[Show abstract][Hide abstract] ABSTRACT: T lymphocyte activation by antigen conditions adaptive immune responses and immunopathologies, but we know little about its variation in humans and its genetic or environmental roots. We analyzed gene expression in CD4+ T cells during unbiased activation or in T helper 17 (TH17) conditions from 348 healthy participants representing European, Asian, and African ancestries. We observed interindividual variability, most marked for cytokine transcripts, with clear biases on the basis of ancestry, and following patterns more complex than simple TH1/2/17 partitions. We identified 39 genetic loci specifically associated in cis with activated gene expression. We further fine-mapped and validated a single-base variant that modulates YY1 binding and the activity of an enhancer element controlling the autoimmune-associated IL2RA gene, affecting its activity in activated but not regulatory T cells. Thus, interindividual variability affects the fundamental immunologic process of T helper activation, with important connections to autoimmune disease.
[Show abstract][Hide abstract] ABSTRACT: Two-way communication between the mammalian nervous and immune systems is increasingly recognized and appreciated. An intriguing example of such crosstalk comes from clinical observations dating from the 1930s: Patients who suffer a stroke and then develop rheumatoid arthritis atypically present with arthritis on only one side, the one not afflicted with paralysis. Here we successfully modeled hemiplegia-induced protection from arthritis using the K/BxN serum-transfer system, focused on the effector phase of inflammatory arthritis. Experiments entailing pharmacological inhibitors, genetically deficient mouse strains, and global transcriptome analyses failed to associate the protective effect with a single nerve quality (i.e., with the sympathetic, parasympathetic, or sensory nerves). Instead, there was clear evidence that denervation had a long-term effect on the limb microvasculature: The rapid and joint-localized vascular leak that typically accompanies and promotes serum-transferred arthritis was compromised in denervated limbs. This defect was reflected in the transcriptome of endothelial cells, the expression of several genes impacting vascular leakage or transendothelial cell transmigration being altered in denervated limbs. These findings highlight a previously unappreciated pathway to dissect and eventually target in inflammatory arthritis.
Proceedings of the National Academy of Sciences of the United States of America. 07/2014;
[Show abstract][Hide abstract] ABSTRACT: Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4(+) and CD8(+) T cells, in the regulation of T and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization; all of which could significantly impact the immune response against self antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell-deficient NOD mice, NOD.Cpa3Cre/+ (Heidelberg), and NOD.KitW-sh/W-sh (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process non-essential and excluding them as potential therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: To extend our understanding of the genetic basis of human immune function and dysfunction, we performed an expression quantitative trait locus (eQTL) study of purified CD4(+) T cells and monocytes, representing adaptive and innate immunity, in a multi-ethnic cohort of 461 healthy individuals. Context-specific cis- and trans-eQTLs were identified, and cross-population mapping allowed, in some cases, putative functional assignment of candidate causal regulatory variants for disease-associated loci. We note an over-representation of T cell-specific eQTLs among susceptibility alleles for autoimmune diseases and of monocyte-specific eQTLs among Alzheimer's and Parkinson's disease variants. This polarization implicates specific immune cell types in these diseases and points to the need to identify the cell-autonomous effects of disease susceptibility variants.
[Show abstract][Hide abstract] ABSTRACT: Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease.
Proceedings of the National Academy of Sciences 04/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Foxp3(+) T regulatory (Treg) cells regulate immune responses and maintain self-tolerance. Recent work shows that Treg cells are comprised of many subpopulations with specialized regulatory functions. Here we identified Foxp3(+) T cells expressing the coinhibitory molecule TIGIT as a distinct Treg cell subset that specifically suppresses proinflammatory T helper 1 (Th1) and Th17 cell, but not Th2 cell responses. Transcriptional profiling characterized TIGIT(+) Treg cells as an activated Treg cell subset with high expression of Treg signature genes. Ligation of TIGIT on Treg cells induced expression of the effector molecule fibrinogen-like protein 2 (Fgl2), which promoted Treg-cell-mediated suppression of T effector cell proliferation. In addition, Fgl2 was necessary to prevent suppression of Th2 cytokine production in a model of allergic airway inflammation. TIGIT expression therefore identifies a Treg cell subset that demonstrates selectivity for suppression of Th1 and Th17 cell but not Th2 cell responses.
[Show abstract][Hide abstract] ABSTRACT: FOXP3(+) regulatory T (Treg) cells enforce immune self-tolerance and homeostasis, and variation in some aspects of Treg function may contribute to human autoimmune diseases. Here, we analyzed population-level Treg variability by performing genome-wide expression profiling of CD4(+) Treg and conventional CD4(+) T (Tconv) cells from 168 donors, healthy or with established type-1 diabetes (T1D) or type-2 diabetes (T2D), in relation to genetic and immunologic screening. There was a range of variability in Treg signature transcripts, some almost invariant, others more variable, with more extensive variability for genes that control effector function (ENTPD1, FCRL1) than for lineage-specification factors like FOXP3 or IKZF2. Network analysis of Treg signature genes identified coregulated clusters that respond similarly to genetic and environmental variation in Treg and Tconv cells, denoting qualitative differences in otherwise shared regulatory circuits whereas other clusters are coregulated in Treg, but not Tconv, cells, suggesting Treg-specific regulation of genes like CTLA4 or DUSP4. Dense genotyping identified 110 local genetic variants (cis-expression quantitative trait loci), some of which are specifically active in Treg, but not Tconv, cells. The Treg signature became sharper with age and with increasing body-mass index, suggesting a tuning of Treg function with repertoire selection and/or chronic inflammation. Some Treg signature transcripts correlated with FOXP3 mRNA and/or protein, suggesting transcriptional or posttranslational regulatory relationships. Although no single transcript showed significant association to diabetes, overall expression of the Treg signature was subtly perturbed in T1D, but not T2D, patients.
Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here, we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or β3-agonist treatment. These animals developed obesity on a high-fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the "browning" of white fat and the healthful effects of subcutaneous adipose tissue.
[Show abstract][Hide abstract] ABSTRACT: Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of "paused" RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire's functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.
Proceedings of the National Academy of Sciences 01/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability to reliably identify pancreatic β-cells would have far reaching implications for a greater understanding of β-cell biology, measurement of β-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic β-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of β-cells. Some of the developed compounds should be particularly useful for basic and translational β-cell research.
[Show abstract][Hide abstract] ABSTRACT: Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.
[Show abstract][Hide abstract] ABSTRACT: Perturbations in endoplasmic reticulum (ER) homeostasis can evoke stress responses leading to aberrant glucose and lipid metabolism. ER dysfunction is linked to inflammatory disorders, but its role in the pathogenesis of autoimmune type 1 diabetes (T1D) remains unknown. We identified defects in the expression of unfolded protein response (UPR) mediators ATF6 (activating transcription factor 6) and XBP1 (X-box binding protein 1) in β cells from two different T1D mouse models and then demonstrated similar defects in pancreatic β cells from T1D patients. Administration of a chemical ER stress mitigator, tauroursodeoxycholic acid (TUDCA), at the prediabetic stage resulted in a marked reduction of diabetes incidence in the T1D mouse models. This reduction was accompanied by (i) a significant decrease in aggressive lymphocytic infiltration in the pancreas, (ii) improved survival and morphology of β cells, (iii) reduced β cell apoptosis, (iv) preserved insulin secretion, and (v) restored expression of UPR mediators. TUDCA's actions were dependent on ATF6 and were lost in mice with β cell-specific deletion of ATF6. These data indicate that proper maintenance of the UPR is essential for the preservation of β cells and that defects in this process can be chemically restored for preventive or therapeutic interventions in T1D.
Science translational medicine 11/2013; 5(211):211ra156. · 10.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells leading to insulin deficiency. While infiltrating immune cells are traditionally considered to negatively impact β-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of β-cells which positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4(+) and CD8(+) T-cells, but not B-cells, selectively promoted β-cell proliferation in vivo independent from the effects of blood glucose, circulating insulin or by modulating apoptosis. Complementary to our in vivo approach co-culture of diabetogenic CD4(+) and CD8(+) T-cells with NOD.RAG1(-/-) islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokine/chemokines (IL-2, IL-6, IL-10, MIP-1α and RANTES) that together enhanced β-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance β-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes.
[Show abstract][Hide abstract] ABSTRACT: Both Foxp3(+)CD4(+) regulatory T cells (Treg cells) and local immune responses in nonlymphoid tissues have long been recognized as important elements of a well-orchestrated immune system, but only recently have these two fields of study begun to intersect. There is growing evidence that Treg cells are present in various nonlymphoid tissues in health and disease, that they have a unique phenotype and that their functions go beyond the classical modulation of immune responses. Thus, tissue Treg cells might add yet another level to classification of the Treg cell compartment into functional and/or phenotypic subtypes. In this Review, we summarize recent findings in this new field, discussing knowns and unknowns about the origin, phenotype, function and memory of nonlymphoid tissue-resident Treg cells.
[Show abstract][Hide abstract] ABSTRACT: Several clinical trials have shown anti-CD3 treatment to be a promising therapy for autoimmune diabetes, but its mechanism of action remains unclear. Foxp3(+) regulatory T cells (Tregs) are likely to be involved, but through unknown mechanistic pathways. We profiled the transcriptional consequences in CD4(+) Tregs and conventional T cells (Tconvs) in the first hours and days after anti-CD3 treatment of NOD mice. Anti-CD3 treatment led to a transient transcriptional response, terminating faster than most Ag-induced responses. Most transcripts were similarly induced in Tregs and Tconvs, but several were differential, in particular, those encoding the IL-7R and transcription factors Id2/3 and Gfi1, upregulated in Tregs but repressed in Tconvs. Because IL-7R was a plausible candidate for driving the homeostatic response of Tregs to anti-CD3, we tested its relevance by supplementation of anti-CD3 treatment with IL-7/anti-IL-7 complexes. Although ineffective alone, IL-7 significantly improved the rate of remission induced by anti-CD3. Four anti-human CD3 mAbs exhibited the same differential effect on IL-7R expression in human as in mouse cells, suggesting that the mechanism also underlies therapeutic effect in human cells, and perhaps a rationale for testing a combination of anti-CD3 and IL-7 for the treatment of recent-onset human type 1 diabetes. Thus, systems-level analysis of the response to anti-CD3 in the early phase of the treatment demonstrates different responses in Tregs and Tconvs, and provides new leads to a mechanistic understanding of its mechanism of action in reverting recent-onset diabetes.
The Journal of Immunology 08/2013; · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alternative splicing (AS) allows increased diversity and orthogonal regulation of the transcriptional products of mammalian genomes. To assess the distribution and variation of alternative splicing across cell lineages of the immune system, we comprehensively analyzed RNA sequencing and microarray data generated by the Immunological Genome Project Consortium. AS is pervasive: 60% of genes showed frequent AS isoforms in T or B lymphocytes, with 7,599 previously unreported isoforms. Distinct cell specificity was observed, with differential exon skipping in 5% of genes otherwise coexpressed in both B and T cells. The distribution of isoforms was mostly all or none, suggesting on/off switching as a frequent mode of AS regulation in lymphocytes. From the identification of differential exon use in the microarray data, clustering of exon inclusion/exclusion patterns across all Immunological Genome Project cell types showed that ∼70% of AS exons are distributed along a common pattern linked to lineage differentiation and cell cycling. Other AS events distinguished myeloid from lymphoid cells or affected only a small set of exons without clear lineage specificity (e.g., Ptprc). Computational analysis predicted specific associations between AS exons and splicing regulators, which were verified by detection of the hnRPLL/Ptprc connection.
Proceedings of the National Academy of Sciences 08/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic, low-grade inflammation of visceral adipose tissue, and systemically, is a critical link between recent strikingly parallel rises in the incidence of obesity and type 2 diabetes. Macrophages have been recognized for some time to be critical participants in obesity-induced inflammation of adipose tissue. Of late, a score of other cell types of the innate and adaptive arms of the immune system have been suggested to play a positive or negative role in adipose tissue infiltrates. This piece reviews the existing data on these new participants; discusses experimental uncertainties, inconsistencies, and complexities; and puts forward a minimalist synthetic scheme.
[Show abstract][Hide abstract] ABSTRACT: Regulatory T (T reg) cells control progression to autoimmune diabetes in the BDC2.5/NOD mouse model by reining in natural killer (NK) cells that infiltrate the pancreatic islets, inhibiting both their proliferation and production of diabetogenic interferon-γ. In this study, we have explored the molecular mechanisms underlying this NK-T reg cell axis, following leads from a kinetic exploration of gene expression changes early after punctual perturbation of T reg cells in BDC2.5/NOD mice. Results from gene signature analyses, quantification of STAT5 phosphorylation levels, cytokine neutralization experiments, cytokine supplementation studies, and evaluations of intracellular cytokine levels collectively argue for a scenario in which T reg cells regulate NK cell functions by controlling the bioavailability of limiting amounts of IL-2 in the islets, generated mainly by infiltrating CD4(+) T cells. This scenario represents a previously unappreciated intertwining of the innate and adaptive immune systems: CD4(+) T cells priming NK cells to provoke a destructive T effector cell response. Our findings highlight the need to consider potential effects on NK cells when designing therapeutic strategies based on manipulation of IL-2 levels or targets.
Journal of Experimental Medicine 05/2013; · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: After infection, many factors coordinate the population expansion and differentiation of CD8(+) effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8(+) T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8(+) effector cells. Long-lived memory CD8(+) cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8(+) effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8(+) T cell immunity.