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The efficacy of immunotherapy is often hindered by the suppression of immune responses via the tumor microenvironment (TME). The presence of cancer cells forces other proximal non-cancerous cells to support tumor growth and persistence. A clear example of this cancerous-to-non-cancerous communication is represented by the accumulation of myeloid-derived suppressor cells (MDSCs) within the TME. Several studies have convergently shown that the overexpression of DNA-methyl-transferase-1 (DNMT1) in these cells results in protection from necroptosis and enhanced accumulation in vivo. Conversely, targeting DNMT1 through hypo-methylating agents has shown promising therapeutic potential by not only reducing the levels of MDSCs but also enhancing cancer immunogenicity and the efficacy of immune checkpoint inhibitors (ICI). Methods: Murine 4T1 (triple-negative breast cancer (TNBC)) and CT26 (colon carcinoma) cell lines were cultured under standard conditions and used to generate tumor models in BALB/c mice. An oncolytic adenovirus expressing a DNMT1-targeting short hairpin RNA (OAd.shDNMT1) was engineered and validated for DNMT1 knockdown and genome-wide methylation reduction. Small extracellular vesicles (sEVs) were isolated from virus-infected cancer cells and characterized for RNA content and uptake by MDSCs. MDSC differentiation and suppressive function were assessed in vitro using flow cytometry and co-culture assays with murine splenocytes. In vivo, tumor-bearing mice received intratumoral OAd.shDNMT1, systemic decitabine, or immune checkpoint inhibitors (anti-Programmed cell Death protein-1), and tumor growth, immune infiltration, and systemic MDSC levels were evaluated. Results: In this study, we report that, by using virally infected TNBC murine cells as a source for shDNMT1-loaded sEVs, OAd.shDNMT1 successfully reduced MDSC levels in vitro and in vivo. Furthermore, the co-administration with ICI resulted in a significant tumor growth reduction in mice bearing poorly immunogenic TNBC 4T1 cells. Also, our treatment promoted antitumor immunity, prolonged survival, and complete tumor eradication in modestly immunogenic colon CT26 cancer cells. Conclusions: This multifaceted strategy, based on OV-mediated immune stimulation and reduction of MDSC levels via sEVs, may improve clinical outcomes and the success of immuno-based regimens for patients facing MDSC-rich and highly aggressive cancer subtypes.

Klebsiella pneumoniae (Kp) is responsible for a wide range of infections, including pneumonia, sepsis, and urinary tract infections. However, the treatment options are limited due to the continuous evolution of drug-resistant and hypervirulent variants. It is crucial to investigate the mechanisms behind the high mortality rate of hypervirulent Kp (hvKp) strains to develop new strategies for preventing hvKp from evading the host¡¯s defenses and improving treatment effectiveness for these fatal infections. In this study, we used a hvKp-induced mouse bacteremia model and performed single-cell RNA sequencing to investigate the effects of hvKp infection. Our findings demonstrated that hvKp infection led to a decrease in lymphocytes (lymphopenia), attributed to impaired proliferation and apoptosis. The infiltration of myeloid-derived suppressor cells (MDSCs) in the infected lungs was confirmed to suppress T cell proliferation, leading to lymphopenia. We further identified that hvKp promotes tryptophan metabolism in infected lungs, enhancing the immunosuppressive activity of MDSCs by inducing the production of the enzyme IDO1. Our ex vivo inhibition experiment revealed that L-kynurenine, a product of tryptophan metabolism, inhibits T-cell proliferation and induces T-cell apoptosis, further suppressing T-cell mediated responses against bacteria. Importantly, when we knocked out the Ido1 gene or inhibited IDO1 expression using a specific inhibitor 1-MT in mice, we observed a significant enhancement in T-cell mediated responses against hvKp. These findings highlight the crucial role of MDSCs in hvKp-induced bacteremia and suggest a promising immunotherapeutic approach by inhibiting IDO1 production to combat infectious diseases. Author summaryKlebsiella pneumoniae is a major cause of health care¨Cassociated infections worldwide, and infections with hvKp is often associated with cytokine storms and lymphopenia, resulting in higher mortality. However, the underlying mechanisms of lymphopenia during hvKp infection is not fully understood. In this study, we demonstrate a mechanism of immunosuppression of T cells in hvKp infection mouse model by combination of single-cell RNA sequencing, flow cytometry and untargeted metabolomic analysis. We confirm MDSCs can suppress T cell proliferation and induce T cell apoptosis by activating tryptophan metabolism during hvKp infection. Specifically, the downstream metabolite kynurenine, which catalyzes by IDO1 in tryptophan metabolism pathway, serves as the signaling molecule in the interactions between MDSCs and T cells. Our findings provide a new insight in understanding the mechanism of how hvKp manipulate the host immune systems and suggest that pharmacological inhibition of IDO1 could be an effective therapeutic strategy for combating hvKp infection.

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway, suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1, via manipulating multiple signaling pathways, induces epithelial cell transformation, MDSC (Myeloid-derived suppressor cells) accumulation, and pRCC development. Interestingly, we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1, a recently developed TEAD inhibitor, impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC. Deletion of upstream tumor suppressors of the Hippo/YAP pathway is frequent in papillary renal cell carcinoma (pRCC). Here, the authors employ a transgenic mouse model, single-cell transcriptomics and public genomic datasets to show that targeting hyperactivated YAP1 prevents neoplastic renal epithelial cell immune evasion and impairs the development of pRCC.