Cytotoxic T lymphocyte antigen 4 (CTLA4) and programmed cell death protein 1 (PD-1) are immune checkpoint proteins expressed in T cells. Although CTLA4 expression was found in multiple tumours including non-small cell lung cancer (NSCLC) tissues and cells, its function in tumour cells is unknown. Recently, PD-1 was found to be expressed in melanoma cells and to promote tumorigenesis. We found that CTLA4 was expressed in a subset of NSCLC cell lines and in a subgroup of cancer cells within the lung cancer tissues. We further found that in NSCLC cells, anti-CTLA4 antibody can induce PD-L1 expression, which is mediated by CTLA4 and the EGFR pathway involving phosphorylation of MEK and ERK. In CTLA4 knockout cells, EGFR knockout cells or in the presence of an EGFR tyrosine kinase inhibitor, anti-CTLA4 antibody was not able to induce PD-L1 expression in NSCLC cells. Moreover, anti-CTLA4 antibody promoted NSCLC cell proliferation in vitro and tumour growth in vivo in the absence of adaptive immunity. These results suggest that tumour cell-intrinsic CTLA4 can regulate PD-L1 expression and cell proliferation, and that anti-CTLA4 antibody, by binding to the tumour cell-intrinsic CTLA4, may result in the activation of the EGFR pathway in cancer cells.

IL-33 and its receptor ST2 play important roles in airway inflammation and contribute to asthma onset and exacerbation. The IL-33/ST2 signaling pathway recruits adapter protein myeloid differentiation primary response 88 (MyD88) to transduce intracellular signaling. MyD88 forms a complex with IL-R-associated kinases (IRAKs), IRAK4 and IRAK2, called the Myddosome (MyD88-IRAK4-IRAK2). The myddosome subsequently activates downstream NF-κB and MAPKs p38 and JNK. We established an asthma-like mouse model by intratracheal administration of IL-33. The IL-33 model has a very similar phenotype compared with the OVA-induced mouse asthma model. The importance of MyD88 in the IL-33/ST2 signaling transduction was demonstrated by the MyD88 knockout mice, which were protected from the IL-33-induced asthma. We synthesized small molecule mimetics of the α-helical domain of IRAK2 with drug-like characteristics based on the recent advances in the designing of α-helix compounds. The mimetics can competitively interfere in the protein-protein interaction between IRAK2 and IRAK4, leading to disruption of Myddosome formation. A series of small molecules were screened using an NF-κB promoter assay in vitro. The lead compound, 7004, was further studied in the IL-33-induced and OVA-induced asthma mouse models in vivo. Compound 7004 can inhibit the IL-33-induced NF-κB activity, disrupt Myddosome formation, and attenuate the proinflammatory effects in asthma-like models. Our data indicate that the Myddosome may represent a novel intracellular therapeutic target for diseases in which IL-33/ST2 plays important roles, such as asthma and other inflammatory diseases.

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.