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Simple SummaryAcute myeloid leukemia (AML) is a challenging blood cancer to treat,with only about 24% of patients surviving for 5 years after diagnosis. A key challenge is that AML cells stick to normal cells in the bone marrow (BM),and these BM cells protect them from chemotherapy. The aim of this project is to find drugs that disrupt AML cell adherence to BM cells and release them into the blood,where chemotherapy will be more effective. To achieve this,we have created a model of adhesive BM and shown that it mimics the drug resistance seen clinically. We have used the model as a testing platform for drugs that disrupt AML cell adhesion. We have shown that the combined targeting of CD44 and FAK,using anti-CD44 and the clinical-grade FAK inhibitor defactinib,inhibits the adhesion of the most primitive AML cells that are associated with drug resistance and disease relapse. AbstractBackground/Objectives: Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy,they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So,the disruption of the BMME,releasing tumor cells into the peripheral circulation,has therapeutic potential. Methods: Using both primary donor AML cells and cell lines,we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance. Results: We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However,some AML cells remained adhered,and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor,defactinib. Importantly,we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures. Conclusions: Taken together,we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody,the combination of this agent with defactinib could be rapidly tested in AML clinical trials. View Publication

Chromosome instability is a prevalent vulnerability of cancer cells that has yet to be fully exploited therapeutically. To identify genes uniquely essential to chromosomally unstable cells,we mined the Cancer Dependency Map for genes essential in tumor cells with high levels of copy number aberrations. We identify and validate KIF18A,a mitotic kinesin,as a vulnerability of chromosomally unstable cancer cells. Knockdown of KIF18A leads to mitotic defects and reduction of tumor growth. Screening of a chemical library for inhibitors of KIF18A enzymatic activity identified a hit that was optimized to yield VLS-1272,which is orally bioavailable,potent,ATP non-competitive,microtubule-dependent,and highly selective for KIF18A versus other kinesins. Inhibition of KIF18A’s ATPase activity prevents KIF18A translocation across the mitotic spindle,resulting in chromosome congression defects,mitotic cell accumulation,and cell death. Profiling VLS-1272 across >100 cancer cell lines demonstrates that the specificity towards cancer cells with chromosome instability differentiates KIF18A inhibition from other clinically tested anti-mitotic drugs. Treatment of tumor xenografts with VLS-1272 results in mitotic defects leading to substantial,dose-dependent inhibition of tumor growth. The strong biological rationale,robust preclinical data,and optimized compound properties enable the clinical development of a KIF18A inhibitor in cancers with high chromosomal instability. Chromosomal instability occurs frequently in cancer,making it an attractive therapeutic target. Here,the authors identify KIF18A as a targetable vulnerability of cancer cells with chromosomal instability and target this using VLS-1272,a selective KIF18A inhibitor. View Publication

Cells die by necrosis due to excessive chemical or thermal stress,leading to plasma membrane rupture,release of intracellular components and severe inflammation. The clearance of necrotic cell debris is crucial for tissue recovery and injury resolution,however,the underlying mechanisms are still poorly understood,especially in vivo. This study examined the role of complement proteins in promoting clearance of necrotic cell debris by leukocytes and their influence on liver regeneration. We found that independently of the type of necrotic liver injury,either acetaminophen (APAP) overdose or thermal injury,complement proteins C1q and (i)C3b were deposited specifically on necrotic lesions via the activation of the classical pathway. Importantly,C3 deficiency led to a significant accumulation of necrotic debris and impairment of liver recovery in mice,which was attributed to decreased phagocytosis of debris by recruited neutrophils in vivo. Monocytes and macrophages also took part in debris clearance,although the necessity of C3 and CD11b was dependent on the specific type of necrotic liver injury. Using human neutrophils,we showed that absence of C3 or C1q caused a reduction in the volume of necrotic debris that is phagocytosed,indicating that complement promotes effective debris uptake in mice and humans. Moreover,internalization of opsonized debris induced the expression of pro-resolving genes in a C3-dependent manner,supporting the notion that debris clearance favors the resolution of inflammation. In summary,complement activation at injury sites is a pivotal event for necrotic debris clearance by phagocytes and determinant for efficient recovery from tissue injury. Graphical Abstract View Publication

Although chimeric antigen receptor (CAR) T cells are effective against B-lineage malignancies,post-CAR relapse is common,and efficacy in other tumors is limited. These challenges may be addressed through rational manipulations to control CAR T cell function. Here we examine the impact of cognate T cell antigen experience on subsequent CD8+ CAR T cell activity. Prior antigen encounter resulted in superior effector function against leukemia expressing low target antigen density at the expense of reduced proliferative capacity and susceptibility to dysfunction at limiting CAR doses. Distinctive temporal transcriptomic and epigenetic profiles in naive-derived and memory-derived CAR T cells identified RUNX family transcription factors as potential targets to augment the function of naive-derived CD8+ CAR T cells. RUNX2 overexpression enhanced antitumor efficacy of mouse CAR T cells,dependent on prior cell state,and heightened human CAR T cell functions. Our data demonstrate that prior antigen experience of CAR T cells determines functional attributes and amenability to transcription factor-mediated functional enhancement. Here,Fry and colleagues examine the impact of antigen experience on subsequent CD8+ CAR T cell activity during the antileukemia response and show that RUNX2 overexpression enhances antitumor activity of these cells. View Publication

Donor-specific antibodies (DSAs) targeting mismatched human leukocyte antigen (HLA) molecules are one of the principal threats to long-term graft survival in solid organ transplantation. However,many patients with long-term circulating DSAs do not manifest rejection responses,suggesting a degree of heterogeneity in their pathogenicity and related functional activity. Immunologic risk stratification of transplant recipients is complicated by challenges intrinsic to defining alloantibody responses that are potentially pathogenic versus those that are not. Thus,a comprehensive understanding of how human alloantibodies target and interact with donor HLA molecules is vital for the development and evaluation of new strategies aimed at reducing antibody-mediated rejection responses. In this study,we employ hydrogen–deuterium exchange–mass spectrometry (HDX–MS),molecular dynamics (MD) simulations,and advanced biochemical and biophysical methodologies to thoroughly characterize a panel of human monoclonal alloantibodies and define the influence of Fc-region biology,antibody binding kinetics,target antigen density,and structural characteristics on their ability to potentiate the forms of immune effector mechanisms that are strongly implicated in transplant rejection. Our findings have significant implications for our understanding of the key biological determinants that underlie the pathogenicity or lack thereof of human alloantibodies. View Publication

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by an overactive immune response,particularly involving excessive production of type I interferons. This overproduction is driven by the phosphorylation of IRF7,a crucial factor in interferon gene activation. Current treatments for SLE are often not very effective and can have serious side effects.MethodsOur study introduces clobenpropit,a histamine analogue,as a potential new therapy targeting the CXCR4 receptor to reduce IRF7 phosphorylation and subsequent interferon production. We employed various laboratory techniques to investigate how clobenpropit interacts with CXCR4 and its effects on immune cells from healthy individuals and SLE patients.ResultsClobenpropit binds effectively to CXCR4,significantly inhibiting IRF7 phosphorylation and reducing interferon production. Additionally,clobenpropit lowered levels of pro-inflammatory cytokines in a mouse model of lupus,demonstrating efficacy comparable to the standard treatment,prednisolone.DiscussionThese results suggest that clobenpropit could be a promising new treatment for SLE,offering a targeted approach with potential advantages over current therapies. View Publication

Achieving the precise targeting of lentiviral vectors (LVs) to specific cell populations is crucial for effective gene therapy,particularly in cancer treatment where the modulation of the tumor microenvironment can enhance anti-tumor immunity. Programmed cell death protein 1 (PD-1) is overexpressed on activated tumor-infiltrating T lymphocytes,including regulatory T cells that suppress immune responses via FOXP3 expression. We developed PD1-targeted LVs by incorporating the anti-PD1 nanobody nb102c3 into receptor-blinded measles virus H and VSV-Gmut glycoproteins. We assessed the retargeting potential of nb102c3 and evaluated transduction efficiency in activated T lymphocytes. FOXP3 expression was suppressed using shRNA delivered by these LVs. Our results demonstrate that PD1-targeted LVs exerted pronounced tropism towards PD1+ cells,enabling the selective transduction of activated T lymphocytes while sparing naive T cells. The suppression of FOXP3 in Tregs reduced their suppressive activity. PD1-targeted glycoprotein H provided greater specificity,whereas the VSV-Gmut,together with the anti-PD1 pseudoreceptor,achieved higher viral titers but was less selective. Our study demonstrates that PD1-targeted LVs may offer a novel strategy to modulate immune responses within the tumor microenvironment with the potential for developing new therapeutic strategies aimed at enhancing anti-tumor immunity. View Publication

AbstractChlamydia genital infection caused by Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. A mouse model has been developed in our laboratory to better understand the effect of cold-induced stress on chlamydia genital infection and immune response. However,the stress mechanism affecting the host response to Chlamydia muridarum genital infection remains unclear. Here,we demonstrate a role for the beta2-adrenergic receptor (β2-AR),which binds noradrenaline and modulates the immune response against chlamydia genital infection in a mouse model. A successful β2-AR homozygous knockout (KO) mouse model was used to study the infection and analyze the immune response. Our data show that stressed mice lacking the β2-AR are less susceptible to C. muridarum genital infection than controls. A correlation was obtained between lower organ load and higher interferon-gamma production by CD4+ and CD8+ cells of the KO mice. Furthermore,exposure of CD4+ T cells to noradrenaline alters the production of cytokines in mice during C. muridarum genital infection. This study suggests that the blockade of β2-AR signaling could be used to increase resistance to chlamydia genital infection. We value the β2-AR KO as a viable model that can provide reproducible results in investigating medical research,including chlamydia genital infection. Deficiency in a receptor leads to a reduced disease of chlamydia in a mouse model. View Publication

IntroductionSystemic sclerosis (SSc) is a complex autoimmune connective tissue disease characterized by microvascular damage,immune system reactivity and progressive fibrosis of skin and internal organs. Interstitial lung disease is the leading cause of death for SSc patients (SSc-ILD),and the process of lung fibrosis involves also circulating monocytes and alveolar macrophages.MethodsCurrent study aimed to identify monocyte/macrophage phenotypes in lung and peripheral blood of SSc-ILD patients by immunostaining and flow cytometry,respectively. Single immunostaining was performed using primary antibodies against CD68 (pan-macrophage marker),CD80,CD86,TLR4 (M1 markers),CD163,CD204,and CD206 (M2 markers). Flow cytometry analysis included the evaluation of CD45,CD14,CD16 (monocyte lineage),CD1c (dendritic lineage),together with M1 and M2 activation markers on circulating monocytes. Protein synthesis of TLR4 and M2 markers was also investigated in cultured monocytes-derived macrophages (MDMs) from SSc-ILD patients by Western Blotting.ResultsLung samples were obtained from 9 SSc-ILD patients (50 ± 9 years old) and 5 control non-SSc patients without lung fibrosis (58 ± 23 years old). Alveolar macrophages (CD68+ cells) showed a significantly higher positivity of M1 and M2 markers in SSc-ILD lung samples than in controls (p<0.05 for CD80,p<0.01 for CD86,p<0.001 for CD68,p<0.0001 for TLR4,CD163,CD204 and CD206). In CD68 positive areas of SSc-ILD samples,a significantly higher percentage of TLR4,CD163,CD204,and CD206 positive cells was observed compared to CD80 and CD86 positive cells (p<0.001 in both cases),suggesting the possible presence of hybrid TLR4+M2 macrophages (CD68+CD80-CD86-TLR4+CD163+CD204+CD206+cells) in SSc-ILD samples. A second cohort of 26 SSc-ILD patients (63 ± 14 years old) and 14 SSc patients without ILD (63 ± 19 years old) was recruited for flow cytometry analysis of circulating monocytes. Again,a significantly higher percentage of hybrid TLR4+M2 monocytes (CD1c-CD80-TLR4+CD163+CD204+CD206+cells) was found in SSc-ILD positive than SSc-ILD negative patients (p<0.05). Moreover,the protein synthesis of TLR4 and M2 markers was also found higher in cultured MDMs obtained from SSc-ILD patients than in MDMs from SSc patients without ILD and this increase was significantly higher for CD163 (p<0.05) and CD206 (p<0.01).ConclusionsThe presence of hybrid TLR4+M2 markers on both circulating monocytes and resident lung macrophages in SSc-ILD patients,is reported for the first time. Therefore,the detection of circulating hybrid TLR4+M2 monocytes in SSc-ILD might represent a further potential biomarker of progressive organ fibrosis,to be searched in blood samples of SSc patients. View Publication

T cell therapies,such as chimeric antigen receptor (CAR) T cells and T cell receptor (TCR) T cells,are a growing class of anti-cancer treatments. However,expansion to novel indications and beyond last-line treatment requires engineering cells' dynamic population behaviors. Here we develop the tools for cellular behavior analysis of T cells from live-cell imaging,a common and inexpensive experimental setup used to evaluate engineered T cells. We first develop a state-of-the-art segmentation and tracking pipeline,Caliban,based on human-in-the-loop deep learning. We then build the Occident pipeline to collect a catalog of phenotypes that characterize cell populations,morphology,movement,and interactions in co-cultures of modified T cells and antigen-presenting tumor cells. We use Caliban and Occident to interrogate how interactions between T cells and cancer cells differ when beneficial knock-outs of RASA2 and CUL5 are introduced into TCR T cells. We apply spatiotemporal models to quantify T cell recruitment and proliferation after interactions with cancer cells. We discover that,compared to a safe harbor knockout control,RASA2 knockout T cells have longer interaction times with cancer cells leading to greater T cell activation and killing efficacy,while CUL5 knockout T cells have increased proliferation rates leading to greater numbers of T cells for hunting. Together,segmentation and tracking from Caliban and phenotype quantification from Occident enable cellular behavior analysis to better engineer T cell therapies for improved cancer treatment. View Publication

Galectin-1 is implicated in several pro-tumourigenic mechanisms and is considered immune-suppressive. The pharmacological inhibition of galectin-1 may be beneficial in cancers in which galectin-1 is overexpressed and driving cancer progression. This study aimed to further characterise the immunosuppressive cytokines influenced by galectin-1 in in vitro immune cell cultures and an in vivo inflammatory model using a recently discovered selective inhibitor of galectin-1,GB1908. To enable a translational approach and link mouse and human pharmacology,anti-CD3/anti-CD28 stimulated T cells cultured from human whole blood and mouse spleens were compared. For in vivo studies of T cell-mediated inflammation,the concanavalin-A (Con-A) mouse model was used to induce a T lymphocyte-driven acute liver injury phenotype. The inhibition of galectin-1 with GB1908 reduced IL-17A,IFNγ and TNFα in a concentration-dependent manner in both mouse and human T cells in vitro. The immunosuppressive cytokines measured in Con-A-treated mice were all upregulated compared to naïve mice. Subsequently,mice treated with GB1908 demonstrated a significant reduction in IL-17A,IFNγ,IL-6 and TNFα compared to vehicle-treated mice. In conclusion,galectin-1 induced the production of several important immune-suppressive cytokines from T cells in vitro and in vivo. This result suggests that,in the context of cancer therapy,a selective galectin-1 could be a viable approach as a monotherapy,or in combination with chemotherapeutic agents and/or checkpoint inhibitors,to enhance the numbers and activity of cytotoxic T cells in the tumour microenvironment of high galectin-1 expressing cancers. View Publication

BackgroundThe characteristics of the tumor immunosuppressive microenvironment represent a major challenge that limits the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy,a tumor ablation system developed in our laboratory,promotes macrophage M1-type polarization and the complete maturation of DCs to remodel the immunosuppressive environment. However,the cells that respond promptly to CTT have not yet been identified. CTT can cause extensive cell death and the release of danger-associated molecular patterns and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. Therefore,we hypothesized that neutrophils are the initial cells that respond to CTT and are involved in the subsequent establishment of antitumor immunity.MethodsIn this study,we examined the kinetics of neutrophil recruitment after CTT via flow cytometry and immunofluorescence staining and explored the effect of neutrophils on the establishment of systemic antitumor immunity by in vivo neutrophil depletion and in vitro co-culture assays.ResultsWe found that CTT led to a rapid and strong proinflammatory neutrophil response,which was essential for the long-term survival of mice. CTT-induced neutrophils promoted the activation of monocytes via reactive oxygen species and further upregulated the expression of IFN-γ and cytotoxic molecules in T and NK cells. Adoptive neutrophil transfer further enhanced the antitumor efficacy of CTT in tumor models of spontaneous and experimental metastasis.ConclusionThese results reveal the important role of neutrophil‒monocyte interactions in the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity. View Publication