Activation of the transcription factor NFAT5 in the tumor microenvironment enforces CD8+ T cell exhaustion.

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. Here we found that NFAT5, an NFAT family transcription factor that lacks an AP-1 docking site, was highly expressed in exhausted CD8+ T cells in the context of chronic infections and tumors but was selectively required in tumor-induced CD8+ T cell exhaustion. Overexpression of NFAT5 in CD8+ T cells reduced tumor control, while deletion of NFAT5 improved tumor control by promoting the accumulation of tumor-specific CD8+ T cells that had reduced expression of the exhaustion-associated proteins TOX and PD-1 and produced more cytokines, such as IFNÉ£ and TNF, than cells with wild-type levels of NFAT5, specifically in the precursor exhausted PD-1+TCF1+TIM-3-CD8+ T cell population. NFAT5 did not promote T cell exhaustion during chronic infection with clone 13 of lymphocytic choriomeningitis virus. Expression of NFAT5 was induced by TCR triggering, but its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. Thus, NFAT5 promoted the exhaustion of CD8+ T cells in a tumor-selective fashion.

Structure-based optimization of type III indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors.

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC50 values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design.

1,2,3-Triazoles as inhibitors of indoleamine 2,3-dioxygenase 2 (IDO2).

Indoleamine 2,3-dioxygenase 2 (IDO2) is a potential therapeutic target for the treatment of diseases that involve immune escape such as cancer. In contrast to IDO1, only a very limited number of inhibitors have been described for IDO2 due to inherent difficulties in expressing and purifying a functionally active, soluble form of the enzyme. Starting from our previously discovered highly efficient 4-aryl-1,2,3-triazole IDO1 inhibitor scaffold, we used computational structure-based methods to design inhibitors of IDO2 which we then tested in cellular assays. Our approach yielded low molecular weight inhibitors of IDO2, the most active displaying an IC50 value of 51µM for mIDO2, and twofold selectivity over hIDO1. These compounds could be useful as molecular probes to investigate the biological role of IDO2, and could inspire the design of new IDO2 inhibitors.

Telomerase inhibitors from cyanobacteria: isolation and synthesis of sulfoquinovosyl diacylglycerols from Microcystis aeruguinosa PCC 7806.

By using the Telospot assay, 27 different extracts of cyanobacteria were evaluated for telomerase inhibition. All extracts showed varying, but significant activity. We selected Microcystis aeruguinosa PCC 7806 to identify the active compound and a bioassay guided fractionation led us to isolate mixtures of sulfoquinovosyl diacylglycerols (SQDGs), which were identified by 2D NMR and MS/MS experiments. Pure SQDG derivatives were then synthesized. The IC(50) values of pure synthetic sulfoquinovosyl dipalmitoylglycerol and the monopalmitoylated derivative against telomerase were determined to be 17 and 40 µM, respectively. A structure-activity relationship study allowed the identification of compounds with modified lipophilic acyl groups that display improved activity.

A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor.

Vectors that facilitate the engineering of T cells that can better harness endogenous immunity and overcome suppressive barriers in the tumour microenvironment would help improve the safety and efficacy of T-cell therapies for more patients. Here we report the design, production and applicability, in T-cell engineering, of a lentiviral vector leveraging an antisense configuration and comprising a promoter driving the constitutive expression of a tumour-directed receptor and a second promoter enabling the efficient activation-inducible expression of a genetic payload. The vector allows for the delivery of a variety of genes to human T cells, as we show for interleukin-2 and a microRNA-based short hairpin RNA for the knockdown of the gene coding for haematopoietic progenitor kinase 1, a negative regulator of T-cell-receptor signalling. We also show that a gene encoded under an activation-inducible promoter is specifically expressed by tumour-redirected T cells on encountering a target antigen in the tumour microenvironment. The single two-gene-encoding vector can be produced at high titres under an optimized protocol adaptable to good manufacturing practices.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs.

T cell receptor (TCR) technologies, including repertoire analyses and T cell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from T cell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

The non-coding RNA TERRA is a natural ligand and direct inhibitor of human telomerase.

Telomeres, the physical ends of eukaryotes chromosomes are transcribed into telomeric repeat containing RNA (TERRA), a large non-coding RNA of unknown function, which forms an integral part of telomeric heterochromatin. TERRA molecules resemble in sequence the telomeric DNA substrate as they contain 5'-UUAGGG-3' repeats near their 3'-end which are complementary to the template sequence of telomerase RNA. Here we demonstrate that endogenous TERRA is bound to human telomerase in cell extracts. Using in vitro reconstituted telomerase and synthetic TERRA molecules we demonstrate that the 5'-UUAGGG-3' repeats of TERRA base pair with the RNA template of the telomerase RNA moiety (TR). In addition TERRA contacts the telomerase reverse transcriptase (TERT) protein subunit independently of hTR. In vitro studies further demonstrate that TERRA is not used as a telomerase substrate. Instead, TERRA acts as a potent competitive inhibitor for telomeric DNA in addition to exerting an uncompetitive mode of inhibition. Our data identify TERRA as a telomerase ligand and natural direct inhibitor of human telomerase. Telomerase regulation by the telomere substrate may be mediated via its transcription.

A cell-based phenotypic library selection and screening approach for the de novo discovery of novel functional chimeric antigen receptors.

Anti-tumor therapies that seek to exploit and redirect the cytotoxic killing and effector potential of autologous or syngeneic T cells have shown extraordinary promise and efficacy in certain clinical settings. Such cells, when engineered to express synthetic chimeric antigen receptors (CARs) acquire novel targeting and activation properties which are governed and orchestrated by, typically, antibody fragments specific for a tumor antigen of interest. However, it is becoming increasingly apparent that not all antibodies are equal in this regard, with a growing appreciation that 'optimal' CAR performance requires a consideration of multiple structural and contextual parameters. Thus, antibodies raised by classical approaches and intended for other applications often perform poorly or not at all when repurposed as CARs. With this in mind, we have explored the potential of an in vitro phenotypic CAR library discovery approach that tightly associates antibody-driven bridging of tumor and effector T cells with an informative and functionally relevant CAR activation reporter signal. Critically, we demonstrate the utility of this enrichment methodology for 'real world' de novo discovery by isolating several novel anti-mesothelin CAR-active scFv candidates.

VEGFR-2 redirected CAR-T cells are functionally impaired by soluble VEGF-A competition for receptor binding.

BACKGROUND: The adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101. METHODS: Primary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups. RESULTS: In line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1+ Ki67- and LAG-3+ Ki67- CAR-T cells in the TME. CONCLUSIONS: This study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function.

Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays an essential role in immunity, neuronal function, and aging through catalysis of the rate-limiting step in the kynurenine pathway of tryptophan metabolism. Many IDO1 inhibitors with different chemotypes have been developed, mainly targeted for use in anti-cancer immunotherapy. Lead optimization of direct heme iron-binding inhibitors has proven difficult due to the remarkable selectivity and sensitivity of the heme-ligand interactions. Here, we present experimental data for a set of closely related small azole compounds with more than 4 orders of magnitude differences in their inhibitory activities, ranging from millimolar to nanomolar levels. We investigate and rationalize their activities based on structural data, molecular dynamics simulations, and density functional theory calculations. Our results not only expand the presently known four confirmed chemotypes of sub-micromolar heme binding IDO1 inhibitors by two additional scaffolds but also provide a model to predict the activities of novel scaffolds.

A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CAR-T-cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ- and CD28-containing endodomain signaling chain. We tested STOP-CAR-T cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CAR-T cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CAR-T cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies.

Author Correction: A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Protein RNA and protein protein interactions mediate association of human EST1A/SMG6 with telomerase.

The human EST1A/SMG6 polypeptide physically interacts with the chromosome end replication enzyme telomerase. In an attempt to better understand hEST1A function, we have started to dissect the molecular interactions between hEST1A and telomerase. Here, we demonstrate that the interaction between hEST1A and telomerase is mediated by protein-RNA and protein-protein contacts. We identify a domain within hEST1A that binds the telomerase RNA moiety hTR while full-length hEST1A establishes in addition RNase-resistant and hTR-independent protein-protein contacts with the human telomerase reverse transcriptase polypeptide (TERT). Conversely, within hTERT, we identify a hEST1A interaction domain, which comprises hTR-binding activity and RNA-independent hEST1A-binding activity. Purified, recombinant hEST1A binds the telomerase RNA moiety (hTR) with high affinity (apparent overall K(d) = 25 nM) but low specificity. We propose that hEST1A assembles specifically with telomerase in the context of the hTR-hTERT ribonucleoprotein, through the high affinity of hEST1A for hTR and specific protein-protein contacts with hTERT.

CD8 Binding of MHC-Peptide Complexes in cis or trans Regulates CD8+ T-cell Responses.

The coreceptor CD8αß can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. Here, we demonstrate that CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation. Using molecular modeling, fluorescence resonance energy transfer experiments on living cells, biochemical and mutational analysis, we show that CD8 binding to pMHC in cis involves a different docking mode and is regulated by posttranslational modifications including a membrane-distal interchain disulfide bond and negatively charged O-linked glycans near positively charged sequences on the CD8ß stalk. These modifications distort the stalk, thus favoring CD8 binding to pMHC in cis. Differential binding of CD8 to pMHC in cis or trans is a means to regulate CD8+ T-cell responses and provides new translational opportunities.

A human homolog of yeast Est1 associates with telomerase and uncaps chromosome ends when overexpressed.

Telomeres protect the eukaryotic chromosome ends from degradation and fusion. They are maintained by the ribonucleoprotein telomerase, the core of which is composed of a reverse transcriptase (TERT) and a RNA subunit. In the yeast Saccharomyces cerevisiae, a third critical telomerase subunit, the Ever Shorter Telomeres 1 (EST1) gene product, recruits or activates telomerase at the 3' end of telomeres. Est1p has so far only been known in budding yeast, and mechanisms that mediate telomerase access and activation in other eukaryotes have remained elusive. Here, we use iterative profile searches to identify homologs of yeast Est1p in a large variety of eukaryotes, including human. One of three human homologs, designated human EST1A (hEST1A), is shown to be associated with most or all active telomerase in HeLa cell extracts. Overexpression of hEST1A induces anaphase bridges due to chromosome-end fusions, and telomeric DNA persists at the fusion points. Thus, overexpression of hEST1A affects telomere capping. The identification of EST1 homologs in a large variety of eukaryotes may indicate that the mechanisms of telomere extension are more conserved than anticipated previously.

Methods for the ex vivo characterization of human CD8+ T subsets based on gene expression and replicative history analysis.

The generation of an antigen-specific T-lymphocyte response is a complex multi-step process. Upon T-cell receptor-mediated recognition of antigen presented by activated dendritic cells, naive T-lymphocytes enter a program of proliferation and differentiation, during the course of which they acquire effector functions and may ultimately become memory T-cells. A major goal of modern immunology is to precisely identify and characterize effector and memory T-cell subpopulations that may be most efficient in disease protection. Sensitive methods are required to address these questions in exceedingly low numbers of antigen-specific lymphocytes recovered from clinical samples, and not manipulated in vitro. We have developed new techniques to dissect immune responses against viral or tumor antigens. These allow the isolation of various subsets of antigen-specific T-cells (with major histocompatibility complex [MHC]-peptide multimers and five-color FACS sorting) and the monitoring of gene expression in individual cells (by five-cell reverse transcription-polymerase chain reaction [RT-PCR]). We can also follow their proliferative life history by flow-fluorescence in situ hybridization (FISH) analysis of average telomere length. Recently, using these tools, we have identified subpopulations of CD8+ T-lymphocytes with distinct proliferative history and partial effector-like properties. Our data suggest that these subsets descend from recently activated T-cells and are committed to become differentiated effector T-lymphocytes.

Induction of interleukin-2 receptor alpha (IL-2Ralpha) expression by interleukin-2: important role of the interleukin-2 receptor beta chain region between the two Stat5 docking sites.

The interleukin-2 receptor alpha (IL-2Ralpha) forms, together with IL-2Rbeta and gammac chains, a high affinity IL-2 receptor that is important for IL-2 responsiveness and normal T cell function. Expression of the IL-2Ralpha gene by T cells is regulated mainly at the transcription level which is transiently activated by antigen and upregulated and then prolonged by stimulation with IL-2. The effect of IL-2 on the IL-2Ralpha gene depends on the activation of the transcription factor Stat5, which acts on an IL-2- responsive enhancer that consists of two Stat5 and an Elf1 binding site. To identify the functional domains of the IL-2 receptor required for the stimulation of IL-2Ralpha gene expression, we introduced, into the CTL44 T cell line, receptor chimeras between the extracellular domain of the IL-9 receptor and the cytoplasmic region of IL-2Rbeta. Analyzing the effect of mutations in the intracellular IL-2Rbeta segment, we found that a minimal receptor containing the Jak boxes and one intact Stat5 docking site (i.e. tyrosine 392 or 510) can, as expected, mediate Stat5 activation, but is unable to stimulate IL-2Ralpha expression. However, when this minimal receptor includes the region between the two tyrosines, its capacity to mediate IL-2Ralpha cell surface expression is restored. These data suggest that the segment between the two Stat5 docking sites of the IL-2Rbeta chain mediates signaling events that, together with Stat5 activation, are essential for the stimulation of IL-2Ralpha gene transcription.

Telomere length homeostasis responds to changes in intracellular dNTP pools.

Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated, and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular deoxyguanosine triphosphate (dGTP) levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms and suggest that telomerase activity is modulated in vivo by dGTP levels.

Specific binding of telomeric G-quadruplexes by hydrosoluble perylene derivatives inhibits repeat addition processivity of human telomerase.

Telomerase is responsible for the immortal phenotype of cancer cells and telomerase inhibition may specifically target cancer cell proliferation. Ligands able to selectively bind to G-quadruplex telomeric DNA have been considered as telomerase inhibitors but their mechanisms of action have often been deduced from a non-quantitative telomerase activity assay (TRAP assay) that involves a PCR step and that does not provide insight on the mechanism of inhibition. Furthermore, quadruplex ligands have also been shown to exert their effects by affecting association of telomere binding proteins with telomeres. Here, we use quantitative direct telomerase activity assays to evaluate the strength and mechanism of action of hydrosoluble perylene diimides (HPDIs). HPDIs contain a perylene moiety and different numbers of positively charged side chains. Side chain features vary with regard to number and distances of the charges. IC(50) values of HPDIs were in the low micromolar (0.5-5 µM) range depending on the number and features of the side chains. HPDIs having four side chains emerged as the best compounds of this series. Analysis of primer elongation products demonstrated that at low HPDI concentrations, telomerase inhibition involved formation of telomeric G-quadruplex structures, which inhibited further elongation by telomerase. At high HPDI concentrations, telomerase inhibition occurred independently of G-quadruplex formation of the substrate. The mechanism of action of HPDIs and their specific binding to G-quadruplex DNA was supported by PAGE analysis, CD spectroscopy and ESI-MS. Finally, competition Telospot experiments with duplex DNA indicated specific binding of HPDIs to the single-stranded telomeric substrates over double stranded DNA, a result supported by competitive ESI-MS. Altogether, our results indicate that HPDIs act by stabilizing G-quadruplex structures in single-stranded telomeric DNA, which in turn prevents repeat addition processivity of telomerase.

Molecular dissection of telomeric repeat-containing RNA biogenesis unveils the presence of distinct and multiple regulatory pathways.

Telomeres are transcribed into telomeric repeat-containing RNA (TERRA), large, heterogeneous, noncoding transcripts which form part of the telomeric heterochromatin. Despite a large number of functions that have been ascribed to TERRA, little is known about its biogenesis. Here, we present the first comprehensive analysis of the molecular structure of TERRA. We identify biochemically distinct TERRA complexes, and we describe TERRA regulation during the cell cycle. Moreover, we demonstrate that TERRA 5' ends contain 7-methylguanosine cap structures and that the poly(A) tail, present on a fraction of TERRA transcripts, contributes to their stability. Poly(A)(-) TERRA, but not poly(A)(+) TERRA, is associated with chromatin, possibly reflecting distinct biological roles of TERRA ribonucleoprotein complexes. In support of this idea, poly(A)(-) and poly(A)(+) TERRA molecules end with distinct sequence registers. We also determine that the bulk of 3'-terminal UUAGGG repeats have an average length of 200 bases, indicating that the length heterogeneity of TERRA likely stems from its subtelomeric regions. Finally, we find that TERRA is regulated during the cell cycle, being lowest in late S phase and peaking in early G(1). Our analyses offer the basis for investigating multiple regulatory pathways that affect TERRA synthesis, processing, turnover, and function.