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SummaryAntibodies represent a primary mediator of protection against respiratory viruses. Serum neutralizing antibodies (NAbs) are often considered a primary correlate of protection. However,detailed antibody profiles including characterization of antibody functions in different anatomic compartments are poorly understood. Here we show that antibody correlates of protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge are different in systemic versus mucosal compartments in rhesus macaques. In serum,NAbs were the strongest correlate of protection and linked to spike-specific binding antibodies and other extra-NAb functions that create a larger protective network. In bronchiolar lavage (BAL),antibody-dependent cellular phagocytosis (ADCP) proved the strongest correlate of protection rather than NAbs. Within BAL,ADCP was linked to mucosal spike-specific immunoglobulin (Ig)G,IgA/secretory IgA,and Fcγ-receptor binding antibodies. Our results support a model in which antibodies with different functions mediate protection at different anatomic sites. Graphical abstract Highlights•Correlates of protection to SARS-CoV-2 Omicron are highly compartment specific•Antibody effector functions are primary correlates of protection at infection site•Mucosal boosting enhances IgA and functionally levered IgG in lower respiratory tract Health sciences; Biological sciences View Publication

Pain and inflammation contribute immeasurably to reduced quality of life,yet modern analgesic and anti-inflammatory therapeutics can cause dependence and side effects. Here,we screened 1444 plant extracts,prepared primarily from native species in California and the United States Virgin Islands,against two voltage-gated K+ channels - T-cell expressed Kv1.3 and nociceptive-neuron expressed Kv7.2/7.3. A subset of extracts both inhibits Kv1.3 and activates Kv7.2/7.3 at hyperpolarized potentials,effects predicted to be anti-inflammatory and analgesic,respectively. Among the top dual hits are witch hazel and fireweed; polymodal modulation of multiple K+ channel types by hydrolysable tannins contributes to their dual anti-inflammatory,analgesic actions. In silico docking and mutagenesis data suggest pore-proximal extracellular linker sequence divergence underlies opposite effects of hydrolysable tannins on different Kv1 isoforms. The findings provide molecular insights into the enduring,widespread medicinal use of witch hazel and fireweed and demonstrate a screening strategy for discovering dual anti-inflammatory,analgesic small molecules. A dual potassium channel functional screen of 1444 plant extracts uncovers unexpected molecular mechanisms underlying the traditional use of witch hazel and fireweed as analgesic,anti-inflammatory medicines. View Publication

A shift of arginine metabolism from polyamine synthesis to nitric oxide synthesis induces reprogramming of macrophages from pro-tumor M2 to anti-tumor M1 types. HER2+ breast tumors have abundant immune-suppressive cells,including M2-type tumor-associated macrophages (TAMs). Although TAMs consist of the immune-stimulatory M1 type and immune-suppressive M2 type,the M1/M2-TAM ratio is reduced in immune-suppressive tumors,contributing to their immunotherapy refractoriness. M1- versus M2-TAM formation depends on differential arginine metabolism,where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- versus M2-TAMs is attributed to different availability of BH4 (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently,we reported that sepiapterin (SEP),the endogenous BH4 precursor,elevates the expression of M1-TAM markers within HER2+ tumors. Here,we show that SEP restores BH4 levels in M2-like macrophages,which then redirects arginine metabolism to NO synthesis and converts M2 type to M1 type. The reprogrammed macrophages exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in the metabolic shift of the HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy. View Publication

Crohn’s disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients,we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as ‘LND’) with high expression of LCN2,NOS2,and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells,confirmed by in-situ RNA and protein imaging,are rare in non-IBD controls but expand in active CD,and actively interact with immune cells and specifically express IBD/CD susceptibility genes,suggesting a possible function in CD immunopathogenesis. Furthermore,we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn’s ileitis and colitis. Crohn’s disease (CD) is a complex disease associated with immune dysregulation. Here the authors use multimodal data to identify and characterize an epithelial cell population,termed ‘LND’ cells,in both terminal ileum and ascending colon,with LND interacting locally with immune cells and potentially contributing to CD pathology. View Publication

Recurrent infections are a hallmark of STAT3 DN hyper-IgE syndrome,a rare immunodeficiency syndrome,and our study suggests that neutrophil-avid nanocarriers have potential for directed delivery of cargo therapeutics to improve neutrophil infection clearance in these patients. Recurrent infections are a hallmark of STAT3 dominant-negative hyper-IgE syndrome (STAT3 HIES),a rare immunodeficiency syndrome previously known as Jobs syndrome,along with elevated IgE levels and impaired neutrophil function. We have been developing nanoparticles with neutrophil trophism that home to the sites of infection via these first-responder leukocytes,named neutrophil-avid nanocarriers (NANs). Here,we demonstrate that human neutrophils can phagocytose nanogels (NGs),a type of NAN,with enhanced uptake after particle serum opsonization,comparing neutrophils from healthy individuals to those with STAT3 HIES,where both groups exhibit NG uptake; however,the patient group showed reduced phagocytosis efficiency with serum-opsonized NANs. Proteomic analysis of NG protein corona revealed complement components,particularly C3,as predominant in both groups. Difference between groups includes STAT3 HIES samples with higher neutrophil protein and lower acute-phase protein expression. The study suggests that despite neutrophil dysfunction in STAT3 HIES,NANs have potential for directed delivery of cargo therapeutics to improve neutrophil infection clearance. View Publication

SummaryA tissue resident-like phenotype in tumor infiltrating T cells can limit systemic anti-tumor immunity. Enhanced systemic anti-tumor immunity is observed in head and neck cancer patients after neoadjuvant PD-L1 immune checkpoint blockade (ICB) and transforming growth factor β (TGF-β) neutralization. Using T cell receptor (TCR) sequencing and functional immunity assays in a syngeneic model of oral cancer,we dissect the relative contribution of these treatments to enhanced systemic immunity. The addition of TGF-β neutralization to ICB resulted in the egress of expanded and exhausted CD8+ tumor infiltrating lymphocytes (TILs) into circulation and greater systemic anti-tumor immunity. This enhanced egress associated with reduced expression of Itgae (CD103) and its upstream regulator Znf683. Circulating CD8+ T cells expressed higher Cxcr3 after treatment,an observation also made in samples from patients treated with dual TGF-β neutralization and ICB. These findings provide the scientific rationale for the use of PD-L1 ICB and TGF-β neutralization in newly diagnosed patients with carcinomas prior to definitive treatment of locoregional disease. Graphical abstract Highlights•TGF-β blockade reduces Znf683 and CD103 in αPDL1-activated TILs•Reduced TIL CD103 expression associates with egress into circulation•The addition of TGF-β blockade to αPDL1 enhances systemic anti-tumor immunity•Circulating CD8+ T cells express greater CXCR3 after dual TGF-β and PDL1 blockade Natural sciences; Biological sciences; Immunology ; Immune response; Systems biology; Cancer systems biology; Cancer View Publication

Epstein-Barr Virus (EBV) is associated with numerous cancers including B cell lymphomas. In vitro,EBV transforms primary B cells into immortalized Lymphoblastoid Cell Lines (LCLs) which serves as a model to study the role of viral proteins in EBV malignancies. EBV induced cellular transformation is driven by viral proteins including EBV-Nuclear Antigens (EBNAs). EBNA-LP is important for the transformation of naïve but not memory B cells. While EBNA-LP was thought to promote gene activation by EBNA2,EBNA-LP Knockout (LPKO) virus-infected cells express EBNA2-activated cellular genes efficiently. Therefore,a gap in knowledge exists as to what roles EBNA-LP plays in naïve B cell transformation. We developed a trans-complementation assay wherein transfection with wild-type EBNA-LP rescues the transformation of peripheral blood- and cord blood-derived naïve B cells by LPKO virus. Despite EBNA-LP phosphorylation sites being important in EBNA2 co-activation; neither phospho-mutant nor phospho-mimetic EBNA-LP was defective in rescuing naïve B cell outgrowth. However,we identified conserved leucine-rich motifs in EBNA-LP that were required for transformation of adult naïve and cord blood B cells. Because cellular PPAR-g coactivator (PGC) proteins use leucine-rich motifs to engage transcription factors including YY1,a key regulator of DNA looping and metabolism,we examined the role of EBNA-LP in engaging transcription factors. We found a significant overlap between EBNA-LP and YY1 in ChIP-Seq data. By Cut&Run,YY1 peaks unique to WT compared to LPKO LCLs occur at more highly expressed genes. Moreover,Cas9 knockout of YY1 in primary B cells prior to EBV infection indicated YY1 to be important for EBV-mediated transformation. We confirmed EBNA-LP and YY1 biochemical association in LCLs by endogenous co-immunoprecipitation and found that the EBNA-LP leucine-rich motifs were required for YY1 interaction in LCLs. We propose that EBNA-LP engages YY1 through conserved leucine-rich motifs to promote EBV transformation of naïve B cells. Author summaryEpstein-Barr Virus (EBV) is associated with various B cell lymphomas,particularly in immunosuppressed individuals. In the absence of a functional immune system,viral latency proteins,including EBV Nuclear Antigens (EBNAs) act as oncoproteins to promote tumorigenesis. EBNA-LP is one of the first viral proteins produced after infection and is important for the transformation of naïve B cells. However,the roles of EBNA-LP during infection are largely undefined. In this study,developed an assay in which the role of wild type and mutant EBNA-LP could be investigated in the context of primary naïve B cells infected with an EBNA-LP Knockout virus. Using this assay,we identified highly conserved leucine-rich motifs within EBNA-LP that are important for transformation of EBV-infected naïve B cells. These conserved motifs associate with the cellular transcription factor YY1,an important transcriptional regulator in B cell development and in many cancers,that we now show is essential for outgrowth of EBV infected B cells. Our study provides further insights into the mechanisms by which EBV transforms naïve B cells. View Publication

SummaryTumor-infiltrating regulatory T cells (TI-Tregs) elicit immunosuppressive effects in the tumor microenvironment (TME) leading to accelerated tumor growth and resistance to immunotherapies against solid tumors. Here,we demonstrate that poly-(ADP-ribose)-polymerase-11 (PARP11) is an essential regulator of immunosuppressive activities of TI-Tregs. Expression of PARP11 correlates with TI-Treg cell numbers and poor responses to immune checkpoint blockade (ICB) in human patients with cancer. Tumor-derived factors including adenosine and prostaglandin E2 induce PARP11 in TI-Tregs. Knockout of PARP11 in the cells of the TME or treatment of tumor-bearing mice with selective PARP11 inhibitor ITK7 inactivates TI-Tregs and reinvigorates anti-tumor immune responses. Accordingly,ITK7 decelerates tumor growth and significantly increases the efficacy of anti-tumor immunotherapies including ICB and adoptive transfer of chimeric antigen receptor (CAR) T cells. These results characterize PARP11 as a key driver of TI-Treg activities and a major regulator of immunosuppressive TME and argue for targeting PARP11 to augment anti-cancer immunotherapies. Graphical abstract Highlights•Tumor-derived factors upregulate PARP11 in the tumor-infiltrating Treg cells•PARP11 supports the immunosuppressive properties of Treg cells•Pharmacologic inhibition of PARP11 inactivates intratumoral Treg cells•PARP11 inhibitor augments the efficacy of immunotherapies Basavaraja et al. demonstrate that induction of PARP11 in the intratumoral regulatory T (Treg) cells is required for their regulatory functions and contributes to the immunosuppressive tumor microenvironment. The selective inhibitor of PARP11 ITK7 inactivates tumor Treg cells and improves the efficacy of immunotherapies against tumors. View Publication

IntroductionInnate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. MethodsTo gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection,we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression,transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. ResultsWe found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover,all ILC subsets displayed a significantly higher frequency of CD69-expressing cells,indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation,activation and homeostasis. In addition,differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. DiscussionOverall,this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection. View Publication

Migrasomes are organelles that are generated by migrating cells. Here we report the key role of neutrophil-derived migrasomes in haemostasis. We found that a large number of neutrophil-derived migrasomes exist in the blood of mice and humans. Compared with neutrophil cell bodies and platelets,these migrasomes adsorb and enrich coagulation factors on the surface. Moreover,they are highly enriched with adhesion molecules,which enable them to preferentially accumulate at sites of injury,where they trigger platelet activation and clot formation. Depletion of neutrophils,or genetic reduction of the number of these migrasomes,significantly decreases platelet plug formation and impairs coagulation. These defects can be rescued by intravenous injection of purified neutrophil-derived migrasomes. Our study reveals neutrophil-derived migrasomes as a previously unrecognized essential component of the haemostasis system,which may shed light on the cause of various coagulation disorders and open therapeutic possibilities. Jiang et al. document an abundance of neutrophil-derived migrasomes in the blood of mice and humans and show that migrasomes are enriched in coagulation factors,accumulate at sites of injury and trigger platelet activation and clot formation. View Publication

Activating interferon responses with STING agonists (STINGa) is a current cancer immunotherapy strategy,and therapeutic modalities that enable tumor-targeted delivery via systemic administration could be beneficial. Here we demonstrate that tumor cell-directed STING agonist antibody-drug-conjugates (STINGa ADCs) activate STING in tumor cells and myeloid cells and induce anti-tumor innate immune responses in in vitro,in vivo (in female mice),and ex vivo tumor models. We show that the tumor cell-directed STINGa ADCs are internalized into myeloid cells by Fcγ-receptor-I in a tumor antigen-dependent manner. Systemic administration of STINGa ADCs in mice leads to STING activation in tumors,with increased anti-tumor activity and reduced serum cytokine elevations compared to a free STING agonist. Furthermore,STINGa ADCs induce type III interferons,which contribute to the anti-tumor activity by upregulating type I interferon and other key chemokines/cytokines. These findings reveal an important role for type III interferons in the anti-tumor activity elicited by STING agonism and provide rationale for the clinical development of tumor cell-directed STINGa ADCs. Activation of the STING pathway can promote anti-tumor immunity. Here the authors generate tumor cell-directed STING agonist antibody-drug conjugates that activate STING in tumor and myeloid cells,promoting anti-tumor innate immune responses in preclinical cancer models. View Publication

The progressive decline of CD8 T cell effector function—also known as terminal exhaustion—is a major contributor to immune evasion in cancer. Yet,the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here,we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis,which mediates cellular adaptations to oxidative stress,directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically,we identify PTGIR,a receptor for the circulating eicosanoid prostacyclin,as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover,lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together,these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states. One Sentence Summary:The KEAP1-NRF2 pathway is hyperactivated in terminally exhausted CD8 T cells and drives T cell dysfunction via transcriptional regulation of the prostacyclin receptor,Ptgir. View Publication