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Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade., Competing Interests: The authors declare no conflict of interest.

Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ from intracellular sites and (ii) an effective block of Ca2+ extrusion. The ability of Ba2+ to release Ca2+ in growth cones that are insensitive to caffeine suggests that Ba2+ may displace Ca2+ from binding sites other than endoplasmic reticulum.