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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

ABSTRACT 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

Background: Preclinical cell tracking is enhanced with a multimodal imaging approach. Bioluminescence imaging (BLI) is a highly sensitive optical modality that relies on engineering cells to constitutively express a luciferase gene. Magnetic particle imaging (MPI) is a newer imaging modality that directly detects superparamagnetic iron oxide (SPIO) particles used to label cells. Here,we compare BLI and MPI for imaging cells in vitro and in vivo. Methods: Mouse 4T1 breast carcinoma cells were transduced to express firefly luciferase,labeled with SPIO (ProMag),and imaged as cell samples after subcutaneous injection into mice. Results: For cell samples,the BLI and MPI signals were strongly correlated with cell number. Both modalities presented limitations for imaging cells in vivo. For BLI,weak signal penetration,signal attenuation,and scattering prevented the detection of cells for mice with hair and for cells far from the tissue surface. For MPI,background signals obscured the detection of low cell numbers due to the limited dynamic range,and cell numbers could not be accurately quantified from in vivo images. Conclusions: It is important to understand the shortcomings of these imaging modalities to develop strategies to improve cellular detection sensitivity. 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

Overcoming resistance to therapy is a major challenge in castration-resistant prostate cancer (CRPC). Lineage plasticity towards a neuroendocrine phenotype enables CRPC to adapt and survive targeted therapies. However,the molecular mechanisms of epigenetic reprogramming during this process are still poorly understood. Here we show that the protein kinase PKCλ/ι-mediated phosphorylation of enhancer of zeste homolog 2 (EZH2) regulates its proteasomal degradation and maintains EZH2 as part of the canonical polycomb repressive complex (PRC2). Loss of PKCλ/ι promotes a switch during enzalutamide treatment to a non-canonical EZH2 cistrome that triggers the transcriptional activation of the translational machinery to induce a transforming growth factor β (TGFβ) resistance program. The increased reliance on protein synthesis creates a synthetic vulnerability in PKCλ/ι-deficient CRPC. The transition of androgen receptor-dependent prostate cancer to a therapy resistant cancer with neuroendocrine phenotype is an important process that remains poorly understood. Here,the authors show that PKCλ/ι-loss promotes epigenetic reprogramming resulting in a TGFβ resistance programme via transcriptional upregulation of translational machinery. 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

BackgroundSepsis survivors exhibit immune dysregulation that contributes to poor long-term outcomes. Phenotypic and functional alterations within the myeloid compartment are believed to be a contributing factor. Here we dissect the cellular and transcriptional heterogeneity of splenic CD11b+Ly6Chigh myeloid cells that are expanded in mice that survive the cecal ligation and puncture (CLP) murine model of polymicrobial sepsis to better understand the basis of immune dysregulation in sepsis survivors.MethodsSham or CLP surgeries were performed on C57BL/6J and BALB/c mice. Four weeks later splenic CD11b+Ly6Chigh cells from both groups were isolated for phenotypic (flow cytometry) and functional (phagocytosis and glycolysis) characterization and RNA was obtained for single-cell RNA-seq (scRNA-seq) and subsequent analysis.ResultsCD11b+Ly6Chigh cells from sham and CLP surviving mice exhibit phenotypic and functional differences that relate to immune function,some of which are observed in both C57BL/6J and BALB/c strains and others that are not. To dissect disease-specific and strain-specific distinctions within the myeloid compartment,scRNA-seq analysis was performed on CD11b+Ly6Chigh cells from C57BL/6J and BALB/c sham and CLP mice. Uniform Manifold Approximation and Projection from both strains identified 13 distinct clusters of sorted CD11b+Ly6Chigh cells demonstrating significant transcriptional heterogeneity and expressing gene signatures corresponding to classical-monocytes,non-classical monocytes,M1- or M2-like macrophages,dendritic-like cells,monocyte-derived dendritic-like cells,and proliferating monocytic myeloid-derived suppressor cells (M-MDSCs). Frequency plots showed that the percentages of proliferating M-MDSCs (clusters 8,11 and 12) were increased in CLP mice compared to sham mice in both strains. Pathway and UCell score analysis in CLP mice revealed that cell cycle and glycolytic pathways were upregulated in proliferating M-MDSCs in both strains. Notably,granule protease genes were upregulated in M-MDSCs from CLP mice. ScRNA-seq analyses also showed that phagocytic pathways were upregulated in multiple clusters including the classical monocyte cluster,confirming the increased phagocytic capacity in CD11b+Ly6Chigh cells from CLP mice observed in ex vivo functional assays in C57BL/6J mice.ConclusionThe splenic CD11b+Ly6Chigh myeloid populations expanded in survivors of CLP sepsis correspond to proliferating cells that have an increased metabolic demand and gene signatures consistent with M-MDSCs,a population known to have immunosuppressive capacity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-024-00970-0. View Publication

While prostaglandin E2 (PGE2) is produced in human tumor microenvironment (TME),its role therein remains poorly understood. Here,we examine this issue by comparative single-cell RNA sequencing of immune cells infiltrating human cancers and syngeneic tumors in female mice. PGE receptors EP4 and EP2 are expressed in lymphocytes and myeloid cells,and their expression is associated with the downregulation of oxidative phosphorylation (OXPHOS) and MYC targets,glycolysis and ribosomal proteins (RPs). Mechanistically,CD8+ T cells express EP4 and EP2 upon TCR activation,and PGE2 blocks IL-2-STAT5 signaling by downregulating Il2ra,which downregulates c-Myc and PGC-1 to decrease OXPHOS,glycolysis,and RPs,impairing migration,expansion,survival,and antitumor activity. Similarly,EP4 and EP2 are induced upon macrophage activation,and PGE2 downregulates c-Myc and OXPHOS in M1-like macrophages. These results suggest that PGE2-EP4/EP2 signaling impairs both adaptive and innate immunity in TME by hampering bioenergetics and ribosome biogenesis of tumor-infiltrating immune cells. Mechanisms of prostaglandin E2 (PGE2)-mediated immunosuppression in the tumor microenvironment (TME) have been previously reported. Here,the authors profile PGE2 functions in human cancer,suggesting that prostaglandin E2-mediated signaling impairs the activity of human CD8+ T cells and macrophages by altering bioenergetics and ribosome biogenesis. View Publication

Acquired immunodeficiency syndrome (AIDS) occurs when HIV depletes CD4+ helper T cells. Some patients develop AIDS slowly or not at all,and are termed long-term non-progressors (LTNP),and while mutations in the HIV-1 Viral Protein R (vpr) gene such as R77Q are associated with LTNP,mechanisms for this correlation are unclear. This study examines the induction of apoptosis,cell cycle arrest,and pro-inflammatory cytokine release in the HUT78 T cell line following infection with replication-competent wild-type strain NL4-3,the R77Q mutant,or a vpr Null mutant. Our results show a significant enhancement of apoptosis and G2 cell cycle arrest in HUT78 cells infected with R77Q,but not with WT NL4-3 or the vpr Null strain. Conversely,HUT78 cells infected with the WT virus show higher levels of necrosis. We also detected lower TNF and IL-6 release after infection with R77Q vs. WT. The apoptotic phenotype was also seen in the CEM cell line and in primary CD4+ T cells. Protein expression of the R77Q vpr variant was low compared to WT vpr,but expression levels alone cannot explain these phenotypes because the Null virus did not show apoptosis or G2 arrest. These results suggest that R77Q triggers a non-inflammatory apoptotic pathway that attenuates inflammation,possibly contributing to LTNP. View Publication

Natural adjuvants have recently garnered interest in the field of vaccinology as their immunostimulatory effects. In this study,we aimed to investigate the potential use of Peyssonnelia caulifera (PC),a marine alga,as a natural adjuvant for an inactivated split A/Puerto Rico/8/1934 H1N1 influenza vaccine (sPR8) in a murine model. We administered PC-adjuvanted vaccines to a murine model via intramuscular prime and boost vaccinations,and subsequently analyzed the induced immunological responses,particularly the production of antigen-specific IgG1 and IgG2a antibodies,memory T and B cell responses,and the protective efficacy against a lethal viral infection. PC extract significantly bolstered the vaccine efficacy,demonstrating balanced Th1/Th2 responses,increased memory T and B cell activities,and improved protection against viral infection. Notably,within 3 days post-vaccination,the PC adjuvant stimulated activation markers on dendritic cells (DCs) and macrophages at the inguinal lymph nodes (ILN),emphasizing its immunostimulatory capabilities. Furthermore,the safety profile of PC was confirmed,showing minimal local inflammation and no significant adverse effects post-vaccination. These findings contribute to our understanding of the immunomodulatory properties of natural adjuvants and suggest the promising roles of natural adjuvants in the development of more effective vaccines for infectious diseases.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-76736-9. View Publication