Absence of ERAP1 in B Cells Increases Susceptibility to Central Nervous System Autoimmunity, Alters B Cell Biology, and Mechanistically Explains Genetic Associations between ERAP1 and Multiple Sclerosis.

Hundreds of genes have been linked to multiple sclerosis (MS); yet, the underlying mechanisms behind these associations have only been investigated in a fraction of cases. Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an endoplasmic reticulum-localized aminopeptidase with important roles in trimming peptides destined for MHC class I and regulation of innate immune responses. As such, genetic polymorphisms in ERAP1 have been linked to multiple autoimmune diseases. In this study, we present, to our knowledge, the first mechanistic studies performed to uncover why polymorphisms in ERAP1 are associated with increased susceptibility to MS. Combining multiple mouse models of CNS autoimmunity with high-dimensional single-cell spectral cytometry, adoptive transfer studies, and integrative analysis of human single-cell RNA-sequencing datasets, we identify an intrinsic defect in B cells as being primarily responsible. Not only are mice lacking ERAP1 more susceptible to CNS autoimmunity, but adoptive transfer of B cells lacking ERAP1 into B cell-deficient mice recapitulates this susceptibility. We found B cells lacking ERAP1 display decreased proliferation in vivo and express higher levels of activation/costimulatory markers. Integrative analysis of single-cell RNA sequencing of B cells from 36 individuals revealed subset-conserved differences in gene expression and pathway activation in individuals harboring the MS-linked K528R ERAP1 single-nucleotide polymorphism. Finally, our studies also led us to create, to our knowledge, the first murine protein-level map of the CNS IL-10+ immune compartment at steady state and during neuroinflammation. These studies identify a role for ERAP1 in the modulation of B cells and highlight this as one reason why polymorphisms in this gene are linked to MS.

SLAMF7 Signaling Reprograms T Cells toward Exhaustion in the Tumor Microenvironment.

T cell exhaustion represents one of the most pervasive strategies tumors employ to circumvent the immune system. Although repetitive, cognate TCR signaling is recognized as the primary driving force behind this phenomenon, and it remains unknown what other forces drive T cell exhaustion in the tumor microenvironment (TME). In this study, we show that activation of the self-ligand SLAMF7 immune receptor on T cells induced STAT1 and STAT3 phosphorylation, expression of multiple inhibitory receptors, and transcription factors associated with T cell exhaustion. Analysis of The Cancer Genome Atlas revealed that SLAMF7 transcript levels were strongly correlated with various inhibitory receptors and that high SLAMF7 expression was indicative of poor survival in clear cell renal cell carcinoma (ccRCC). Targeted reanalysis of a CyTOF dataset, which profiled the TME in 73 ccRCC patients, revealed cell-type-specific SLAMF7 expression patterns, strong correlations between exhausted T cells and SLAMF7+ tumor-associated macrophages (TAMs), and a unique subset of SLAMF7highCD38high TAMs. These SLAMF7highCD38high TAMs showed the strongest correlations with exhausted T cells and were an independent prognostic factor in ccRCC. Confirmatory ex vivo coculture studies validated that SLAMF7-SLAMF7 interactions between murine TAMs and CD8+ T cells induce expression of multiple inhibitory receptors. Finally, mice lacking SLAMF7 show restricted growth of B16-F10 tumors, and CD8+ T cells from these mice express less PD-1 and TOX and exhibited an impaired ability to progress through the exhaustion developmental trajectory to terminal exhaustion. These findings suggest that SLAMF7 might play an important role in modulating T cell function in the TME.

ERAP1 is a critical regulator of inflammasome-mediated proinflammatory and ER stress responses.

BACKGROUND: In addition to its role in antigen presentation, recent reports establish a new role for endoplasmic reticulum aminopeptidase 1 (ERAP1) in innate immunity; however, the mechanisms underlying these functions are not fully defined. We previously confirmed that loss of ERAP1 functions resulted in exaggerated innate immune responses in a murine in vivo model. Here, we investigated the role of ERAP1 in suppressing inflammasome pathways and their dependence on ER stress responses. RESULTS: Using bone marrow-derived macrophages (BMDMs), we found that loss of ERAP1 in macrophages resulted in exaggerated production of IL-1ß and IL-18 and augmented caspase-1 activity, relative to wild type macrophages. Moreover, an in vivo colitis model utilizing dextran sodium sulfate (DSS) confirmed increased levels of proinflammatory cytokines and chemokines in the colon of DSS treated ERAP1-/- mice as compared to identically stimulated WT mice. Interestingly, stimulated ERAP1-/- BMDMs and CD4+ T cells simultaneously demonstrated exaggerated ER stress, assessed by increased expression of ER stress-associated genes, a state that could be reverted to WT levels with use of the ER stress inhibitor Tauroursodeoxycholic acid (TUDCA). CONCLUSIONS: Together, these results not only suggest that ERAP1 is important for regulating inflammasome dependent innate immune response pathways in vivo, but also propose a mechanism that underlies these changes, that may be associated with increased ER stress due to lack of normal ERAP1 functions.

SLAMF7 modulates B cells and adaptive immunity to regulate susceptibility to CNS autoimmunity.

BACKGROUND: Multiple sclerosis (MS) is a chronic, debilitating condition characterized by CNS autoimmunity stemming from a complex etiology involving both environmental and genetic factors. Our current understanding of MS points to dysregulation of the immune system as the pathogenic culprit, however, it remains unknown as to how the many genes associated with increased susceptibility to MS are involved. One such gene linked to MS susceptibility and known to regulate immune function is the self-ligand immune cell receptor SLAMF7. METHODS: We subjected WT and SLAMF7-/- mice to multiple EAE models, compared disease severity, and comprehensively profiled the CNS immune landscape of these mice. We identified all SLAMF7-expressing CNS immune cells and compared the entire CNS immune niche between genotypes. We performed deep phenotyping and in vitro functional studies of B and T cells via spectral cytometry and BioPlex assays. Adoptive transfer studies involving the transfer of WT and SLAMF7-/- B cells into B cell-deficient mice (µMT) were also performed. Finally, B-T cell co-culture studies were performed, and a comparative cell-cell interaction network derived from scRNA-seq data of SLAMF7+ vs. SLAMF7- human CSF immune cells was constructed. RESULTS: We found SLAMF7-/- mice to be more susceptible to EAE compared to WT mice and found SLAMF7 to be expressed on numerous CNS immune cell subsets. Absence of SLAMF7 did not grossly alter the CNS immune landscape, but allowed for altered immune cell subset infiltration during EAE in a model-dependent manner. Global lack of SLAMF7 expression increased myeloid cell activation states along with augmented T cell anti-MOG immunity. B cell profiling studies revealed increased activation states of specific plasma and B cell subsets in SLAMF7-/- mice during EAE, and functional co-culture studies determined that SLAMF7-/- B cells induce exaggerated T cell activation. Adoptive transfer studies revealed that the increased susceptibility of SLAMF7-/- mice to EAE is partly B cell dependent and reconstruction of the human CSF SLAMF7-interactome found B cells to be critical to cell-cell communication between SLAMF7-expressing cells. CONCLUSIONS: Our studies have identified novel roles for SLAMF7 in CNS immune regulation and B cell function, and illuminate underpinnings of the genetic association between SLAMF7 and MS.

SLAMF7 Is a Critical Negative Regulator of IFN-α-Mediated CXCL10 Production in Chronic HIV Infection.

Current advances in combined antiretroviral therapy have rendered HIV infection a chronic, manageable disease; however, the problem of persistent immune activation still remains despite treatment. The immune cell receptor SLAMF7 has been shown to be upregulated in diseases characterized by chronic immune activation. In this study, we studied the function of the SLAMF7 receptor in immune cells of HIV patients and the impacts of SLAMF7 signaling on peripheral immune activation. We observed increased frequencies of SLAMF7+ PBMCs in HIV+ individuals in a clinical phenotype-dependent manner, with discordant and long-term nonprogressor patients showing elevated SLAMF7 levels, and elite controllers showing levels comparable to healthy controls. We also noted that SLAMF7 was sensitive to IFN-⍺ stimulation, a factor elevated during HIV infection. Further studies revealed SLAMF7 to be a potent inhibitor of the monocyte-derived proinflammatory chemokine CXCL10 (IP-10) and other CXCR3 ligands, except in a subset of HIV+ patients termed SLAMF7 silent (SF7S). Studies utilizing small molecule inhibitors revealed that the mechanism of CXCL10 inhibition is independent of known SLAMF7 binding partners. Furthermore, we determined that SLAMF7 activation on monocytes is able to decrease their susceptibility to HIV-1 infection in vitro via downregulation of CCR5 and upregulation of the CCL3L1 chemokine. Finally, we discovered that neutrophils do not express SLAMF7, are CXCL10+ at baseline, are able to secrete CXCL10 in response to IFN-⍺ and LPS, and are nonresponsive to SLAMF7 signaling. These findings implicate the SLAMF7 receptor as an important regulator of IFN-⍺-driven innate immune responses during HIV infection.

Targeted inhibition of the deubiquitinating enzymes, USP14 and UCHL5, induces proteotoxic stress and apoptosis in Waldenström macroglobulinaemia tumour cells.

Deubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome-associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b-AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient-derived WM tumour cells. Importantly, b-AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib. In silico modelling identified protein residues that were critical for the binding of b-AP15 with USP14 or UCHL5 and proteasome enzyme activity assays confirmed that b-AP15 does not affect the proteolytic capabilities of the 20S proteasome ß-subunits. In vitro toxicity from b-AP15 appeared to result from a build-up of ubiquitinated proteins and activation of the endoplasmic reticulum stress response in WM cells, an effect that also disrupted the mitochondria. Focused transcriptome profiling of b-AP15-treated WM cells revealed modulation of several genes regulating cell stress and NF-κB signalling, the latter whose protein translocation and downstream target activation was reduced by b-AP15 in vitro. This is the first report to define the effects and underlying mechanisms associated with inhibition of USP14 and UCHL5 DUB activity in WM tumour cells.

Fundamentals to therapeutics: Epigenetic modulation of CD8+ T Cell exhaustion in the tumor microenvironment.

In the setting of chronic antigen exposure in the tumor microenvironment (TME), cytotoxic CD8+ T cells (CTLs) lose their immune surveillance capabilities and ability to clear tumor cells as a result of their differentiation into terminally exhausted CD8+ T cells. Immune checkpoint blockade (ICB) therapies reinvigorate exhausted CD8+ T cells by targeting specific inhibitory receptors, thus promoting their cytolytic activity towards tumor cells. Despite exciting results with ICB therapies, many patients with solid tumors still fail to respond to such therapies and patients who initially respond can develop resistance. Recently, through new sequencing technologies such as the assay for transposase-accessible chromatin with sequencing (ATAC-seq), epigenetics has been appreciated as a contributing factor that enforces T cell differentiation toward exhaustion in the TME. Importantly, specific epigenetic alterations and epigenetic factors have been found to control CD8+ T cell exhaustion phenotypes. In this review, we will explain the background of T cell differentiation and various exhaustion states and discuss how epigenetics play an important role in these processes. Then we will outline specific epigenetic changes and certain epigenetic and transcription factors that are known to contribute to CD8+ T cell exhaustion. We will also discuss the most recent methodologies that are used to study and discover such epigenetic modulations. Finally, we will explain how epigenetic reprogramming is a promising approach that might facilitate the development of novel exhausted T cell-targeting immunotherapies.

Adenoviral delivery of an immunomodulatory protein to the tumor microenvironment controls tumor growth.

Targeted modulation of the immune system against tumors can achieve responses in otherwise refractory cancers, which has spurred efforts aimed at optimizing such strategies. To this end, we have previously investigated cancer immunotherapy approaches using recombinant adenovirus vectors, as well as via modulation of the self-ligand receptor SLAMF7. Here, we present a gene transfer-based immunotherapy approach using targeted expression of a SLAMF7-Fc fusion construct directly into tumors at high concentrations via a recombinant adenoviral vector (Ad-SF7-Fc). Using multiple murine cancer models, we show that Ad-SF7-Fc can induce tumor control via augmentation of innate immunity; specifically, induction of type I interferons and activation of dendritic cells (DCs) and macrophages. Analogously, we find that modulating SLAMF7 signaling via an adenoviral vector expressing its intracellular adaptor, EAT-2, is also capable of inducing tumor control. Finally, we employ a novel in vivo prediction approach and dataset integration with machine learning to dissect how Ad-SF7-Fc modulates cell-type-specific responses in the tumor microenvironment to achieve tumor control. Thus, our novel combinatorial cancer immunotherapy highlights the benefit of multimodal immune modulation and lays a framework for combination with complementary approaches capable of inducing adaptive immune responses.

Inhibition of the Myocardin-Related Transcription Factor Pathway Increases Efficacy of Trametinib in NRAS-Mutant Melanoma Cell Lines.

The Ras/MEK/ERK pathway has been the primary focus of targeted therapies in melanoma; it is aberrantly activated in almost 80% of human cutaneous melanomas (≈50% BRAFV600 mutations and ≈30% NRAS mutations). While drugs targeting the MAPK pathway have yielded success in BRAFV600 mutant melanoma patients, such therapies have been ineffective in patients with NRAS mutant melanomas in part due to their cytostatic effects and primary resistance. Here, we demonstrate that increased Rho/MRTF-pathway activation correlates with high intrinsic resistance to the MEK inhibitor, trametinib, in a panel of NRAS mutant melanoma cell lines. A combination of trametinib with the Rho/MRTF-pathway inhibitor, CCG-222740, synergistically reduced cell viability in NRAS mutant melanoma cell lines in vitro. Furthermore, the combination of CCG-222740 with trametinib induced apoptosis and reduced clonogenicity in SK-Mel-147 cells, which are highly resistant to trametinib. These findings suggest a role of the Rho/MRTF-pathway in intrinsic trametinib resistance in a subset of NRAS mutant melanoma cell lines and highlight the therapeutic potential of concurrently targeting the Rho/MRTF-pathway and MEK in NRAS mutant melanomas.

ERAP1 deficient mice have reduced Type 1 regulatory T cells and develop skeletal and intestinal features of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a prototypical sero-negative autoimmune disease that affects millions worldwide. Single nucleotide polymorphisms in the Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) gene have been linked to AS via GWAS studies, however, the exact mechanism as to how ERAP1 contributes to pathogenesis of AS is not understood. We undertook µCT imaging and histologic analysis to evaluate bone morphology of the axial skeletons of ERAP1-/- mice and discovered the hallmark skeletal features of AS in these mice, including spinal ankylosis, osteoporosis, and spinal inflammation. We also confirmed the presence of spontaneous intestinal dysbiosis and increased susceptibility to Dextran Sodium Sulfate (DSS)-induced colitis in ERAP1-/- mice, however the transfer of healthy microbiota from wild type mice via cross-fostering experiments did not resolve the skeletal phenotypes of ERAP1-/- mice. Immunological analysis demonstrated that while ERAP1-/- mice had normal numbers of peripheral Foxp3+ Tregs, they had reduced numbers of both "Tr1-like" regulatory T cells and tolerogenic dendritic cells, which are important for Tr1 cell differentiation. Together, our data suggests that ERAP1-/- mice may serve as a useful animal model for studying pathogenesis of intestinal, skeletal, and immunological manifestations of Ankylosing Spondylitis.

Immunophenotyping of Waldenströms macroglobulinemia cell lines reveals distinct patterns of surface antigen expression: potential biological and therapeutic implications.

Waldenströms macroglobulinemia (WM) is a subtype of Non-Hodgkin's lymphoma in which the tumor cell population is markedly heterogeneous, consisting of immunoglobulin-M secreting B-lymphocytes, plasmacytoid lymphocytes and plasma cells. Due to rarity of disease and scarcity of reliable preclinical models, many facets of WM molecular and phenotypic architecture remain incompletely understood. Currently, there are 3 human WM cell lines that are routinely used in experimental studies, namely, BCWM.1, MWCL-1 and RPCI-WM1. During establishment of RPCI-WM1, we observed loss of the CD19 and CD20 antigens, which are typically present on WM cells. Intrigued by this observation and in an effort to better define the immunophenotypic makeup of this cell line, we conducted a more comprehensive analysis for the presence or absence of other cell surface antigens that are present on the RPCI-WM1 model, as well as those on the two other WM cell lines, BCWM.1 and MWCL-1. We examined expression of 65 extracellular and 4 intracellular antigens, comprising B-cell, plasma cell, T-cell, NK-cell, myeloid and hematopoietic stem cell surface markers by flow cytometry analysis. RPCI-WM1 cells demonstrated decreased expression of CD19, CD20, and CD23 with enhanced expression of CD28, CD38 and CD184, antigens that were differentially expressed on BCWM.1 and MWCL-1 cells. Due to increased expression of CD184/CXCR4 and CD38, RPCI-WM1 represents a valuable model in which to study the effects anti-CXCR4 or anti-CD38 targeted therapies that are actively being developed for treatment of hematologic cancers. Overall, differences in surface antigen expression across the 3 cell lines may reflect the tumor clone population predominant in the index patients, from whom the cell lines were developed. Our analysis defines the utility of the most commonly employed WM cell lines as based on their immunophenotype profiles, highlighting unique differences that can be further studied for therapeutic exploit.