Acidity suppresses CD8 + T-cell function by perturbing IL-2, mTORC1, and c-Myc signaling.

CD8 + T cells have critical roles in tumor control, but a range of factors in their microenvironment such as low pH can suppress their function. Here, we demonstrate that acidity restricts T-cell expansion mainly through impairing IL-2 responsiveness, lowers cytokine secretion upon re-activation, and reduces the cytolytic capacity of CD8 + T cells expressing low-affinity TCR. We further find decreased mTORC1 signaling activity and c-Myc levels at low pH. Mechanistically, nuclear/cytoplasmic acidification is linked to mTORC1 suppression in a Rheb-, Akt/TSC2/PRAS40-, GATOR1- and Lkb1/AMPK-independent manner, while c-Myc levels drop due to both decreased transcription and higher levels of proteasome-mediated degradation. In addition, lower intracellular levels of glutamine, glutamate, and aspartate, as well as elevated proline levels are observed with no apparent impact on mTORC1 signaling or c-Myc levels. Overall, we suggest that, due to the broad impact of acidity on CD8 + T cells, multiple interventions will be required to restore T-cell function unless intracellular pH is effectively controlled.

Combining SiRPα decoy-coengineered T cells and antibodies augments macrophage-mediated phagocytosis of tumor cells.

The adoptive transfer of T cell receptor-engineered (TCR-engineered) T cells (ACT) targeting the HLA-A2-restricted cancer-testis epitope NY-ESO-1157-165 (A2/NY) has yielded favorable clinical responses against several cancers. Two approaches to improve ACT are TCR affinity optimization and T cell coengineering to express immunomodulatory molecules that can exploit endogenous immunity. By computational design we previously developed a panel of binding-enhanced A2/NY-TCRs including A97L, which augmented the in vitro function of gene-modified T cells as compared with WT. Here, we demonstrated higher persistence and improved tumor control by A97L-T cells. In order to harness macrophages in tumors, we further coengineered A97L-T cells to secrete a high-affinity signal regulatory protein α (SiRPα) decoy (CV1) that blocks CD47. While CV1-Fc-coengineered A97L-T cells mediated significantly better control of tumor outgrowth and survival in Winn assays, in subcutaneous xenograft models the T cells, coated by CV1-Fc, were depleted. Importantly, there was no phagocytosis of CV1 monomer-coengineered T cells by human macrophages. Moreover, avelumab and cetuximab enhanced macrophage-mediated phagocytosis of tumor cells in vitro in the presence of CV1 and improved tumor control upon coadministration with A97L-T cells. Taken together, our study indicates important clinical promise for harnessing macrophages by combining CV1-coengineered TCR-T cells with targeted antibodies to direct phagocytosis against tumor cells.

Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8+ T cells.

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ tumor-infiltrating lymphocytes away from exhausted effector states remains an elusive goal. Our work provides evidence that orthogonal gene engineering of T cells to secrete an interleukin (IL)-2 variant binding the IL-2Rßγ receptor and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic effector state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host and led-in the absence of lymphodepletion or exogenous cytokine support-to high levels of engraftment and tumor regression. Our work unlocks a new opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors.

Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A.

Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level.

Expression and Purification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA/protein co-expression in order to express and purify RNA by affinity in native condition. As a proof-of-concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution.

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg(2+) drives compaction of the RNA structure and that, in the absence of Mg(2+), SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology.

RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

RNA-methyltransferase TrmA is a dual-specific enzyme responsible for C5-methylation of uridine in both tmRNA and tRNA.

In bacteria, trans-translation rescues stalled ribosomes by the combined action of tmRNA (transfer-mRNA) and its associated protein SmpB. The tmRNA 5' and 3' ends fold into a tRNA-like domain (TLD), which shares structural and functional similarities with tRNAs. As in tRNAs, the UUC sequence of the T-arm of the TLD is post-transcriptionally modified to m (5)UψC. In tRNAs of gram-negative bacteria, formation of m (5)U is catalyzed by the SAM-dependent methyltransferase TrmA, while formation of m (5)U at two different positions in rRNA is catalyzed by distinct site-specific methyltransferases RlmC and RlmD. Here, we show that m (5)U formation in tmRNAs is exclusively due to TrmA and should be considered as a dual-specific enzyme. The evidence comes from the lack of m (5)U in purified tmRNA or TLD variants recovered from an Escherichia coli mutant strain deleted of the trmA gene. Detection of m (5)U in RNA was performed by NMR analysis.

Targeting the proangiogenic VEGF-VEGFR protein-protein interface with drug-like compounds by in silico and in vitro screening.

Protein-protein interactions play a central role in medicine, and their modulation with small organic compounds remains an enormous challenge. Because it has been noted that the macromolecular complexes modulated to date have a relatively pronounced binding cavity at the interface, we decided to perform screening experiments over the vascular endothelial growth factor receptor (VEGFR), a validated target for antiangiogenic treatments with a very flat interface. We focused the study on the VEGFR-1 D2 domain, and 20 active compounds were identified. These small compounds contained a (3-carboxy-2-ureido)thiophen unit and had IC(50) values in the low micromolar range. The most potent compound inhibited the VEGF-induced VEGFR-1 transduction pathways. Our findings suggest that our best hit may be a promising scaffold to probe this macromolecular complex and for the development of treatments of VEGFR-1-dependent diseases.

Tether influence on the binding properties of tRNALys3 ligands designed by a fragment-based approach.

A small library of 1,5-triazole derivatives linking a diaminocyclopentadiol and aromatic ketones has been prepared and screened using NMR and fluorescent techniques against tRNA(Lys)(3), the HIV reverse transcription primer. The comparison of their binding properties to those of their 1,4-triazole isomers, previously discovered in a fragment-based approach, outlines the influence of the linker on affinity and binding selectivity in such an approach.

1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin.

In eubacteria, the formyl group of nascent polypeptides is removed by peptide deformylase protein (PDF). This is the reason why PDF has received special attention in the course of the search for new antibacterial agents. We observed by NMR that actinonin, a natural inhibitor, induced drastic changes in the HSQC spectrum of E. coli PDF. We report here the complete NMR chemical shift assignments of PDF resonances bound to actinonin.

Design of tRNA(Lys)3 ligands: fragment evolution and linker selection guided by NMR spectroscopy.

A fragment-based approach for the synthesis of ligands of tRNA(Lys) (3), the HIV reverse-transcription primer, is described. The use of NMR spectroscopy has proved to be very useful in this approach, not only to detect low-affinity complexes between small compounds and RNA, but also to provide information on their binding mode and on the way they can be connected. This NMR-spectroscopy-guided analysis enabled us to design micromolar ligands after the optimisation and connection of millimolar fragments with an appropriate linker. The influence of the linker region on the binding affinity and selectivity outlines the importance of having a flexible assemblage strategy with a variety of linkers in such an approach.