技术资料

Interferon regulatory factor 1 (IRF1) regulates diverse biological functions,including modulation of cellular responses involved in tumorigenesis. Genetic mutations and altered IRF1 function are associated with several cancers. Although the function of IRF1 in the immunobiology of cancer is emerging,IRF1-specific mechanisms regulating tumorigenesis and tissue homeostasis in vivo are not clear. Here,we found that mice lacking IRF1 were hypersusceptible to colorectal tumorigenesis. IRF1 functions in both the myeloid and epithelial compartments to confer protection against AOM/DSS-induced colorectal tumorigenesis. We further found that IRF1 also prevents tumorigenesis in a spontaneous mouse model of colorectal cancer. The attenuated cell death in the colons of Irf1-/- mice was due to defective pyroptosis,apoptosis,and necroptosis (PANoptosis). IRF1 does not regulate inflammation and the inflammasome in the colon. Overall,our study identified IRF1 as an upstream regulator of PANoptosis to induce cell death during colitis-associated tumorigenesis. View Publication

Personalized cancer therapy is based on a patient's tumor lineage,histopathology,expression analyses,and/or tumor DNA or RNA analysis. Here,we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care,we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care. Kodack et al. report on the development of cancer models from tumor biopsies and technologies toward a functional approach for personalized medicine. They describe the ability to reliably test drug response in patient-derived samples of mixed cell populations. In doing so,they show that patient biopsy cultures may predict patient clinical responses. View Publication

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy,but the overall metabolic properties of HSCs remain elusive. Using combined approaches,including single-cell RNA sequencing (RNA-seq),we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed,but not quiescent,HSCs relied readily on glycolysis. Notably,in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant. View Publication

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate,we performed global transcriptome profiling in treatment-na{\{i}}ve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells sensitized them to TKIs in vitro and markedly eliminated long-term repopulating LSCs and infiltrating blast cells conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity sensitizing therapy-resistant cells to TKIs. Thus miR-185 presents as a potential predictive biomarker and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients." View Publication

S100A12 is a calcium-binding protein of the S100 subfamily of myeloid-related proteins that acts as an alarmin to induce a pro-inflammatory innate immune response. It has been linked to several chronic inflammatory diseases,however its role in the common oral immunopathology periodontitis is largely unknown. Previous in vitro monoculture experiments indicate that S100A12 production decreases during monocyte differentiation stages,while the regulation within tissue is poorly defined. This study evaluated S100A12 expression in monocyte subsets,during monocyte-to-macrophage differentiation and following polarization,both in monoculture and in a tissue context,utilizing a three-dimensional co-culture oral tissue model. Further,we explored the involvement of S100A12 in periodontitis by analyzing its expression in peripheral circulation and gingival tissue,as well as in saliva. We found that S100A12 expression was higher in classical than in non-classical monocytes. S100A12 expression and protein secretion declined significantly during monocyte-to-macrophage differentiation,while polarization of monocyte-derived macrophages had no effect on either. Peripheral monocytes from periodontitis patients had higher S100A12 expression than monocytes from controls,a difference particularly observed in the intermediate and non-classical monocyte subsets. Further,monocytes from periodontitis patients displayed an increased secretion of S100A12 compared with monocytes from controls. In oral tissue cultures,monocyte differentiation resulted in increased S100A12 secretion over time,which further increased after inflammatory stimuli. Likewise,S100A12 expression was higher in gingival tissue from periodontitis patients where monocyte-derived cells exhibited higher expression of S100A12 in comparison to non-periodontitis tissue. In line with our findings,patients with severe periodontitis had significantly higher levels of S100A12 in saliva compared to non-periodontitis patients,and the levels correlated to clinical periodontal parameters. Taken together,S100A12 is predominantly secreted by monocytes rather than by monocyte-derived cells. Moreover,S100A12 is increased in inflamed tissue cultures,potentially as a result of enhanced production by monocyte-derived cells. This study implicates the involvement of S100A12 in periodontitis pathogenesis,as evidenced by increased S100A12 expression in inflamed gingival tissue,which may be due to altered circulatory monocytes in periodontitis. View Publication

BACKGROUND Leucine rich repeat kinase 2 (LRRK2) and SNCA are genetically linked to late-onset Parkinson's disease (PD). Aggregated $\alpha$-synuclein pathologically defines PD. Recent studies identified elevated LRRK2 expression in pro-inflammatory CD16+ monocytes in idiopathic PD,as well as increased phosphorylation of the LRRK2 kinase substrate Rab10 in monocytes in some LRRK2 mutation carriers. Brain-engrafting pro-inflammatory monocytes have been implicated in dopaminergic neurodegeneration in PD models. Here we examine how $\alpha$-synuclein and LRRK2 interact in monocytes and subsequent neuroinflammatory responses. METHODS Human and mouse monocytes were differentiated to distinct transcriptional states resembling macrophages,dendritic cells,or microglia,and exposed to well-characterized human or mouse $\alpha$-synuclein fibrils. LRRK2 expression and LRRK2-dependent Rab10 phosphorylation were measured with monoclonal antibodies,and myeloid cell responses to $\alpha$-synuclein fibrils in R1441C-Lrrk2 knock-in mice or G2019S-Lrrk2 BAC mice were evaluated by flow cytometry. Chemotaxis assays were performed with monocyte-derived macrophages stimulated with $\alpha$-synuclein fibrils and microglia in Boyden chambers. RESULTS $\alpha$-synuclein fibrils robustly stimulate LRRK2 and Rab10 phosphorylation in human and mouse macrophages and dendritic-like cells. In these cells,$\alpha$-synuclein fibrils stimulate LRRK2 through JAK-STAT activation and intrinsic LRRK2 kinase activity in a feed-forward pathway that upregulates phosphorylated Rab10. In contrast,LRRK2 expression and Rab10 phosphorylation are both suppressed in microglia-like cells that are otherwise highly responsive to $\alpha$-synuclein fibrils. Corroborating these results,LRRK2 expression in the brain parenchyma occurs in pro-inflammatory monocytes infiltrating from the periphery,distinct from brain-resident microglia. Mice expressing pathogenic LRRK2 mutations G2019S or R1441C have increased numbers of infiltrating pro-inflammatory monocytes in acute response to $\alpha$-synuclein fibrils. In primary cultured macrophages,LRRK2 kinase inhibition dampens $\alpha$-synuclein fibril and microglia-stimulated chemotaxis. CONCLUSIONS Pathologic $\alpha$-synuclein activates LRRK2 expression and kinase activity in monocytes and induces their recruitment to the brain. These results predict that LRRK2 kinase inhibition may attenuate damaging pro-inflammatory monocyte responses in the brain. View Publication

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent antitumor effects in B-cell malignancies including acute lymphoblastic leukemia (ALL),but antigen loss remains the major cause of treatment failure. To mitigate antigen escape and potentially improve the durability of remission,we developed a dual-targeting approach using an optimized,bispecific CAR construct that targets both CD19 and BAFF-R. CD19/BAFF-R dual CAR T cells exhibited antigen-specific cytokine release,degranulation,and cytotoxicity against both CD19-/- and BAFF-R-/- variant human ALL cells in vitro. Immunodeficient mice engrafted with mixed CD19-/- and BAFF-R-/- variant ALL cells and treated with a single dose of CD19/BAFF-R dual CAR T cells experienced complete eradication of both CD19-/- and BAFF-R-/- ALL variants,whereas mice treated with monospecific CD19 or BAFF-R CAR T cells succumbed to outgrowths of CD19-/BAFF-R+ or CD19+/BAFF-R- tumors,respectively. Further,CD19/BAFF-R dual CAR T cells showed prolonged in vivo persistence,raising the possibility that these cells may have the potential to promote durable remissions. Together,our data support clinical translation of BAFF-R/CD19 dual CAR T cells to treat ALL. View Publication

Solid tumor cells have an altered metabolism that can protect them from cytotoxic lymphocytes. The anti-diabetic drug metformin modifies tumor cell metabolism and several clinical trials are testing its effectiveness for the treatment of solid cancers. The use of metformin in hematologic cancers has received much less attention,although allogeneic cytotoxic lymphocytes are very effective against these tumors. We show here that metformin induces expression of Natural Killer G2-D (NKG2D) ligands (NKG2DL) and intercellular adhesion molecule-1 (ICAM-1),a ligand of the lymphocyte function-associated antigen 1 (LFA-1). This leads to enhance sensitivity to cytotoxic lymphocytes. Overexpression of anti-apoptotic Bcl-2 family members decrease both metformin effects. The sensitization to activated cytotoxic lymphocytes is mainly mediated by the increase on ICAM-1 levels,which favors cytotoxic lymphocytes binding to tumor cells. Finally,metformin decreases the growth of human hematological tumor cells in xenograft models,mainly in presence of monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy. View Publication

Intronic single-nucleotide polymorphisms (SNPs) in the ANKRD55 gene are associated with the risk for multiple sclerosis (MS) and rheumatoid arthritis by genome-wide association studies (GWAS). The risk alleles have been linked to higher expression levels of ANKRD55 and the neighboring IL6ST (gp130) gene in CD4+ T lymphocytes of healthy controls. The biological function of ANKRD55,its role in the immune system,and cellular sources of expression other than lymphocytes remain uncharacterized. Here,we show that monocytes gain capacity to express ANKRD55 during differentiation in immature monocyte-derived dendritic cells (moDCs) in the presence of interleukin (IL)-4/granulocyte-macrophage colony-stimulating factor (GM-CSF). ANKRD55 expression levels are further enhanced by retinoic acid agonist AM580 but downregulated following maturation with interferon (IFN)-$\gamma$ and lipopolysaccharide (LPS). ANKRD55 was detected in the nucleus of moDC in nuclear speckles. We also analyzed the adjacent IL6ST,IL31RA,and SLC38A9 genes. Of note,in healthy controls,MS risk SNP genotype influenced ANKRD55 and IL6ST expression in immature moDC in opposite directions to that in CD4+ T cells. This effect was stronger for a partially correlated SNP,rs13186299,that is located,similar to the main MS risk SNPs,in an ANKRD55 intron. Upon analysis in MS patients,the main GWAS MS risk SNP rs7731626 was associated with ANKRD55 expression levels in CD4+ T cells. MoDC-specific ANKRD55 and IL6ST mRNA levels showed significant differences according to the clinical form of the disease,but,in contrast to healthy controls,were not influenced by genotype. We also measured serum sgp130 levels,which were found to be higher in homozygotes of the protective allele of rs7731626. Our study characterizes ANKRD55 expression in moDC and indicates monocyte-to-dendritic cell (Mo-DC) differentiation as a process potentially influenced by MS risk SNPs. View Publication

Heterologous immunity,when the memory T cell response elicited by one pathogen recognizes another pathogen,has been offered as a contributing factor for the high variability in coronavirus disease 2019 (COVID-19) severity outcomes. Here we demonstrate that sensitization with bacterial peptides can induce heterologous immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) derived peptides and that vaccination with the SARS-CoV-2 spike protein can induce heterologous immunity to bacterial peptides. Using in silico prediction methods,we identified 6 bacterial peptides with sequence homology to either the spike protein or non-structural protein 3 (NSP3) of SARS-CoV-2. Notwithstanding the effects of bystander activation,in vitro co-cultures showed that all individuals tested (n=18) developed heterologous immunity to SARS-CoV-2 peptides when sensitized with the identified bacterial peptides. T cell recall responses measured included cytokine production (IFN-$\gamma$,TNF,IL-2),activation (CD69) and proliferation (CellTrace). As an extension of the principle of heterologous immunity between bacterial pathogens and COVID-19,we tracked donor responses before and after SARS-CoV-2 vaccination and measured the cross-reactive T cell responses to bacterial peptides with similar sequence homology to the spike protein. We found that SARS-CoV-2 vaccination could induce heterologous immunity to bacterial peptides. These findings provide a mechanism for heterologous T cell immunity between common bacterial pathogens and SARS-CoV-2,which may explain the high variance in COVID-19 outcomes from asymptomatic to severe. We also demonstrate proof-of-concept that SARS-CoV-2 vaccination can induce heterologous immunity to pathogenic bacteria derived peptides. 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

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