SUMMARY The antitumor effector T helper 1 (Th1) and Th17 cells represent two T cell paradigms: short-lived cytolytic Th1 cells and “stem cell-like” memory Th17 cells. We report that Th9 cells represent a third paradigm—they are less-exhausted, fully cytolytic, and hyperproliferative. Only tumor-specific Th9 cells completely eradicated advanced tumors, maintained a mature effector cell signature with cytolytic activity as strong as Th1 cells, and persisted as long as Th17 cells in vivo. Th9 cells displayed a unique Pu.1-Traf6-NF-κB activation-driven hyperproliferative feature, suggesting a persistence mechanism rather than an antiapoptotic strategy. Th9 antitumor efficacy depended on interleukin-9 and upregulated expression of Eomes and Traf6. Thus, tumor-specific Th9 cells are a more effective CD4+ T cell subset for adoptive cancer therapy.

CD8+ T cells can be polarized into IL-9–secreting (Tc9) cells. Ma et al. show that Tc9 cell differentiation is associated with a low cholesterol reprogramming profile, and manipulating cholesterol content in polarizing Tc9 cells significantly affects Tc9 differentiation and antitumor response.

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Previous studies have established the link between aberrant microRNA (miRNA) expression and hypoxia in various neoplasms. However, how these hypoxia-related miRNAs modulate tumor progression is still unclear. Therefore, the patterns of miRNA in colorectal carcinoma cell lines in response to hypoxia or not were first screened and the hypoxia-induced repression of the miR-15-16 cluster was confirmed. Then, this repression was found to be associated with high tumor stage and poor prognosis in colorectal carcinoma and is shown to promote tumor angiogenesis and metastasis by the loss of restriction of its target gene, fibroblast growth factor-2 (FGF2). Moreover, the general and alterative promoters of the miR-15-16 host (deleted in lymphocytic leukemia 2, DLEU2) were mapped, and three c-Myc/Max binding sites in response to the hypoxia-induced repression of miR-15-16 were further identified. Finally, an enhanced stability of c-Myc/Max heterodimer promoted by increased hypoxia-inducible factor-2α (HIF-2α) was validated, and we also verified that the enhancement contributed to the hypoxia-induced repression of miR-15-16. In brief, the c-Myc-mediated transcriptional repression of miR-15-16 in hypoxia is induced by increased HIF-2α and promoted tumor angiogenesis and hematogenous metastasis by the further loss of post-transcriptional inhibition of FGF2. Our study provides a better understanding of the coping mechanisms in response to tumor hypoxia and may be helpful in developing an effective prognostic marker or treatment target against solid tumors.

IL-9-producing CD4+ (Th9) cells are a subset of CD4+ T-helper cells that are endowed with powerful antitumor capacity. Both IL-4 and TGF-β have been reported to be indispensable for Th9 cell-priming and differentiation. Here we show, by contrast, that Th9 cell development can occur in the absence of TGF-β signaling. When TGF-β was replaced by IL-1β, the combination of IL-1β and IL-4 efficiently promoted IL-9-producing T cells (Th9IL-4+IL-1β). Th9IL-4+ IL-1β cells are phenotypically distinct T cells compared to classic Th9 cells (Th9IL-4+TGF-β) and other Th cells, and are enriched for IL-1 and NF-κB gene signatures. Inhibition of NF-κB but not TGF-β-signaling negates IL-9 production by Th9IL-4+IL-1β cells. Furthermore, when compared with classic Th9IL-4+TGF-β cells, Th9IL-4+IL-1β cells are less exhausted, exhibit cytotoxic T effector gene signature and tumor killing function, and exert a superior antitumor response in a mouse melanoma model. Our study thus describes an alternative pathway for Th9 cell differentiation and provides a potential avenue for antitumor therapies.

Purpose: The aims of this work were to investigate the antitumor effect of IFNg gene transfer on human nasopharyngeal carcinoma (NPC) and to assess the potential of minicircle vector for antitumor gene therapy. Experimental Design: We developed a recombinant minicircle vector carrying the human IFNg gene and evaluated the effects of minicircle-mediated IFNg gene transfer on NPC cell lines in vitro and on xenografts in vivo.Results: Relative to p2AC31-IFNg, minicircle-mediated IFNg gene transfer in vitro resulted in 19-to 102-fold greater IFNg expression levels in transfected cells (293, NIH 3T3, CNE-1, CNE-2, and C666-1) and inhibited the growth of CNE-1, CNE-2, and C666-1cells more efficiently, reducing relative growth rates to 7.1 F1.6%, 2.7 F 1.0%, and 6.1 F1.6%, respectively. Flow cytometry and caspase-3 activity assays suggested that the antiproliferative effects of IFNg gene transfer on NPC cell lines could be attributed to G 0 -G 1 arrest and apoptosis. Minicircle-mediated intratumoral IFNg expression in vivo was 11 to 14 times higher than p2AC31-IFNg in CNE-2-and C666-1-xenografted mice and lasted for 21 days. Compared with p2AC31-IFNg treatment, minicircle-IFNg treatment significantly increased survival and achieved inhibition rates of 77.5% and 83%, respectively. Conclusions: Our data indicate that IFNg gene transfer exerts antiproliferative effects on NPC cells in vitro and leads to a profound antitumor effect in vivo. Minicircle-IFNg is more efficient than corresponding conventional plasmids due to its capability of mediating long-lasting high levels of IFNg gene expression. Therefore, minicircle-mediated IFNg gene transfer is a promising novel approach in the treatment of NPC.

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A 15 kDa ribonuclease (RNase) was purified from dried fruiting bodies of the wild edible mushroom Armillaria luteo-virens. The simple 4-step purification protocol involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on SP-Sepharose and a final gel filtration by FPLC on Superdex-75. The RNase was unadsorbed on Affi-gel blue gel, but adsorbed on DEAE-cellulose and SP-Sepharose. The N-terminal amino acid sequence of purified RNase was AGVQYKLTILLV, which showed low sequence homology to those of previously reported RNases. The optimal pH and temperature of the enzyme were very close to 4.0 and 70 degrees C, respectively. The enzyme showed considerably high ribonucleolytic activity and broad specificity towards polyhomoribonucleotides, with a specificity of poly(U) > poly(C) > poly (G) > poly(A). The ribonucleolytic activities towards poly(U), poly(C), poly(G) and poly(A) were 279.5, 184.1, 69.9 and 52.3 U/mg, respectively.