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The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical \"patrolling\" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

Stress can induce expression of norepinephrine, which can enhance neuroinflammation. Shaked, Hedrick and colleagues show that the transcriptional repressor Nr4a1 limits this stress-induced response by suppressing expression of tyrosine hydroxylase required for the synthesis of norepinephrine. The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo . In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Immune surveillance in tissues is mediated by a long-lived subset of tissue-resident memory T cells (Trm cells). A putative subset of tissue-resident long-lived stem cells is characterized by the ability to efflux Hoechst dyes and is referred to as side population (SP) cells. Here, we have characterized a subset of SP T cells (Tsp cells) that exhibit a quiescent (G0) phenotype in humans and mice. Human Trm cells in the gut and BM were enriched in Tsp cells that were predominantly in the G0 stage of the cell cycle. Moreover, in histone 2B-GFP mice, the 2B-GFP label was retained in Tsp cells, indicative of a slow-cycling phenotype. Human Tsp cells displayed a distinct gene-expression profile that was enriched for genes overexpressed in Trm cells. In mice, proteins encoded by Tsp signature genes, including nuclear receptor subfamily 4 group A member 1 (NR4A1) and ATP-binding cassette (ABC) transporters, influenced the function and differentiation of Trm cells. Responses to adoptive transfer of human Tsp cells into immune-deficient mice and plerixafor therapy suggested that human Tsp cell mobilization could be manipulated as a potential cellular therapy. These data identify a distinct subset of human T cells with a quiescent/slow-cycling phenotype, propensity for tissue enrichment, and potential to mobilize into circulation, which may be harnessed for adoptive cellular therapy.

ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT ( i NKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7 −/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function.

Several unconventional T cell populations, including γδ T cells and regulatory T cells, are selected by recognition of self antigen in the thymus. Craft and colleagues add T H -17 cells to the list of T cell subsets enriched by self-reactivity. Interleukin 17 (IL-17)-producing CD4 + helper T cells (T H -17 cells) share a developmental relationship with Foxp3 + regulatory T cells (T reg cells). Here we show that a T H -17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-β (TGF-β). Like previously described T H -17 cells, the T H -17 cells that developed in the thymus expressed the transcription factor RORγt and the IL-23 receptor. These cells also expressed α 4 β 1 integrins and the chemokine receptor CCR6 and were recruited to the lung, gut and liver. In the liver, these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus, T H -17 cells, like T reg cells, can be selected by self antigens in the thymus.

The NR4A nuclear receptor family member Nr4a1 is strongly induced in thymocytes undergoing selection and has been shown to control the development of T reg cells; however the role of Nr4a1 in CD8 + T cells remains undefined. Here we report a novel role for Nr4a1 in regulating the development and frequency of CD8 + T cells through direct transcriptional control of Runx3 . We discovered that Nr4a1 recruits the corepressor, CoREST to suppress Runx3 expression in CD8 + T cells. Loss of Nr4a1 results in increased Runx3 expression in thymocytes which consequently causes a 2-fold increase in the frequency and total number of intrathymic and peripheral CD8 + T cells. Our findings establish Nr4a1 as a novel and critical player in the regulation of CD8 T cell development through the direct suppression of Runx3 .

ICVB-1042 is an oncolytic adenovirus containing modifications to enhance replication, lysis, and viral spreading in tumor cells. The anti-tumor activity, immune activation, tropism, selectivity, and mechanism of action were evaluated in preparation for a first-in-human study. ICVB-1042 was at least 100-fold more cytotoxic in A549 cells than in normal primary cells tested, demonstrating its high tumor selectivity and a low likelihood of targeting primary tissues. ICVB-1042 administered to mice intravenously or intratumorally was effective in reducing tumor burden. Its intravenous administration also inhibited tumor growth in orthotopic models. ICVB-1042 was well tolerated in mice compared to HAdV-C5 (Wt Ad5), with reduced liver sequestration, supporting safety of the drug for systemic delivery. These preclinical data demonstrating the safety and potency of ICVB-1042 for treatment of various solid tumors support the ongoing clinical investigation (NCT05904236). ICVB-1042, an oncolytic adenovirus containing modifications to enhance replication, lysis, and viral spreading in tumor cells, demonstrated safety and potency in multiple tumor cell lines and mouse models of solid tumors.

BackgroundViral entry into target cells through cell surface receptor-viral protein interactions is a crucial step in oncolytic virotherapy. The broad tropism and high transduction efficiency of adenoviruses (Ads) have contributed to their extensive employment in gene therapy and as oncolytic viruses (OVs). The commonly used Ad type-5 (Ad5) utilizes the coxsackie adenovirus receptor (CAR) for cell entry. However, CAR downregulation during cancer progression1,2 limits the therapeutic efficacy of OVs reliant on this surface protein. Group B Ads use CD46 (a ubiquitously expressed receptor frequently overexpressed in cancer)3,4 for cell entry, thereby presenting an opportunity to equip OVs with chimeric fibers to enhance tropism to malignant cells. We engineered ICVB-1042, a potent, selective, and systemically available OV, with an Ad5/Ad34 chimeric fiber to enable viral entry via CD46 instead of CAR proteins. Here, we demonstrate the cell entry requirements for ICVB-1042 compared to ICVB-421, an Ad5-derived virus with wild-type fiber and capsid.MethodsCD46 and CAR knockout (CD46- and CAR-) A549 human lung carcinoma cell lines were generated using CRISPR editing. The cell lines were exposed to ICVB-1042 or ICVB-421, and the yellow fluorescent reporter protein (YPET) expression was analyzed in viable CD46+ and CD46- cells. Viral cytotoxicity was measured using cell index, a cell viability surrogate, in CD46-, CAR-, and CD46+ cells. The percentage of cytolysis was quantified relative to controls (no virus). Mouse LL/2 cells expressing transgenic human CD46 were exposed to a replication-independent YPET-expressing vector with the capsid of ICVB-1042 to determine viral entry.ResultsA dramatic increase in YPET expression in CD46+ cells was observed post-infection with ICVB-1042, suggesting that CD46 expression is a requisite for ICVB-1042 cell entry (figure 1A-B). ICVB-421 infection was not strongly associated with CD46 expression, indicating that the absence of CD46 did not impair ICVB-421 tumor cell entry. ICVB-1042 infection induced 100% cytolysis in all cell lines faster than ICVB-421 but with a lower rate in CD46- cells (figure 1C), demonstrating that CD46 deficiency induces resistance to ICVB-1042-induced tumor killing. High YPET fluorescence intensity in mouse LL/2 cells harboring human CD46 transgenes was observed, denoting that human CD46 expression by LL/2 cells enabled transduction by a vector version of ICVB-1042 (figure 2A-B).ConclusionsThese results demonstrate that replacing wild-type fiber with the chimeric Ad5/Ad34 fiber of ICVB-1042 results in a potent OV that relies on CD46 expression for tumor entry.ReferencesSachs MD, Rauen KA, Ramamurthy M, et al. Integrin alpha(v) and coxsackie adenovirus receptor expression in clinical bladder cancer. Urology. 2002;60:531–536.Wunder T, Schmid K, Wicklein D, et al. Expression of the coxsackie adenovirus receptor in neuroendocrine lung cancers and its implications for oncolytic adenoviral infection. Cancer Gene Ther. 2013;20:25–32.Russell S. CD46: a complement regulator and pathogen receptor that mediates links between innate and acquired immune function. Tissue Antigens. 2004;64:111–118.Elvington M, Liszewski MK, Atkinson JP. CD46 and oncologic interactions: friendly fire against cancer. Antibodies (Basel). 2020;9:59.Abstract 1216 Figure 1(A) Flow cytometry analysis of YPET expression in CD46 knockout cell lines; the A549 parental cell line with endogenous CD46 expression (CD46+) was used as a positive control. (B) Plots showing percentage of YPET-expressing CD46+ and CD46- cells (46.5% in CD46+ versus 1.9% in CD46- cells; p=0.0005, t-test). (C) Cell viability was measured at baseline and every 30 minutes for 144 hours following ICVB-1042 or ICVB-421infection. MOI: multiplicity of infection; 24hpi: 24 hours post infection.[Figure omitted. See PDF]Abstract 1216 Figure 2(A) Representative images showing YPET fluorescence in mouse lung carcinoma LL/2 cells (generated via piggyBac transposition) following infection of a replication-independent YPET-expressing vector of ICVB-1042. (B) Plots showing average YPET fluorescence signal intensity (with background fluorescence subtracted). MOI: multiplicity of infection; 1085 and Clone 5 are constructs expressing CD46 isoforms in parental LL/2 cell lines.[Figure omitted. See PDF]

Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1 and Nr4a1 -deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset.