Polygenic risk associated with Alzheimer's disease and other traits influences genes involved in T cell signaling and activation.

Introduction: T cells, known for their ability to respond to an enormous variety of pathogens and other insults, are increasingly recognized as important mediators of pathology in neurodegeneration and other diseases. T cell gene expression phenotypes can be regulated by disease-associated genetic variants. Many complex diseases are better represented by polygenic risk than by individual variants. Methods: We first compute a polygenic risk score (PRS) for Alzheimer's disease (AD) using genomic sequencing data from a cohort of Alzheimer's disease (AD) patients and age-matched controls, and validate the AD PRS against clinical metrics in our cohort. We then calculate the PRS for several autoimmune disease, neurological disorder, and immune function traits, and correlate these PRSs with T cell gene expression data from our cohort. We compare PRS-associated genes across traits and four T cell subtypes. Results: Several genes and biological pathways associated with the PRS for these traits relate to key T cell functions. The PRS-associated gene signature generally correlates positively for traits within a particular category (autoimmune disease, neurological disease, immune function) with the exception of stroke. The trait-associated gene expression signature for autoimmune disease traits was polarized towards CD4+ T cell subtypes. Discussion: Our findings show that polygenic risk for complex disease and immune function traits can have varying effects on T cell gene expression trends. Several PRS-associated genes are potential candidates for therapeutic modulation in T cells, and could be tested in in vitro applications using cells from patients bearing high or low polygenic risk for AD or other conditions.

Clonal CD8 T Cells Accumulate in the Leptomeninges and Communicate with Microglia in Human Neurodegeneration.

Murine studies have highlighted a crucial role for immune cells in the meninges in surveilling the central nervous system (CNS) and influencing neuroinflammation. However, how meningeal immunity is altered in human neurodegeneration and its effects on CNS inflammation is understudied. We performed the first single-cell analysis of the transcriptomes and T cell receptor (TCR) repertoire of 104,635 immune cells from 55 postmortem human brain and leptomeningeal tissues from donors with neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. RNA and TCR sequencing from paired leptomeninges and brain allowed us to perform lineage tracing to identify the spatial trajectory of clonal T cells in the CNS and its borders. We propose that T cells activated in the brain emigrate to and establish residency in the leptomeninges where they likely contribute to impairments in lymphatic drainage and remotely to CNS inflammation by producing IFNγ and other cytokines. We identified regulatory networks local to the meninges including NK cell-mediated CD8 T cell killing which likely help to control meningeal inflammation. Collectively, these findings provide not only a foundation for future studies into brain border immune surveillance but also highlight important intercellular dynamics that could be leveraged to modulate neuroinflammation.

Stat1 is an inducible transcriptional repressor of neural stem cells self-renewal program during neuroinflammation.

A central issue in regenerative medicine is understanding the mechanisms that regulate the self-renewal of endogenous stem cells in response to injury and disease. Interferons increase hematopoietic stem cells during infection by activating STAT1, but the mechanisms by which STAT1 regulates intrinsic programs in neural stem cells (NSCs) during neuroinflammation is less known. Here we explored the role of STAT1 on NSC self-renewal. We show that overexpressing Stat1 in NSCs derived from the subventricular zone (SVZ) decreases NSC self-renewal capacity while Stat1 deletion increases NSC self-renewal, neurogenesis, and oligodendrogenesis in isolated NSCs. Importantly, we find upregulation of STAT1 in NSCs in a mouse model of multiple sclerosis (MS) and an increase in pathological T cells expressing IFN-γ rather than interleukin 17 (IL-17) in the cerebrospinal fluid of affected mice. We find IFN-γ is superior to IL-17 in reducing proliferation and precipitating an abnormal NSC phenotype featuring increased STAT1 phosphorylation and Stat1 and p16ink4a gene expression. Notably, Stat1-/- NSCs were resistant to the effect of IFN-γ. Lastly, we identified a Stat1-dependent gene expression profile associated with an increase in the Sox9 transcription factor, a regulator of self-renewal. Stat1 binds and transcriptionally represses Sox9 in a transcriptional luciferase assay. We conclude that Stat1 serves as an inducible checkpoint for NSC self-renewal that is upregulated during chronic brain inflammation leading to decreased self-renewal. As such, Stat1 may be a potential target to modulate for next generation therapies to prevent progression and loss of repair function in NSCs/neural progenitors in MS.

Clonal CD8 T cells in the leptomeninges are locally controlled and influence microglia in human neurodegeneration.

Recent murine studies have highlighted a crucial role for the meninges in surveilling the central nervous system (CNS) and influencing CNS inflammation. However, how meningeal immunity is altered in human neurodegeneration and its potential effects on neuroinflammation is understudied. In the present study, we performed single-cell analysis of the transcriptomes and T cell receptor repertoire of 72,576 immune cells from 36 postmortem human brain and leptomeninges tissues from donors with neurodegenerative diseases including amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. We identified the meninges as an important site of antigen presentation and CD8 T cell activation and clonal expansion and found that T cell activation in the meninges is a requirement for infiltration into the CNS. We further found that natural killer cells have the potential to negatively regulate T cell activation locally in the meninges through direct killing and are one of many regulatory mechanisms that work to control excessive neuroinflammation.

Distinct Th17 effector cytokines differentially promote microglial and blood-brain barrier inflammatory responses during post-infectious encephalitis.

Group A Streptococcus (GAS) infections can cause neuropsychiatric sequelae in children due to post-infectious encephalitis. Multiple GAS infections induce migration of Th17 lymphocytes from the nose into the brain, which are critical for microglial activation, blood-brain barrier (BBB) and neural circuit impairment in a mouse disease model. How endothelial cells (ECs) and microglia respond to GAS infections, and which Th17-derived cytokines are essential for these responses are unknown. Using single-cell RNA sequencing and spatial transcriptomics, we found that ECs downregulate BBB genes and microglia upregulate interferon-response, chemokine and antigen-presentation genes after GAS infections. Several microglial-derived chemokines were elevated in patient sera. Administration of a neutralizing antibody against interleukin-17A (IL-17A), but not ablation of granulocyte-macrophage colony-stimulating factor (GM-CSF) in T cells, partially rescued BBB dysfunction and microglial expression of chemokine genes. Thus, IL-17A is critical for neuropsychiatric sequelae of GAS infections and may be targeted to treat these disorders.

GW5074 Increases Microglial Phagocytic Activities: Potential Therapeutic Direction for Alzheimer's Disease.

Microglia, the resident immune cells of the central nervous system (CNS), are responsible for maintaining homeostasis in the brain by clearing debris and are suggested to be inefficient in Alzheimer's Disease (AD), a progressive neurodegenerative disorder for which there is no disease-modifying drug. Besides pathological approaches, unbiased evidence from genome-wide association studies (GWAS) and gene network analysis implicate genes expressed in microglia that reduce phagocytic ability as susceptibility genes for AD. Thus, a central feature toward AD therapy is to increase the microglial phagocytic activities while maintaining synaptic integrity. Here, we developed a robust unbiased high content screening assay to identify potential therapeutics which can reduce the amyloid-beta (Aß1-42) load by increasing microglial uptake ability. Our screen identified the small-molecule GW5074, an inhibitor of c-RAF, a serine/threonine kinase, which significantly increased the Aß1-42 clearance activities in human monocyte-derived microglia-like (MDMi) cells, a microglia culture model that recapitulates many genetic and phenotypic aspects of human microglia. Notably, GW5074 was previously reported to be neuroprotective for cerebellar granule cells and cortical neurons. We found that GW5074 significantly increased the expression of key AD-associated microglial molecules known to modulate phagocytosis: TYROBP, SIRPß1, and TREM2. Our results demonstrated that GW5074 is a potential therapeutic for AD, by targeting microglia.

Genotype-phenotype correlation of T-cell subtypes reveals senescent and cytotoxic genes in Alzheimer's disease.

Recent studies identifying expression quantitative trait loci (eQTLs) in immune cells have uncovered important links between disease risk alleles and gene expression trends in monocytes, T cells and other cell types. However, these studies are generally done with young, healthy subjects, limiting the utility of their findings for age-related conditions such as Alzheimer's disease (AD). We have performed RNA sequencing on four T-cell subsets in genome-wide genotyped and well-characterized AD subjects and age- and sex-matched controls from the Religious Orders Study/Memory and Aging Project. We correlated gene expression data with AD neuropathological traits and with single-nucleotide polymorphisms to detect eQTLs. We identified several significant genes involved in T-cell senescence and cytotoxicity, consistent with T-cell RNA sequencing studies in aged/AD cohorts. We identified unexpected eQTLs previously associated with neuropsychiatric disease traits. Finally, we discovered that pathways related to axon guidance and synaptic function were enriched among trans-eQTLs in coding regions of the genome. Our data strengthen the potential link between T-cell senescence and age-related neurodegenerative disease. In addition, our eQTL data suggest that T-cell phenotypes may influence neuropsychiatric disorders and can be influenced by genes involved in neurodevelopmental processes.

T Cells: A Growing Universe of Roles in Neurodegenerative Diseases.

T cells play a central role in homeostasis and host defense against infectious diseases. T cell dysregulation can lead to recognizing self-antigens as foreign antigens, causing a detrimental autoimmune response. T cell involvement in multiple sclerosis (MS), long understood to be an autoimmune-mediated neurodegenerative disease, is well characterized. More recently, a role for T cells has also been identified for the neurodegenerative diseases Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Interestingly, several alleles and variants of human leukocyte antigen (HLA) genes have been classified as AD and PD risk genes. HLA codes for components of major histocompatibility complex (MHC) class I or class II, both of which are expressed by microglia, the innate immune cells of the central nervous system (CNS). Thus, both microglia and T cells may potentially interact in an antigen-dependent or independent fashion to shape the inflammatory cascade occurring in neurodegenerative diseases. Dissecting the antigen specificity of T cells may lead to new options for disease-modifying treatments in neurodegenerative diseases. Here, we review the current understanding of T cells in neurodegenerative diseases. We summarize the subsets of T cells, their phenotype and potential functions in animal models and in human studies of neurodegenerative diseases.

Single cell RNA sequencing of human microglia uncovers a subset associated with Alzheimer's disease.

The extent of microglial heterogeneity in humans remains a central yet poorly explored question in light of the development of therapies targeting this cell type. Here, we investigate the population structure of live microglia purified from human cerebral cortex samples obtained at autopsy and during neurosurgical procedures. Using single cell RNA sequencing, we find that some subsets are enriched for disease-related genes and RNA signatures. We confirm the presence of four of these microglial subpopulations histologically and illustrate the utility of our data by characterizing further microglial cluster 7, enriched for genes depleted in the cortex of individuals with Alzheimer's disease (AD). Histologically, these cluster 7 microglia are reduced in frequency in AD tissue, and we validate this observation in an independent set of single nucleus data. Thus, our live human microglia identify a range of subtypes, and we prioritize one of these as being altered in AD.

IL-27: An endogenous constitutive repressor of human monocytes.

Interleukin (IL)-27 is a pleiotropic cytokine that initially was described as being pro-inflammatory and an inducer of T helper (Th)1 cells. In contrast, it has also been described as an anti-inflammatory cytokine in that it suppresses pro-inflammatory Th17 cells and induces anti-inflammatory IL-10 producing T regulatory (Tr)1 cells. While the majority of studies have been focused on the effects of IL-27 on T cells, human antigen-presenting cells express high levels of the IL-27 receptor ex vivo, in addition to being the major producer of IL-27. We report here that human monocytes are repressed by endogenous IL-27, in that the addition of an anti-IL-27 neutralizing antibody increases the production of pro-inflammatory cytokines ex vivo. We observed that neutralizing monocyte-derived IL-27 leads to increased IL-17A production by CD4+ T cells and a down-regulation of the IL-17 modulating ectonucleotidase CD39 on monocytes. The locus that contains the IL27 gene has been linked to susceptibility for type 1 diabetes (T1D). Interestingly, ex vivo monocytes from subjects with T1D produce more IL-27 suggesting this upregulation of IL-27 acts as a negative feedback loop to attempt to counterbalance the pro-inflammatory immune response in the disease state. In summary, we provide evidence that IL-27 is an endogenous regulator of human monocytes and has consequences on CD4+ T cell phenotype, particularly Th17 cells.

A novel Tmem119-tdTomato reporter mouse model for studying microglia in the central nervous system.

Microglia are resident immune cells of the central nervous system (CNS). The exact role of microglia in CNS disorders is not clear due to lack of tools to discriminate between microglia and infiltrating myeloid cells. Here, we present a novel reporter mouse model targeting a microglia-specific marker, TMEM119, for studying microglia in health and disease. By placing a reporter cassette (GSG-3xFlag-P2A-tdTomato) between the coding sequence of exon 2 and 3'UTR of the Tmem119 gene using CRISPR/Cas9 technology, we generated a Tmem119-tdTomato knock-in mouse strain. Gene expression assay showed no difference of endogenous Tmem119 in the CNS of Tmem119tdTomato/+ relative to wild-type mice. The cells expressing tdTomato were recognized by immunofluorescence staining using commercially available anti-TMEM119 antibodies. Additionally, immunofluorescence and flow cytometry techniques revealed that tdTomato+ cells are detected throughout the CNS, but not in peripheral tissues of Tmem119tdTomato/+ mice. Aging does not influence TMEM119 expression as tdTomato+ cells were detectable in the CNS of older mice (300 and 540 days old). Further immunofluorescence characterization shows that tdTomato+ cells colocalize with Iba1+ cells in the brain, but not with neurons, astrocytes or oligodendrocytes. Moreover, flow cytometry analysis of brain tissues of adult mice demonstrates that the majority of microglia CD45loCD11b+ cells (96.3%) are tdTomato-positive; and a minority of infiltrating CD45hiCD11b+ myeloid cells (5.3%) are also tdTomato-positive, which we further characterized and found that tdTomato expression is in part of choroid plexus macrophages but not in meningeal and perivascular macrophages. Functionally, using an acute injury model, we measured time-lapse activation of tdTomato-labeled microglia by transcranial two-photon microscopy in live Tmem119tdTomato/+ mice. Taken together, the Tmem119-tdTomato reporter mouse model is a valuable tool to specifically study the role of microglia in health and disease.

Opposite functions of STAT3 and Smad3 in regulating Tiam1 expression in Th17 cells.

We recently showed that Tiam1 expression is induced in pro-inflammatory T helper 17 (Th17) cells differentiated with interleukin (IL)-6 and TGF-ß1, and together with Rac1 promote Th17 cell development and autoimmunity in a mouse model of multiple sclerosis. Here we found that STAT3 and Smad3, downstream transcription factors of IL-6 and TGF-ß1, respectively, play opposing roles in regulating Tiam1 transcription in CD4+ T-cells. While IL-6-STAT3 signaling promotes Tiam1 expression, TGF-ß1-Smad3 induces the opposite outcome. At the Tiam1 promoter, both STAT3 and Smad3 bind to the Tiam1 promoter in Th17 cells. However, STAT3 induces Tiam1 promoter activity whereas Smad3 competes with STAT3 and inhibits its activity. Our findings uncover the complexity of STAT3/Smad3 signaling in regulating Tiam1 expression and Th17 cells.

Notch-1 Inhibition Promotes Immune Regulation in Transplantation Via Regulatory T Cell-Dependent Mechanisms.

BACKGROUND: Transplantation is the treatment of choice for many patients with end-stage organ disease. Despite advances in immunosuppression, long-term outcomes remain suboptimal, hampered by drug toxicity and immune-mediated injury, the leading cause of late graft loss. The development of therapies that promote regulation while suppressing effector immunity is imperative to improve graft survival and minimize conventional immunosuppression. Notch signaling is a highly conserved pathway pivotal to T-cell differentiation and function, rendering it a target of interest in efforts to manipulate T cell-mediated immunity. METHODS: We investigated the pattern of Notch-1 expression in effector and regulatory T cells (Tregs) in both murine and human recipients of a solid-organ transplant. Using a selective human anti-Notch-1 antibody (aNotch-1), we examined the effect of Notch-1 receptor inhibition in full major histocompatibility complex-mismatch murine cardiac and lung transplant models, and in a humanized skin transplant model. On the basis of our findings, we further used a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. RESULTS: We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 in comparison with conventional T cells, both in mice with transplants and in the peripheral blood of patients with transplants. In the murine cardiac transplant model, peritransplant administration of aNotch-1 (days 0, 2, 4, 6, 8, and 10) significantly prolonged allograft survival in comparison with immunoglobulin G-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intragraft conventional T cells, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1-treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mammalian target of rapamycin pathway and increased the phosphorylation of STAT5 (signal transducer and activator of transcription 5) in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent suppressive capacity than Notch-1high Tregs. Last, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) signaling. CONCLUSIONS: Our data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1.

A transcriptomic atlas of aged human microglia.

With a rapidly aging global human population, finding a cure for late onset neurodegenerative diseases has become an urgent enterprise. However, these efforts are hindered by the lack of understanding of what constitutes the phenotype of aged human microglia-the cell type that has been strongly implicated by genetic studies in the pathogenesis of age-related neurodegenerative disease. Here, we establish the set of genes that is preferentially expressed by microglia in the aged human brain. This HuMi_Aged gene set captures a unique phenotype, which we confirm at the protein level. Furthermore, we find this gene set to be enriched in susceptibility genes for Alzheimer's disease and multiple sclerosis, to be increased with advancing age, and to be reduced by the protective APOEε2 haplotype. APOEε4 has no effect. These findings confirm the existence of an aging-related microglial phenotype in the aged human brain and its involvement in the pathological processes associated with brain aging.

Foxo1 Promotes Th9 Cell Differentiation and Airway Allergy.

T helper 9 (Th9) cells are effector CD4+ T cells that are characterized by the production of interleukin-9 (IL-9) and have been associated with allergic responses. Here, we found that the expression of the transcription factor forkhead box O1 (Foxo1) was induced in Th9 and Foxo1 plays a crucial role in the differentiation of Th9 cells. Pharmacological inhibition of Foxo1 or genetic disruption of Foxo1 in CD4+ T cells caused a reduction in IL-9 expression while upregulating IL-17A and IFNγ production. Furthermore, chromatin immunoprecipitation (ChIP) followed by luciferase assays revealed direct binding of Foxo1 to both the Il9 and Irf4 promoters and induces their transactivation. Lastly, adoptive transfer of Th9 cells into lungs induced asthma-like symptoms that were ameliorated by Foxo1 inhibitor, AS1842856. Together, our findings demonstrate a novel regulator of Th9 cells with a direct implication in allergic inflammation.

MS AHI1 genetic risk promotes IFNγ+ CD4+ T cells.

OBJECTIVE: To study the influence of the Abelson helper integration site 1 (AHI1) locus associated with MS susceptibility on CD4+ T cell function. METHODS: We characterized the chromatin state of T cells in the MS-associated AHI1 linkage disequilibrium (LD) block. The expression and the role of the AHI1 variant were examined in T cells from genotyped healthy subjects who were recruited from the PhenoGenetic Project, and the function of AHI1 was explored using T cells from Ahi1 knockout mice. RESULTS: Chromatin state analysis reveals that the LD block containing rs4896153, which is robustly associated with MS susceptibility (odds ratio 1.15, p = 1.65 × 10-13), overlaps with strong enhancer regions that are present in human naive and memory CD4+ T cells. Relative to the rs4896153A protective allele, the rs4896153T susceptibility allele is associated with decreased AHI1 mRNA expression, specifically in naive CD4+ T cells (p = 1.73 × 10-74, n = 213), and we replicate this effect in an independent set of subjects (p = 2.5 × 10-9, n = 32). Functional studies then showed that the rs4896153T risk variant and the subsequent decreased AHI1 expression were associated with reduced CD4+ T cell proliferation and a specific differentiation into interferon gamma (IFNγ)-positive T cells when compared with the protective rs4896153A allele. This T cell phenotype was also observed in murine CD4+ T cells with genetic deletion of Ahi1. CONCLUSIONS: Our findings suggest that the effect of the AHI1 genetic risk for MS is mediated, in part, by enhancing the development of proinflammatory IFNγ+ T cells that have previously been implicated in MS and its mouse models.

A human microglia-like cellular model for assessing the effects of neurodegenerative disease gene variants.

Microglia are emerging as a key cell type in neurodegenerative diseases, yet human microglia are challenging to study in vitro. We developed an in vitro cell model system composed of human monocyte-derived microglia-like (MDMi) cells that recapitulated key aspects of microglia phenotype and function. We then used this model system to perform an expression quantitative trait locus (eQTL) study examining 94 genes from loci associated with Alzheimer's disease, Parkinson's disease, and multiple sclerosis. We found six loci (CD33, PILRB, NUP160, LRRK2, RGS1, and METTL21B) in which the risk haplotype drives the association with both disease susceptibility and altered expression of a nearby gene (cis-eQTL). In the PILRB and LRRK2 loci, the cis-eQTL was found in the MDMi cells but not in human peripheral blood monocytes, suggesting that differentiation of monocytes into microglia-like cells led to the acquisition of a cellular state that could reveal the functional consequences of certain genetic variants. We further validated the effect of risk haplotypes at the protein level for PILRB and CD33, and we confirmed that the CD33 risk haplotype altered phagocytosis by the MDMi cells. We propose that increased LRRK2 gene expression by MDMi cells could be a functional outcome of rs76904798, a single-nucleotide polymorphism in the LRKK2 locus that is associated with Parkinson's disease.

Human regulatory T cells undergo self-inflicted damage via granzyme pathways upon activation.

Tregs hold great promise as a cellular therapy for multiple immunologically mediated diseases, given their ability to control immune responses. The success of such strategies depends on the expansion of healthy, suppressive Tregs ex vivo and in vivo following the transfer. In clinical studies, levels of transferred Tregs decline sharply in the blood within a few days of the transfer. Tregs have a high rate of apoptosis. Here, we describe a new mechanism of Treg self-inflicted damage. We show that granzymes A and -B (GrA and GrB), which are highly upregulated in human Tregs upon stimulation, leak out of cytotoxic granules to induce cleavage of cytoplasmic and nuclear substrates, precipitating apoptosis in target cells. GrA and GrB substrates were protected from cleavage by inhibiting granzyme activity in vitro. Additionally, we show - by using cytometry by time of flight (CYTOF) - an increase in GrB-expressing Tregs in the peripheral blood and renal allografts of transplant recipients undergoing rejection. These GrB-expressing Tregs showed an activated phenotype but were significantly more apoptotic than non-GrB expressing Tregs. This potentially novel finding improves our understanding of Treg survival and suggests that manipulating Gr expression or activity might be useful for designing more effective Treg therapies.