The tumor microenvironment (TME) enhances regulatory T (T _reg ) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as T _reg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying T _reg fitness. We demonstrate that TME-specific stressors including transforming growth factor–β (TGF-β), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 ( Usp22 ) and Usp21 , by regulating TGF-β, HIF, and mTOR signaling, respectively, to maintain T _reg fitness. Simultaneous deletion of both USPs in T _reg cells largely diminishes TME-induced Foxp3 up-regulation, alters T _reg metabolic signatures, impairs T _reg -suppressive function, and alleviates T _reg suppression on cytotoxic CD8 ⁺ T cells. Furthermore, we developed the first Usp22 -specific small-molecule inhibitor, which dramatically reduced intratumoral T _reg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying T _reg fitness and identify Usp22 as an antitumor therapeutic target that inhibits T _reg adaptability in the TME.

Treg differentiation, maintenance, and function are controlled by the transcription factor FoxP3, which can be destabilized under inflammatory or other pathological conditions. Tregs can be destabilized under inflammatory or other pathological conditions, but the underlying mechanisms are not fully defined. Herein, we show that inflammatory cytokines induce ER stress response, which destabilizes Tregs by suppressing FoxP3 expression, suggesting a critical role of the ER stress response in maintaining Treg stability. Indeed, genetic deletion of Hrd1, an E3 ligase critical in suppressing the ER stress response, leads to elevated expression of ER stress–responsive genes in Treg and largely diminishes Treg suppressive functions under inflammatory condition. Mice with Treg-specific ablation of Hrd1 displayed massive multiorgan lymphocyte infiltration, body weight loss, and the development of severe small intestine inflammation with aging. At the molecular level, the deletion of Hrd1 led to the activation of both the ER stress sensor IRE1α and its downstream MAPK p38. Pharmacological suppression of IRE1α kinase, but not its endoribonuclease activity, diminished the elevated p38 activation and fully rescued the stability of Hrd1-null Tregs. Taken together, our studies reveal ER stress response as a previously unappreciated mechanism underlying Treg instability and that Hrd1 is crucial for maintaining Treg stability and functions through suppressing the IRE1α-mediated ER stress response.

Hantaan virus (HTNV) is a major agent causing hemorrhagic fever with renal syndrome (HFRS). Although the pathogenesis of HFRS is unclear, some reports have suggested that the abundant production of proinflammatory cytokines and uncontrolled inflammatory responses may contribute to the development of HFRS. CXCL10 is one of these cytokines and is found to be involved in the pathogenesis of many virus infectious diseases. However, the role of CXCL10 in the pathogenesis of HFRS and the molecular regulation mechanism of CXCL10 in HTNV infection remain unknown. In this study, we report that CXCL10 expresses highly in the HFRS patients’ sera and the elevated CXCL10 is positively correlated with the severity of HFRS. We find that HTNV, a single-strand RNA virus, can act as a double-strand RNA to activate the TLR3, RIG-I, and MDA-5 signaling pathways. Through the downstream transcription factors of these pathways, NF-κB and IRF7, which bind directly to the CXCL10’s promoter, the expression of CXCL10 is increased. Our results may help to better understand the role of CXCL10 in the development of HFRS and may provide some novel insights into the immune response of HTNV infection.

Hantaan virus (HTNV) infection can cause a severe lethal hemorrhagic fever with renal syndrome (HFRS) in humans. CD8⁺T cells play a critical role in combating HTNV infections. However, the contributions of different CD8⁺T cell subsets to the immune response against viral infection are poorly understood. Here, we identified a novel subset of CD8⁺T cells characterized by the CD8^lowCD100⁻phenotype in HFRS patients. The CD8^lowCD100⁻subset accounted for a median of 14.3% of the total CD8⁺T cells in early phase of HFRS, and this percentage subsequently declined in the late phase of infection, whereas this subset was absent in healthy controls. Furthermore, the CD8^lowCD100⁻cells were associated with high activation and expressed high levels of cytolytic effector molecules and exhibited a distinct expression profile of effector CD8⁺T cells (CCR7^+/−CD45RA⁻CD127^highCD27^intCD28^lowCD62L⁻). When stimulated with specific HTNV nucleocapsid protein-derived peptide pools, most responding CD8⁺cells (gamma interferon [IFN-γ] positive and/or tumor necrosis factor alpha [TNF-α] positive) were CD8^lowCD100⁻cells. The frequency of CD8^lowCD100⁻cells among HTNV-specific CD8⁺T cells was higher in milder cases than in more severe cases. Importantly, the proportion of the CD8^lowCD100⁻subset among CD8⁺T cells in early phase of HFRS was negatively correlated with the HTNV viral load, suggesting that CD8^lowCD100⁻cells may be associated with viral clearance. The contraction of the CD8^lowCD100⁻subset in late phase of infection may be related to the consistently high expression levels of PD-1. These results may provide new insights into our understanding of CD8⁺T cell-mediated protective immunity as well as immune homeostasis after HTNV infection in humans.IMPORTANCECD8⁺T cells play important roles in the antiviral immune response. We found that the proportion of CD8^lowCD100⁻cells among CD8⁺T cells from HFRS patients was negatively correlated with the HTNV viral load, and the frequency of CD8^lowCD100⁻cells among virus-specific CD8⁺T cells was higher in milder HFRS cases than in more severe cases. These results imply a beneficial role for the CD8^lowCD100⁻cell subset in viral control during human HTNV infection.

Background: As a cellular membrane triggering receptor, CD226 is involved in the NK cell- or CTL-mediated lysis of tumor cells of different origin, including freshly isolated tumor cells and tumor cell lines. Here, we evaluated soluble CD226 (sCD226) levels in sera, and membrane CD226 (mCD226) expression on peripheral blood mononuclear cells (PBMC) from cancer patients as well as normal subjects, and demonstrated the possible function and origin of the altered sCD226, which may provide useful information for understanding the mechanisms of tumor escape and for immunodiagnosis and immunotherapy.Results: Soluble CD226 levels in serum samples from cancer patients were significantly higher than those in healthy individuals (P< 0.001), while cancer patients exhibited lower PBMC mCD226 expression than healthy individuals (P< 0.001). CD226-Fc fusion protein could significantly inhibit the cytotoxicity of NK cells against K562 cells in a dose-dependent manner. Furthermore, three kinds of protease inhibitors could notably increase mCD226 expression on PMA-stimulated PBMCs and Jurkat cells with a decrease in the sCD226 level in the cell culture supernatant.Conclusion: These findings suggest that sCD226 might be shed from cell membranes by certain proteases, and, further, sCD226 may be used as a predictor for monitoring cancer, and more important, a possible immunotherapy target, which may be useful in clinical application.

Cell-free DNA (cf-DNA) in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS), depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV) load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.