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With the approval of the antibody-drug conjugate enfortumab vedotin (EV), NECTIN4 has emerged as a bona fide therapeutic target in urothelial carcinoma (UC). Here, we report the development of a NECTIN4-directed chimeric antigen receptor (CAR) T cell, which exhibits reactivity across cells expressing a range of endogenous NECTIN4, with enhanced activity in high expressors. We demonstrate that the PPARγ pathway, critical for luminal differentiation, transcriptionally controls NECTIN4 , and that the PPARγ agonist rosiglitazone primes and augments NECTIN4 expression, thereby increasing sensitivity to NECTIN4-CAR T cell-mediated killing. NECTIN4-CAR T cells have potent anti-tumor activity even against EV resistant cells, which largely retain NECTIN4 expression, including in a post-EV biopsy cohort. Our results elucidate a therapeutically actionable mechanism that UC cells use to control NECTIN4 expression and suggest therapeutic approaches that leverage PPARγ agonists for rational combinations with NECTIN4-targeting agents in UC, as well as future potential treatment options for EV-refractory patients. Subject terms: Bladder cancer, Cancer immunotherapy, Cancer therapeutic resistance, Oncology, Bladder cancer

In bone marrow, cell numbers are balanced between production and loss. After chemotherapy, blood cell counts decrease initially but later recover as hematopoietic progenitor cells expand, although the mechanisms underlying this recovery are still unclear. We investigated the influence of red blood cells (RBCs) on hematopoietic stem cell (HSC) function during bone marrow recovery. Following chemotherapy, RBC concentrations in bone marrow peaked on day 5 posttreatment, coinciding with the recovery of hematopoiesis. Coculture of HSCs with RBCs resulted in a significant increase in hematopoiesis. Direct contact between RBCs and HSCs was essential for enhancement of hematopoiesis, and HSCs precultured with RBCs resulted in greater numbers of donor‐derived mature hematopoietic cells after transplantation. RNA‐sequencing analysis showed that Hes1 was the most significantly upregulated transcription factor in RBC coculture, and the response to RBC‐induced hematopoiesis of Hes1‐deficient HSCs was reduced. These findings imply a role of RBCs and Hes1 in the enhancement of hematopoietic recovery following bone marrow stress.

If iPS cells can be established easily and efficiently using freshly collected blood cells, it will enhance regenerative and personalized medicine. While reports of iPS derivation from blood-derived endothelial progenitor cells using RNA have been documented, none have been reported from peripheral blood-derived mononuclear cells (PBMCs). In this study, we established a method to generate iPS cells from PBMCs using synthetic RNAs and found that MDM4, which suppresses p53, improved reprogramming efficiency. Subject terms: Reprogramming, Induced pluripotent stem cells

Despite the success of antiretroviral therapy in suppressing plasma viremia in people living with human immunodeficiency virus type-1 (HIV-1), persistent viral RNA expression in tissue reservoirs is observed and can contribute to HIV-1-induced immunopathology and comorbidities. Infection of long-lived innate immune cells, such as tissue-resident macrophages and microglia may contribute to persistent viral RNA production and chronic inflammation. We recently reported that de novo cytoplasmic expression of HIV-1 intron-containing RNA (icRNA) in macrophages and microglia leads to MDA5 and MAVS-dependent innate immune sensing and induction of type I IFN responses, demonstrating that HIV icRNA is a pathogen-associated molecular pattern (PAMP). In this report, we show that cytoplasmic expression of HIV-1 icRNA also induces NLRP1 inflammasome activation and IL-1β secretion in macrophages and microglia in an RLR- and endosomal TLR-independent manner. Infection of both macrophages and microglia with either replication-competent or single-cycle HIV-1 induced IL-1β secretion, which was attenuated when cytoplasmic expression of viral icRNA was prevented. While IL-1β secretion was blocked by treatment with caspase-1 inhibitors or knockdown of NLRP1 or caspase-1 expression in HIV-infected macrophages, overexpression of NLRP1 significantly enhanced IL-1β secretion in an HIV-icRNA-dependent manner. Immunoprecipitation analysis revealed interaction of HIV-1 icRNA, but not multiply-spliced HIV-1 RNA, with NLRP1, suggesting that HIV-1 icRNA sensing by NLRP1 is sufficient to trigger inflammasome activation. Together, these findings reveal a pathway of NLRP1 inflammasome activation induced by de novo expressed HIV icRNA in HIV-infected myeloid cells.

Particulate matters such as diesel exhaust particles induce oxidative stress in cells and thereby have a negative impact on health. The aim of this study was to test whether the membrane-permeable, anti-inflammatory metabolite 4-Octyl Itaconate can counteract the oxidative stress induced by diesel exhaust particles and to analyze the downstream-regulated pathways both in human nasal epithelial cells and PBMCs. Human nasal epithelial cells were cultured from nasal swabs, and the response of the cells to diesel exhaust particles either alone or in combination with 4-Octyl Itaconatee was investigated using RNA sequencing, qPCR, and cytokine measurement. The presence of reactive oxygen species in the cells was analyzed using CellROX staining and flow cytometric DCFDA assay. Diesel exhaust particles caused an upregulation of CYP1A1 in nasal epithelial cells. The administration of 4-Octyl Itaconate reduced the reactive oxygen species and increased the expression of antioxidant genes regulated by the transcription factor NRF2, which was also confirmed in PBMCs. IL-6 secretion from NEC was elevated by diesel exhaust particles and reduced when 4-Octyl Itaconate was administered. 4-Octyl Itaconate can reduce the diesel-exhaust-particle-induced oxidative damage by the activation of NRF2-regulated antioxidative pathways.

Advancing research in oncology highlights the inverse correlation between antibiotic treatment and the positive outcomes of immune checkpoint inhibitor (ICI) administration, confirming once more the importance of microbiota and microbiota-derived compounds as complementary tools for treating cancer. Among the immune checkpoints, the CD200 cell surface glycoprotein has gained attention for its role in promoting self-tolerance and potentially facilitating tumor growth through interaction with the CD200R1 receptor. We developed a robust AlphaLISA-based screening to identify human gut microbiota-derived proteins that may interact with CD200R1 and screened a library of 10,966 gut bacterial proteins. The antitumor activity of BOC1 was investigated in vitro by cytokine analysis, mixed lymphocyte reactions, and myeloid-derived suppressor cell (MDSC)–T-cell suppression assay. AlphaFold modeling was used to predict potential interaction points between BOC1 and CD200R1. We successfully identified BOC1, a protein from the Bacteroides genus, showing better affinity than the natural ligand, CD200, toward the CD200R1 receptor. BOC1 induces cytokine secretion by monocyte-derived dendritic cells (MoDCs) and enhances CD8 + /CD4 + T-cell populations and IFNγ production, highlighting its potent immunostimulatory properties. BOC1 also negatively impacts the differentiation of MDSCs, maintaining an immature monocytic profile (high CD14 and HLA-DR expression) and restoring T-cell proliferation even at low (10 nM) concentration. Mutation of amino acids within the N-terminal region of BOC1 reduces binding to CD200R1, supporting the importance of this region for a possible interaction with CD200R1. The immunostimulatory properties of BOC1 observed in vitro are compatible with an ICI-like behavior of this bacterial protein. Given that neither the CD200 protein nor the anti-CD200 antibody is able to compete with BOC1 for binding to CD200R1, and as supported by AlphaFold modeling predictions, CD200 and BOC1 might target different regions of CD200R1.