Mice that lack IL-15 or the IL-15R α-chain (IL-15Rα) are deficient in peripheral CD8+, but not in CD4+, T cells. This CD8+ T cell-specific deficiency has now been investigated further by characterization of a new strain of IL-15Rα−/− mice. The adult mutant mice exhibited a specific reduction in the percentage of CD8-single positive TCRhigh thymocytes. The expression of Bcl-2 was reduced in both CD8+ thymocytes and naive T cells of the mutant animals, and the susceptibility of these cells to death was increased. Memory CD8+ cells were profoundly deficient in IL-15Rα−/−mice, and the residual memory-like CD8+ cells contained a high percentage of dead cells and failed to up-regulate Bcl-2 expression compared with naive CD8+ cells. Moreover, exogenous IL-15 both up-regulated the level of Bcl-2 in and reduced the death rate of wild-type and mutant CD8+ T cells activated in vitro. These results indicate that IL-15 and IL-15Rα regulate the expression of Bcl-2 in CD8+ T cells at all developmental stages. The reduced Bcl-2 content in CD8+ cells might result in survival defect and contribute to the reduction of CD8+ cells in IL-15Rα−/−mice.

Programmed necrosis, necroptosis, is considered to be a highly immunogenic activity, often mediated via the release of damage-associated molecular patterns (DAMPs). Interestingly, enhanced macroautophagic/autophagic activity is often found to be accompanied by necroptosis. However, the possible role of autophagy in the immunogenicity of necroptotic death remains largely obscure. In this study, we investigated the possible mechanistic correlation between phytochemical shikonin-induced autophagy and the shikonin-induced necroptosis for tumor immunogenicity. We show that shikonin can instigate RIPK1 (receptor [TNFRSF]-interacting serine-threonine kinase 1)- and RIPK3 (receptor-interacting serine-threonine kinase 3)-dependent necroptosis that is accompanied by enhanced autophagy. Shikonin-induced autophagy can directly contribute to DAMP upregulation. Counterintuitively, among the released and ectoDAMPs, only the latter were shown to be able to activate the cocultured dendritic cells (DCs). Interruption of autophagic flux via chloroquine further upregulated ectoDAMP activity and resultant DC activation. For potential clinical application, DC vaccine preparations treated with tumor cells that were already pretreated with chloroquine and shikonin further enhanced the antimetastatic activity of 4T1 tumors and reduced the effective dosage of doxorubicin. The enhanced immunogenicity and vaccine efficacy obtained via shikonin and chloroquine cotreatment of tumor cells may thus constitute a compelling strategy for developing cancer vaccines via the use of a combinational drug treatment.

In this study, we investigated the functional outcomes of CD4

Antivascular immunity may provide long-term protection by preventing neovascularization that precedes tumor progression. Although the tumorigenesis promoted by EBV-encoded oncogene latent membrane protein 1 derived from Taiwanese nasopharyngeal carcinoma (N-LMP1) has been demonstrated, the potential of N-LMP1 for inducing immune surveillance remains elusive. In this article, we describe the immunogenicity of N-LMP1 (1510) and its induction of antivascular immunity in a transplantable tumor model in immunocompetent BALB/c mice. The immunogenicity of N-LMP1 was evaluated on the basis of tumor rejection following immunization. The impact of the immunization on the dynamics of tumor angiogenesis was assessed by temporal noninvasive dynamic contrast-enhanced magnetic resonance imaging and was further confirmed by histologic study and vascular count. Through the experiments of in vivo depletion and adoptive transfer, CD4 T cells were identified as effectors that depend on IFN-γ for tumor prevention. The response was further verified by the identification of an MHC H-2 I-Ed–restricted peptide derived from N-LMP1 and by the immunization of mice with N-LMP1 peptide–loaded dendritic cells. These studies provide insight into N-LMP1–specific immunity in vivo, which suggests that CD4 T cells may play an important role in angiogenic surveillance against LMP1–associated cancer via tumor stroma targeting.

Introduction Metabolic reprogramming is one of the hallmarks of cancer, to support their needs for massive growth and proliferation. One major metabolic reprogramming is from oxidative phosphorylation to aerobic glycolysis, a well-documented phenomenon known as the Warburg effect. A key enzyme in this process is hexokinase 2 (HK2), which catalyses the first step of glucose metabolism and is overexpressed in many cancer types. Unlike HK1, which is ubiquitously expressed in normal cells, HK2 is required for cancer initiation and transformation even though their catalytic activity is highly similar. HK2 in cancer cells is attached to the outer mitochondrial membrane via the VDAC1 channel. VDAC1/HK2 association blocks pro-apoptotic signals, is less sensitive to feedback inhibition by the HK product, glucose-6-phosphate, as well as allows a continuous flux of mitochondrial ATP to HK, leading to apoptosis prevention and a high rate of glycolysis.Temporal high HK2 expression, and binding to VDAC, is also found in a variety of activated immune cells to support their changing metabolic needs. Detachment of HK2 from VDAC1 in activated immune cells leads to a range of responses ranging from glycolysis inhibition, NLRP3-mediated inflammasome activation, and metabolic reprogramming to activate immune pathways. Material and methods A novel small molecule VDAC/HK2 modulator, VDA-1102, is being developed as a bi-functional drug for the treatment of solid tumours -triggering apoptosis in cancer cells while simultaneously enhancing an immunemediated anti-tumour response by regulating immune cell metabolism. Results and discussions In vitro studies established that VDA-1102 selectively detaches HK2, but not HK1, from VDAC1 leading to cancer cell apoptosis, glycolysis inhibition, and prevention of cancer cell proliferation. In vivo efficacy studies demonstrated significant tumour growth delay and prolongation of survival in syngeneic solid tumour models. Analysis of tumor-associated macrophages in vivo indicated a treatmentinduced change in these macrophage phenotype from M2 to M1.

TCRγδ+ cells are enriched in the intestine mucosa and constitute approximately half of the intestinal intraepithelial lymphocytes (iIEL) in mice. They are likely activated by self and foreign Ags in situ, but little is known about how the activated γδ iIEL are regulated. In the iIEL compartment, IL-2 is produced by activated TCRαβ+ iIEL, and IL-15 message is detected in iIEL and in the epithelial cells. We found surface expression of IL-2 as well as IL-15Rs on activated γδ iIEL, and examined the effects of IL-2 and IL-15 on the survival and death of γδ iIEL during secondary stimulation through TCR. We found that both cytokines supported growth of the restimulated γδ iIEL, but exerted different effects on their survival. A significant higher number of live cells were recovered from the γδ iIEL cultures restimulated in IL-15 than in IL-2. Quantitation of apoptotic cells showed more cell death in the IL-2 group than in the IL-15 group. The cell death was associated with restimulation through TCR and was not caused by insufficient growth factor, thus representing activation-induced cell death. Western blot analyses found no difference in the levels of Bcl-2 and Bax proteins between the two groups. However, the level of Bcl-xL protein diminished with time in the IL-2 group whereas the level was sustained in the IL-15 group, which may contribute to the pro-survival effect of IL-15. These results demonstrated that the survival of activated γδ iIEL is differentially regulated by IL-2 and IL-15.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine. We have generated a novel codon optimized murine GM-CSF sequences as an adjuvant. The codon optimized murine GM-CSF plasmids significantly increased GM-CSF expression levels in all cells tested, including murine, monkey, and human cell lines. Here we show that coadministration of plasmids encoding the codon optimized GM-CSF sequence with the HPV-16 E7 DNA vaccine resulted in complete tumor regression in 100% of the C57BL/6 mice tested. Consistent with these findings, coadministarton of these plasmids elicited E7-specific CTL responses and a protective immune response against challenge of E7- expressing TC-1 tumors in mice. Histological analysis revealed that the tumor destruction occurred at the margins of the tumor, where there was a heavy lymphocytic infiltrate. We also created a TC-1 tumors expressing E7 and GM-CSF genes. Combining a GM-CSF secreting TC-1 vaccine with a systemic DNA vaccination successfully treated >90% of the mice bearing an established tumor but showed no resistance to an unrelated ovarian cancer. This novel codon optimized GM-CSF plasmid may prove a practical molecular strategy for overcoming this limitation of DNA vaccines and potentiating immune responses to vaccines as well as other immunotherapeutic strategies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3833.

Tumor immunogenicity can be characterized by the exposure of damage‐associated molecular patterns (DAMPs) on cell surface, which can recruit dendritic cells (DCs) and enhance DC maturation. Recent studies have demonstrated that chemotherapy‐induced autophagy may positively regulate immunogenic cell death (ICD). We recently showed that shikonin (SHK) can confer anti‐tumor activity and be employed as an adjuvant of DC‐based tumor vaccine via the induction of ICD. In this study, we investigated possible mechanistic correlation between SHK‐induced autophagy and SHK‐induced ICD for tumor immunogenicity. SHK enhanced ROS‐related cell death and increased the exposure of DAMP proteins on cell surface of test cells. SHK‐treated tumor cells activated the phagocytosis activity and the release of IL‐1β and IL‐6 from dendritic cells. SHK‐treated tumor cells as tumor vaccine reduced tumor growth and prolonged the survival of test mice. SHK reduced the proteasome activity to result in accumulating ubiquitinylated proteins and skewed to induce autophagy formation in tumor cells. The aggregation of SHK‐induced, ubiquitinylated proteins were found to co‐localize with p62 protein. Blockage of autolysosome formation via chloroquine (CQ) in SHK‐treated tumor cells further enhanced the expression of DAMPs on tumor cell surface. In addition, CQ and SHK cotreated tumor cells further enhanced the phagocytosis activity and CD40 and TLR4 expression of DC. The much enhanced immunogenicity and efficacy of SHK and CQ may create a compelling strategy, i.e., via the use of a combinational treatment, for developing DC‐based cancer vaccines.