Vascular wall homeostasis depends on coordinated interactions between endothelial cells, smooth muscle cells, extracellular matrix, and circulating immune cells. Non-classical monocytes (Ly6C low nMo) mediate immune surveillance of the vascular wall and are essential for maintaining vessel integrity during physiological and pathological stress. An orphan nuclear receptor Nr4a1 is a lineage-defining regulator of nMo survival and patrolling. We have previously shown that the E2 sub-domain within the Nr4a1 super-enhancer region is essential for regulating Ly6C low nMo functions. Here, we hypothesized that disruption of Nr4a1-E2-dependent nMo surveillance compromises vascular structure and adaptive remodeling under stress. To test this, we examined vascular homeostasis in Nr4a1-E2-deficient mice across ischemic and tumor-associated conditions. Loss of Nr4a1-E2 resulted in a marked reduction of circulating nMo, increased systemic inflammatory and soluble adhesion marker profiles in serum, including IL1a ( p-val=0.02 ), ICAM-1 ( p-val=0.008 ), E-Selectin ( p-val=0.009 ), PECAM-1 ( p-val=0.0003 ), and L-Selectin ( p-val=0.006 ) in Nr4a1-E2-KO compared to wild-type mice. At baseline, mason’s trichrome analysis of the aortic wall revealed a significant reduction in fibrosis in E2-deficient mice, indicating altered extracellular matrix organization. E2-deficient mice also exhibited significantly impaired ( p-val=0.02 ) perfusion recovery compared with wild-type, as assessed by Doppler imaging in hindlimb ischemic model. E2-deficient mice showed increased endothelium-independent vasorelaxation in mesenteric arteries, indicating altered smooth muscle function. Under B16F10 tumor-induced vascular stress, bulk RNA sequencing of flow-sorted CD31 + lung endothelial cells revealed extensive transcriptional reprogramming, characterized by altered expression of genes governing vascular tone. Pathway analysis identified disruptions in integrin-mediated cell interactions, collagen biosynthesis and degradation, and ECM organization processes critical for vascular stability and stress adaptation. Together, our findings demonstrate that loss of Nr4a1-E2-dependent nMo surveillance establishes a structurally fragile and functionally dysregulated vascular homeostasis that is poorly adapted to both ischemic and tumor-associated stress.

Oncolytic HSV-1 (oHSV) treatment induces Notch signaling and myelosuppression in the tumor microenvironment (TME) of preclinical cancer models. Clinically, the Notch ligand JAG1 was upregulated in recurrent high-grade glioma patients treated with the oHSV CAN-3110 and correlated with poor prognosis. To better understand endogenous JAG1-mediated signaling in glioma cells and tumor-associated macrophages (TAMs), we engineered a JAG1-antagonizing oHSV (OD-0J1) and interrogated its impact on cancer and myeloid cells in the tumor microenvironment. OD-0J1 antagonized JAG1-mediated Notch signaling and suppressed tumor growth in athymic nude and humanized mice, an effect reliant on Notch signaling in tumor cells. Kinome profiling revealed that OD-0J1 treatment suppressed CDK1, resulting in activation of the G2/M cell cycle checkpoint. Cell cycle arrest led to senescence and correlated with increased reactive oxygen species, p62 and autophagosome accumulation, and senescence-associated β-galactosidase activity. OD-0J1-induced senescence resulted in increased production of inflammatory chemokines and DAMPs, such as IL-1β, HMGB1, and extracellular ATP. Co-culturing macrophages with OD-0J1-infected tumor cells led to stimulation of chemotactic and pro-inflammatory pathways, as well as increased Fc receptor activation. Single-cell RNA sequencing and flow cytometric analysis of F4/80+ cells isolated from tumors showed a shift from tumor-supporting TAMs to inflammatory macrophages upon OD-0J1 treatment. Heightened EGFR activation in senescent cells was a mechanism to escape cell death, which created a unique opportunity for cetuximab as a senolytic agent. Combination therapy reduced EGFR signaling and induced macrophage-mediated antibody-dependent cellular cytotoxicity, thereby increasing the anti-tumor therapeutic efficacy of OD-0J1.

Cytotoxic chemotherapy primarily targets rapidly proliferating cancer cells but also depletes normal myeloid cells. The resulting cell loss triggers reactive myelopoiesis, a compensatory process in which hematopoietic stem and progenitor cells in the bone marrow (BM) regenerate myeloid lineages. We previously showed that the alkylating agent cyclophosphamide (CTX) induces myelopoiesis, leading to the expansion of immunosuppressive monocytes in mice. However, the molecular features and clinical relevance of these cells remain poorly understood. Here, we report the emergence of immunosuppressive monocytes in the peripheral blood of lymphoma patients receiving CTX-containing chemotherapy. To gain mechanistic insight into CTX-induced myelopoiesis, we performed single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) on BM monocytes from CTX-treated mice. These analyses revealed a heterogeneous monocyte population and demonstrated that CTX skews myelopoiesis toward the generation of neutrophil-like monocytes (NeuMo). Moreover, CTX-induced NeuMo cells, enriched within the CXCR4+CX3CR1- monocyte subset, exhibited potent T cell-suppressive activity. Using the NeuMo gene signature, reanalysis of public scRNA-seq datasets identified a transcriptionally similar monocyte subset in chemotherapy-treated cancer patients. Collectively, our findings suggest that the expansion of NeuMo cells following chemotherapy represents a conserved immunoregulatory feedback mechanism with potential impact on tumor response to chemoimmunotherapy.

GSEA pathways enriched in OD-0J1 compared to OD

Humanized mice engraftment and human PBMC cytokine analysis

HIV-associated mortality has been reduced by antiretroviral therapies (ART), but prolonged ART usage by people living with HIV (PLWH) is associated with frailty and poor healthspan. Mechanisms driving this phenomenon are not fully known, but clinical and preclinical studies suggest that HIV and ART may drive aberrant activation of the aryl hydrocarbon receptor (AhR) by kynurenine (KYN), an endogenous metabolite of tryptophan. Therefore, we investigated whether the combination of an HIV-like phenotype (Tg26 mice) and treatment with ART (emtricitabine; FTC) in female mice alters skeletal muscle homeostasis in an AhR-dependent manner to promote premature muscle aging phenotypes. Short-term FTC treatment increased serum KYN:tryptophan ratio and activated AhR signaling in skeletal muscle of Tg26 mice, although the study duration was not sufficient to induce significant FTC-related functional decline. FTC, alone or in combination with other ART (tenofovir alafenamide and tenofovir disproxil fumarate), activated AhR and induced senescence of female myoblasts in a manner comparable to KYN. Sequencing-based studies revealed targets and pathways related to the impacts of an HIV phenotype and ART in female skeletal muscle, including Gnas (encoding Gsα protein, critical for muscle glucose metabolism), inflammatory pathways, and lipid metabolism. Our studies suggest that the combined presence of HIV viral proteins and exposure to ART induced activation of AhR-mediated signaling in female muscle, as well as widespread changes across the skeletal muscle transcriptome and methylation landscape that may contribute to development of muscle dysfunction. This suggests AhR may represent a novel target for addressing persistent disparities in healthspan for PLWH.

Pediatric brain tumors are widely considered non-immunogenic and do not respond to checkpoint blockade. We have developed a novel strategy to break immune tolerance to tumor by combining: (i) IDO pathway-inhibitor indoximod to promote dendritic cell maturation and antigen cross-presentation, and (ii) moderately intense oral chemotherapy to release a wave of tumor antigens. We hypothesize this cyclic chemo-immunotherapy combination stimulates normally quiescent T cells to become activated, clonally expand, and travel to tumor via the bloodstream to mount anti-tumor responses. We analyzed longitudinal blood samples from 52 patients with pediatric CNS tumors treated with indoximod plus either chemotherapy or chemotherapy plus BTK-inhibitor ibrutinib from trials NCT02502708, NCT04049669, NCT05106296. Patients in this cross-sectional “training cohort” were chosen to represent a wide range of tumor types, molecular risk factors, and overall survival. Blood samples (2-11 samples/patient depending on treatment duration) were analyzed by paired single-cell TCR and single-cell RNA sequencing. For each sample, a “Clonal Expansion Index” (CEI) was calculated as the sum of the number of T cells derived from treatment-expanded TCR clonotypes, normalized to the total number of T cells. High peak CEI values were predictive of longer overall survival and indicative of robust treatment-induced (pharmacodynamic) clonal expansion of predominantly CD8+ effector T cells. Many of these responding T cells were clonally linked to an early stem-like precursor population distinctly different from conventional “precursor-exhausted” T cells (Tpex). Treatment-expanded clones making up the CEI were an average of 100-fold more likely to be found in pre-treatment tumor biopsies by TCR-beta sequencing than random naïve T cells from the same patient; and up to 80% of CD8+ CEI T cells in circulation matched clonotypes found in tumor biopsies while on treatment. To our knowledge, this is the first report of immunotherapy-induced activation and clonal expansion of endogenous T cells in children.

BACKGROUND: Although combination antiretroviral therapy has increased life expectancy in people living with HIV, it has led to a marked increase in the prevalence of hypertension, the cause of which is unknown. Despite combination antiretroviral therapy, HIV-derived proteins remain expressed and produced by CD4 + T lymphocytes in people living with HIV. However, their contribution to HIV-associated hypertension and impaired endothelium-dependent relaxation remains ill defined. METHODS: Here, we tested the hypothesis that CD4 + T cells expressing viral proteins contribute to endothelial dysfunction and hypertension using the Tg26 mouse model of HIV that expresses 7 of the 9 HIV proteins under the long terminal repeat promoter. We used male and female mice, bone marrow transplantation (BMT), adoptive transfer of CD4 + T cells, and aorta specimen discarded from people living with HIV. RESULTS: We reported that intact Tg26 mice and mice receiving BMT (Tg26→WT) or CD4 + T cells from Tg26 mice display impaired endothelium-dependent relaxation and hypertension. Conversely, BMT from WT mice into Tg26 mice, inhibition of T cell activation, and CD4 + T cell depletion restored endothelial function and blood pressure in Tg26 mice. Cytokine profiling revealed that Tg26 mice, Tg26→WT, and Tg26 CD4 + T cells consistently exhibit high interleukin 1α (IL-1α) levels with no significant increase in other cytokines, whereas BMT from WT mice into Tg26 mice reduced IL-1α levels. IL-1α neutralization reduced blood pressure and restored endothelial function in Tg26 mice. To investigate the role of CD4 + T cells and IL-1α in endothelial dysfunction, we developed an aorta-immune cell coculture system. Exposure of WT aortas to Tg26 CD4 + T cells impaired endothelium-dependent relaxation, which was blocked by IL-1α–neutralizing antibody. While investigating the mechanisms of endothelial dysfunction, we reported that Tg26 mice, Tg26→WT aorta exhibit high NADPH oxidase (NOX) 1 expression. IL-1α exposure increased NOX1 in human microvascular endothelial cells, and NOX1 blockade restored endothelial function in Tg26 and Tg26→WT arteries, whereas NOX1 deficiency protected against Tg26 BMT-induced impaired endothelium-dependent relaxation and hypertension. Aortas from people living with HIV exhibit high NOX1 levels, and exposure of human aorta to Tg26 T cells increased NOX1 expression. CONCLUSIONS: We provide the first evidence that CD4 + T cells expressing HIV viral proteins induced hypertension through IL-1α–mediated increases in vascular NOX1, which impairs endothelial function in males and females.

The aryl hydrocarbon receptor (AhR) is proposed to mediate the frailty-promoting effects of the tryptophan metabolite kynurenine, which increases with age in mice and humans. The goal of the current study was to test whether administration of pharmacological AhR inhibitors, BAY2416964 and CH-223191, could abrogate musculoskeletal decline in aging mice. Female C57BL/6 mice (18 months old) were treated with vehicle (VEH) or 30 mg/kg BAY2416964 (BAY) via daily oral gavage 5 days/week for 8 weeks. A second AhR antagonist, CH-223191, was administered to 16-month-old male and female C57BL/6 mice via intraperitoneal injections (3.3 mg/kg) 3 days/week for 12 weeks. While grip strength declined over time in VEH-treated mice, BAY preserved grip strength in part by improving integrity of neuromuscular junctions (NMJs), an effect replicated during in vitro studies with siRNA against AhR. Cortical bone mass was also greater in BAY- than VEH-treated mice. Similarly, CH-223191 treatment improved cortical bone and showed beneficial effects in skeletal muscle, including reducing oxidative stress as compared with VEH-treated animals. Transcriptomic and proteomic data from BAY-treated mice supported a positive impact of BAY on molecular targets that affect NMJ function. Taken together, these data support AhR as a therapeutic target for improving musculoskeletal health during aging.

Background The autonomic nervous system (ANS) plays a key role in regulating tumor development and therapy resistance in various solid tumors. Within the ANS, the sympathetic nervous system (SNS) is typically associated with protumor effects. However, whether the SNS influences the antitumor efficacy of intratumoral injections of oncolytic herpes simplex virus (oHSV) in solid tumors remains unknown.Methods In this study, we examined SNS innervation and its interaction with immune cell infiltration in both human and murine triple-negative breast cancer models during intratumoral oHSV injections and SNS blockade on oHSV’s antitumor activity.Results Intratumor oHSV injection promotes SNS innervation accompanied by CD45+cell infiltration in both the human MDA-MB-468 orthotopic model and the murine 4T1 mammary tumor model. Mechanistically, tumor-secreted factors vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-β) and transcription factors (CREB, AP-1, MeCP2, and REST), which promote SNS innervation, were found to be upregulated in oHSV-treated tumors. Combining the SNS antagonist, a β-blocker, with oHSV significantly increased immune cell infiltration, particularly CD8+T cells in oHSV-treated 4T1 tumors. Single-cell messenger RNA sequencing revealed that oHSV injection upregulated a specific population of perivascular macrophages (pvMacs) expressing high levels of VEGFA, CD206, CCL3, and CCL4, which suppress T-cell activation. The use of a β-blocker reduced the infiltration of oHSV-induced pvMacs, transition to inflammatory macrophages expressing Hexb, enhancing the diversity of T-cell receptor clonotypes. Further analysis suggested that TGF-β signaling within the tumor partially mediates SNS activation in the 4T1 model.Conclusion Our findings demonstrate that combining a β-blocker with oHSV significantly enhances the antitumor efficacy of oHSV in breast cancer by targeting TGF-β-mediated SNS innervation and immunosuppression.

Background Emerging clinical and mouse tumor data indicate that tumor cells induce immune suppression in an anatomical site-specific manner. In lung metastases, tumor cell programmed death-ligand 1 (PD-L1) engages myeloid cell programmed cell death protein 1 to activate SHP2 to suppress type I interferon (IFN-I) expression to repress Cxcl9 expression to impair cytotoxic T lymphocyte (CTL) tumor recruitment. Loss of IFN-I expression thus underlies tumor immune evasion in lung metastases niche. We aimed at testing the hypothesis that forcing tumor cells to express IFNα2 activates Cxcl9 expression to increase CTL tumor recruitment to suppress lung metastasis.Methods Codon usage-optimized IFNα2-encoding DNA was designed and cloned to plasmid. IFNα2-encoding messenger RNA (mRNA) was synthesized. The plasmid DNA and mRNA were encapsulated into DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate)-cholesterol to generate lipid nanoparticle (LNP)-encapsulated mouse IFNα2 (LNP-mIFNα2), human IFNα2 plasmid, and mouse IFNα2 mRNA (LNP-mIFNα2-mRNA). Mouse breast tumor spontaneous lung metastasis, mouse melanoma experimental lung metastasis, and human colon tumor experimental lung metastasis humanized mouse models were used to determine LNP-encapsulated IFNα2-encoding plasmid and mRNA efficacy in IFNα2 expression and antitumor immunity, toxicity, and mechanism of action in vivo.Results LNP-encapsulated IFNα2-encoding plasmid primarily accumulated in tumor-bearing lungs in mice. LNP-IFNα2 therapy produces mouse IFNα2 protein in mouse tumor-bearing mice and human IFNα2 protein in human tumor-bearing humanized mice to suppress lung metastasis, respectively. Similarly, LNP-mIFNα2-mRNA therapy produces mouse IFNα2 protein and suppressed lung metastasis in tumor-bearing mice. The increased IFNα2 protein activates Cxcl9 expression and increases T cell infiltration in lung metastases. LNP-IFNα2 therapy did not induce liver toxicity and inflammatory cytokines. In human patients with cancer, IFN-I pathway activation is correlated with CXCL9 expression and T cell expansion after PD-(L)1 immune checkpoint inhibitor immunotherapy. Mechanistically, LNP-delivered IFNα2 suppresses tumor lung metastasis through upregulating Cxcl9 in tumor-bearing mice.Conclusions Our findings determine that LNP-encapsulated IFNα2-encoding plasmid DNA and mRNA are effective agents in restoring IFNα2 expression to activate Cxcl9 expression to enhance T cell tumor recruitment to suppress tumor lung metastasis. LNP-IFNα2 is potentially a safe and yet effective third-generation IFNα2 agent for human cancer immunotherapy to treat patients with lung metastasis.

Background Glioblastoma (GBM) is an aggressive malignant brain-tumor that invades adjacent normal brain tissue. Unlike other solid tumors, GBM is infiltrated by various normal brain cells. Methods We analyzed tumor invasion in the murine GSC005 glioma model using both immunodeficient and immunocompetent mice, focusing on the role of host-intrinsic and therapeutic interferon signaling in regulating glioblastoma (GBM) invasion. Results In this study, we observed that mouse GBM tumor GSC005 grown in immunodeficient (RAG1-KO, NSG) mice exhibited a more invasive phenotype compared to those in immunocompetent C57BL/6J mice. Immunofluorescence staining revealed the presence of vimentin + and GFAP + cells at the tumor-border interface. Bulk mRNA-seq analysis showed that GSC005 tumors in NSG mice displayed an upregulated mesenchymal signature, characterized by epithelial-to-mesenchymal transition (EMT), and downregulation of type-I and type-II interferon signaling. Our data further suggests that host-intrinsic and therapeutic type-I interferon promotes, while type-II interferon inhibits, the GBM mesenchymal signature. CD73, a key regulator of the EMT process, was found to be upregulated in GSC005 tumors in NSG mice compared to C57BL/6J mice. Mechanistic studies revealed that type-I interferon increases CD73 expression in both tumor and stromal cells, such as tumor-associated astrocytes (mAS), while type-II interferon suppresses CD73 in mAS. Functional assays indicated that CD73 modulates both type-I and type-II interferon signaling-mediated GBM invasion. Conclusion These findings suggest that therapies inducing type-I or type-II interferon signaling in GBM may reciprocally regulate CD73-mediated mesenchymal transitions, impacting GBM invasion.