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Introduction: Severe respiratory illness is the most prominent manifestation of patients infected with SARS-CoV-2, and yet the molecular mechanisms underlying severe lung disease in COVID-19 affected patients still require elucidation. Human leukocyte antigen class I (HLA-I) expression is crucial for antigen presentation and the host's response to SARS-CoV-2., Methods: To gain insights into the immune response and molecular pathways involved in severe lung disease, we performed immunopeptidomic and proteomic analyses of lung tissues recovered at four COVID-19 autopsy and six non-COVID-19 transplants., Results: We found signals of tissue injury and regeneration in lung fibroblast and alveolar type I/II cells, resulting in the production of highly immunogenic self-antigens within the lungs of COVID-19 patients. We also identified immune activation of the M2c macrophage as the primary source of HLA-I presentation and immunogenicity in this context. Additionally, we identified 28 lung signatures that can serve as early plasma markers for predicting infection and severe COVID-19 disease. These protein signatures were predominantly expressed in macrophages and epithelial cells and were associated with complement and coagulation cascades., Discussion: Our findings emphasize the significant role of macrophage-mediated immunity in the development of severe lung disease in COVID-19 patients., Competing Interests: DK is a scientific advisor for Immunitrack and Breakbio. DK owns equity in Affimed N.V., Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Celldex Therapeutics, Editas Medicine, Gilead Sciences, Immunitybio, ImmunoGen, IMV, Lexicon Pharmaceuticals, Neoleukin Therapeutics. BeiGene, a Chinese biotech company, supported unrelated SARS COV-2 research at TIGL. EK has an unrelated financial interest in Novartis AG. EK receives unrelated research funding from Bayer AG and 10x Genomics, Inc. CW receives funding support from Pharmacyclics and holds equity in BioNTech, Inc. SY holds equity in Yihui Bio, Inc. RK receives grant support from Boehringer Ingelheim and Bayer, LH receives grants from Boehringer Ingelheim and has received personal consulting fees from Boehringer Ingelheim, Pliant Therapeutics, Bioclinica, Abbvie and Biogen Idec. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Yin, Klaeger, Chea, Carulli, Rachimi, Black, Filbin, Hariri, Knipe, Padera, Stevens, Lane, Carr, Wu, Kim and Keskin.)

Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors., (© 2023. The Author(s).)

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.

Within the tumour microenvironment, CD4 + T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules 1,2 , but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4 + T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4 + T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4 + T regulatory (T Reg ) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4 + T Reg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4 + T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4 + T Reg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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.)

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape 1,2 . Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape 3-5 . We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution 6 . SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.

The Musca domestica salivary gland hypertrophy virus (MdSGHV) substantially enlarges the house fly's salivary glands and prevents or delays ovarian development in its adult host, but the effect that MdSGHV has on the house fly's food consumption is currently unknown. Using house flies from a laboratory-reared colony, we evaluated the effect of MdSGHV infection on food consumption over a 7-d period. Both treatment (virus-infected) and control (saline-injected) flies were provided with a choice of 8% sucrose solution and 4% powdered milk solution to determine food preferences. Quantities of each solution consumed were measured every 24 h for each fly to measure food consumptions. Infected house flies were shown to consume less overall of both solutions than house flies injected with saline. The largest consumption discrepancy was seen between female house flies. Healthy female flies with developing ovaries continued to consume a sugar and protein diet, whereas infected female flies fed predominantly on a sugar diet. Additionally, infected male and female flies consumed significantly lower quantities of protein and sucrose than control flies. This suggests that MdSGHV has a negative consumption effect (e.g., hunger, starvation) on its host. Thus, differences in food consumption of infected and control flies probably represent differences in the nutritional requirements of flies resulting from viral infection., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.)