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Uveal melanoma (UM) is the most prevalent cancer of the eye in adults, driven by activating mutation of GNAQ/GNA11; however, there are limited therapies against UM and metastatic UM (mUM). Here, we perform a high-throughput chemogenetic drug screen in GNAQ-mutant UM contrasted with BRAF-mutant cutaneous melanoma, defining the druggable landscape of these distinct melanoma subtypes. Across all compounds, darovasertib demonstrates the highest preferential activity against UM. Our investigation reveals that darovasertib potently inhibits PKC as well as PKN/PRK, an AGC kinase family that is part of the "dark kinome." We find that downstream of the Gαq-RhoA signaling axis, PKN converges with ROCK to control FAK, a mediator of non-canonical Gαq-driven signaling. Strikingly, darovasertib synergizes with FAK inhibitors to halt UM growth and promote cytotoxic cell death in vitro and in preclinical metastatic mouse models, thus exposing a signaling vulnerability that can be exploited as a multimodal precision therapy against mUM.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Despite the widespread use of SARS-CoV-2-specific monoclonal antibody (mAb) therapy for the treatment of acute COVID-19, the impact of this therapy on the development of SARS-CoV-2-specific T cell responses has been unknown, resulting in uncertainty as to whether anti-SARS-CoV-2 mAb administration may result in failure to generate immune memory. Alternatively, it has been suggested that SARS-CoV-2-specific mAb may enhance adaptive immunity to SARS-CoV-2 via a "vaccinal effect." Bamlanivimab (Eli Lilly and Company) is a recombinant human IgG1 that was granted FDA emergency use authorization for the treatment of mild to moderate COVID-19 in those at high risk for progression to severe disease. Here, we compared SARS-CoV-2-specific CD4+ and CD8+ T cell responses of 95 individuals from the ACTIV-2/A5401 clinical trial 28 days after treatment with bamlanivimab versus placebo. SARS-CoV-2-specific T cell responses were evaluated using activation-induced marker assays in conjunction with intracellular cytokine staining. We demonstrate that most individuals with acute COVID-19 developed SARS-CoV-2-specific T cell responses. Overall, our findings suggest that the quantity and quality of SARS-CoV-2-specific T cell memory were not diminished in individuals who received bamlanivimab for acute COVID-19. Receipt of bamlanivimab during acute COVID-19 neither diminished nor enhanced SARS-CoV-2-specific cellular immunity.

Both SARS-CoV-2 infections and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of 2 pools of experimentally defined SARS-CoV-2 T cell epitopes that, in combination with spike, were used to discriminate 4 groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status. The overall T cell-based classification accuracy was 89.2% and 88.5% in the experimental and validation cohorts. This scheme was applicable to different mRNA vaccines and different lengths of time post infection/post vaccination and yielded increased accuracy when compared to serological readouts. T cell responses from breakthrough infections were also studied and effectively segregated from vaccine responses, with a combined performance of 86.6% across all 239 subjects from the 5 groups. We anticipate that a T cell-based immunodiagnostic scheme to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccinations and for establishing SARS-CoV-2 correlates of protection.

Head and neck squamous cell carcinoma (HNSCC) represents a highly prevalent and deadly malignancy worldwide. The prognosis for locoregionally advanced HNSCC has not appreciably improved over the past 30 years despite advances in surgical, radiation, and targeted therapies and less than 20% of HNSCC patients respond to recently approved immune checkpoint inhibitors. The Hippo signaling pathway, originally discovered as a mechanism regulating tissue growth and organ size, transduces intracellular and extracellular signals to regulate the transcriptional co-activators YAP and TAZ. Alterations in the Hippo pathway resulting in persistent YAP and TAZ activation have emerged as major oncogenic drivers. Our analysis of the human HNSCC oncogenome revealed multiple genomic alterations impairing Hippo signaling and activating YAP and TAZ, which in turn contribute to HNSCC development. This includes mutations and deletions of the FAT1 gene (29%) and amplification of the WWTR1 (encoding TAZ, 14%) and YAP1 genes (8%), together representing one of the most genetically altered signaling mechanisms in this malignancy. Here, we discuss key elements of the mammalian Hippo pathway, detail mechanisms by which perturbations in Hippo signaling promote HNSCC initiation and progression and outline emerging strategies to target Hippo signaling vulnerabilities as part of novel multimodal precision therapies for HNSCC.

The emergence of SARS-CoV-2 variants with evidence of antibody escape highlight the importance of addressing whether the total CD4+ and CD8+ T cell recognition is also affected. Here, we compare SARS-CoV-2-specific CD4+ and CD8+ T cells against the B.1.1.7, B.1.351, P.1, and CAL.20C lineages in COVID-19 convalescents and in recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. The total reactivity against SARS-CoV-2 variants is similar in terms of magnitude and frequency of response, with decreases in the 10%-22% range observed in some assay/VOC combinations. A total of 7% and 3% of previously identified CD4+ and CD8+ T cell epitopes, respectively, are affected by mutations in the various VOCs. Thus, the SARS-CoV-2 variants analyzed here do not significantly disrupt the total SARS-CoV-2 T cell reactivity; however, the decreases observed highlight the importance for active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution.

BackgroundCoronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach.MethodOver 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions.FindingsThe 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity.InterpretationThe ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.FundingThis work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S.C, cchiCooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists.One sentence summaryThe host immune response in COVID-19.

Understanding immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics and vaccines and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥6 months after infection. Immunoglobulin G (IgG) to the spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month after symptom onset. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with a half-life of 3 to 5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens and precisely measure virus-specific CD4+ and CD8+ T cells, we study epitope-specific T cell responses of 99 convalescent coronavirus disease 2019 (COVID-19) cases. The SARS-CoV-2 proteome is probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of human leukocyte antigen (HLA) alleles for class II responses. For HLA class I, we study an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identify several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance are observed, which differ for CD4+ T cells, CD8+ T cells, and antibodies. The class I and class II epitopes are combined into epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4+ and CD8+ T cells.

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4 + T cells and CD8 + T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4 + T cell, and CD8 + T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4 + and CD8 + T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4 + T cells, CD8 + T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4 + and CD8 + T cells.

BackgroundDespite increased utilization of hepatitis C virus-infected (HCV+) organs for transplantation into HCV-uninfected recipients, there is lack of standardization in HCV-related patient education/consent and limited data on financial and social impact on patients.MethodsWe conducted a survey on patients with donor-derived HCV infection at our center transplanted between 4/1/2017 and 11/1/2019 to assess: why patients chose to accept HCV+ organ(s), the adequacy of their pre-transplant HCV education and informed consent process, financial issues related to copays after discharge, and social challenges they faced.ResultsAmong 49 patients surveyed, transplanted organs included heart (n = 19), lung (n = 9), kidney (n = 11), liver (n = 4), heart/kidney (n = 4), and liver/kidney (n = 2). Many recipients accepted an HCV-viremic (HCV-V) organ due to perceived reduction in waitlist time (n = 33) and/or trust in their physician's recommendation (n = 29). Almost all (n = 47) felt that pre-transplant education and consent was appropriate. Thirty patients had no copay for direct-acting antivirals (DAA) for HCV, including 21 with household income $1000. Two patients reported feeling isolated due to HCV infection and eight reported higher than anticipated medication costs. Patients' biggest concern was potential HCV transmission to partners (n = 18) and family/friends (n = 15). Overall almost all (n = 47) patients reported a positive experience with HCV-V organ transplantation.ConclusionWe demonstrate that real-world patient experiences surrounding HCV-V organ transplantation have been favorable. Almost all patients report comprehensive HCV-related pre-transplant consent and education. Additionally, medication costs and social isolation/exclusion were not barriers to the use of these organs.

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.

Many unknowns exist about human immune responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. SARS-CoV-2-reactive CD4+ T cells have been reported in unexposed individuals, suggesting preexisting cross-reactive T cell memory in 20 to 50% of people. However, the source of those T cells has been speculative. Using human blood samples derived before the SARS-CoV-2 virus was discovered in 2019, we mapped 142 T cell epitopes across the SARS-CoV-2 genome to facilitate precise interrogation of the SARS-CoV-2-specific CD4+ T cell repertoire. We demonstrate a range of preexisting memory CD4+ T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underlie at least some of the extensive heterogeneity observed in coronavirus disease 2019 (COVID-19) disease.

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4+ T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells (TIC) at single cell resolution. TIC displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. YAP-mediated TIC programs included the activation of oncogenic transcriptional networks and mTOR signaling, and the recruitment of myeloid cells to the invasive front contributing to tumor infiltration. TIC transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

T cells are involved in protective immunity against numerous viral infections. Data regarding functional roles of human T cells in SARS-CoV-2 (SARS2) viral clearance in primary COVID-19 are limited. To address this knowledge gap, samples were assessed for associations between SARS2 upper respiratory tract viral RNA levels and early virus-specific adaptive immune responses for 95 unvaccinated clinical trial participants with acute primary COVID-19 aged 18-86 years old, approximately half of whom were considered high risk for progression to severe COVID-19. Functionality and magnitude of acute SARS2-specific CD4 and CD8 T cell responses were evaluated, in addition to antibody responses. Most individuals with acute COVID-19 developed SARS2-specific T cell responses within 6 days of COVID-19 symptom onset. Early CD4 T cell and CD8 T cell responses were polyfunctional, and both strongly associated with reduced upper respiratory tract SARS2 viral RNA, independent of neutralizing antibody titers. Overall, these findings provide evidence for protective roles for circulating SARS2-specific CD4 and CD8 T cells during acute COVID-19.

Therapeutic monoclonal antibodies (mAbs) have been studied in humans, but the impact on immune memory of mAb treatment during an ongoing infection has remained unclear. We evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific reduction in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating memory B cell responses to infection, and single mAb administration can continue to impact memory B cell responses to additional antigen exposures months later., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The emergence of SARS-CoV-2 variants with evidence of antibody escape highlight the importance of addressing whether the total CD4 + and CD8 + T cell recognition is also affected. Here, we compare SARS-CoV-2-specific CD4 + and CD8 + T cells against the B.1.1.7, B.1.351, P.1, and CAL.20C lineages in COVID-19 convalescents and in recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. The total reactivity against SARS-CoV-2 variants is similar in terms of magnitude and frequency of response, with decreases in the 10%-22% range observed in some assay/VOC combinations. A total of 7% and 3% of previously identified CD4 + and CD8 + T cell epitopes, respectively, are affected by mutations in the various VOCs. Thus, the SARS-CoV-2 variants analyzed here do not significantly disrupt the total SARS-CoV-2 T cell reactivity; however, the decreases observed highlight the importance for active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution., Competing Interests: A. Sette is a consultant for Gritstone, Flow Pharma, CellCarta, Arcturus, Oxfordimmunotech, and Avalia. S.C. is a consultant for Avalia. All of the other authors declare no competing interests. LJI has filed for patent protection for various aspects of vaccine design and identification of specific epitopes., (© 2021 The Author(s).)