技术资料

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

BACKGROUND Thrombocytopenia leading to life-threatening excessive bleeding can be treated by platelet transfusion. Currently,such treatments are totally dependent on donor-derived platelets. To support future applications in the use of in vitro-derived platelets,we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival,megakaryocyte ploidy and TPO-mediated activation in mice,but its role in human megakaryocytes and platelets has not been examined. METHODS To analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation,conditional Lyn-deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn-deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC-derived megakaryocytes. RESULTS Temporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes,as measured by the rate and extent of appearance of CD41+ CD42+ cells. Lyn-deficient megakaryocytes also demonstrated improved hemostatic effectiveness,as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally,Lyn-deficient megakaryocytes produced increased numbers of platelet-like particles (PLP) in vitro. CONCLUSIONS Conditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro-derived platelets may contribute to both their efficiency of production and their ability to support hemostasis. View Publication

Immunogenicity following inactivated SARS-CoV-2 vaccination among solid organ transplant recipients has not been assessed. Seventy-five patients (37 kidney transplant [KT] recipients and 38 healthy controls) received two doses,at 4-week intervals,of an inactivated whole-virus SARS-CoV-2 vaccine. SARS-CoV-2-specific humoral (HMI) and cell-mediated immunity (CMI) were measured before,4 weeks post-first dose,and 2 weeks post-second dose. The median (IQR) age of KT recipients was 50 (42-54) years and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median (IQR) time since transplant was 4.5 (2-9.5) years. Among 35 KT patients,the median (IQR) of anti-RBD IgG level measured by CLIA after vaccination was not different from baseline,but was significantly lower than in controls (2.4 [1.1-3.7] vs. 1742.0 [747.7-3783.0] AU/ml,p < .01) as well as percentages of neutralizing antibody inhibition measured by surrogate viral neutralization test (0 [0-0] vs. 71.2 [56.8-92.2]%,p < .01). However,the median (IQR) of SARS-CoV-2 mixed peptides-specific T cell responses measured by ELISpot was significantly increased compared with baseline (30 [4-120] vs. 12 [0-56] T cells/106  PBMCs,p = .02) and not different from the controls. Our findings revealed weak HMI but comparable CMI responses in fully vaccinated KT recipients receiving inactivated SARS-CoV-2 vaccination compared to immunocompetent individuals (Thai Clinical Trials Registry,TCTR20210226002). View Publication

Although critical for host defense,innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS,compared to ARDS from other respiratory pathogens,is unclear. Moreover,mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics,we discovered that,compared to bacterial ARDS,COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils,altered IFNactive neutrophils,downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion,potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19. View Publication

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells,suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells,but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration,we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells,we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1,which led to a high-affinity LFA-1 conformation,decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME,through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1,represent a new strategy to improve the efficiency of immunotherapy for cancer treatment. View Publication

BACKGROUND Monocytes are thought to be involved in venous thrombosis but the role of individual monocyte subpopulations on thrombus formation,clot inflammation,and degradation is an important unresolved issue. We investigate the role of inflammatory Ly6Chi monocytes in deep vein thrombosis and their potential therapeutic impact. METHODS Frequencies and compositions of blood monocytes were analyzed by flow cytometry in CCR2-/- (C-C chemokine receptor type 2) and wild-type mice of different ages and after treatment with the NR4A1 (nuclear receptor group 4 family A member 1,Nur77) agonist CnsB (cytosporone B). TF (tissue factor) sufficient and deficient Ly6Chi monocytes were adoptively transferred into aged CCR2-/- mice. Thrombus formation and size were followed by ultrasound over a 3-week period after surgical reduction of blood flow (stenosis) in the inferior vena cava. RESULTS Reduced numbers of peripheral monocytes in aged (>30 w) CCR2-/- mice are accompanied by reduced thrombus formation after inferior vena cava ligation. Reducing the number of inflammatory Ly6Chi monocytes in wild-type mice by CsnB treatment before ligation,similarly suspends clotting,while later treatment (d1 or d4) reduces thrombus growth and accelerates resolution. We describe how changes in inflammatory monocyte numbers affect the gradual differentiation of monocytes in thrombi and show that only tissue factor-competent Ly6Chi monocytes restore thrombosis in aged CCR2-/- mice. CONCLUSIONS We conclude that the number of inflammatory Ly6Chi monocytes controls deep vein thrombosis formation,growth,and resolution and can be therapeutically manipulated with a NR4A1 agonist at all disease stages. 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