Efficient Regeneration of Human Vα24+ Invariant Natural Killer T Cells and Their Anti-Tumor Activity In Vivo.

Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24+ iNKT) cells, which have been shown to act as cellular adjuvants and thus exert anti-tumor activity in mice and humans, and to re-differentiate the iNKT cell-derived iPSCs into functional iNKT cells. These iPSC-derived iNKT cells (iPS-Vα24+ iNKT cells) can be activated by ligand-pulsed dendritic cells (DCs) and produce a large amount of interferon-γ upon activation, as much as parental Vα24+ iNKT cells, but exhibit even better cytotoxic activity against various tumor cell lines. The iPS-Vα24+ iNKT cells possess significant anti-tumor activity in tumor-bearing mice and can activate autologous NK cells upon activation by ligand-pulsed DCs in the NOG mouse model in vivo, further extending their therapeutic potential. This study thus provides a first proof of concept for the clinical application of human iPS-Vα24+ iNKT cells for cancer immunotherapy. Stem Cells 2016;34:2852-2860.

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone-2 exposure in Xenopus laevis from embryo to juvenile.

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone-2 (BP-2). X. laevis embryos were exposed in flow-through conditions to 0, 1.5, 3.0 or 6.0 mg l-1 BP-2 until 2 months post-metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l-1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration-dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP-2-induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l-1 ) and 100% of the genetic males in the 3.0 and 6.0 mg l-1 treatments experiencing complete male-to-female sex reversal. Concentration-dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen-responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.

Granzyme A Stimulates pDCs to Promote Adaptive Immunity via Induction of Type I IFN.

Granzyme A (GzmA), together with perforin, are well-known for their cytotoxic activity against tumor or virus-infected cells. In addition to this cytotoxic function, GzmA stimulates several immune cell types and induces inflammation in the absence of perforin, however, its effect on the dendritic cell (DC) is unknown. In the current study, we showed that recombinant GzmA induced the phenotypic maturation of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), but not their apoptosis. Particularly, GzmA made pDCs more functional, thus leading to production of type I interferon (IFN) via the TLR9-MyD88 pathway. We also demonstrated that GzmA binds TLR9 and co-localizes with it in endosomes. When co-administered with antigen, GzmA acted as a powerful adjuvant for eliciting antigen-specific cytotoxic CD8+ T lymphocytes (CTLs) that protected mice from tumor challenge. The induction of CTL was completely abolished in XCR1+ DC-depleted mice, whereas it was reduced to less than half in pDC-depleted or IFN-α/ß receptor knockout mice. Thus, CTL cross-priming was dependent on XCR1+cDC and also type I IFN, which was produced by GzmA-activated pDCs. These results indicate that GzmA -stimulated pDCs enhance the cross-priming activity of cDCs in situ. We also showed that the adjuvant effect of GzmA is superior to CpG-ODN and LPS. Our findings highlight the ability of GzmA to bridge innate and adaptive immune responses via pDC help and suggest that GzmA may be useful as a vaccine adjuvant.

In vivo dendritic cell targeting cellular vaccine induces CD4+ Tfh cell-dependent antibody against influenza virus.

An induction of long-term cellular and humoral immunity is for the goal of vaccines, but the combination of antigens and adjuvant remain unclear. Here, we show, using a cellular vaccine carrying foreign protein antigen plus iNKT cell glycolipid antigen, designated as artificial adjuvant vector cells (aAVCs), that mature XCR1- DCs in situ elicit not only ordinal antigen-specific CD4+T cells, but also CD4+ Tfh and germinal center, resulted in inducing long-term antibody production. As a mechanism for leading the long-term antibody production by aAVC, memory CD4+ Tfh cells but not iNKTfh cells played an important role in a Bcl6 dependent manner. To develop it for influenza infection, we established influenza hemagglutinin-carrying aAVC (aAVC-HA) and found that all the mice vaccinated with aAVC-HA were protected from life-threatening influenza infection. Thus, the in vivo DC targeting therapy by aAVC would be useful for protection against viral infection.

Enhanced responses of the chorda tympani nerve to sugars in the ventromedial hypothalamic obese rat.

Sweet taste sensitivity in obese rats with lesions of the ventromedial hypothalamus (VMH) was studied by examining chorda tympani nerve responses to various taste stimuli including sugars. In the early progressive phase of obesity (2 wk after creating VMH lesions), there was no significant difference in the nerve responses to any taste stimulus between sham-operated and VMH-lesioned rats. In contrast, in the late phase of obesity (15-18 wk after VMH lesions), the magnitude of responses to sugars (except for fructose) was prominently greater than that in age-matched controls. High-fat diet-induced obese rats and streptozotocin-diabetic rats also showed greater chorda tympani nerve responses to sugars as was observed in VMH-lesioned obese rats, indicating that VMH lesions might not be specifically related to the enhanced gustatory neural responses to sugars. Although it has been demonstrated that the enhanced responses of the chorda tympani nerve to sugars in genetically diabetic db/db mice is largely attributable to the lack of the direct suppressive effect of leptin on the taste receptor cells, plasma leptin levels were not correlated with the changes in chorda tympani responsiveness to sugars in these models of obesity and diabetes. Accordingly, our results suggest that some chronic factors, including high blood glucose, inefficiency of insulin action, or leptin resistance may be related to the enhancement of chorda tympani nerve responses to sugars.