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Purpose: The importance of the DNA damage response in mediating effects of radiotherapy (RT) has galvanized efforts to target this pathway with radiosensitizers. Yet early clinical trials of this approach have failed to yield a benefit in unselected populations. We hypothesized that ataxia-telangiectasia mutated (Atm)-null tumors would demonstrate genotype-specific synergy between RT and an inhibitor of the DNA damage response protein ataxia-telangiectasia and Rad3-related (ATR) kinase., Experimental Design: We investigated the synergistic potential of the ATR inhibitor (ATRi) RP-3500 and RT in two Atm-null and isogenic murine models, both in vitro and in vivo. Staining of γ-H2AX foci, characterization of the immune response via flow cytometry, and tumor rechallenge experiments were performed to elucidate the mechanism of interaction. To examine genotype specificity, we tested the interaction of ATRi and RT in a Brca1-null model. Finally, patients with advanced cancer with ATM alterations were enrolled in a phase I/II clinical trial to validate preclinical findings., Results: Synergy between RP-3500 and RT was confirmed in Atm-null lines in vitro, characterized by an accumulation of DNA double-strand breaks. In vivo, Atm-null tumor models had higher rates of durable control with RT and ATRi than controls. In contrast, there was no synergy in tumors lacking Brca1. Analysis of the immunologic response indicated that efficacy is largely mediated by cell-intrinsic mechanisms. Lastly, early results from our clinical trial showed complete responses in patients., Conclusions: Genotype-directed radiosensitization with ATRi and RT can unleash significant therapeutic benefit and could represent a novel approach to develop more effective combinatorial synthetic cytotoxic RT-based treatments. See related commentary by Schrank and Colbert, p. 5505., (©2024 American Association for Cancer Research.)

Cell-intrinsic mechanisms of immunogenicity in ovarian cancer (OC) are not well understood. Damaging mutations in the SWI/SNF chromatin remodeling complex, such as SMARCA4 (BRG1), are associated with improved response to immune checkpoint blockade; however, the mechanism by which this occurs is unclear. We found that SMARCA4 loss in OC models resulted in increased cancer cell-intrinsic immunogenicity, characterized by up-regulation of long-terminal RNA repeats, increased expression of interferon-stimulated genes, and up-regulation of antigen presentation machinery. Notably, this response was dependent on STING, MAVS, and IRF3 signaling but was independent of the type I interferon receptor. Mouse ovarian and melanoma tumors with SMARCA4 loss demonstrated increased infiltration and activation of cytotoxic T cells, NK cells, and myeloid cells in the tumor microenvironment. These results were recapitulated in BRG1 inhibitor-treated SMARCA4- proficient tumor models, suggesting that modulation of chromatin remodeling through targeting SMARCA4 may serve as a strategy to overcome cancer immune evasion.

Chlamydia muridarum (Cm) has reemerged as a moderately prevalent infectious agent in research mouse colonies. Despite its' experimental use, few studies evaluate Cm's effects on immunocompetent mice following its natural route of infection. A Cm field isolate was administered (orogastric gavage) to 8-week-old female BALB/cJ (C) mice. After confirming shedding (through 95d), these mice were cohoused with naïve C57BL/6J (B6), C, and Swiss (J:ARC[S]) mice (n=28/strain) for 30 days. Cohoused mice (n=3-6 exposed and 1-6 control/strain) were evaluated 7, 14, 21, 63, 120, and 180 days post-cohousing (DPC) via hemograms, serum biochemistry analysis, fecal qPCR, histopathology, and Cm MOMP immunohistochemistry. Immunophenotyping was performed on spleen (B6, C, S; n=6/strain) and intestines (B6; n=6) at 14 and 63 DPC. Serum cytokine concentrations were measured (B6; n=6 exposed and 2 control) at 14 and 63 DPC. All B6 mice were shedding Cm by 3 through 180 DPI. One of 3 C and 1 of 6 S mice began shedding Cm at 3 and 14 DPC, respectively, with the remaining shedding thereafter. Clinical pathology was nonremarkable. Minimal-to-moderate enterotyphlocolitis and gastrointestinal associated lymphoid tissue (GALT) hyperplasia was observed in 15 and 47 of 76 Cm-infected mice, respectively. Cm antigen was frequently detected in GALT-associated surface intestinal epithelial cells. Splenic immunophenotyping revealed increased monocytes and shifts in T cell population subsets in all strains/timepoints. Gastrointestinal immunophenotyping (B6) revealed sustained increases in total inflammatory cells and elevated cytokine production in innate lymphoid cells and effector T cells (large intestine). Elevated concentrations of pro-inflammatory cytokines were detected in the serum (B6). Results demonstrate that while clinical disease was not appreciated, 3 commonly utilized strains of mice are susceptible to chronic enteric Cm infection which may alter various immune responses. Considering the widespread use of mice to model GI disease, institutions should consider excluding Cm from their colonies., Competing Interests: Conflicts of Interest Kenneth Henderson, Cheryl Woods, and Panagiota Momtsios are employees of Charles River Laboratories, a company that produces and distributes research models and provides diagnostic services. The other authors have no competing interest to declare.

Introduction: In vivo studies of cancer biology and assessment of therapeutic efficacy are critical to advancing cancer research and ultimately improving patient outcomes. Murine cancer models have proven to be an invaluable tool in pre-clinical studies. In this context, multi-parameter flow cytometry is a powerful method for elucidating the profile of immune cells within the tumor microenvironment and/or play a role in hematological diseases. However, designing an appropriate multi-parameter panel to comprehensively profile the increasing diversity of immune cells across different murine tissues can be extremely challenging., Methods: To address this issue, we designed a panel with 13 fixed markers that define the major immune populations -referred to as the backbone panel- that can be profiled in different tissues but with the option to incorporate up to seven additional fluorochromes, including any marker specific to the study in question., Results: This backbone panel maintains its resolution across different spectral flow cytometers and organs, both hematopoietic and non-hematopoietic, as well as tumors with complex immune microenvironments., Discussion: Having a robust backbone that can be easily customized with pre-validated drop-in fluorochromes saves time and resources and brings consistency and standardization, making it a versatile solution for immuno-oncology researchers. In addition, the approach presented here can serve as a guide to develop similar types of customizable backbone panels for different research questions requiring high-parameter flow cytometry panels., Competing Interests: WX has received research support from Stemline Therapeutics. SL serves on the scientific advisory board and has equity in ORIC Pharmaceuticals, Blueprint Medicines, Mirimus Inc, Senecea Therapeutics, Faeth Therapeutics, and PMV Pharmaceuticals. RL is a scientific advisor to Imago, Mission Bio, Zentalis, Ajax, Auron, Prelude, C4 Therapeutics, and Isoplexis, and sits on the supervisory board of Qiagen. RL has also received research support from Ajax, Zentalis, and Abbvie, and has consulted for Incyte, Janssen, Novartis, and AstraZeneca. Additionally, RL has received honoraria from AstraZeneca and Kura for invited lectures, and from Gilead for grant reviews. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Longhini, Fernández-Maestre, Kennedy, Wereski, Mowla, Xiao, Lowe, Levine and Gardner.)

Human induced pluripotent stem cells (hiPSCs) were differentiated into a specific mesoderm subset characterized by KDR + CD56 + APLNR + (KNA + ) expression. KNA + cells had high clonal proliferative potential and specification into endothelial colony-forming cell (ECFCs) phenotype. KNA + cells differentiated into perfused blood vessels when implanted subcutaneously into the flank of nonobese diabetic/severe combined immunodeficient mice and when injected into the vitreous of type 2 diabetic mice ( db/db mice). Transcriptomic analysis showed that differentiation of hiPSCs derived from diabetics into KNA + cells was sufficient to change baseline differences in gene expression caused by the diabetic status and reprogram diabetic cells to a pattern similar to KNA + cells derived from nondiabetic hiPSCs. Proteomic array studies performed on retinas of db/db mice injected with either control or diabetic donor-derived KNA + cells showed correction of aberrant signaling in db/db retinas toward normal healthy retina. These data provide "proof of principle" that KNA + cells restore perfusion and correct vascular dysfunction in db/db mice.

Background: Emerging evidence of antibody-independent functions, as well as the clinical efficacy of anti-CD20 depleting therapies, helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis., Objective: To investigate whether CD19 + B cells may share expression of the serine-protease granzyme-B (GzmB), resembling classical cytotoxic CD8 + T lymphocytes, in the peripheral blood from relapsing-remitting MS (RRMS) patients., Methods: In this study, 104 RRMS patients during different treatments and 58 healthy donors were included. CD8, CD19, Runx3, and GzmB expression was assessed by flow cytometry analyses., Results: RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8 + GzmB + T lymphocytes when compared to healthy volunteers. An increase in circulating CD19 + GzmB + B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA), untreated RRMS patients, and healthy donors but not when compared to interferon-β (IFN). Moreover, regarding Runx3, the transcriptional factor classically associated with cytotoxicity in CD8 + T lymphocytes, the expression of GzmB was significantly higher in CD19 + Runx3 + -expressing B cells when compared to CD19 + Runx3 - counterparts in RRMS patients., Conclusions: CD19 + B cells may exhibit cytotoxic behavior resembling CD8 + T lymphocytes in MS patients during different treatments. In the future, monitoring "cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions., Competing Interests: LS received a research grant from Biogen and a consultation honorarium from Biogen and Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Boldrini, Marques, Quintiliano, Moraes, Stella, Longhini, Santos, Andrade, Ferrari, Damasceno, Carneiro, Brandão, Farias and Santos.)

Aims/hypothesis: Hypothalamic inflammation and sympathetic nervous system hyperactivity are hallmark features of the metabolic syndrome and type 2 diabetes. Hypothalamic inflammation may aggravate metabolic and immunological pathologies due to extensive sympathetic activation of peripheral tissues. Loss of somatostatinergic (SST) neurons may contribute to enhanced hypothalamic inflammation., Methods: The present data show that leptin receptor-deficient (db/db) mice exhibit reduced hypothalamic SST neurons, particularly in the periventricular nucleus. We model this finding, using adeno-associated virus delivery of diphtheria toxin subunit A (DTA) driven by an SST-cre system to deplete these neurons in Sst cre/gfp mice (SST-DTA)., Results: SST-DTA mice exhibit enhanced hypothalamic c-Fos expression and brain inflammation as demonstrated by microglial and astrocytic activation. Bone marrow from SST-DTA mice undergoes skewed haematopoiesis, generating excess granulocyte-monocyte progenitors and increased proinflammatory (C-C chemokine receptor type 2; CCR2 hi ) monocytes. SST-DTA mice exhibited a 'diabetic retinopathy-like' phenotype: reduced visual function by optokinetic response (0.4 vs 0.25 cycles/degree; SST-DTA vs control mice); delayed electroretinogram oscillatory potentials; and increased percentages of retinal monocytes. Finally, mesenteric visceral adipose tissue from SST-DTA mice was resistant to catecholamine-induced lipolysis, displaying 50% reduction in isoprenaline (isoproterenol)-induced lipolysis compared with control littermates. Importantly, hyperglycaemia was not observed in SST-DTA mice., Conclusions/interpretation: The isolated reduction in hypothalamic SST neurons was able to recapitulate several hallmark features of type 2 diabetes in disease-relevant tissues., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Neurofilament Light (NfL) chain levels in both cerebrospinal fluid (CSF) and serum have been correlated with the reduction of axonal damage in multiple sclerosis (MS) patients treated with Natalizumab (NTZ). However, little is known about the function of plasmacytoid cells in NTZ-treated MS patients., Objective: To evaluate CSF NfL, serum levels of soluble-HLA-G (sHLA-G), and eventual tolerogenic behavior of plasmacytoid dendritic cells (pDCs) in MS patients during NTZ treatment., Methods: CSF NfL and serum sHLA-G levels were measured using an ELISA assay, while pDCs (BDCA-2 + ) were accessed through flow cytometry analyses., Results: CSF levels of NfL were significantly reduced during NTZ treatment, while the serum levels of sHLA-G were increased. Moreover, NTZ treatment enhanced tolerogenic (HLA-G + , CD274 + , and HLA-DR + ) molecules and migratory (CCR7 + ) functions of pDCs in the peripheral blood., Conclusion: These findings suggest that NTZ stimulates the production of molecules with immunoregulatory function such as HLA-G and CD274 programmed death-ligand 1 (PD-L1) which may contribute to the reduction of axonal damage represented by the decrease of NfL levels in patients with MS., Competing Interests: LS received a research grant from BIOGEN and a consultation honorarium from BIOGEN and ROCHE. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moraes, Boldrini, Dionete, Andrade, Longhini, Santos, Lima, Silva, Dias Carneiro, Quintiliano, Ferrari, Damasceno, Pradella, Farias, Tilbery, Domingues, Senne, Fernandes, von Glehn, Brandão, Stella and Santos.)

Objective: Characterization of partial remission using the insulin dose-adjusted HbA1c (IDAA1c) ≤ 9 definition in a multiethnic Brazilian population of children and adolescents with type 1 diabetes (T1D), in addition with the determination of both Class II HLA genotype and autoantibodies., Methods: We analyzed the prevalence of partial remission in 51 new-onset T1D patients with a median time follow-up of 13 months from diagnosis. For this study, anti-GAD65, anti-IA2 and HLA class II genotyping were considered., Results: Partial remission occurred in 41.2% of T1D patients until 3 months after diagnosis, mainly in those aged 5-15 years. We have demonstrated a significant increase in the haplotypes of class II HLA DRB1*0301-DQB1*0201 in children and adolescents with a partial remission phase of the disease (42.9% vs 21.7% in non-remitters, P = .0291). This haplotype was also associated with the reduction of anti-IA2 antibodies production. Homozygote DRB1*03-DQB1*0201/DRB1*03-DQB1*0201 children had the lowest prevalence of IA-2A antibodies (P = .0402). However, this association does not correlate with the time of the remission phase., Conclusion: Although the number of patients studied was reduced, our data suggested that the association between genetics and decrease in antibody production to certain islet auto-antigen may contribute, at least in part, to the remission phase of T1D., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Rationale: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function., Objective: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function., Methods and Results: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2 -/y , Akita (type 1 diabetes mellitus), and ACE2 -/y -Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2 -/y -Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2 -/y -Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium., Conclusions: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2 -/y -Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to β oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

Macrophage activation is implicated in the development of pulmonary fibrosis by generation of profibrotic molecules. Although NADPH oxidase 4 (NOX4) is known to contribute to pulmonary fibrosis, its effects on macrophage activation and mitochondrial redox signaling are unclear. Here, we show that NOX4 is crucial for lung macrophage profibrotic polarization and fibrotic repair after asbestos exposure. NOX4 was elevated in lung macrophages from subjects with asbestosis, and mice harboring a deletion of NOX4 in lung macrophages were protected from asbestos-induced fibrosis. NOX4 promoted lung macrophage profibrotic polarization and increased production of profibrotic molecules that induce collagen deposition. Mechanistically, NOX4 further augmented mitochondrial ROS production and induced mitochondrial biogenesis. Targeting redox signaling and mitochondrial biogenesis prevented the profibrotic polarization of lung macrophages by reducing the production of profibrotic molecules. These observations provide evidence that macrophage NOX4 is a potentially novel therapeutic target to halt the development of asbestos-induced pulmonary fibrosis.

Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans., Competing Interests: The authors have declared that no competing interests exist.

Exosomes may represent an interesting antigenic pulse for new forms of anti-tumor immunotherapy. We evaluated exosomes from serum of patients with acute myeloid leukemia (AML) as an antigenic source for dendritic cells (DC) and the effects upon antitumor cytotoxicity, assessed by the percentage of specific lysis of K562 leukemic cells in co-cultures. Surprisingly, incubation of exosomes with DCs decreased lysis of K562, which may correspond to a mechanism of tumor evasion in vivo. However, when immature DCs were pulsed with exosomes purified from K562 culture supernatants, the lysis of target cells was notably enhanced, associated with a substantial increase in the expression of the maturation marker CD83. Thus, the development of vaccines using patients' exosomes would probably add no benefits to the treatment of AML; alternately, exosomes from cultured cells may represent an effective way for maturing DCs into a cytotoxic phenotype, without the immunosuppression observed with patients' exosomes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)