. Using an in vitro culture model yielding human mo-DCs and mo-Macs closely resembling those found in vivo in ascites, we show that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DCs and mo-Macs, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Macs. AhR deficiency also impaired the in vivo differentiation of mouse mo-DCs. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DCs and mo-Macs are controlled by distinct transcription factors and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) and monocytes play a central role in pathogen sensing, phagocytosis, and antigen presentation and consist of multiple specialized subtypes. However, their identities and interrelationships are not fully understood. Using unbiased single-cell RNA sequencing (RNA-seq) of ~2400 cells, we identified six human DCs and four monocyte subtypes in human blood. Our study reveals a new DC subset that shares properties with plasmacytoid DCs (pDCs) but potently activates T cells, thus redefining pDCs; a new subdivision within the CD1C + subset of DCs; the relationship between blastic plasmacytoid DC neoplasia cells and healthy DCs; and circulating progenitor of conventional DCs (cDCs). Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease., (Copyright © 2017, American Association for the Advancement of Science.)

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation., (Copyright © 2017 Elsevier Inc. All rights reserved.)