Cell membrane-anchored and tumor-targeted IL-12 T-cell therapy destroys cancer-associated fibroblasts and disrupts extracellular matrix in heterogenous osteosarcoma xenograft models.

BACKGROUND: The 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. METHOD: In 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. RESULTS: RNA 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. CONCLUSIONS: This 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.

FGL2-targeting T cells exhibit antitumor effects on glioblastoma and recruit tumor-specific brain-resident memory T cells.

Although tissue-resident memory T (TRM) cells specific for previously encountered pathogens have been characterized, the induction and recruitment of brain TRM 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+ TRM cells that prevent glioblastoma recurrence. These CD8+ TRM 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+ TRM 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+ TRM cells may have promising implications for the prevention of brain tumor recurrence.

Potential role of serum hypoxia-inducible factor 1alpha as a biomarker of delayed cerebral ischemia and poor clinical outcome after human aneurysmal subarachnoid hemorrhage: A prospective, longitudinal, multicenter, and observational study.

Objective: Hypoxia-inducible factor 1alpha (HIF-1α) functions as a crucial transcriptional mediator in hypoxic and ischemic brain response. We endeavored to assess the prognostic significance of serum HIF-1α in human aneurysmal subarachnoid hemorrhage (aSAH). Methods: In this prospective, longitudinal, multicenter, and observational study of 257 patients with aSAH and 100 healthy controls, serum HIF-1α levels were quantified. Univariate analyses, followed by multivariate analyses, were performed to discern the relationship between serum HIF-1α levels and severity and delayed cerebral ischemia (DCI) plus poststroke 6-month poor outcome [extended Glasgow outcome scale (GOSE) scores of 1-4]. Predictive efficiency was determined under the receiver operating characteristic (ROC) curve. Results: There were significantly increased serum HIF-lα levels after aSAH, in comparison to controls (median, 288.0 vs. 102.6 pg/ml; P < 0.001). Serum HIF-lα levels were independently correlated with Hunt-Hess scores [ß, 78.376; 95% confidence interval (CI): 56.446-100.305; P = 0.001] and modified Fisher scores (ß, 52.037; 95% CI: 23.461-80.614; P = 0.002). Serum HIF-lα levels displayed significant efficiency for discriminating DCI risk [area under ROC curve (AUC), 0.751; 95% CI: 0.687-0.815; P < 0.001] and poor outcome (AUC, 0.791; 95% CI: 0.736-0.846; P < 0.001). Using the Youden method, serum HIF-1α levels >229.3 pg/ml predicted the development of DCI with 92.3% sensitivity and 48.4% specificity and serum HIF-1α levels >384.0 pg/ml differentiated the risk of a poor prognosis with 71.4% sensitivity and 81.1% specificity. Serum HIF-1α levels >229.3 pg/ml were independently predictive of DCI [odds ratio (OR), 3.061; 95% CI: 1.045-8.965; P = 0.041] and serum HIF-1α levels >384.0 pg/ml were independently associated with a poor outcome (OR, 2.907; 95% CI: 1.403-6.024; P = 0.004). The DCI predictive ability of their combination was significantly superior to those of Hunt-Hess scores (AUC, 0.800; 95% CI: 0.745-0.855; P = 0.039) and modified Fisher scores (AUC, 0.784; 95% CI: 0.726-0.843; P = 0.004). The prognostic predictive ability of their combination substantially exceeded those of Hunt-Hess scores (AUC, 0.839; 95% CI: 0.791-0.886; P < 0.001) and modified Fisher scores (AUC, 0.844; 95% CI: 0.799-0.890; P < 0.001). Conclusion: Elevated serum HIF-lα levels after aSAH, in independent correlation with stroke severity, were independently associated with DCI and 6-month poor outcome, substantializing serum HIF-lα as a potential prognostic biomarker of aSAH.

Serum Soluble Scavenger Receptor A Levels are Associated with Delayed Cerebral Ischemia and Poor Clinical Outcome After Aneurysmal Subarachnoid Hemorrhage: A Prospective Observational Study.

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.

A microfluidics-based method for isolation and visualization of cells based on receptor-ligand interactions.

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.

Serum NOX4 as a Promising Prognostic Biomarker in Association with 90-Day Outcome of Severe Traumatic Brain Injury.

Purpose: 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). Methods: 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. Results: 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). Conclusion: 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.

Membrane-Anchored and Tumor-Targeted IL12 (attIL12)-PBMC Therapy for Osteosarcoma.

PURPOSE: Chimeric antigen receptor (CAR) T-cell therapy has shown great promise for treating hematologic malignancies but requires a long duration of T-cell expansion, is associated with severe toxicity, and has limited efficacy for treating solid tumors. We designed experiments to address those challenges. EXPERIMENTAL DESIGN: We generated a cell membrane-anchored and tumor-targeted IL12 (attIL12) to arm peripheral blood mononuclear cells (PBMC) instead of T cells to omit the expansion phase for required CAR T cells. RESULTS: This IL12-based attIL12-PBMC therapy showed significant antitumor efficacy in both heterogeneous osteosarcoma patient-derived xenograft tumors and metastatic osteosarcoma tumors with no observable toxic effects. Mechanistically, attIL12-PBMC treatment resulted in tumor-restricted antitumor cytokine release and accumulation of attIL12-PBMCs in tumors. It also induced terminal differentiation of osteosarcoma cells into bone-like cells to impede tumor growth. CONCLUSIONS: In summary, attIL12-PBMC therapy is safe and effective against osteosarcoma. Our goal is to move this treatment into a clinical trial. Owing to the convenience of the attIL12-PBMC production process, we believe it will be feasible.

Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors.

BACKGROUND: Adoptive 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. METHODS: We 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. RESULTS: attIL12-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. CONCLUSIONS: This novel approach sheds light on the safe application of IL-12-based T-cell therapy for large and heterogeneous solid tumors.

WSX1 act as a tumor suppressor in hepatocellular carcinoma by downregulating neoplastic PD-L1 expression.

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 GSK3ß inhibition. Activated GSK3ß 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/GSK3ß/PD-L1 pathway. Our results may yield insights into the host homeostatic control of immune response and benefit the development of cancer immunotherapies.

Regulation of tumor immune suppression and cancer cell survival by CXCL1/2 elevation in glioblastoma multiforme.

The invasiveness and high immune suppression of glioblastoma multiforme (GBM) produce poor survival of afflicted patients. Unfortunately, in the past decades, no therapeutic approach has remarkably improved the survival time of patients with GBM. Our analysis of the TCGA database and brain tumor tissue arrays indicated that CXCL1 and CXCL2 overexpression is closely associated with GBM's aggressiveness. Our results showed that elevation of CXCL1 or CXCL2 facilitated myeloid cell migration and simultaneously disrupted CD8+ T cell accumulation at tumor sites, causing accelerated tumor progression. Yet, blockade of CXCL1/2 significantly prevented myeloid-derived suppressor cell migration and thereby increased CD8+ T cell accumulation in vitro and in vivo. CXCL1/2 also promoted the paracrine factor S100A9 and further activated Erk1/2 and p70S60k, whereas blocking CXCL1/2 down-regulated these prosurvival factors. The combination of targeting CXCL1/2 and standard temozolomide chemotherapy improved upon the antitumor efficacy of chemotherapy alone, extending the overall survival time in GBM.

Lysine acetylation of NKG2D ligand Rae-1 stabilizes the protein and sensitizes tumor cells to NKG2D immune surveillance.

Shedding, loss of expression, or internalization of natural killer group 2, member D (NKG2D) ligands from the tumor cell surface leads to immune evasion, which is associated with poor prognosis in patients with cancer. In many cancers, matrix metalloproteinases cause the proteolytic shedding of NKG2D ligands. However, it remained unclear how to protect NKG2D ligands from shedding. Here, we showed that the shedding of the mouse NKG2D ligand Rae-1 can be prevented by two critical acetyltransferases, GCN5 and PCAF, which acetylate the lysine residues of Rae-1 to avoid shedding both in vitro and in vivo. In contrast, mutations at lysines 80 and 87 of Rae-1 abrogated this acetylation and thereby desensitized tumor cells to NKG2D-dependent immune surveillance. Notably, the protein levels of GCN5 correlated with the expression levels of the human NKG2D ligand ULPB1 in a human tumor tissue microarray and, more importantly, with prolonged overall survival in many cancers. Our results suggest that the acetylation of Rae-1 protein at lysines 80 and 87 by GCN5 and PCAF protects Rae-1 from shedding so as to activate NKG2D-dependent immune surveillance. This discovery may shed light on new targets for NKG2D immunotherapy in cancer treatment.

Induction of NKG2D ligand expression on tumor cells by CD8+ T-cell engagement-mediated activation of nuclear factor-kappa B and p300/CBP-associated factor.

The ligands for the natural killer group 2 (NKG2D) protein render tumor cells susceptible to NKG2D-dependent immune cell attack. However, cancer cells escape from immune surveillance by downregulating NKG2D ligands. We previously discovered that engagement of activated CD8+ T cells and tumor cells induces NKG2D ligands on tumor cells, but the underlying mechanism remains to be defined. Both in vivo mouse tumor models and in vitro cell assays were performed to study the downstream signaling. Our results supported the notion that, upon engagement with the cognate receptors, CD137 ligand and CD40 initiates activation of nuclear factor-kappa B (NF-κB) signaling in tumor cells even in the absence of CD8+ T cells. Like tumor and CD8+ T cell contact-dependent NKG2D ligand induction, this CD137L/CD40-mediated signaling activation was associated with elevated levels of acetyltransferase P300/CBP-associated factor (PCAF), whereas inhibition of phosphorylated NF-κB abrogated PCAF induction. Although stimulation of CD137L/CD40-mediated signaling is vital, inflammatory cytokines, including interferon gamma (IFNγ) and TNFα, also facilitate NKG2D ligand-induced immune surveillance via both facilitating T-cell chemotaxis and CD137L/CD40 induced NF-κB/PCAF activation. Collectively, our results unveil a novel mechanism of NKG2D ligand upregulation involving reverse signaling of CD40 and CD137L on tumor cells which, along with inflammatory cytokines IFNγ and TNFα, stimulate downstream NF-κB and PCAF activation. Understanding this mechanism may help in development of induced NKG2D ligand-dependent T-cell therapy against cancers.

Tumor-targeted IL-12 combined with tumor resection yields a survival-favorable immune profile.

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 (IFNγHiCD8HiFOXP3LowCD33Low) 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 IFNγHiCD8HiFOXP3LowCD33Low immune profile, eliminated relapse and metastasis, and prolonged overall survival.

T-cell Homing Therapy for Reducing Regulatory T Cells and Preserving Effector T-cell Function in Large Solid Tumors.

Purpose: Infused autologous tumor-infiltrating lymphocytes (TIL) and tumor-targeted chimeric antigen receptor (CAR) T cells typically surround malignant lesions or penetrate small tumor nodules but fail to penetrate large solid tumors, significantly compromising their antitumor impact. Strategies to overcome this primary challenge are largely required.Experimental Design: We tested the effects of IL12 plus doxorubicin on T-cell penetration and efficacy in solid tumors in a murine lung cancer model, a murine breast carcinoma lung metastasis model, and two human xenograft tumor models bearing large tumors (>10 mm).Results: Intriguingly, this simple approach increased the numbers, the distribution, and the depth of penetration of infused CD8+ T cells in these tumors, including both TILs and CAR T cells. This combined treatment halted tumor progression and significantly extended survival time. Studies of the underlying mechanism revealed multiple effects. First, the combined treatment maintained the high ratios of immune-stimulatory receptors to immune-inhibitory receptors on infiltrated CD8+ T cells, reduced the accumulation of immunosuppressive regulatory T cells, and enhanced the numbers of T-bet+ effector T cells in the tumors. Second, doxorubicin induced chemokines CXCL9 and CXCL10, which may attract NKG2D+CD8+ T cells to tumors, and this effect was boosted by IL12-induced IFNγ accumulation in tumors, promoting the penetration of NKG2D+CD8+ T cells.Conclusions: The deep penetration of infused T cells associated with combined IL12 plus doxorubicin yielded striking therapeutic effects in murine and human xenograft solid tumors. This approach might broaden the application of T-cell therapy to a wider range of solid tumors. Clin Cancer Res; 24(12); 2920-34. ©2018 AACRSee related commentary by Berraondo et al., p. 2716.

Induction of NKG2D Ligands on Solid Tumors Requires Tumor-Specific CD8+ T Cells and Histone Acetyltransferases.

NKG2D-mediated immune surveillance is crucial for inhibiting tumor growth and metastases. Malignant tumor cells often downregulate NKG2D ligands to escape from immune surveillance. High-profile studies have shown that restoring NKG2D ligand expression via genetic engineering inhibits tumor formation and progression. However, no effective in vivo approaches are available to restore these ligands across different types of solid tumors because the classic stress signal-dependent induction of this ligand in vitro is transient and has rarely been duplicated in solid tumors in vivo We found that coadministration of an immune stimulatory signal (IL12) and chemotherapy (doxorubicin) restored the NKG2D ligand Rae-1 in multiple tumor types, including a human tumor model. The restored expression of NKG2D ligands was associated with tumor cell death and delay of tumor progression in vivo Induction of tumor-specific NKG2D ligands required the engagement of CD8+ T cells and was regulated by the histone acetyltransferases GCN5 and PCAF. The tumor-specific restoration of NKG2D ligands in a variety of tumor models, including a human tumor model, resulted in NKG2D-dependent tumor regression and extended survival time. The elucidation of a CD8+ T cell-dependent mechanism suggests that activated NKG2D+CD8+ T-cell therapy alone may be able to restore the NKG2D ligand in tumors. Cancer Immunol Res; 5(4); 300-11. ©2017 AACR.

Potential Function of Exogenous Vimentin on the Activation of Wnt Signaling Pathway in Cancer Cells.

Cancer cell signaling, growth, morphology, proliferation and tumorigenic potential are largely depending on the signaling molecules present naturally in the tumor microenvironment and the identification of key molecules that drive the tumor progression is critical for the development of new modalities for the prevention of tumor progression. High concentrations of vimentin in the blood of cancer patients have been reported, however the function of blood circulating vimentin remains unknown. Here, we investigated the functional role of exogenously supplemented vimentin on colon cancer cells and examined the Wnt Signaling activation and cancer cell invasion. Vimentin when supplemented to the cancer cells remained bound to the surface of the cancer cells. Furthermore, bound vimentin activates Wnt signaling pathway as detectable by increased ß-catenin accumulation in the nucleus with concomitant activation of ß-catenin-dependent transcription of Wnt signaling downstream targets. Functionally, there was an increase in the rate of cellular invasion in these cancer cells upon binding with vimentin. Our results thus suggest that free vimentin in the tumor microenvironment acts as a positive regulator of the ß-catenin signaling pathway, thus providing a basis for cancer invasive properties.

Interleukin-30 (IL27p28) alleviates experimental sepsis by modulating cytokine profile in NKT cells.

BACKGROUND & AIMS: Sepsis is an acute systemic inflammatory response to infection associated with high patient mortality (28-40%). We hypothesized that interleukin (IL)-30, a novel cytokine protecting mice against liver injury resulting from inflammation, would generate a protective effect against systemic inflammation and sepsis-induced death. METHODS: Sepsis was induced by lipopolysaccharide (LPS) or cecal ligation and puncture (CLP). The inhibitory effects of IL-30 on septic inflammation and associated therapeutic effects were determined in wild-type, IL30 (p28)(-/-), IL10(-/-), and CD1d(-/-) mice. RESULTS: Mice treated with pIL30 gene therapy or recombinant IL-30 protein (rIL30) were protected from LPS-induced septic shock or CLP-induced polymicrobial sepsis and showed markedly less liver damage and lymphocyte apoptosis than control septic mice. The resulting reduction in mortality was mediated through attenuation of the systemic pro-inflammatory response and augmentation of bacterial clearance. Mice lacking IL-30 were more sensitive to LPS-induced sepsis. Natural killer-like T cells (NKT) produced much higher levels of IL-10 and lower levels of interferon-gamma and tumor necrosis factor-alpha in IL-30-treated septic mice than in control septic mice. Likewise, deficiency in IL-10 or NKT cells abolished the protective role of IL-30 against sepsis. Furthermore, IL-30 induced IL-10 production in purified and LPS-stimulated NKT cells. Blocking IL-6R or gp130 inhibited IL-30 mediated IL-10 production. CONCLUSIONS: IL-30 is important in modulating production of NKT cytokines and subsequent NKT cell-mediated immune regulation of other cells. Therefore, IL-30 has a role in prevention and treatment of sepsis via modulation of cytokine production by NKT.

Regulation of NKG2D+CD8+ T-cell-mediated antitumor immune surveillance: Identification of a novel CD28 activation-mediated, STAT3 phosphorylation-dependent mechanism.

The natural killer (NK) group 2D (NKG2D) receptor, which displays on mouse and human NK cells, activates CD8+ T cells and small subsets of other T cells. NKG2D+CD8+ T cells play critical roles in both innate and adaptive immunity upon engagement with NKG2D ligands to eliminate tumor and infected cells. Despite the important role of NKG2D+CD8+ T cells in immune surveillance, the mechanisms of how NKG2D expression on CD8+ T cells is regulated remain poorly defined. We treated mouse and human CD8+ T cells with CD80 recombinant protein, plus a pharmacologic model with small molecular inhibitors to determine which signaling pathway leads to NKG2D regulation on CD8+T cells. This study revealed that CD28 activation gives rise to sustained NKG2D expression on both mouse and human CD8+ T cells in a signal transducer and activator of transcription 3 (STAT3) phosphorylation-dependent manner. Further, we found that CD28 activation stimulated sustained activation of the tyrosine kinase Lck, which recruits and triggers Janus kinase/STAT3 signaling to phosphorylate STAT3, and in turn increases NKG2D expression. Moreover, NKG2D induction on CD8+ T cells exerts cytolytic activity against target tumor cells in vitro, as well as significantly improves the antitumor therapeutic effects in vivo in an NKG2D-dependent manner. Taken together, these results elucidated a novel mechanism of NKG2D regulation by phosphorylated STAT3 (pSTAT3) on CD8+ T cells upon CD28 activation. This mechanism may shed light on the effectiveness of CD80-based, NKG2D-dependent antitumor immunotherapy.

The Duality of Fgl2 - Secreted Immune Checkpoint Regulator Versus Membrane-Associated Procoagulant: Therapeutic Potential and Implications.

Fibrinogen-like protein 2 (Fgl2), a member of the fibrinogen family, can be expressed as a membrane-associated protein with coagulation activity or in a secreted form possessing unique immune suppressive functions. The biological importance of Fgl2 is evident within viral-induced fibrin depositing inflammatory diseases and malignancies and provides a compelling rationale for Fgl2 expression to not only be considered as a disease biomarker but also as a therapeutic target. This article will provide a comprehensive review of the currently known biological properties of Fgl2 and clarifies future scientific directives.