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WSX1, a receptor subunit for IL-27, is widely expressed in immune cells and closely involved in immune response, but its function in nonimmune cells remains unknown. Here we report that WSX1 is highly expressed in human hepatocytes but downregulated in hepatocellular carcinoma (HCC) cells. Using NRAS/AKT -derived spontaneous HCC mouse models, we reveal an IL-27–independent tumor-suppressive effect of WSX1 that largely relies on CD8 + T-cell immune surveillance via reducing neoplastic PD-L1 expression and the associated CD8 + T-cell exhaustion. Mechanistically, WSX1 transcriptionally downregulates an isoform of PI3K—PI3Kδ and thereby inactivates AKT, reducing AKT-induced GSK3β inhibition. Activated GSK3β then boosts PD-L1 degradation, resulting in PD-L1 reduction. Overall, we demonstrate that WSX1 is a tumor suppressor that reinforces hepatic immune surveillance by blocking the PI3Kδ/AKT/GSK3β/PD-L1 pathway. Our results may yield insights into the host homeostatic control of immune response and benefit the development of cancer immunotherapies. The biological functions of WSX1, the alpha subunit of the interleukin-27 receptor, in non-immune cells are largely unknown. Here, the authors propose an IL-27-independent tumor suppressor role for WSX1 in hepatocytes, showing that WSX1 restricts tumor progression by down-regulating PD-L1 expression in tumour cells and maximizing T cell mediated antitumor immune responses.

Although tissue-resident memory T (T RM ) cells specific for previously encountered pathogens have been characterized, the induction and recruitment of brain T RM cells following immune therapy has not been observed in the context of glioblastoma. Here, we show that T cells expressing fibrinogen-like 2 (FGL2)–specific single-chain variable fragments (T-αFGL2) can induce tumor-specific CD8 + T RM cells that prevent glioblastoma recurrence. These CD8 + T RM cells display a highly expanded T cell receptor repertoire distinct from that found in peripheral tissue. When adoptively transferred to the brains of either immunocompetent or T cell-deficient naïve mice, these CD8 + T RM cells reject glioma cells. Mechanistically, T-αFGL2 cell treatment increased the number of CD69 + CD8 + brain-resident memory T cells in tumor-bearing mice via a CXCL9/10 and CXCR3 chemokine axis. These findings suggest that tumor-specific brain-resident CD8 + T RM cells may have promising implications for the prevention of brain tumor recurrence. Glioblastoma is an aggressive type of cancer with poor patient prognosis. Here, the authors show that T cells armed with an FGL2-specific scFV can induce antitumour responses mediated by tissue-resident memory T cells in the brain.

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip–based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor’s affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.

Receptor-ligand binding has been analyzed at the protein level using isothermal titration calorimetry and surface plasmon resonance and at the cellular level using interaction-associated downstream gene induction/suppression. However, no currently available technique can characterize this interaction directly through visualization. In addition, all available assays require a large pool of cells; no assay capable of analyzing receptor-ligand interactions at the single-cell level is publicly available. Here, we describe a new microfluidic chip–based technique for analyzing and visualizing these interactions at the single-cell level. First, a protein is immobilized on a glass slide and a low-flow-rate pump is used to isolate cells that express receptors that bind to the immobilized ligand. Specifically, we demonstrate the efficacy of this technique by immobilizing biotin-conjugated FGL2 on an avidin-coated slide chip and passing a mixture of GFP-labeled wild-type T cells and RFP-labeled FcγRIIB-knockout T cells through the chip. Using automated scanning and counting, we found a large number of GFP+ T cells with binding activity but significantly fewer RFP+ FcγRIIB-knockout T cells. We further isolated T cells expressing a membrane-anchored, tumor-targeted IL-12 based on the receptor’s affinity to vimentin to confirm the versatility of our technique. This protocol allows researchers to isolate receptor-expressing cells in about 4 hours for further downstream processing.

BackgroundThe extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) play major roles in tumor progression, metastasis, and the poor response of many solid tumors to immunotherapy. CAF-targeted chimeric antigen receptor-T cell therapy cannot infiltrate ECM-rich tumors such as osteosarcoma.MethodIn this study, we used RNA sequencing to assess whether the recently invented membrane-anchored and tumor-targeted IL-12-armed (attIL12) T cells, which bind cell-surface vimentin (CSV) on tumor cells, could destroy CAFs to disrupt the ECM. We established an in vitro model of the interaction between osteosarcoma CAFs and attIL12-T cells to uncover the underlying mechanism by which attIL12-T cells penetrate stroma-enriched osteosarcoma tumors.ResultsRNA sequencing demonstrated that attIL12-T cell treatment altered ECM-related gene expression. Immunohistochemistry staining revealed disruption or elimination of high-density CAFs and ECM in osteosarcoma xenograft tumors following attIL12-T cell treatment, and CAF/ECM density was inversely correlated with T-cell infiltration. Other IL12-armed T cells, such as wild-type IL-12-targeted or tumor-targeted IL-12-T cells, did not disrupt the ECM because this effect depended on the engagement between CSV on the tumor cell and its ligand on the attIL12-T cells. Mechanistic studies found that attIL12-T cell treatment elevated IFNγ production on interacting with CSV+ tumor cells, suppressing transforming growth factor beta secretion and in turn upregulating FAS-mediated CAF apoptosis. CAF destruction reshaped the tumor stroma to favor T-cell infiltration and tumor inhibition.ConclusionsThis study unveiled a novel therapy—attIL12-T cells—for targeting CAFs/ECM. These findings are highly relevant to humans because CAFs are abundant in human osteosarcoma.

Objective: Scavenger receptor A (SRA), a pattern recognition molecule, is implicated in immune response after acute brain injury. We strived to identify serum soluble SRA (sSRA) as a potential biomarker of prognosis after aneurysmal subarachnoid hemorrhage (aSAH). Methods: In this prospective observational study, we quantified serum sSRA levels of 131 aSAH patients and 131 healthy controls. A poor outcome was defined as extended Glasgow outcome scale (GOSE) scores of 1-4 at 90 days after injury. Relations of serum sSRA levels to severity, delayed cerebral ischemia (DCI) and poor outcome were assessed using multivariate analysis. Predictive efficiency was determined via area under receiver operating characteristic curve (AUC). Results: Serum sSRA levels were markedly higher in aSAH patients than in controls (median, 2.9 ng/mL versus 1.0 ng/mL; P < 0.001). Serum sSRA levels were independently correlated with Hunt-Hess scores (beta, 0.569; 95% confidence interval (CI), 0.244-0.894; P = 0.001), modified Fisher scores (beta, 0.664; 95% CI, 0.254-1.074; P = 0.002) and 90-day GOSE scores (beta, -0.275; 95% CI, -0.440-0.110; P = 0.005). Serum sSRA levels independently predicted DCI (odds ratio, 1.305; 95% CI, 1.012-1.687; P = 0.040) and a poor outcome (odds ratio, 2.444; 95% CI, 1.264-4.726; P = 0.008), as well as showed significant accuracy for the discrimination of DCI (AUC, 0.753; 95% CI, 0.649-0.857; P < 0.001) and a poor outcome (AUC, 0.800; 95% CI, 0.721-0.880; P < 0.001). Its combination with Hunt-Hess scores and modified Fisher scores displayed significantly improved AUCs for predicting DCI and poor outcome, as compared to any of them (all P < 0.05). Conclusion: There is a significant elevation of serum sSRA levels after aSAH, which in close correlation with illness severity, are independently associated with DCI and poor clinical outcome after aSAH. Hypothetically, SRA may regulate immune response in acute brain injury after aSAH and serum sSRA is presumed to be a potential prognostic biomarker of aSAH. Keywords: aneurysm, subarachnoid hemorrhage, scavenger receptor A, delayed cerebral ischemia, prognosis, severity, biomarkers, mechanism

BackgroundAdoptive T-cell transfer has become an attractive therapeutic approach for hematological malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) exhibits potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors.MethodsWe generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and ttIL-12 (attIL12) and a cell membrane-anchored and tumor-targeted ttIL-12 (attIL12) armed T cells, chimeric antigen receptor-T cells, and T cell receptor-T (TCR-T) cells with each. We compared the safety and efficacy of these armed T cells in treating osteosarcoma patient-derived xenograft tumors and mouse melanoma tumors after intravenous infusions of the armed T cells.ResultsattIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells targeted tumor cells expressing cell-surface vimentin, enriching effector T cell and interferon γ production in tumors, which in turn stimulates dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects.ConclusionsThis novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors.

Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is related to brain oxidative stress. We attempted to examine the association between serum NOX4 levels, severity and prognosis of severe traumatic brain injury (sTBI). We measured serum NOX4 levels in 105 patients with sTBI. Trauma severity was assessed using Glasgow coma scale (GCS) and Rotterdam computed tomography (CT) classification. Study outcome data on death and worst outcome (Glasgow outcome scale score of 1-3) were collected at 90 days after trauma. Multivariate analyses were performed to determine independent factors for overall survival and worst outcome. Area under receiver operating characteristic curve (AUC) was estimated to assess prognostic predictive ability. Serum NOX4 levels were tightly correlated with GCS score (t=-5.843, P < 0.001) and Rotterdam CT score (t = 4.231, P < 0.001). During 90 days of follow-up, 50 participants (47.6%) experienced a worse outcome, 28 (26.7%) died and the mean overall survival time was 71.9 days (95% confidence interval (CI), 65.7-78.1 days). Serum NOX4 was independently associated with an increased risk of short overall survival (hazard ratio, 1.129; 95% CI, 1.039-1.228) or worse outcome (odds ratio, 1.053; 95% CI, 1.014-1.095). Serum NOX4 levels had a certain predictive value for the patient's risk of mortality (AUC, 0.803; 95% CI, 0.714-0.874) or worse outcome (AUC, 0.780; 95% CI, 0.689-0.855). Moreover, its AUC was in the range of GCS score and Rotterdam CT score (both P > 0.05) and it significantly improved their AUCs (both P < 0.05). Serum NOX4 levels in the acute phase of sTBI were associated with trauma severity, an increased risk of mortality and worse outcome, suggesting that serum NOX4 could be an important prognostic factor for sTBI.

Background Although accumulated evidence provides a strong scientific premise for using immune profiles to predict survival in patients with cancer, a universal immune profile across tumor types is still lacking, and how to achieve a survival-associated immune profile remains to be evaluated. Methods We analyzed datasets from The Cancer Genome Atlas to identify an immune profile associated with prolonged overall survival in multiple tumor types and tested the efficacy of tumor cell-surface vimentin–targeted interleukin 12 (ttIL-12) in inducing that immune profile and prolonging survival in both mouse and patient-derived xenograft tumor models. Results We identified an immune profile (IFNγ Hi CD8 Hi FOXP3 Low CD33 Low ) associated with prolonged overall survival across several human tumor types. ttIL-12 in combination with surgical resection of the primary tumor transformed tumors to this immune profile. Intriguingly, this immune profile transformation led to inhibition of metastasis and to prolonged survival in both mouse and patient-derived xenograft malignant models. Wild-type IL-12 combined with surgery was significantly less effective. In the IL-12–sensitive C3H mouse strain, in fact, wild-type IL-12 and surgery resulted in shorter overall survival than in mice treated with control pDNA; this surprising result is believed to be attributable to IL-12 toxicity, which was absent in the mice treated with ttIL-12. The ttIL-12–induced immune profile associated with longer overall survival was also associated with a greater accumulation of CD8 + T cells and reduced infiltration of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. The underlying mechanism for this transformation by ttIL-12 treatment was induction of expression of CXCL9 and reduction of expression of CXCL2 and CCL22 in tumors. Conclusions ttIL-12 when combined with surgery led to conversion to the IFNγ Hi CD8 Hi FOXP3 Low CD33 Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.

Tumor-targeted antibody therapy is one of the safest biological therapeutics for cancer patients, but it is often ineffective at inducing direct tumor cell death and is ineffective against resistant tumor cells. Currently, the antitumor efficacy of antibody therapy is primarily achieved by inducing indirect tumor cell death, such as antibody-dependent cell cytotoxicity. Our study reveals that Herceptin conjugates, if generated via the crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), are capable of engendering human epidermal growth factor receptor 2 (Her2) positive tumor cells death. Using a high-performance liquid chromatography (HPLC) system, three peaks with estimated molecular weights of antibody monomer, dimer, and trimer were isolated. Both Herceptin trimer and dimer separated by HPLC induced significant levels of necrotic tumor cell death, although the trimer was more effective than the dimer. Notably, the Herceptin trimer also induced Herceptin-resistant tumor cell death. Surprisingly different from the known cell death mechanism that often results from antibody treatment, the Herceptin trimer elicited effective and direct tumor cell death via a novel mechanism: programmed cell necrosis. In Her2-positive cells, inhibition of necrosis pathways significantly reversed Herceptin trimer-induced cell death. In summary, the Herceptin trimer reported herein harbors great potential for overcoming tumor cell resistance to Herceptin treatment.