Epigenetic signature of human immune aging in the GESTALT study.

Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4+ and CD8+ T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1ß) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.

TCF-1 Is Required for CD4 T Cell Persistence Functions during AlloImmunity.

The transcription factor T cell factor-1 (TCF-1) is encoded by Tcf7 and plays a significant role in regulating immune responses to cancer and pathogens. TCF-1 plays a central role in CD4 T cell development; however, the biological function of TCF-1 on mature peripheral CD4 T cell-mediated alloimmunity is currently unknown. This report reveals that TCF-1 is critical for mature CD4 T cell stemness and their persistence functions. Our data show that mature CD4 T cells from TCF-1 cKO mice did not cause graft versus host disease (GvHD) during allogeneic CD4 T cell transplantation, and donor CD4 T cells did not cause GvHD damage to target organs. For the first time, we showed that TCF-1 regulates CD4 T cell stemness by regulating CD28 expression, which is required for CD4 stemness. Our data showed that TCF-1 regulates CD4 effector and central memory formation. For the first time, we provide evidence that TCF-1 differentially regulates key chemokine and cytokine receptors critical for CD4 T cell migration and inflammation during alloimmunity. Our transcriptomic data uncovered that TCF-1 regulates critical pathways during normal state and alloimmunity. Knowledge acquired from these discoveries will enable us to develop a target-specific approach for treating CD4 T cell-mediated diseases.

TCF-1 negatively regulates the suppressive ability of canonical and noncanonical Tregs.

Regulatory T cells are suppressive immune cells used in various clinical and therapeutic applications. Canonical regulatory T cells express CD4, FOXP3, and CD25, which are considered definitive markers of their regulatory T-cell status when expressed together. However, a subset of noncanonical regulatory T cells expressing only CD4 and FOXP3 have recently been described in some infection contexts. Using a unique mouse model for the first time demonstrated that the TCF-1 regulation of regulatory T-cell suppressive function is not limited to the thymus during development. Our data showed that TCF-1 also regulated regulatory T cells' suppressive ability in secondary organs and graft-vs-host disease target organs as well as upregulating noncanonical regulatory T cells. Our data demonstrated that TCF-1 regulates the suppressive function of regulatory T cells through critical molecules like GITR and PD-1, specifically by means of noncanonical regulatory T cells. Our in vitro approaches show that TCF-1 regulates the regulatory T-cell effector-phenotype and the molecules critical for regulatory T-cell migration to the site of inflammation. Using in vivo models, we show that both canonical and noncanonical regulatory T cells from TCF-1 cKO mice have a superior suppressive function, as shown by their ability to control conventional T-cell proliferation, avert acute graft-vs-host disease, and limit tissue damage. Thus, for the first time, we provide evidence that TCF-1 negatively regulates the suppressive ability of canonical and noncanonical regulatory T cells. These findings provide evidence that TCF-1 is a novel target for developing strategies to treat alloimmune disorders.

TCF-1 regulates NKG2D expression on CD8 T cells during anti-tumor responses.

Cancer immunotherapy relies on improving T cell effector functions against malignancies, but despite the identification of several key transcription factors (TFs), the biological functions of these TFs are not entirely understood. We developed and utilized a novel, clinically relevant murine model to dissect the functional properties of crucial T cell transcription factors during anti-tumor responses. Our data showed that the loss of TCF-1 in CD8 T cells also leads to loss of key stimulatory molecules such as CD28. Our data showed that TCF-1 suppresses surface NKG2D expression on naïve and activated CD8 T cells via key transcriptional factors Eomes and T-bet. Using both in vitro and in vivo models, we uncovered how TCF-1 regulates critical molecules responsible for peripheral CD8 T cell effector functions. Finally, our unique genetic and molecular approaches suggested that TCF-1 also differentially regulates essential kinases. These kinases, including LCK, LAT, ITK, PLC-γ1, P65, ERKI/II, and JAK/STATs, are required for peripheral CD8 T cell persistent function during alloimmunity. Overall, our molecular and bioinformatics data demonstrate the mechanism by which TCF-1 modulated several critical aspects of T cell function during CD8 T cell response to cancer. Summary Figure: TCF-1 is required for persistent function of CD8 T cells but dispensable for anti-tumor response. Here, we have utilized a novel mouse model that lacks TCF-1 specifically on CD8 T cells for an allogeneic transplant model. We uncovered a molecular mechanism of how TCF-1 regulates key signaling pathways at both transcriptomic and protein levels. These key molecules included LCK, LAT, ITK, PLC-γ1, p65, ERK I/II, and JAK/STAT signaling. Next, we showed that the lack of TCF-1 impacted phenotype, proinflammatory cytokine production, chemokine expression, and T cell activation. We provided clinical evidence for how these changes impact GVHD target organs (skin, small intestine, and liver). Finally, we provided evidence that TCF-1 regulates NKG2D expression on mouse naïve and activated CD8 T cells. We have shown that CD8 T cells from TCF-1 cKO mice mediate cytolytic functions via NKG2D.

Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD.

BACKGROUND: Although ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear. METHODS: We explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff). RESULTS: We found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators. CONCLUSIONS: We propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.

DNA methylation signatures reveal that distinct combinations of transcription factors specify human immune cell epigenetic identity.

Epigenetic reprogramming underlies specification of immune cell lineages, but patterns that uniquely define immune cell types and the mechanisms by which they are established remain unclear. Here, we identified lineage-specific DNA methylation signatures of six immune cell types from human peripheral blood and determined their relationship to other epigenetic and transcriptomic patterns. Sites of lineage-specific hypomethylation were associated with distinct combinations of transcription factors in each cell type. By contrast, sites of lineage-specific hypermethylation were restricted mostly to adaptive immune cells. PU.1 binding sites were associated with lineage-specific hypo- and hypermethylation in different cell types, suggesting that it regulates DNA methylation in a context-dependent manner. These observations indicate that innate and adaptive immune lineages are specified by distinct epigenetic mechanisms via combinatorial and context-dependent use of key transcription factors. The cell-specific epigenomics and transcriptional patterns identified serve as a foundation for future studies on immune dysregulation in diseases and aging.

Human Wnt/ß-Catenin Regulates Alloimmune Signaling during Allogeneic Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most widely applied forms of adoptive immunotherapy for the treatment of hematological malignancies. Detrimental graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia (GVL) effects occurring after allo-HSCT are largely mediated by alloantigen-reactive donor T cells in the graft. Separating GVHD from GVL effects is a formidable challenge, and a greater understanding of donor T cell biology is required to accomplish the uncoupling of GVHD from GVL. Here, we evaluated the role of ß-catenin in this process. Using a unique mouse model of transgenic overexpression of human ß-catenin (Cat-Tg) in an allo-HSCT model, we show here that T cells from Cat-Tg mice did not cause GVHD, and surprisingly, Cat-Tg T cells maintained the GVL effect. Donor T cells from Cat-Tg mice exhibited significantly lower inflammatory cytokine production and reduced donor T cell proliferation, while upregulating cytotoxic mediators that resulted in enhanced cytotoxicity. RNA sequencing revealed changes in the expression of 1169 genes for CD4, and 1006 genes for CD8+ T cells involved in essential aspects of immune response and GVHD pathophysiology. Altogether, our data suggest that ß-catenin is a druggable target for developing therapeutic strategies to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

Neuroinflammation is associated with infiltration of T cells in Lewy body disease and α-synuclein transgenic models.

BACKGROUND: α-Synuclein (α-syn) is a pre-synaptic protein which progressively accumulates in neuronal and non-neuronal cells in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. Recent evidence suggests that aberrant immune activation may be involved in neurodegeneration in PD/DLB. While previous studies have often focused on the microglial responses, less is known about the role of the peripheral immune system in these disorders. METHODS: To understand the involvement of the peripheral immune system in PD/DLB, we evaluated T cell populations in the brains of α-syn transgenic (tg) mice (e.g., Thy1 promoter line 61) and DLB patients. RESULTS: Immunohistochemical analysis showed perivascular and parenchymal infiltration by CD3+/CD4+ helper T cells, but not cytotoxic T cells (CD3+/CD8+) or B cells (CD20+), in the neocortex, hippocampus, and striatum of α-syn tg mice. CD3+ cells were found in close proximity to the processes of activated astroglia, particularly in areas of the brain with significant astrogliosis, microgliosis, and expression of pro-inflammatory cytokines. In addition, a subset of CD3+ cells co-expressed interferon γ. Flow cytometric analysis of immune cells in the brains of α-syn tg mice revealed that CD1d-tet+ T cells were also increased in the brains of α-syn tg mice suggestive of natural killer T cells. In post-mortem DLB brains, we similarly detected increased numbers of infiltrating CD3+/CD4+ T cells in close proximity with blood vessels. CONCLUSION: These results suggest that infiltrating adaptive immune cells play an important role in neuroinflammation and neurodegeneration in synucleinopathies and that modulating peripheral T cells may be a viable therapeutic strategy for PD/DLB.

Dynamic regulation of chromatin organizer SATB1 via TCR-induced alternative promoter switch during T-cell development.

The chromatin organizer SATB1 is highly enriched in thymocytes and is essential for T-cell development. Although SATB1 regulates a large number of genes important for T-cell development, the mechanism(s) regulating expression of SATB1 during this process remain elusive. Using chromatin immune precipitation-seq-based occupancy profiles of H3K4me3 and H3Kme1 at Satb1 gene locus, we predicted four different alternative promoters of Satb1 in mouse thymocytes and characterized them. The expression of Satb1 transcript variants with distinct 5' UTRs occurs in a stage-specific manner during T-cell development and is dependent on TCR signaling. The observed discrepancy between the expression levels of SATB1 mRNA and protein in developing thymocytes can be explained by the differential translatability of Satb1 transcript variants as confirmed by polysome profiling and in vitro translation assay. We show that Satb1 alternative promoters exhibit lineage-specific chromatin accessibility during T-cell development from progenitors. Furthermore, TCF1 regulates the Satb1 P2 promoter switch during CD4SP development, via direct binding to the Satb1 P2 promoter. CD4SP T cells from TCF1 KO mice exhibit downregulation of P2 transcript variant expression as well as low levels of SATB1 protein. Collectively, these results provide unequivocal evidence toward alternative promoter switch-mediated developmental stage-specific regulation of SATB1 in thymocytes.

Immune system development and age-dependent maintenance in Klotho-hypomorphic mice.

Circulating Klotho peptide hormone has anti-aging activity and affects tissue maintenance. Hypomorphic mutant Klotho [kl/kl] mice on C57BL/6xC3H, BALB/c and 129 genetic backgrounds, show decreased Klotho expression that correlate with accelerated aging including pre-mature death due to abnormally high levels of serum vitamin D. These mice also show multiple impairments in the immune system. However, it remains unresolved if the defects in the immune system stem from decreased Klotho expression or high vitamin D levels in the serum. Transfer of the kl/kl allele to pure C57BL/6 genetic background [B6-kl/kl] significantly reduced expression of Klotho at all ages. Surprisingly, B6-kl/kl mice showed normalized serum vitamin D levels, amelioration of severe aging-related phenotypes and normal lifespan. This paper reports a detailed analysis of the immune system in B6-kl/kl mice in the absence of detrimental levels of serum vitamin D. Remarkably, the data reveal that in the absence of overt systemic stress, such as abnormally high vitamin D levels, reduced expression of Klotho does not have a major effect on the generation and maintenance of the immune system.

Chronic Mild Gut Inflammation Accelerates Brain Neuropathology and Motor Dysfunction in α-Synuclein Mutant Mice.

Emerging findings suggest that Parkinson's disease (PD) pathology (α-synuclein accumulation) and neuronal dysfunction may occur first in peripheral neurons of the autonomic nervous system including the enteric branches of the vagus nerve. The risk of PD increases greatly in people over the age of 65, a period of life in which chronic inflammation is common in many organ systems including the gut. Here we report that chronic mild focal intestinal inflammation accelerates the age of disease onset in α-synuclein mutant PD mice. Wild-type and PD mice treated with 0.5% dextran sodium sulfate (DSS) in their drinking water for 12 weeks beginning at 3 months of age exhibited histological and biochemical features of mild gut inflammation. The age of onset of motor dysfunction, evaluated using a rotarod test, gait analysis, and grip strength measurements, was significantly earlier in DSS-treated PD mice compared to control PD mice. Levels of the dopaminergic neuron marker tyrosine hydroxylase in the striatum and numbers of dopaminergic neurons in the substantia nigra were reduced in PD mice with gut inflammation. Levels of total and phosphorylated α-synuclein were elevated in enteric and brain neurons in DSS-treated PD mice, suggesting that mild gut inflammation accelerates α-synuclein pathology. Markers of inflammation in the colon and brain, but not in the blood, were elevated in DSS-treated PD mice, consistent with retrograde transneuronal propagation of α-synuclein pathology and neuroinflammation from the gut to the brain. Our findings suggest that interventions that reduce gut inflammation may prove beneficial in the prevention and treatment of PD.

Senolytic therapy alleviates Aß-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model.

Neuritic plaques, a pathological hallmark in Alzheimer's disease (AD) brains, comprise extracellular aggregates of amyloid-beta (Aß) peptide and degenerating neurites that accumulate autolysosomes. We found that, in the brains of patients with AD and in AD mouse models, Aß plaque-associated Olig2- and NG2-expressing oligodendrocyte progenitor cells (OPCs), but not astrocytes, microglia, or oligodendrocytes, exhibit a senescence-like phenotype characterized by the upregulation of p21/CDKN1A, p16/INK4/CDKN2A proteins, and senescence-associated ß-galactosidase activity. Molecular interrogation of the Aß plaque environment revealed elevated levels of transcripts encoding proteins involved in OPC function, replicative senescence, and inflammation. Direct exposure of cultured OPCs to aggregating Aß triggered cell senescence. Senolytic treatment of AD mice selectively removed senescent cells from the plaque environment, reduced neuroinflammation, lessened Aß load, and ameliorated cognitive deficits. Our findings suggest a role for Aß-induced OPC cell senescence in neuroinflammation and cognitive deficits in AD, and a potential therapeutic benefit of senolytic treatments.

Noncoding variations in Cyp24a1 gene are associated with Klotho-mediated aging phenotypes in different strains of mice.

In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging-related phenotypes caused by Klotho down-regulation. The hypomorphic Klotho [kl/kl] allele that decreases Klotho expression in C3H, BALB/c, 129, and C57BL/6 genetic backgrounds substantially increases 1,25(OH)2D3 levels in the sera of susceptible C3H, BALB/c, and 129, but not C57BL/6 mice. This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super-enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6-kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.

Temporal regulation of Wnt/ß-catenin signaling is important for invariant NKT cell development and terminal maturation.

The Wnt/ß-catenin signaling pathway plays important roles during various cellular functions including survival and proliferation of immune cells. The critical role of this pathway in conventional T cell development is established but little is known about its contributions to innate T cell development. In this study, we found that ß-catenin level, an indication of the strength of Wnt/ß-catenin signaling, is regulated during invariant NKT (iNKT) cell development. ß-catenin levels were greatly increased during iNKT cell selection from double positive thymocytes to Stage 0 of iNKT cell development and during subsequent development to Stage 1. Thereafter, ß-catenin levels decrease from Stage 2, which is essential for the terminal maturation of iNKT cells. Failure to dampen Wnt/ß-catenin signaling as in mice expressing a stabilized active form of ß-catenin (CATtg) resulted in increased Stage 2 and decreased Stage 3 iNKT cells. Inefficient transition from Stage 2 to 3 in CATtg iNKT cells seems to be contributed by poor expression of IL-15R (CD122) and transcription factor T-bet, both of which are necessary for terminal maturation of iNKT cells in the thymus. Consequently, IFN-γ+ iNKT cells were greatly reduced in CATtg mice. Together, our findings reveal that proper regulation of ß-catenin and in turn Wnt signaling plays an important role in the terminal maturation and function of iNKT cells.

Regulation of SATB1 during thymocyte development by TCR signaling.

T lymphocyte development and differentiation is a multi-step process that begins in the thymus and completed in the periphery. Sequential development of thymocytes is dependent on T cell receptor (TCR) signaling and an array of transcription factors. In this study we show that special AT-rich binding protein 1 (SATB1), a T lineage-enriched chromatin organizer and regulator, is induced in response to TCR signaling during early thymocyte development. SATB1 expression profile coincides with T lineage commitment and upregulation of SATB1 correlates with positive selection of thymocytes. CD4 thymocytes exhibit a characteristic bimodal expression pattern that corresponds to immature and mature CD4 thymocytes. We also demonstrate that GATA3, the key transcriptional regulator of αß T cells positively regulates SATB1 expression in thymocytes suggesting an important role for SATB1 during T cell development.

Cell-autonomous requirement for TCF1 and LEF1 in the development of Natural Killer T cells.

Natural killer T (NKT) cells develop from common CD4(+) CD8(+) thymocyte precursors. Transcriptional programs that regulate the development of NKT cells in the thymus development remain to be fully delineated. Here, we demonstrate a cell-intrinsic requirement for transcription factors TCF1 and LEF1 for the development of all subsets of NKT cells. Conditional deletion of TCF1 alone results in a substantial reduction in NKT cells. The remaining NKT cells are eliminated when TCF1 and LEF1 are both deleted. These data reveal an essential role for TCF1 and LEF1 in development of NKT cells.

ß-Catenin is required for the differentiation of iNKT2 and iNKT17 cells that augment IL-25-dependent lung inflammation.

BACKGROUND: Invariant Natural Killer T (iNKT) cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. iNKT cells differentiate and acquire functional characteristics during development in the thymus. However, the correlation between development of iNKT cells in the thymus and role in lung inflammation remains unknown. In addition, transcriptional control of differentiation of iNKT cells into iNKT cell effector subsets in the thymus during development is also unclear. In this report we show that ß-catenin dependent mechanisms direct differentiation of iNKT2 and iNKT17 subsets but not iNKT1 cells. METHODS: To study the role for ß-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of ß-catenin in a well-established mouse model for IL-25-dependen lung inflammation. RESULTS: Specifically, we demonstrate that conditional deletion of ß-catenin permitted development of mature iNKT1 cells while impeding maturation of iNKT2 and 17 cells. A role for ß-catenin expression in promoting iNKT2 and iNKT17 subsets was confirmed when we noted that enforced transgenic expression of ß-catenin in iNKT cell precursors enhanced the frequency and number of iNKT2 and iNKT17 cells at the cost of iNKT1 cells. This effect of expression of ß-catenin in iNKT cell precursors was cell autonomous. Furthermore, iNKT2 cells acquired greater capability to produce type-2 cytokines when ß-catenin expression was enhanced. DISCUSSION: This report shows that ß-catenin deficiency resulted in a profound decrease in iNKT2 and iNKT17 subsets of iNKT cells whereas iNKT1 cells developed normally. By contrast, enforced expression of ß-catenin promoted the development of iNKT2 and iNKT17 cells. It was important to note that the majority of iNKT cells in the thymus of C57BL/6 mice were iNKT1 cells and enforced expression of ß-catenin altered the pattern to iNKT2 and iNKT17 cells suggesting that ß-catenin may be a major factor in the distinct pathways that critically direct differentiation of iNKT effector subsets. CONCLUSIONS: Thus, we demonstrate that ß-catenin expression in iNKT cell precursors promotes differentiation toward iNKT2 and iNKT17 effector subsets and supports enhanced capacity to produce type 2 and 17 cytokines which in turn augment lung inflammation in mice.