Alternative Splicing Coupled with Transcript Degradation Modulates OAS1g Antiviral Activity

At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g (Oas1g), a key component of the 2-5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show a multitude of alternative splicing events predicted to lead to decay exist and thus, have the potential to play a significant role in the regulation of gene expression in innate immunity

Functional TCR T cell screening using single-cell droplet microfluidics

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies

T cell antigen discovery via signaling and antigen-presenting bifunctional receptors

CD8^+ T cells recognize and eliminate tumors in an antigen-specific manner. Despite progress in characterizing the antitumor T cell repertoire and function, the identification of target antigens remains a challenge. Here we describe the use of chimeric receptors called signaling and antigen-presenting bifunctional receptors (SABRs) in a cell-based platform for T cell receptor (TCR) antigen discovery. SABRs present an extracellular complex comprising a peptide and major histocompatibility complex (MHC), and induce intracellular signaling via a TCR-like signal after binding with a cognate TCR. We devised a strategy for antigen discovery using SABR libraries to screen thousands of antigenic epitopes. We validated this platform by identifying the targets recognized by public TCRs of known specificities. Moreover, we extended this approach for personalized neoantigen discovery

Functional TCR T cell screening using single-cell droplet microfluidics

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies

Requirement of the phosphatidylinositol 3-kinase/Akt signaling pathway for the effect of nicotine on interleukin-1beta-induced chondrocyte apoptosis in a rat model of osteoarthritis

National Natural Science Foundation of China [81072015]; Natural Science Foundation of Fujian, China [2010D007]; Medical Innovation Foundation of Fujian, China [2011-CXB-36]; Xiamen Science and Technology Key program grant, Fujian, ChinaChondrocyte apoptosis is mainly responsible for the progressive degeneration of cartilage in osteoarthritis (OA). Interleukin-1beta (IL-1 beta) was widely used as a modulating and chondrocyte apoptosis-inducing agent. Nicotine is able to confer resistance to apoptosis and promote cell survival in some cell lines, but its regulatory mechanism is ambiguous. We aimed to investigate the effect of nicotine on IL-1 beta-induced chondrocyte apoptosis and the mechanism underlying how nicotine antagonizes IL-1 beta-induced apoptosis of rat chondrocytes. Chondrocytes isolated from newborn rat joints were exposed to IL-1 beta. The cell viability was analyzed by the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay, and the apoptotic cells were counted with DAPI staining. The levels of Akt, phosphorylated-Akt (p-Akt) and downstream protein targets of Akt were detected by western blotting. The results showed that nicotine neutralized the effect of IL-1 beta on chondrocytes by activating PI3K/Akt signaling pathways, including the PI3K/Akt/Bcl-2 pathway, to block IL-1 beta-induced cell apoptosis and the PI3K/Akt/p70S6K (p70S6 kinase)/S6 pathway for promoting protein synthesis, modulating its downstream effectors such as TIMP-1 and MMP-13. Activation of the PI3K/Akt pathway is, in part, required for the effect of nicotine on IL-1 beta-induced chondrocyte apoptosis in a rat model of osteoarthritis. (C) 2012 Elsevier Inc. All rights reserved

Kinetic Inference Resolves Epigenetic Mechanism of Drug Resistance in Melanoma

We resolved a mechanism connecting tumor epigenetic plasticity with non-genetic adaptive resistance to therapy, with MAPK inhibition of BRAF-mutant melanomas providing the model. These cancer cells undergo multiple, reversible drug-induced cell-state transitions, ultimately yielding a drug-resistant mesenchymal-like phenotype. A kinetic series of transcriptome and epigenome data, collected over two months of drug treatment and release, revealed changing levels of thousands of genes and extensive chromatin remodeling. However, a 3-step computational algorithm greatly simplified the interpretation of these changes, and revealed that the whole adaptive process was controlled by a gene module activated within just three days of treatment, with RelA driving chromatin remodeling to establish an epigenetic program encoding long-term phenotype changes. These findings were confirmed across several patient-derived cell lines and in melanoma patients under MAPK inhibitor treatment. Co-targeting BRAF and histone-modifying enzymes arrests adaptive transitions towards drug tolerance in epigenetically plastic melanoma cells and may be exploited therapeutically

T cell antigen discovery via trogocytosis

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide–major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide–MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy