Alarmins are endogenous mediators that are elicited rapidly in response to danger signals, enhancing innate and adaptive immune responses by promoting the recruitment and maturation of antigen-presenting cells (APCs). The nucleosome-binding protein HMGN1 is a potent alarmin that binds TLR4 and induces antigen-specific Th1 immune responses, but its contributions to antitumor immunity have not been explored. We found that ovalbumin (OVA)-expressing EG7 mouse thymoma cells grew much faster in Hmgn1-deficient mice than littermate-matched controls. Tumor-bearing Hmgn1-/- mice generated fewer OVA-specific CD8 cells in the spleen than EG7-bearing Hmgn1+/+ mice, suggesting that HMGN1 supported T cell-mediated antitumor immunity. Additionally, EG7 tumors expressing HMGN1grew more slowly than control EG7 tumors, suggesting greater resistance to HMGN1-expressing tumors. This resistance relied on T cell mediated immunity because it was abolished by in vivo depletion of CD4+ and CD8+ T cells. Moreover, Mice vaccinated with a DNA vector expressing an HMGN1-gp100 fusion protein manifested gp100-specific, Th1-polarized immune responses, acquiring resistance to challenge with mouse B16F1 melanoma. Overall, our findings show that HMGN1 contributes to antitumor immunity and it may offer an effective adjuvant to heighten responses to cancer vaccines.

Viruses disrupt the host cell microRNA (miRNA) network to facilitate their replication. Human T-cell leukemia virus type I (HTLV-1) replication relies on the clonal expansion of its host CD4+ and CD8+ T cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4+ phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ–miRNA axis promotes cell proliferation and genetic instability, as indicated by comet assays that showed increased numbers of DNA-strand breaks. Expression profiling of miRNA revealed that infected CD4+ cells, but not CD8+ T cells, overexpressed oncogenic miRNAs, including miR17 and miR21. HBZ activated these miRNAs via a posttranscriptional mechanism. These effects were alleviated by knocking down miR21 or miR17 and by ectopic expression of OBFC2A, a DNA-damage factor that is downregulated by miR17 and miR21 in HTLV-1–infected CD4+ T cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells. Cancer Res; 74(21); 1–12. ©2014 AACR.

Viruses disrupt the host cell microRNA network to facilitate their replication. HTLV-1 replication relies on the clonal expansion of its host CD4+ and CD8+ T-cells, yet this virus causes adult T-cell leukemia/lymphoma (ATLL) that typically has a CD4+ phenotype. The viral oncoprotein Tax, which is rarely expressed in ATLL cells, has long been recognized for its involvement in tumor initiation by promoting cell proliferation, genetic instability, and miRNA dysregulation. Meanwhile, HBZ is expressed in both untransformed infected cells and ATLL cells, and is involved in sustaining cell proliferation and silencing virus expression. Here, we show that an HBZ/miRNA axis promotes cell proliferation and genetic instability as indicated by comet assays that showed increased numbers of DNA strand breaks. Expression profiling of miRNA revealed that infected CD4+ cells, but not CD8+ T-cells, overexpressed oncogenic miRNAs, including miR-17 and miR-21. HBZ activated these miRNAs via a post-transcriptional mechanism. These effects were alleviated by knocking down miR-21 or miR-17 and by ectopic expression of OBFC2A, a DNA damage factor that is down-regulated by miR-17 and miR-21 in HTLV-1 infected CD4+ T-cells. These findings extend the oncogenic potential of HBZ and suggest that viral expression might be involved in the remarkable genetic instability of ATLL cells.

T cells of the T helper (Th)17 subset offer promise in adoptive T-cell therapy for cancer. However, current protocols for ex vivo programming of Th17 cells, which include TGFβ exposure, increase the expression of CD39 and CD73, two cell surface ATP ectonucleotidases that reduce T-cell effector functions and promote immunosuppression. Here, we report that ATP-mediated suppression of IFNγ production by Th17 cells can be overcome by genetic ablation of CD73 or by using IL1β instead of TGFβ to program Th17 cells ex vivo. Th17 cells cultured in IL1β were also highly polyfunctional, expressing high levels of effector molecules and exhibiting superior short-term control of melanoma in mice, despite reduced stem cell-like properties. TGFβ addition at low doses that did not upregulate CD73 expression but induced stemness properties drastically improved the antitumor effects of IL1β-cultured Th17 cells. Effector properties of IL1β-dependent Th17 cells were likely related to their high glycolytic capacity, since ex vivo programming in pyruvate impaired glycolysis and antitumor effects. Overall, we show that including TGFβ in ex vivo cultures used to program Th17 cells blunts their immunotherapeutic potential and demonstrate how this potential can be more fully realized for adoptive T-cell therapy. Cancer Res; 74(21); 1–12. ©2014 AACR.

T helper (Th)-17 subsets hold promise in adoptive T cell transfer therapy for cancer. However, ex vivo programming of Th17 cells in presence of TGF-β increases cell surface expression of ectonucleotidases CD39 and CD73, that in turn increases susceptibility to immunosuppression and reduces effector functions. Our data shows that ATP mediated suppression of IFN-γ production by Th17 cells can be overcome either by genetic ablation of CD73 or by generating TGF-β independent Th17 in presence of IL-1β. Th17 cells cultured in IL-1β are also highly polyfunctional, express high level of effector molecules and exhibit better short-term control of B16-F10 murine melanoma, despite reduced stem cell like properties. Adding TGF-β at low dose that does not up regulate CD73 expression, but induces stemness, drastically improves anti-tumor function of IL-1β cultured Th17 cells. It is likely that effector property of IL-1β dependent Th17 is due to their high glycolytic capacity, since generating IL-1β dependent Th17 cells in pyruvate containing media impaired glycolysis and its anti-tumor potential. Thus, our data suggests that due to induction of ectonucleotidase expression by TGF-β, ex vivo culture conditions for generating Th17 cells need to be reconsidered for exploiting their full potential in adoptive T cell therapy.