Scientific Resources

The integrated stress response (ISR) facilitates cellular adaptation to unfavorable conditions by reprogramming the cellular response. ISR activation was reported in neurological disorders and solid tumors; however,the function of ISR and its role as a possible therapeutic target in hematological malignancies still remain largely unexplored. Previously,we showed that the ISR is activated in chronic myeloid leukemia (CML) cells and correlates with blastic transformation and tyrosine kinase inhibitor (TKI) resistance. Moreover,the ISR was additionally activated in response to imatinib as a type of protective internal signaling. Here,we show that ISR inhibition combined with imatinib treatment sensitized and more effectively eradicated leukemic cells both in vitro and in vivo compared to treatment with single agents. The combined treatment specifically inhibited the STAT5 and RAS/RAF/MEK/ERK pathways,which are recognized as drivers of resistance. Mechanistically,this drug combination attenuated both interacting signaling networks,leading to BCR-ABL1- and ISR-dependent STAT5 activation. Consequently,leukemia engraftment in patient-derived xenograft mice bearing CD34+ TKI-resistant CML blasts carrying PTPN11 mutation responsible for hyperactivation of the RAS/RAF/MAPK and JAK/STAT5 pathways was decreased upon double treatment. This correlated with the downregulation of genes related to the RAS/RAF/MAPK,JAK/STAT5 and stress response pathways and was associated with lower expression of STAT5-target genes regulating proliferation,viability and the stress response. Collectively,these findings highlight the effect of imatinib plus ISRIB in the eradication of leukemic cells resistant to TKIs and suggest potential clinical benefits for leukemia patients with TKI resistance related to RAS/RAF/MAPK or STAT5 signaling. We propose that personalized treatment based on the genetic selection of patients carrying mutations that cause overactivation of the targeted pathways and therefore make their sensitivity to such treatment probable should be considered as a possible future direction in leukemia treatment. View Publication

Rationale: IgA can induce activation of neutrophils which are the most abundant cell type in blood,but the development of IgA as therapeutic has been confounded by its short half-life and a weak ability to recruit NK cells as effector cells. Therefore,we generated an X-shaped antibody (X-body) based on the principle of molecular self-assembly that combines the activities of both IgG and IgA,which can effectively recruit and activate NK cells,macrophages,and neutrophils to kill tumor cells. Methods: X-body was generated by using a self-assembly strategy. The affinity of the X-body with the antigen and Fc receptors was tested by surface plasmon resonance. The shape of X-body was examined using negative staining transmission electron microscopy. The tumor cell killing activity of X-body was assessed in vitro and in multiple syngeneic mouse models. To explore the mechanism of X-body,tumor-infiltrating immune cells were analyzed by single-cell RNA-seq and flow cytometry. The dependence of neutrophil,macrophage,and NK cells for the X-body efficacy was confirmed by in vivo depletion of immune cell subsets. Results: The X-body versions of rituximab and trastuzumab combined the full spectrum activity of IgG and IgA and recruited NK cells,macrophages,and neutrophils as effector cells for eradication of tumor cells. Treatment with anti-hCD20 and anti-hHER2 X-bodies leads to a greater reduction in tumor burden in tumor-bearing mice compared with the IgA or IgG counterpart,and no obvious adverse effect is observed upon X-body treatment. Moreover,the X-body has a serum half-life and drug stability comparable to IgG. Conclusions: The X-body,as a myeloid-cell-centered therapeutic strategy,holds promise for the development of more effective cancer-targeting therapies than the current state of the art. View Publication

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts,which eventually blocks signal propagation and often induces receptor internalization. However,there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2,GRK3,GRK5,GRK6) simultaneously,but we have very limited knowledge about their interplay in primary immune cells. In particular,we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type,and (b) one specific GPCR between several immune cell subsets. To address this issue,we generated mouse models of single,combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils,T cells,B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes,many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands,whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types,and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly,we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis,which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together,our findings demonstrate the complexity of GRK functions in immune cells,which go beyond GPCR desensitization in specific leukocyte types. Furthermore,they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset. View Publication

Normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study,we tested the hypothesis that NDGs from healthy donors preferentially inhibit T helper 1 (Th1) cells and investigated the myeloid-derived suppressive effect in different T-cell populations. We found that NDG-induced suppression of T-cell proliferation was contact dependent,mediated by integrin CD11b,and dependent on NDG-production of reactive oxygen species (ROS). The suppression was rapid and occurred within the first few hours of coculture. The suppression did not influence the CD8+/CD4+ ratio indicating an equal sensitivity in these populations. We further analyzed the CD4+ T helper subsets and found that NDGs induced a loss of Th1 surface marker,CD183,that was unrelated to ligand-binding to CD183. In addition,we analyzed the Th1,Th2,and Th17 cytokine production and found that all cytokine groups were suppressed when T-cells were incubated with NDGs. We therefore concluded that NDGs do not preferentially suppress Th1-cells. Instead,NDGs generally suppress Th cells and cytotoxic T-cells but specifically downregulate the Th1 marker CD183. View Publication

Facile and sensitive detection and isolation of circulating tumor cells (CTCs) was achieved using the aptamer-targeted magnetic nanoparticles (Apt-MNPs) in conjugation with a microfluidic device. Apt-MNPs were developed by the covalent attachment of anti-MUC1 aptamer to the silica-coated magnetic nanoparticles via the glutaraldehyde linkers. Apt-MNPs displayed high stability and functionality after 6 months of storage at 4 °C. The specific microfluidic device consisting of mixing,sorting and separation modules was fabricated through conventional photo- and soft-lithography by using polydimethylsiloxane. The capture efficiency of Apt-MNPs was first studied in vitro on MCF-7 and MDA-MB-231 cancer cell lines in the bulk and microfluidic platforms. The cell capture yields of more than 91% were obtained at the optimum condition after 60 minutes of exposure to 50 $\mu$g mL-1 Apt-MNPs with 10 to 106 cancer cells in different media. CTCs were also isolated efficiently from the blood samples of breast cancer patients and successfully propagated in vitro. The isolated CTCs were further characterized using immunofluorescence staining. The overall results indicated the high potential of the present method for the detection and capture of CTCs. View Publication

A breakdown in cellular homeostasis is thought to drive na{\{i}}ve T cell aging however the link between na{\"{i}}ve T cell homeostasis and aging in humans is poorly understood. To better address this we developed a lymphoid organoid system that maintains resting na{\"{i}}ve T cells for more than 2 weeks in conjunction with high CD45RA expression. Deep phenotypic characterization of na{\"{i}}ve T cells across age identified reduced CD45RA density as a hallmark of aging. A conversion from CD45RAhigh naive cells to a CD45RAlow phenotype was reproduced within our organoid system by structural breakdown but not by stromal cell aging or reduced lymphocyte density and mediated by alternative CD45 splicing. Together these data suggest that external influences within the lymph node microenvironment may cause phenotypic conversion of na{\"{i}}ve T cells in older adults." View Publication

BACKGROUND CD8+ T cells are important for protective immunity against intracellular pathogens. Excessive amounts of antigen and/or inflammatory signals often lead to the gradual deterioration of CD8+ T cell function,a state called exhaustion". However the association between CD8+ T cell exhaustion and acute respiratory distress syndrome (ARDS) has not been studied. This study was conducted to elucidate how CD8+ T cells and inhibitory receptors were related to the clinical prognosis of ARDS. METHODS A prospective observational study in an emergency department enrolled patients who were diagnosed with sepsis-associated ARDS according to the sepsis-3 criteria and Berlin definition. Peripheral blood samples were collected within 24??h post recruitment. CD8+ T cell count proliferation ratio cytokine secretion and the expression of coinhibitory receptors were assayed. RESULTS Sixty-two patients with ARDS met the inclusion criteria. CD8+ T cell counts and proliferation rates were dramatically decreased in non-surviving ARDS patients. Increasing programmed cell death 1 (PD-1) expression on the CD8+ T cell surface was seen in patients with worse organ function while an increasing level of T cell immunoglobulin mucin-3 (Tim-3) was associated with a longer duration of the shock. Kaplan-Meier analysis showed that low CD8+ T cell percentages and increased inhibitory molecule expression were significantly associated with a worse survival rate. CONCLUSIONS CD8+ T cells and coinhibitory receptors are promising independent prognostic markers of sepsis-induced ARDS and increased CD8+ T cell exhaustion is significantly correlated with poor prognosis." View Publication

BACKGROUND There is an unmet need for developing faithful animal models for preclinical evaluation of immunotherapy. The current approach to generate preclinical models for immunotherapy evaluation has been to transplant CD34+ cells from umbilical cord blood into immune-deficient mice followed by implantation of patient derived tumor cells. However,current models are associated with high tumor rejection rate secondary to the allograft vs. tumor response from human leukocyte antigen (HLA) mismatches. We herein report the first development of a novel,humanized patient-derived xenograft (PDX) model using autologous CD34+ cells from bone marrow aspirate obtained from a patient with metastatic clear cell renal cell carcinoma (mRCC) from whom a PDX had been developed. CASE DESCRIPTION This is a 68-year-old Caucasian man diagnosed with mRCC with metastasis to the liver in 2014. He was treated with sunitinib +/- AGS-003 and underwent a cytoreductive right nephrectomy,left adrenalectomy and partial liver resection. PDX was generated using resected nephrectomy specimen. After surgery,patient received multiple lines of standard of care therapy including sunitinib,axitinib,bevacizumab,everolimus and cabozantinib. While progressing on cabozantinib,he was treated with nivolumab. Seven years after initiation of nivolumab,and 4 years after stopping systemic therapy,he remains in complete remission. To generate autologous PDX model,bone marrow aspirate was performed and CD34+ hematopoietic stem/progenitor cells (HSPCs) were isolated and injected into 150 rad irradiated non-obese diabetic scid gamma null (NSG) mice. At 11 weeks post-transplant,the matched patient PDX was injected subcutaneously into the humanized mice and the mice were treated with nivolumab. CONCLUSIONS Our case represents successful therapy of nivolumab in mRCC. Furthermore,HPSCs obtained from a single bone marrow aspirate were able to reconstitute an immune system in the mice that allowed nivolumab to inhibit the tumor growth of PDX and recapitulated the durable remission observed in the patient with nivolumab. We observed the reconstitution of human T cells,B cells and natural killer (NK) cells and unlike the humanized mouse model using cord blood,our model system eliminates the tumor rejection from mis-matched HLA. Our autologous humanized renal cell carcinoma (RCC) PDX model provides an effective tool to study immunotherapy in a preclinical setting. View Publication

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here,we investigated the role of arginase 1 (ARG1) in V$\kappa$*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in EY-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of V$\kappa$*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However,arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether,these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors. View Publication

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore,depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However,identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here,we describe GS-1811,a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs,and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets,demonstrating a window for selective depletion of Tregs in the tumor. Importantly,we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy,which is dependent on Treg depleting activity,and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity. View Publication

BACKGROUND Novel therapies are urgently needed for ovarian cancer (OC),the fifth deadliest cancer in women. Preclinical work has shown that DNA methyltransferase inhibitors (DNMTis) can reverse the immunosuppressive tumor microenvironment in OC. Inhibiting DNA methyltransferases activate transcription of double-stranded (ds)RNA,including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger type I interferon (IFN) signaling,recruiting host immune cells to kill the tumor cells. Adenosine deaminase 1 (ADAR1) is induced by IFN signaling and edits mammalian dsRNA with an A-to-I nucleotide change,which is read as an A-to-G change in sequencing data. These edited dsRNAs cannot be sensed by dsRNA sensors,and thus ADAR1 inhibits the type I IFN response in a negative feedback loop. We hypothesized that decreasing ADAR1 editing would enhance the DNMTi-induced immune response. METHODS Human OC cell lines were treated in vitro with DNMTi and then RNA-sequenced to measure RNA editing. Adar1 was stably knocked down in ID8 Trp53-/- mouse OC cells. Control cells (shGFP) or shAdar1 cells were tested with mock or DNMTi treatment. Tumor-infiltrating immune cells were immunophenotyped using flow cytometry and cell culture supernatants were analyzed for secreted chemokines/cytokines. Mice were injected with syngeneic shAdar1 ID8 Trp53-/- cells and treated with tetrahydrouridine/DNMTi while given anti-interferon alpha and beta receptor 1,anti-CD8,or anti-NK1.1 antibodies every 3 days. RESULTS We show that ADAR1 edits transposable elements in human OC cell lines after DNMTi treatment in vitro. Combining ADAR1 knockdown with DNMTi significantly increases pro-inflammatory cytokine/chemokine production and sensitivity to IFN-$\beta$ compared with either perturbation alone. Furthermore,DNMTi treatment and Adar1 loss reduces tumor burden and prolongs survival in an immunocompetent mouse model of OC. Combining Adar1 loss and DNMTi elicited the most robust antitumor response and transformed the immune microenvironment with increased recruitment and activation of CD8+ T cells. CONCLUSION In summary,we showed that the survival benefit from DNMTi plus ADAR1 inhibition is dependent on type I IFN signaling. Thus,epigenetically inducing transposable element transcription combined with inhibition of RNA editing is a novel therapeutic strategy to reverse immune evasion in OC,a disease that does not respond to current immunotherapies. View Publication

BACKGROUND Systemic sclerosis (SSc) is a multiple-organ disease characterized by vascular damage,autoimmunity,and tissue fibrosis. Organ injuries such as interstitial lung diseases (ILD),resulting from inflammatory and fibrosis processes,lead to poor prognosis. Although autoantibodies are detected in the serum of patients with SSc,the mechanisms by which immune cells are involved in tissue inflammation and fibrosis is not fully understood. Recent studies have revealed carcinoembryonic antigen related cell adhesion molecule (CEACAM)-positive monocytes are involved in murine bleomycin-induced lung fibrosis. We investigated CEACAM-positive monocytes in patients with SSc to clarify the role of monocytes in the pathogenesis of SSc. METHODS The proportion of of CEACAM-positive classical monocytes in healthy controls (HCs) and patients with rheumatoid arthritis (RA) and SSc was evaluated using flow cytometry. The correlation between the proportion of CEACAM-positive monocytes and clinical parameters was analyzed in patients with SSc. Gene expression microarrays were performed in CEACAM-positive and negative monocytes in patients with SSc. Infiltration of CEACAM-positive monocytes into scleroderma skin was evaluated by immunohistochemical staining. RESULTS The proportion of CEACAM-positive classical monocytes was increased in patients with early SSc within 2 years after diagnosis,which positively correlated with ESR,serum IgG,and serum KL-6 and negatively correlated with %forced vital capacity. The percentage of CEACAM-positive monocytes decreased after immunosuppressive therapy. CEACAM6-positive cells among classical monocytes were significantly increased in patients with SSc compared with HCs and patients with rheumatoid arthritis. SSc serum induced CEACAM6 expression on monocytes from HCs. Functionally,CEACAM-positive monocytes produced higher levels of TNF-$\alpha$ and IL-1$\beta$ compared to CEACAM-negative cells and showed activation of the NF-$\kappa$B pathway. Furthermore,CEACAM6-positive monocytes infiltrated the dermis of SSc. CONCLUSIONS CEACAM-positive monocytes showed inflammatory phenotypes and may be involved in the tissue inflammation and fibrosis in early SSc. CEACAM-positive monocytes may be one of biomarkers to detect patients with progressive ILD,requiring therapeutic intervention. View Publication