fit of the cytokine treatment in mice with RMA-S tumors was completely abrogated if the mice were NK-depleted, demonstrating that the effect of the cytokines depended on NK cells ( Figure 1C).The efficacy of cytokine treatments in mice bearing RMA-S tumors did not apply to mice bearing RMA tumors, which are similar to RMA-S cells except that they express high amounts of MHC class I and are therefore resistant to NK cells ( Figure 1B, bottom panel). The survival time in mice with RMA tumors did not change when the mice were treated with cytokines, and was similarly rapid to that in untreated mice with RMA-S tumors.Recently, Levin and colleagues described the "superkine" H9, an engineered version of IL-2, which functions independently of the α chain (CD25) of the IL-2 receptor. Compared with WT IL-2, H9 exhibits much more activity on NK cells and T cells. In vivo, H9 stimulated rejection of B16F10 melanoma tumors in B6 mice (13), but the role of NK cells in rejection has not been investigated. We tested whether H9 induces NK-dependent rejection of MHC class I-deficient tumors by implanting high doses of RMA-S or RMA cells and initiating H9 treatment after 7 days. Similar to the results with IL-12+IL-18 treatment, H9 resulted in improved survival of RMA-S-bearing mice, but had no effect in RMA-bearing mice (Figure 2, A and B). Notably, when mice were depleted of NK cells, the efficacy of H9 treatment was abolished (Figure 2A). These results show that H9 exerts its antitumor effect against MHC class I-deficient tumor cells in an NK cell-dependent fash-

Structured RNAs embedded in the untranslated regions (UTRs) of messenger RNAs can regulate gene expression. In bacteria, control of a metabolite gene is mediated by the self-cleaving activity of a ribozyme embedded in its 5′ UTR 1 . This discovery has raised the question of whether generegulating ribozymes also exist in eukaryotic mRNAs. Here we show that highly active hammerhead ribozymes 2,3 are present in the 3′ UTRs of rodent C-type lectin type II (Clec2) genes 4-7 . Using a hammerhead RNA motif search with relaxed delimitation of the non-conserved regions, we detected ribozyme sequences in which the invariant regions, in contrast to the previously identified continuous hammerheads 8-10 , occur as two fragments separated by hundreds of nucleotides. Notably, a fragment pair can assemble to form an active hammerhead ribozyme structure between the translation termination and the poly-adenylation signals within the 3′ UTR. We demonstrate that this hammerhead structure can self-cleave both in vitro and in vivo, and is able to reduce protein expression in mouse cells. These results indicate that an unrecognized mechanism of posttranscriptional gene regulation involving association of discontinuous ribozyme sequences within an mRNA may be modulating the expression of several CLEC2 proteins that function in bone remodelling and the immune response of several mammals.The hammerhead ribozyme is a small, self-cleaving motif composed of a three-helical junction with a core of invariant nucleotides required for activity. To identify hammerhead ribozymes in mammalian mRNAs, we searched mRNA sequence databases using a pattern descriptor that allowed for insertions of up to 5,000 nucleotides at the ends of stem 1 or stem 3 ( Supplementary Fig. 1) 11,12 . Three hammerhead ribozymes were identified in the 3′ UTRs of known rodent mRNAs. Two are found embedded in the transcripts of mouse Clec2d (osteoclast inhibitory lectin, also known as Ocil, Clr-b and Clec2d8) 13 and its paralogue Clec2e (also known as Clra and Clec2d7) 14 , genes that belong to a group of phylogenetically related sequences within the natural killer receptor gene complex of chromosome 6. The third ribozyme is found in rat CLEC2D11 (ref. 7)-a homologue of mouse Clec2d-which resides in the syntenic natural killer receptor gene complex region on chromosome 4. We extended our search to the genomic sequences of other organisms using the UCSC genome browser's comparative genomics tool 15 . Alignments using the natural killer receptor gene complex regions of mouse and rat led to the identification of nine candidate hammerhead ribozymes: four in the 3′ regions of predicted rat, horse and platypus Clec2-like genes, and five in the unannotated regions of five other mammalian genomes ( Supplementary Fig. 2 Unlike most known self-cleaving RNA motifs that are contiguous 8-10,16-19 , the hammerhead ribozymes identified here (referred to as CLEC2 ribozymes) are split into two fragments separated by a long ribozyme-unrelated insertion in the stem-1-capping loop. Th...

This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The ''ratchet model,'' based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes.ratchet ͉ tRNA ͉ hybrid state R ibosomes, the ribonucleoprotein complexes responsible for protein synthesis, are always found to be composed of two subunits (1), called 30S and 50S in bacteria and archaea and 40S and 60S in eukarya. The underlying reason for this two-subunit architecture was addressed by Spirin (2) and Bretscher (3), who independently proposed the involvement of intersubunit movement in translocation of tRNAs and mRNA through the ribosome. Bretscher (3) depicted translocation as a two-step process involving an intermediate state in which the ends of the peptidyltRNA are bound to different sites on the two ribosomal subunits. Twenty years later, direct experimental evidence for hybrid states of tRNA binding as intermediates in translocation came from chemical probing studies (4). According to the hybridstates model, translocation involves independent, sequential movement of the two extremities of tRNA. In the first step, after peptide bond formation, the acceptor ends of the A-and P-site tRNAs move into the 50S P and E sites, respectively, forming the A/P and P/E hybrid states. In the second step, whose catalysis requires elongation factor (EF)-G-dependent GTP hydrolysis, the anticodon ends of the tRNAs move from the 30S A and P sites into the P and E sites, coupled with movement of the mRNA. Implicit in this mechanism was the suggestion of relative movement of the 30S and 50S subunits, in which the classical and hybrid states correspond to two different conformational states of the ribosome (4, 5), recalling the proposals of Spirin (2) and Bretscher (3).More recently, cryo-EM reconstructions have provided direct visualization of trapped tRNA-ribosome complexes, in which the ribosomal subunits have an altered rotational orientation compared with that of the classical state (6, 7). Vacant ribosomes, or ones containing peptidyl-tRNA bound to the P site, had similar subunit orientations and tRNA binding states to those observed crystallographically for classical-state ribosomes (8). In complexes containing the translocational EF-G bound with either a nonhydrolyzable GTP anal...

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.