Deorphanizing a novel GPCR expressed by tissue resident memory cells

About This Grant

PROJECT SUMMARY Lung infections remain a significant source of morbidity and mortality across all age groups. The recent SARS- CoV-2 pandemic underscores the critical need for the scientific community to urgently develop strategies that enhance mucosal immunity against a wide range of pathogens. While vaccines offer protection against severe disease, their effectiveness are not often durable and for many infections, such as tuberculosis, they are not effective. Therefore, gaining a deeper understanding of the mechanisms supporting long-term protective immunity at barrier sites, like the respiratory tract, is vital for developing therapies that boost mucosal immune responses. Tissue-resident memory T cells (TRM cells) have emerged as the guardians of barrier immunity. TRM cells are a distinct population of memory T cells that reside within tissues and respond immediately against pathogens invading barrier tissues, thus representing the first-line of defense. Dr. Vijayanand’s team (LJI PI) has long-standing expertise in human lung TRM cells having shown their importance in driving natural and anti-PD1 therapy-induced T cell responses in lung cancer. Given the profound importance of TRM cells in protective immunity, the generation and maintenance of robust TRM responses is considered important for the success of vaccines aimed at preventing severe lung infections. However, currently, there are no therapeutic options for boosting or maintaining natural and vaccine-induced TRM responses in the lungs. Our transcriptomic studies have identified a novel orphan G-protein coupled receptor (GPCR) that is expressed at high levels in lung TRM cells. Using genetic knock-out models, we found that this GPCR plays a key role in the development of lung TRM cells. In this proposal, our objective is to de-orphanize this GPCR. We have already detected ligand activity in murine thymic extracts using the b-arrestin recruitment assay in HEK293 cells. In Aim 1, Dr. Changlu Liu’s team (PI) at SBP, will identify and purify the ligand from thymic extracts and thymic-derived cell types, as described in their previous work to deorphanize other GPCR like GPR183 and GPCR135. We will purify the ligand from thymic tissue through organic extractions followed by High-performance liquid chromatography (HPLC). Fractions with ligand activity will be analyzed by mass spectrometry to identify the nature and sequence of the ligand. The putative ligand will then be tested in a dose-dependent manner to determine affinity and specificity of the ligand to the orphan GPCR. In Aim 2, we will test functional activity of the putative GPCR ligand in primary T cells. We initially test in vitro the functional activities of thymic tissue/cellular extracts in T cells. Then, we will perform similar studies using the purified ligand and determine its activity on T cell signaling, activation, proliferation, cytokine production and TRM cell generation in vivo. Studies in this aim will identify the physiological ligand that can enhance the activity of an important target (GPCR) in TRM cells. This ligand or its analogues have the potential to enhance the generation of lung TRM cells, thereby boosting protective mucosal immune responses in humans.