PVRIG is Expressed on Stem-Like T Cells in Dendritic Cell-Rich Niches in Tumors and Its Blockade May Induce Immune Infiltration in Non-Inflamed Tumors.

Cancers that are poorly immune infiltrated pose a substantial challenge, with current immunotherapies yielding limited clinical success. Stem-like memory T cells (TSCM) have been identified as a subgroup of T cells that possess strong proliferative capacity and that can expand and differentiate following interactions with dendritic cells (DCs). In this study, we explored the pattern of expression of a recently discovered inhibitory receptor poliovirus receptor-related immunoglobulin domain protein (PVRIG) and its ligand, poliovirus receptor-related ligand 2 (PVRL2), in the human tumor microenvironment. Using spatial and single-cell RNA transcriptomics data across diverse cancer indications, we found that among the T-cell checkpoints, PVRIG is uniquely expressed on TSCM and PVRL2 is expressed on DCs in immune aggregate niches in tumors. PVRIG blockade could therefore enhance TSCM-DC interactions and efficiently drive T-cell infiltration to tumors. Consistent with these data, following PVRIG blockade in patients with poorly infiltrated tumors, we observed immune modulation including increased tumor T-cell infiltration, T-cell receptor (TCR) clonality, and intratumoral T-cell expansion, all of which were associated with clinical benefit. These data suggest PVRIG blockade as a promising strategy to induce potent antitumor T-cell responses, providing a novel approach to overcome resistance to immunotherapy in immune-excluded tumors.

ILDR2-Fc Is a Novel Regulator of Immune Homeostasis and Inducer of Antigen-Specific Immune Tolerance.

ILDR2 is a member of the Ig superfamily, which is implicated in tricellular tight junctions, and has a putative role in pancreatic islet health and survival. We recently found a novel role for ILDR2 in delivering inhibitory signals to T cells. In this article, we show that short-term treatment with ILDR2-Fc results in long-term durable beneficial effects in the relapsing-remitting experimental autoimmune encephalomyelitis and NOD type 1 diabetes models. ILDR2-Fc also promotes transplant engraftment in a minor mismatch bone marrow transplantation model. ILDR2-Fc displays a unique mode of action, combining immunomodulation, regulation of immune homeostasis, and re-establishment of Ag-specific immune tolerance via regulatory T cell induction. These findings support the potential of ILDR-Fc to provide a promising therapeutic approach for the treatment of autoimmune diseases.

ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

Follow the leader: preference for specific amino acids directly following the initial methionine in proteins of different organisms.

It is well established that the vast majority of proteins of all taxonomical groups and species are initiated by an AUG codon, translated into the amino acid methionine (Met). Many attempts were made to evaluate the importance of the sequences surrounding the initiation codon, mostly focusing on the RNA sequence. However, the role and importance of the amino acids following the initiating Met residue were rarely investigated, mostly in bacteria and fungi. Herein, we computationally examined the protein sequences of all major taxonomical groups represented in the Swiss-Prot database, and evaluated the preference of each group to specific amino acids at the positions directly following the initial Met. The results indicate that there is a species-specific preference for the second amino acid of the majority of protein sequences. Interestingly, the preference for a certain amino acid at the second position changes throughout evolution from lysine in prokaryotes, through serine in lower eukaryotes, to alanine in higher plants and animals.

The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity.

NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytotoxicity thus providing an "alternative self" mechanism for MHC class I inhibition.

Peptides modulating conformational changes in secreted chaperones: from in silico design to preclinical proof of concept.

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helix-helix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNFalpha, IL-1beta, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNFalpha, IL-6, and IFNgamma by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.

Activation of relaxin-related receptors by short, linear peptides derived from a collagen-containing precursor.

In a screening effort based on algorithmic predictions for novel G-protein-coupled receptor (GPCR) peptide activators, we were able to identify and examine two novel peptides (P59 and P74) which are short, linear, and derived from a natural, previously unidentified precursor protein containing a collagen-like repeat. Both peptides seemed to show an apparent cAMP-related effect on CHO-K1 cells transiently transfected with either LGR7 or LGR8, usually after treatment with cAMP-generating forskolin, compared to the same cells treated with forskolin plus relaxin. This activation was not found for the relaxin-3 receptor (GPR135). In a set of follow-up experiments, both peptides were found to stimulate cAMP production, mostly upon initial stimulation of cAMP production by 5 micro M forskolin in cells transfected with either LGR7 or LGR8. In a dye-free cell impedance GPCR activation assay, we were able to show that these peptides were also able to activate a cellular response mediated by these receptors. Although untransfected CHO-K1 cells showed some cellular activation by both relaxin and at least one of our newly discovered peptides, both LGR7- and LGR8-transfected cells showed a stronger response, indicating stimulation of a cellular pathway through activation of these receptors. In conclusion, we were able to show that these newly discovered peptides, which have no similarity to any member of the relaxin-insulin-like peptide family, are potential ligands for the relaxin-related family of receptors and as such might serve as novel candidates for relaxin-related therapeutic indications. Both peptides are linear and were found to be active after being chemically synthesized.

Genomic fossils as a snapshot of the human transcriptome.

Processed pseudogenes (PPGs) are cDNA sequences that were generated through reverse transcription of mature, spliced mRNAs and have subsequently been reinserted at a new genomic location. These cDNA sequences are usually no longer transcribed and are considered "dead on arrival." Here we show that PPGs can be used to generate a map of the transcriptome. By analyzing thousands of human PPGs, we were able to discover hundreds of transcript variants so far unidentified. An experimental verification of a subset of these variants by RT-PCR indicates that most of them are still active in the human transcriptome. Furthermore, we demonstrate that PPGs can enable the identification of ancient splice variants that were expressed ancestrally but are now extinct. Our results show that the genome itself carries a "virtual cDNA library" that can readily be used to analyze both present and ancestral transcripts. Our approach can be applied to sequenced metazoan genomes to computationally annotate splicing variation even when expressed sequences are unavailable.

Transcription-mediated gene fusion in the human genome.

Transcription of a gene usually ends at a regulated termination point, preventing the RNA-polymerase from reading through the next gene. However, sporadic reports suggest that chimeric transcripts, formed by transcription of two consecutive genes into one RNA, can occur in human. The splicing and translation of such RNAs can lead to a new, fused protein, having domains from both original proteins. Here, we systematically identified over 200 cases of intergenic splicing in the human genome (involving 421 genes), and experimentally demonstrated that at least half of these fusions exist in human tissues. We showed that unique splicing patterns dominate the functional and regulatory nature of the resulting transcripts, and found intergenic distance bias in fused compared with nonfused genes. We demonstrate that the hundreds of fused genes we identified are only a subset of the actual number of fused genes in human. We describe a novel evolutionary mechanism where transcription-induced chimerism followed by retroposition results in a new, active fused gene. Finally, we provide evidence that transcription-induced chimerism can be a mechanism contributing to the evolution of protein complexes.

Evolution of multicellularity in Metazoa: comparative analysis of the subcellular localization of proteins in Saccharomyces, Drosophila and Caenorhabditis.

A comparison of the subcellular assignments of proteins between the unicellular Saccharomyces cerevisiae and the multicellular Drosophila melanogaster and Caenorhabditis elegans was performed using a computational tool for the prediction of subcellular localization. Nine subcellular compartments were studied: (1) extracellular domain, (2) cell membrane, (3) cytoplasm, (4) endoplasmic reticulum, (5) Golgi apparatus, (6) lysosome, (7) peroxisome, (8) mitochondria, and (9) nucleus. The transition to multicellularity was found to be characterized by an increase in the total number of proteins encoded by the genome. Interestingly, this increase is distributed unevenly among the subcellular compartments. That is, a disproportionate increase in the number of proteins in the extracellular domain, the cell membrane, and the cytoplasm is observed in multicellular organisms, while no such increase is seen in other subcellular compartments. A possible explanation involves signal transduction. In terms of protein numbers, signal transduction pathways may be roughly described as a pyramid with an expansive base in the extracellular domain (the numerous extracellular signal proteins), progressively narrowing at the cell membrane and cytoplasmic levels, and ending in a narrow tip consisting of only a handful of transcription modulators in the nucleus. Our observations suggest that extracellular signaling interactions among metazoan cells account for the uneven increase in the numbers of proteins among subcellular compartments during the transition to multicellularity.

Large-scale protein annotation through gene ontology.

Recent progress in genomic sequencing, computational biology, and ontology development has presented an opportunity to investigate biological systems from a unique perspective, that is, examining genomes and transcriptomes through the multiple and hierarchical structure of Gene Ontology (GO). We report here our development of GO Engine, a computational platform for GO annotation, and analysis of the resultant GO annotations of human proteins. Protein annotation was centered on sequence homology with GO-annotated proteins and protein domain analysis. Text information analysis and a multiparameter cellular localization predictive tool were also used to increase the annotation accuracy, and to predict novel annotations. The majority of proteins corresponding to full-length mRNA in GenBank, and the majority of proteins in the NR database (nonredundant database of proteins) were annotated with one or more GO nodes in each of the three GO categories. The annotations of GenBank and SWISS-PROT proteins are available to the public at the GO Consortium web site.