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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

Acute systemic inflammation critically alters the function of the immune system,often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus,systemic inflammation results in acute thymic atrophy and,consequently,impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here,we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy,while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs),while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs),where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture,indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis,TL1A,and IL-18 synergistically increased GM-CSF levels in the NTOC,which was mainly produced by thymic ILC1s. In support,TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade,revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together,our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner. View Publication

Humanized mice are limited in terms of modeling human immunity,particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated,genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells,followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system,including marginal zone B cells,germinal center B cells,follicular helper T cells and neutrophils,and develop well-formed lymph nodes and intestinal lymphoid tissue,including Peyer’s patches,and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses,entailing somatic hypermutation,class-switch recombination,and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination,THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain,with blood incretion of human cytokines,including APRIL,BAFF,TGF-β,IL-4 and IFN-γ,all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses,THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics. Humanized mice have been a valuable tool for modeling human immunology but are limited in their ability to model human antibody responses. Here the authors present their THX humanized mouse that does model human antibody responses and test its suitability for vaccination and autoimmunity studies. 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

Presepsin (P-SEP) is a specific biomarker for sepsis. Monocytes produce P-SEP by phagocytosing neutrophil extracellular traps (NETs). Herein,we investigated whether M1 macrophages (M1 MΦs) are the primary producers of P-SEP after NET phagocytosis. We co-cultured M1 MΦs and NETs from healthy participants,measured P-SEP levels in the culture medium supernatant,and detected P-SEP using western blotting. When NETs were co-cultured with M1 MΦs,the P-SEP level of the culture supernatant was high. Notably,we demonstrated,for the first time,the intracellular kinetics of P-SEP production by M1 MΦs via NET phagocytosis: M1 MΦs produced P-SEP intracellularly 15 min after NET phagocytosis and then released it extracellularly. In a sepsis mouse model,the blood NET ratio and P-SEP levels,detected using ELISA,were significantly increased (p < 0.0001). Intracellular P-SEP analysis via flow cytometry demonstrated that lung,liver,and kidney MΦs produced large amounts of P-SEP. Therefore,we identified these organs as the origin of M1 MΦs that produce P-SEP during sepsis. Our data indicate that the P-SEP level reflects the trend of NETs,suggesting that monitoring P-SEP can be used to both assess NET-induced organ damage in the lungs,liver,and kidneys during sepsis and determine treatment efficacy. View Publication

Recently developed small-molecule inhibitors of the lysosomal protease dipeptidyl peptidase 1 (DPP1),also known as cathepsin C (CatC),can suppress suppurative inflammation in vivo by blocking the processing of zymogenic (pro-) forms of neutrophil serine proteases (NSPs),including neutrophil elastase,proteinase 3,and cathepsin G. DPP1 also plays an important role in activating granzyme serine proteases that are expressed by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Therefore,it is critical to determine whether DPP1 inhibition can also cause off-target suppression of CTL/NK-cell-mediated killing of virus-infected or malignant cells. Herein,we demonstrate that the processing of human granzymes A and B,transitioning from zymogen to active proteases,is not solely dependent on DPP1. Thus,the killing of target cells by primary human CD8+ T cells,NK cells,and gene-engineered anti-CD19 CAR T cells was not blocked in vitro even after prior exposure to high concentrations of the reversible DPP1 inhibitor brensocatib. Consistent with this observation,the turnover of model granzyme A/B peptide substrates in the human CTL/NK cell lysates was not significantly reduced by brensocatib. In contrast,preincubation with brensocatib almost entirely abolished (>90%) both the cytotoxic activity of mouse CD8+ T cells and granzyme substrate turnover. Overall,our finding that the effects of DPP1 inhibition on human cytotoxic lymphocytes are attenuated in comparison to those of mice indicates that granzyme processing/activation pathways differ between mice and humans. Moreover,the in vitro data suggest that human subjects treated with reversible DPP1 inhibitors,such as brensocatib,are unlikely to experience any appreciable deficits in CTL/NK-cell-mediated immunities. 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

SummarySepsis survivors are at high risk for infection-related rehospitalization and mortality for years following the resolution of the acute septic event. These infection-causing microorganisms generally do not cause disease in immunocompetent hosts,suggesting that the post-septic immune response is compromised. Given the importance of CD4 T cells in the development of long-lasting protective immunity,we analyzed their post-septic function. Here we showed that sepsis induced chronic increased and non-specific production of IL-17 by CD4 T cells,resulting in the inability to mount an effective immune response to a secondary pneumonia challenge. Altered cell function was associated with metabolic reprogramming,characterized by mitochondrial dysfunction and increased glycolysis. This metabolic reprogramming began during the acute septic event and persisted long after sepsis had resolved. Our findings reveal cell metabolism as a potential therapeutic target. Given the critical role of cell metabolism in the physiological and pathophysiological processes of immune cells,these findings reveal a potential new therapeutic target to help mitigate sepsis survivors’ susceptibility to secondary infections. Graphical abstract Highlights•Sepsis survivors demonstrate dysfunctional CD4 T cell immunity•Sepsis induces persistent mitochondrial dysfunction in CD4 T cells•Post-septic CD4 T cells are highly glycolytic and exhibit a Th17 phenotype•Sepsis impairs the CD4 T cell recall response Physiology; Molecular biology; Immunology; Components of the immune system View Publication