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.

APOE and immunity: Research highlights.

INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.

Notch receptors and Smad3 signaling cooperate in the induction of interleukin-9-producing T cells.

Interleukin 9 (IL-9) is a pleiotropic cytokine that can regulate autoimmune responses by enhancing regulatory CD4(+)FoxP3(+) T regulatory (Treg) cell survival and T helper 17 (Th17) cell proliferation. Here, we analyzed the costimulatory requirements for the induction of Th9 cells, and demonstrated that Notch pathway cooperated with TGF-ß signaling to induce IL-9. Conditional ablation of Notch1 and Notch2 receptors inhibited the development of Th9 cells. Notch1 intracellular domain (NICD1) recruited Smad3, downstream of TGF-ß cytokine signaling, and together with recombining binding protein (RBP)-Jκ bound the Il9 promoter and induced its transactivation. In experimental autoimmune encephalomyelitis (EAE), Jagged2 ligation regulated clinical disease in an IL-9-dependent fashion. Signaling through Jagged2 expanded Treg cells and suppressed EAE when administered before antigen immunization, but worsened EAE when administered concurrently with immunization by favoring Th17 cell expansion. We propose that Notch and Smad3 cooperate to induce IL-9 and participate in regulating the immune response.

Deconvolving the contributions of cell-type heterogeneity on cortical gene expression.

Complexity of cell-type composition has created much skepticism surrounding the interpretation of bulk tissue transcriptomic studies. Recent studies have shown that deconvolution algorithms can be applied to computationally estimate cell-type proportions from gene expression data of bulk blood samples, but their performance when applied to brain tissue is unclear. Here, we have generated an immunohistochemistry (IHC) dataset for five major cell-types from brain tissue of 70 individuals, who also have bulk cortical gene expression data. With the IHC data as the benchmark, this resource enables quantitative assessment of deconvolution algorithms for brain tissue. We apply existing deconvolution algorithms to brain tissue by using marker sets derived from human brain single cell and cell-sorted RNA-seq data. We show that these algorithms can indeed produce informative estimates of constituent cell-type proportions. In fact, neuronal subpopulations can also be estimated from bulk brain tissue samples. Further, we show that including the cell-type proportion estimates as confounding factors is important for reducing false associations between Alzheimer's disease phenotypes and gene expression. Lastly, we demonstrate that using more accurate marker sets can substantially improve statistical power in detecting cell-type specific expression quantitative trait loci (eQTLs).

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.

Rheumatoid arthritis-associated RBPJ polymorphism alters memory CD4+ T cells.

Notch signaling has recently emerged as an important regulator of immune responses in autoimmune diseases. The recombination signal-binding protein for immunoglobulin kappa J region (RBPJ) is a transcriptional repressor, but converts into a transcriptional activator upon activation of the canonical Notch pathway. Genome-wide association studies of rheumatoid arthritis (RA) identified a susceptibility locus, rs874040(CC), which implicated the RBPJ gene. Here, chromatin state mapping generated using the chromHMM algorithm reveals strong enhancer regions containing DNase I hypersensitive sites overlapping the rs874040 linkage disequilibrium block in human memory, but not in naïve CD4(+) T cells. The rs874040 overlapping this chromatin state was associated with increased RBPJ expression in stimulated memory CD4(+) T cells from healthy subjects homozygous for the risk allele (CC) compared with memory CD4(+) T cells bearing the protective allele (GG). Transcriptomic analysis of rs874040(CC) memory T cells showed a repression of canonical Notch target genes IL (interleukin)-9, IL-17 and interferon (IFN)γ in the basal state. Interestingly, activation of the Notch pathway using soluble Notch ligand, Jagged2-Fc, induced IL-9 and IL-17A while delta-like 4Fc, another Notch ligand, induced higher IFNγ expression in the rs874040(CC) memory CD4(+) T cells compared with their rs874040(GG) counterparts. In RA, RBPJ expression is elevated in memory T cells from RA patients compared with control subjects, and this was associated with induced inflammatory cytokines IL-9, IL-17A and IFNγ in response to Notch ligation in vitro. These findings demonstrate that the rs874040(CC) allele skews memory T cells toward a pro-inflammatory phenotype involving Notch signaling, thus increasing the susceptibility to develop RA.

Monoallelic expression of the human FOXP2 speech gene.

The recent descriptions of widespread random monoallelic expression (RMAE) of genes distributed throughout the autosomal genome indicate that there are more genes subject to RMAE on autosomes than the number of genes on the X chromosome where X-inactivation dictates RMAE of X-linked genes. Several of the autosomal genes that undergo RMAE have independently been implicated in human Mendelian disorders. Thus, parsing the relationship between allele-specific expression of these genes and disease is of interest. Mutations in the human forkhead box P2 gene, FOXP2, cause developmental verbal dyspraxia with profound speech and language deficits. Here, we show that the human FOXP2 gene undergoes RMAE. Studying an individual with developmental verbal dyspraxia, we identify a deletion 3 Mb away from the FOXP2 gene, which impacts FOXP2 gene expression in cis. Together these data suggest the intriguing possibility that RMAE impacts the haploinsufficiency phenotypes observed for FOXP2 mutations.

CD33: increased inclusion of exon 2 implicates the Ig V-set domain in Alzheimer's disease susceptibility.

We previously demonstrated that the Alzheimer's disease (AD) associated risk allele, rs3865444(C), results in a higher surface density of CD33 on monocytes. Here, we find alternative splicing of exon 2 to be the primary mechanism of the genetically driven differential expression of CD33 protein. We report that the risk allele, rs3865444(C), is associated with greater cell surface expression of CD33 in both subjects of European and African-American ancestry and that there is a single haplotype influencing CD33 surface expression. A meta-analysis of the two populations narrowed the number of significant SNPs in high linkage disequilibrium (LD) (r(2) > 0.8) with rs3865444 to just five putative causal variants associated with increased protein expression. Using gene expression data from flow-sorted CD14(+)CD16(-) monocytes from 398 healthy subjects of three populations, we show that the rs3865444(C) risk allele is strongly associated with greater expression of CD33 exon 2 (pMETA = 2.36 × 10(-60)). Western blotting confirms increased protein expression of the full-length CD33 isoform containing exon 2 relative to the rs3865444(C) allele (P < 0.0001). Of the variants in strong LD with rs3865444, rs12459419, which is located in a putative SRSF2 splice site of exon 2, is the most likely candidate to mediate the altered alternative splicing of CD33's Immunoglobulin V-set domain 2 and ultimately influence AD susceptibility.

A TREM1 variant alters the accumulation of Alzheimer-related amyloid pathology.

OBJECTIVE: Genome-wide association studies have linked variants in TREM2 (triggering receptor expressed on myeloid cells 2) and TREML2 with Alzheimer disease (AD) and AD endophenotypes. Here, we pursue a targeted analysis of the TREM locus in relation to cognitive decline and pathological features of AD. METHODS: Clinical, cognitive, and neuropathological phenotypes were collected in 3 prospective cohorts on aging (n = 3,421 subjects). Our primary analysis was an association with neuritic plaque pathology. To functionally characterize the associated variants, we used flow cytometry to measure TREM1 expression on monocytes. RESULTS: We provide evidence that an intronic variant, rs6910730(G) , in TREM1, is associated with an increased burden of neuritic plaques (p = 3.7 × 10(-4) ), diffuse plaques (p = 4.1 × 10(-3) ), and Aß density (p = 2.6 × 10(-3) ) as well as an increased rate of cognitive decline (p = 5.3 × 10(-3) ). A variant upstream of TREM2, rs7759295(C) , is independently associated with an increased tau tangle density (p = 4.9 × 10(-4) ), an increased burden of neurofibrillary tangles (p = 9.1 × 10(-3) ), and an increased rate of cognitive decline (p = 2.3 × 10(-3) ). Finally, a cytometric analysis shows that the TREM1 rs6910730(G) allele is associated with decreased TREM1 expression on the surface of myeloid cells (p = 1.7 × 10(-3) ). INTERPRETATION: We provide evidence that 2 common variants within the TREM locus are associated with pathological features of AD and aging-related cognitive decline. Our evidence suggests that these variants are likely to be independent of known AD variants and that they may work through an alteration of myeloid cell function.

Polyfunctional responses by human T cells result from sequential release of cytokines.

The release of cytokines by T cells defines a significant part of their functional activity in vivo, and their ability to produce multiple cytokines has been associated with beneficial immune responses. To date, time-integrated end-point measurements have obscured whether these polyfunctional states arise from the simultaneous or successive release of cytokines. Here, we used serial, time-dependent, single-cell analysis of primary human T cells to resolve the temporal dynamics of cytokine secretion from individual cells after activation ex vivo. We show that multifunctional, Th1-skewed cytokine responses (IFN-γ, IL-2, TNFα) are initiated asynchronously, but the ensuing dynamic trajectories of these responses evolve programmatically in a sequential manner. That is, cells predominantly release one of these cytokines at a time rather than maintain active secretion of multiple cytokines simultaneously. Furthermore, these dynamic trajectories are strongly associated with the various states of cell differentiation suggesting that transient programmatic activities of many individual T cells contribute to sustained, population-level responses. The trajectories of responses by single cells may also provide unique, time-dependent signatures for immune monitoring that are less compromised by the timing and duration of integrated measures.

Distinct microglial transcriptomic signatures within the hippocampus.

Microglia, the resident immune cells of the brain, are crucial in the development of the nervous system. Recent evidence demonstrates that microglia modulate adult hippocampal neurogenesis by inhibiting cell proliferation of neural precursors and survival both in vitro and in vivo, thus maintaining a balance between cell division and cell death in the neural stem cell pool. There are increasing reports suggesting these microglia found in neurogenic niches differ from their counterparts in non-neurogenic areas. Here, we present evidence that hippocampal microglia exhibit transcriptomic heterogeneity, with some cells expressing genes associated with neurogenesis. By comprehensively profiling myeloid lineage cells in the hippocampus using single cell RNA-sequencing, we have uncovered a small, yet distinct population of microglia which exhibit depletion in genes associated with homeostatic microglia and enrichment of genes associated with phagocytosis. Intriguingly, this population also expresses a gene signature with substantial overlap with previously characterized phenotypes, including disease associated microglia (DAM), a particularly unique and compelling microglial state.

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.

Increased frequencies of myelin oligodendrocyte glycoprotein/MHC class II-binding CD4 cells in patients with multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the CNS resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin-reactive T cells in peripheral blood. Using T cell cloning techniques, a modest increase in the frequency of myelin-reactive T cells in patients as compared with control subjects was observed. In this study, we investigated whether myelin oligodendrocyte glycoprotein (MOG)-specific T cells could be detected and their frequency was measured using DRB1*0401/MOG(97-109(107E-S)) tetramers in MS subjects and healthy controls expressing HLA class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG-reactive T cells upon MOG peptide stimulation of PMBCs. MOG(97-109)-reactive CD4(+) T cells, isolated with DRB1*0401/MOG(97-109) tetramers, and after a short-term culture of PMBCs with MOG(97-109) peptides, were detected more frequently from patients with MS as compared with healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG(97-109(107E-S)) tetramer(+) cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects using class II tetramers, and the frequency of MOG(97-109)-reactive T cells is greater in patients with MS as compared with healthy controls.

Alzheimer's Disease Genetics: A Dampened Microglial Response?

Alzheimer's disease (AD) is a debilitating age-related neurodegenerative condition. Unbiased genetic studies have implicated a central role for microglia, the resident innate immune cells of the central nervous system, in AD pathogenesis. On-going efforts are clarifying the biology underlying these associations and the microglial pathways that are dysfunctional in AD. Several genetic risk factors converge to decrease the function of activating microglial receptors and increase the function of inhibitory receptors, resulting in a seemingly dampened microglial phenotype in AD. Moreover, many of these microglial proteins that are genetically associated with AD appear to interact and share pathways or regulatory mechanisms, presenting several points of convergence that may be strategic targets for therapeutic intervention. Here, we review some of these studies and their implications for microglial participation in AD pathogenesis.

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.

TGF-beta induces IL-9 production from human Th17 cells.

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-beta and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-beta during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-beta to the Th17-inducing cytokines (IL-1beta, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-beta-dependent coexpression of IL-9 and IL-17 was identified to be IL-1beta. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1beta. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9(+)IL-17(+) cells. These data demonstrate the presence of IL-17(+)IL-9(+) CD4 cells induced by IL-1beta that may play a role in human autoimmune disease.

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells.

The development of T helper (T(H))17 and regulatory T (T(reg)) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-beta alone induces FoxP3(+) T(reg) cells, but together with IL-6 or IL-21 induces T(H)17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of T(H)17 cells and T(reg) function. IL-9 predominantly produced by T(H)17 cells, synergizes with TGF-beta1 to differentiate naïve CD4(+) T cells into T(H)17 cells, while IL-9 secretion by T(H)17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3(+) CD4(+) T(reg) cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nT(regs) in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on T(H)17 and T(regs) is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.

The aging immune system in Alzheimer's and Parkinson's diseases.

The neurodegenerative diseases Alzheimer's disease (AD) and Parkinson's disease (PD) both have a myriad of risk factors including genetics, environmental exposures, and lifestyle. However, aging is the strongest risk factor for both diseases. Aging also profoundly influences the immune system, with immunosenescence perhaps the most prominent outcome. Through genetics, mouse models, and pathology, there is a growing appreciation of the role the immune system plays in neurodegenerative diseases. In this review, we explore the intersection of aging and the immune system in AD and PD.

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.