B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor expressed on T cells and B cells that is structurally and functionally related to programmed death protein-1 (PD-1), a well-established cancer immunotherapy target. Binding of T cell specific BTLA with its ligand HVEM on antigen-presenting cells triggers tyrosine phosphorylation of the intracellular tail of BTLA, and ultimately leads to suppression of the T cell response. There is growing interest in targeting BTLA for cancer immunotherapy, yet very little is known about its biochemical mechanism and potential crosstalk with PD-1. Using a well-defined liposome reconstitution system with purified recombinant proteins, we quantitatively compared the biochemical specificities of BTLA and PD-1 and dissected the roles of each tyrosine residues within BTLA using purified BTLA proteins containing point mutants. These experiments reveal unexpected differences in BTLA and PD-1 specificities, which might underlie their functional disparities.

Programmed Death Ligand 1 (PD-L1) overexpressed on cancer cells can help tumors evade the immune system by triggering the coinhibitory receptor Programmed Death 1 (PD-1) on T-cells, subsequently inhibiting T-cell activity. PD-L1 binding to PD-1 is reciprocally regulated by two proteins, CD80 and CKLF-like MARVEL-containing Transmembrane 6 (CMTM6). CD80 is a key immunostimulatory ligand that binds PD-L1 in cis on the same membrane, and blocks PD-L1:PD-1 inhibitory signaling. Conversely, CMTM6 is a multi-pass transmembrane protein that prevents the degradation of PD-L1, and thereby promotes PD-L1:PD-1 inhibitory signaling. However, the biochemical nature of CMTM6:PD-L1 interaction, and how it may be influenced by CD80, is poorly defined. Here, I established an intact cell-based, quantitative, and functional assay of CMTM6 to determine the molecular requirements for PD-L1 and CMTM6 interaction, and its potential regulation by CD80. Using domain-swapping and cellular reconstitution, I found that PD-L1 chimeras with the transmembrane (TM), extracellular (EC), or intracellular (IC) domains of PD-L1 swapped with that of the CMTM6 non-client PD-L2, were all upregulated by CMTM6, but to a lesser extent than wild-type PD-L1. The TM replacement showed the greatest impairment in CMTM6-mediated upregulation, suggesting that CMTM6 primarily interacts with the TM of PD-L1. Finally, co-expression of CD80 did not affect the magnitude of the CMTM6 upregulation of PD-L1, suggesting that cis-CD80:PD-L1 interaction does not perturb CMTM6:PD-L1 interaction. These data provide a better understanding of the trimolecular relationship between PD-L1, CMTM6, and CD80.

Fibrosis is a condition characterized by tissue overgrowth, hardening, and /or scarring. It is typically caused by excess deposition of extracellular matrix (ECM) components. Fibroblasts play an important role in the maintenance and reabsorption of the ECM, and thus can be critical mediators of the condition. Protein Tyrosine Phosphatase Receptor Type Gamma (PTPRG) is highly expressed in fibroblasts, and so we want to delve into how PTPRG activation and inactivation may play a role in the condition. The phosphatase activity of receptor-type protein tyrosine phosphatases (RPTPs) is widely thought to be regulated through dimerization, however, the dimerization of PTPRG in fibroblast activity has not been seen in primary cells. Thus, we used FRET microscopy on a primary PTPRG knockout murine dermal fibroblast cell line (mDF) transfected with PTPRG mutant constructs to observe full-length protein dimerization in a cellular context. We also performed biochemical observations of protein activity through phosphatase activity experiments. We found that WT and several mutants of PTPRG dimerize in mDFs. A dimerization-inactivating mutant exhibits less dimerization in cells and in vitro.

The ability of T lymphocytes to detect and eliminate cancer and viral infection is restricted by pathological subversion of the co-inhibitory receptor signaling pathways which normally function to restrain the immune response and preserve "self" tolerance. As a result, cancer treatments employing therapeutic antibodies to block the T cell co-inhibitory receptors PD-1 or CTLA4 have seen dramatic clinical success over the past decade. However, despite extensive efforts to improve and predict patient responses to these therapies, additional advances have been slow to materialize and severe autoimmune side effects remain common. To more effectively manipulate the curative potential of adaptive immunity to eliminate cancer and infectious disease, a greater understanding of how T cell co-receptor signaling operates from the level of single cells and molecules to that of the entire system is critical but remains incomplete. To advance understanding of T cell signaling at the molecular and cellular level, an APEX2 peroxidase-based proximity labeling approach is developed and applied to identify novel effectors and substrates of co-inhibitory immune receptors by mass spectrometry. To clarify how CTLA4 coordinates cellular and system level regulation of immune responses, an alternative mechanism is described in which CTLA4 acts in a cell intrinsic manner to regulate availability of the B7 co-stimulatory ligands on the surface of activated T cells. In this model, CTLA4 control of immune homeostasis is achieved in part by directing cis-endocytosis of T cell B7 ligands which are both endogenously expressed and inherently acquired via trogocytosis upon contact with activating APCs. Importantly, these APC-derived innate inflammatory signals targeted by CTLA4 are displayed to other T cells alongside co-acquired peptide antigen during an immune response. A model is presented in which this pathway thereby allows CTLA4 to exert dual cell intrinsic and extrinsic regulatory function to more efficiently operate at the interface between innate and adaptive immunity and between single T cells and the lymphocyte collective.

Parkinson’s Disease (PD) is an age-related neurodegenerative disease characterized by tremors, bradykinesia, and rigidity. While several pharmaceutical therapies have been developed to address PD symptoms, there are no preventative treatment or therapies available. To address this gap, Kainos Medicine, Inc. developed KM-819, a FAF-1 inhibitor currently in phase 2 clinical trial1,2. In order to understand the mechanistic effects of this drug in humans early in the disease course, this research utilizes an iPSC model derived from Gaucher Disease (GD) patients – a population shown to acquire PD at significantly higher rates than the general population3. Specifically, live-cell imaging techniques are used to explore the quantity and morphology of lysosomes and mitochondria in GD and healthy control (HC) cell lines, with and without KM-819 treatment. This model system has been explored previously; 24h treatment with 1-20µM KM-819 was found to increase lysosome size and motility, with the most significant improvement consistently occurring around 1µM4. In order to explore the efficacy of KM-819 at low dosages, cells were treated for 24h with 0.1 µM KM-819 and imaged using LysoTracker. Consistent with previous findings, lysosome size and quantity both increased, even at low dosage. Next, the mitochondrial system was explored as it is frequently implicated in PD. Initial experiments focused on establishing baseline measurements for untreated cells; morphological analysis indicated that GD mitochondria present with increased fragmentation and reduced network complexity compared to HC cells. Treating GD cells with 0.1µM of KM-819 for 24h showed significant reductions in mitochondria fragmentation and increased network complexity.

Alzheimer’s Disease (AD), along with several related neurodegenerative diseases called tauopathies, are characterized by neurofibrillary tangles (NFTs) consist of aggregated tau proteins in the brain, leading to cognitive dysfunctions and neurodegeneration. Accumulation of DNA damage has been found in AD brains, which is thought to underlie neurodegeneration. Recent studies from our collaborators have shown that AD patient brains have decreased activity of a DNA repair enzyme – polynucleotide kinase 3'-phosphatase (PNKP) – compared to healthy brains. Dysfunction of PNKP has been implicated in the pathophysiology of other neurodegenerative diseases, including Huntington’s Disease (HD) and spinocerebellar ataxia type 3 (SCA3). In a previous study on SCA3, increasing the expression of PNKP rescued DNA damage accumulation and neurodegenerative phenotypes in an animal model. However, the role of PNKP in context of tau-mediated neurodegeneration is unknown. In this study, we aimed to characterize the role of PNKP in tauopathies using a Drosophila model. We generated transgenic fly lines for wild-type Drosophila PNKP (dPNKP.WT) and several dPNKP mutants. Our results showed that overexpression of dPNKP.WT in human tau (hTau)-transgenic flies rescued the rough eye phenotype and motor dysfunction. These findings show that increasing PNKP expression ameliorates tau-mediated neurotoxicity in flies, suggesting that enhancing PNKP level or activity may be a therapeutic strategy for tauopathies including AD.

Tumor associated macrophages (TAMs) are a major component in the tumor microenvironment. TAMs are associated with poor prognosis in solid tumors and promote an immunosuppressive environment, which can result from persistent inflammation. Preclinical studies suggest boosting existing immunotherapies by reprogramming TAMs from a pro-tumor phenotype towards an anti-tumor one. To better target these TAMs, we must look at the complexes that regulate them. The BAF complex, an ATP-dependent chromatin remodeler, regulates gene expression by altering the position of nucleosomes to promote gene expression. ARID1A, a core protein subunit of cBAF plays a vital role in the epigenetic regulation of inflammatory genes and the differentiation of myeloid cells. Little is known about the effects of Arid1a loss in TAMs and its implications in cancer. We sought to recapitulate the effects of Arid1a loss on macrophages in vitro, investigate the mechanisms driving the associated phenotype, and assess phagocytotic function in Arid1a-deficient (Arid1aKO) TAMs. Using an in vitro system of bone marrow derived macrophages (BMDMs), we observed that Arid1aKO macrophages upregulated the inhibitory ligand PD-L1 (programmed death ligand-1) and CD86. This upregulation is both JAK1/2 and type I interferon signaling independent and operates through a cell intrinsic mechanism. Furthermore, we found that Arid1aKO TAMs displayed similar phagocytic capabilities to Arid1a-intact TAMs. Our results demonstrate that upregulation of PD-L1 and CD86 in macrophages that occurs upon Arid1a loss may explain enhanced response to anti-PD-L1 immunotherapy. In the future, these results can help further direct preclinical models/strategies aimed at reprogramming macrophages as a cancer therapeutic.

Currently, prostate adenocarcinoma (PRAD) is the second most common cancer in males, and the metastatic progression of prostate tumors to the bone accounts for a large majority of prostate cancer-related deaths. As such, identifying potential therapeutic targets to prevent prostate cancer progression proves to be vital. The low-molecular-weight protein tyrosine phosphatase (LMPTP) has been found to be highly expressed in metastatic hormone-naïve prostate cancer patients and is associated with lower survival rates. However, the molecular mechanism by which LMPTP promotes prostate cancer growth and metastatic development remains unclear. In this paper, we confirmed the oncogenic nature of LMPTP in prostate cancer growth in vitro and in vivo using both CRISPR-Cas9-mediated knockout (KO) of LMPTP and inhibition of LMPTP via an orally bio-available LMPTP inhibitor. Additionally, the use of a soft agar colony formation assay, Matrigel invasion chamber assay, and in vivo intraosseous metastasis model further implicated LMPTP in metastatic prostate cancer development. Through phosphoproteomic and metabolomic analyses we uncovered a possible glutathione deficiency and compensatory model in LMPTP KO cells that would explain the role of LMPTP in prostate cancer progression. Our findings implicate LMPTP in prostate cancer progression which taken together with our confirmation that loss of LMPTP responds synergistically with current prostate cancer drugs establishes LMPTP as a potential drug target for prostate cancer patients.