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PURPOSE Chronic inflammation contributes to tumor initiation,progression,and immune escape. Neutrophils are the major component of inflammatory response and participate in the tumorigenesis process. However,compared to other immune cells in the tumor microenvironment of laryngeal squamous cell carcinoma (LSCC),neutrophils,especially the tumor-associated neutrophils (TANs),have not yet been comprehensively explored. The mechanism for regulating the crosstalk between TANs and tumor cells still remains unclear. MATERIALS AND METHODS The distribution profiles and phenotypic features of neutrophils and other inflammatory immune cell populations from a large LSCC patient cohort were systemically analyzed. Co-culturing of peripheral blood associated neutrophils (PANs) and TANs with PBMCs was performed,and the immunosuppression effect on T-cells was examined. RESULTS LSCC microenvironment is highly inflammatory with remarkable TANs infiltration,which is often associated with unfavorable prognosis and advanced clinical stage. We find that TANs in LSCC display morphologically immature and lower apoptosis,exhibit distinctively immunosuppressive phenotype of high PD-L1,and suppress CD8+ T lymphocytes proliferation and activation. We subsequently discover that PD-L1+TANs induced by LSCC-derived GM-CSF potently impair CD8+ T-cells proliferation and cytokines production function,which are partially blocked by a PD-L1-neutralizing antibody. Clinical data further support GM-CSF as an unfavorable prognostic biomarker and reveal a potential association with inflammatory immune cell infiltration,in particular neutrophils. CONCLUSION Tumor-infiltrating PD-L1+ neutrophils induced by LSCC-derived GM-CSF suppress T cell proliferation and activation in the inflammatory microenvironment of LSCC and predict unfavorable prognosis. These TANs cripple antitumor T cell immunity and promote tumor progression. Our findings provide a basis for targeting PD-L1+TANs or GM-CSF as a new immunotherapeutic strategy for LSCC. View Publication

BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers,leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet,the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing,P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B),leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings,P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover,analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects,suggesting P62 as a new immunomodulatory target for treating atherosclerosis. View Publication

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance,however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly,a misfolded P53 was described in AML blasts that do not harbor mutations in TP53,emphasizing the dynamic equilibrium between wild-type (WT) and pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC suggesting that while a pre-leukemic mutation can predispose for P53 misfolding additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53 specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention." View Publication

BACKGROUND Emerging evidence of antibody-independent functions,as well as the clinical efficacy of anti-CD20 depleting therapies,helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. OBJECTIVE To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB),resembling classical cytotoxic CD8+ T lymphocytes,in the peripheral blood from relapsing-remitting MS (RRMS) patients. METHODS In this study,104 RRMS patients during different treatments and 58 healthy donors were included. CD8,CD19,Runx3,and GzmB expression was assessed by flow cytometry analyses. RESULTS RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA),untreated RRMS patients,and healthy donors but not when compared to interferon-$\beta$ (IFN). Moreover,regarding Runx3,the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes,the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. CONCLUSIONS CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future,monitoring cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions." View Publication

CD8+ T cell longevity regulated by metabolic activity plays important roles in cancer immunotherapy. Although in vitro-polarized,transferred IL-9-secreting CD8+ Tc9 (cytotoxic T lymphocyte subset 9) cells exert greater persistence and antitumor efficacy than Tc1 cells,the underlying mechanism remains unclear. Here,we show that tumor-infiltrating Tc9 cells display significantly lower lipid peroxidation than Tc1 cells in several mouse models,which is strongly correlated with their persistence. Using RNA-sequence and functional validation,we found that Tc9 cells exhibited unique lipid metabolic programs. Tc9 cell-derived IL-9 activated STAT3,upregulated fatty acid oxidation and mitochondrial activity,and rendered Tc9 cells with reduced lipid peroxidation and resistance to tumor- or ROS-induced ferroptosis in the tumor microenvironment. IL-9 signaling deficiency,inhibiting STAT3,or fatty acid oxidation increased lipid peroxidation and ferroptosis of Tc9 cells,resulting in impaired longevity and antitumor ability. Similarly,human Tc9 cells also exhibited lower lipid peroxidation than Tc1 cells and tumor-infiltrating CD8+ T cells expressed lower IL9 and higher lipid peroxidation- and ferroptosis-related genes than circulating CD8+ T cells in patients with melanoma. This study indicates that lipid peroxidation regulates Tc9 cell longevity and antitumor effects via the IL-9/STAT3/fatty acid oxidation pathway and regulating T cell lipid peroxidation can be used to enhance T cell-based immunotherapy in human cancer. View Publication

T cell engagers represent a novel promising class of cancer-immunotherapies redirecting T cells to tumor cells and have some promising outcomes in the clinic. These molecules can be associated with a mode-of-action related risk of cytokine release syndrome (CRS) in patients. CRS is characterized by the rapid release of pro-inflammatory cytokines such as TNF-$\alpha$,IFN-$\gamma$,IL-6 and IL-1$\beta$ and immune cell activation eliciting clinical symptoms of fever,hypoxia and hypotension. In this work,we investigated the biological mechanisms triggering and amplifying cytokine release after treatment with T cell bispecific antibodies (TCBs) employing an in vitro co-culture assay of human PBMCs or total leukocytes (PBMCs + neutrophils) and corresponding target antigen-expressing cells with four different TCBs. We identified T cells as the triggers of the TCB-mediated cytokine cascade and monocytes and neutrophils as downstream amplifier cells. Furthermore,we assessed the chronology of events by neutralization of T-cell derived cytokines. For the first time,we demonstrate the contribution of neutrophils to TCB-mediated cytokine release and confirm these findings by single-cell RNA sequencing of human whole blood incubated with a B-cell depleting TCB. This work could contribute to the construction of mechanistic models of cytokine release and definition of more specific molecular and cellular biomarkers of CRS in the context of treatment with T-cell engagers. In addition,it provides insight for the elaboration of prophylactic mitigation strategies that can reduce the occurrence of CRS and increase the therapeutic index of TCBs. View Publication

Diffuse alveolar hemorrhage (DAH),although rare,is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans,although increasingly neutrophils,NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model,we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress,namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model,with accompanying reduction in IFN-driven genes and pathology. Lastly,coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH. View Publication

BACKGROUND AND AIMS Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However,the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light,immunohistochemistry,and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets,including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2),were found to be significantly enriched in hypoxia-high tumor regions. On the other hand,the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular,we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies. View Publication

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here,we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset,correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFN? and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model,we further demonstrate that pDCs,while not supporting SARS-CoV-2 replication,directly sense the virus and in response produce multiple antiviral (interferons: IFN? and IFN?1) and inflammatory (IL-6,IL-8,CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways,we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs),whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response,but not the IL-6 response,suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection. View Publication

Germinal centres (GC) are lymphoid structures in which B cells acquire affinity-enhancing somatic hypermutations (SHM),with surviving clones differentiating into memory B cells (MBCs) and long-lived bone marrow plasma cells1-5 (BMPCs). SARS-CoV-2 mRNA vaccination induces a persistent GC response that lasts for at least six months in humans6-8. The fate of responding GC B cells as well as the functional consequences of such persistence remain unknown. Here,we detected SARS-CoV-2 spike protein-specific MBCs in 42 individuals who had received two doses of the SARS-CoV-2 mRNA vaccine BNT162b2 six month earlier. Spike-specific IgG-secreting BMPCs were detected in 9 out of 11 participants. Using a combined approach of sequencing the B cell receptors of responding blood plasmablasts and MBCs,lymph node GC B cells and plasma cells and BMPCs from eight individuals and expression of the corresponding monoclonal antibodies,we tracked the evolution of 1,540 spike-specific B cell clones. On average,early blood spike-specific plasmablasts exhibited the lowest SHM frequencies. By contrast,SHM frequencies of spike-specific GC B cells increased by 3.5-fold within six months after vaccination. Spike-specific MBCs and BMPCs accumulated high levels of SHM,which corresponded with enhanced anti-spike antibody avidity in blood and enhanced affinity as well as neutralization capacity of BMPC-derived monoclonal antibodies. We report how the notable persistence of the GC reaction induced by SARS-CoV-2 mRNA vaccination in humans culminates in affinity-matured long-term antibody responses that potently neutralize the virus. View Publication

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However,it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study,we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C,which is the master regulator of Th17 cells. In contrast,inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-? and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore,inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-? by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4,inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally,treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis. View Publication

Rationale: T cell therapeutic strategy using CD19-targeting chimeric antigen receptor (CAR) is a revolutionary,novel,and successful treatment for B-cell malignancies. However,the dependency on T-cell mediated cytotoxicity restricts CAR-T therapy as a patient-specific individualized therapy with severe side effects,such as cytokine release syndrome (CRS). Whether a non-T-cell based universal cellular therapy can substitute CAR-T therapy is largely unknown. Methods: Various artificial antigen-recognizing cells were prepared to determine whether non-T-cell-derived CD19-scFv bearing effector cells could cause target cell death. A universal strategy for CRS-free cellular therapeutics was proposed,utilizing artificial antigen-recognizing cells (AARC),which can be manufactured universally and routinely as off-the-shelf" mesenchymal stromal cells (MSCs) or other types of non-autologous cells expressing anergic CARs. Results: We demonstrated that T-lymphocytic and non-lymphocytic cells could cause CD19 internalization and subsequent depletion when armed with a CD19-recognizing moiety. This CD19 antigen depletion could efficiently induce T-cell independent apoptosis in target cancer cells whose survival depends on CD19 expression suggesting that CD19 antigen depletion constitutes a crucial tumor destroying mechanism for CD19-CAR-T especially for its long-term efficacy. Conclusion: Our results uncovered an unrecognized CAR-T cytotoxicity and antigen loss mechanism and provided new insights into a shift from unique patient-specific autologous therapeutics to universal and standardized allogeneic treatment." View Publication