Cold-shock proteome of myoblasts reveals role of RBM3 in promotion of mitochondrial metabolism and myoblast differentiation.

Adaptation to hypothermia is important for skeletal muscle cells under physiological stress and is used for therapeutic hypothermia (mild hypothermia at 32 °C). We show that hypothermic preconditioning at 32 °C for 72 hours improves the differentiation of skeletal muscle myoblasts using both C2C12 and primary myoblasts isolated from 3 month and 18-month-old mice. We analyzed the cold-shock proteome of myoblasts exposed to hypothermia (32 °C for 6 and 48 h) and identified significant changes in pathways related to RNA processing and central carbon, fatty acid, and redox metabolism. The analysis revealed that levels of the cold-shock protein RBM3, an RNA-binding protein, increases with both acute and chronic exposure to hypothermic stress, and is necessary for the enhanced differentiation and maintenance of mitochondrial metabolism. We also show that overexpression of RBM3 at 37 °C is sufficient to promote mitochondrial metabolism, cellular proliferation, and differentiation of C2C12 and primary myoblasts. Proteomic analysis of C2C12 myoblasts overexpressing RBM3 show significant enrichment of pathways involved in fatty acid metabolism, RNA metabolism and the electron transport chain. Overall, we show that the cold-shock protein RBM3 is a critical factor that can be used for controlling the metabolic network of myoblasts.

Tumor membrane-based vaccine immunotherapy in combination with anti-CTLA-4 antibody confers protection against immune checkpoint resistant murine triple-negative breast cancer.

Triple-negative breast cancer (TNBC) afflicts women at a younger age than other breast cancers and is associated with a worse clinical outcome. This poor clinical outcome is attributed to a lack of defined targets and patient-to-patient heterogeneity in target antigens and immune responses. To address such heterogeneity, we tested the efficacy of a personalized vaccination approach for the treatment of TNBC using the 4T1 murine TNBC model. We isolated tumor membrane vesicles (TMVs) from homogenized 4T1 tumor tissue and incorporated glycosyl phosphatidylinositol (GPI)-anchored forms of the immunostimulatory B7-1 (CD80) and IL-12 molecules onto these TMVs to make a TMV vaccine. Tumor-bearing mice were then administered with the TMV vaccine either alone or in combination with immune checkpoint inhibitors. We show that TMV-based vaccine immunotherapy in combination with anti-CTLA-4 mAb treatment upregulated immunomodulatory cytokines in the plasma, significantly improved survival, and reduced pulmonary metastasis in mice compared to either therapy alone. The depletion of CD8+ T cells, but not CD4+ T cells, resulted in the loss of efficacy. This suggests that the vaccine acts via tumor-specific CD8+ T cell immunity. These results suggest TMV vaccine immunotherapy as a potential enhancer of immune checkpoint inhibitor therapies for metastatic triple-negative breast cancer.

Plasma membrane vesicles decorated with glycolipid-anchored antigens and adjuvants via protein transfer as an antigen delivery platform for inhibition of tumor growth.

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity.

Cancer CARtography: charting out a new approach to cancer immunotherapy.

Evaluation of: Davila ML, Riviere I, Wang X et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 6(224), 224ra25 (2014). Recently, chimeric antigen receptor (CAR) T-cell immunotherapy has entered clinical trials in patients with relapsed or refractory B-cell acute lymphoblastic leukemia. 19-28z CAR T cells express a fusion protein comprised of an anti-CD19 mAb fused with CD28 costimulatory and CD3-zeta-chain signaling domains. The current paper demonstrates that administration of 19-28z CAR T cells in patients with relapsed or refractory B-ALL in a Phase I clinical trial has led to 88% of patients undergoing complete remission. Despite the benefits, CAR T-cell therapy is associated with cytokine release syndrome toxicities. The authors demonstrated criteria to diagnose severe cytokine release syndrome (sCRS) and treated sCRS with either high-dose steroids or with tocilizumab, an IL-6 receptor-specific mAb. Although both alleviated sCRS, steroid treatment negated the beneficial effects of CAR T-cell therapy, whereas tocilizumab did not. Taken together, CAR T-cell immunotherapy can be used as a safe and effective approach against tumors with known tumor-associated antigens.