Research

 

Linking genotype, cell function and pathology in inflammatory diseases


The past years have been marked by a leap forward in our understanding of the genetic basis of many inflammatory diseases. Genome-wide association studies (GWAS) have provided insight into the “genetic hardware” of inflammatory diseases. These studies have brought into the spotlight many genes linked to signaling pathways that were not known to be involved in disease pathogenesis, pointing to new directions in the study of disease mechanisms. The current challenge is to correlate the genetic variants with the effector mechanisms implicated in pathogenesis, to allow translation of the genetic data into novel diagnostics and treatment strategies. We have investigated the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4+ T lymphocyte subsets involved in chronic inflammatory disease.
We recently found that the effector functions of Th17 and Th1 cells in SpA patients are under combinatorial control by multiple SNPs at genes associated with the IL-23/Th17 pathway. SpA patients carrying risk-associated alleles of genes in this pathway expressed the highest levels of genes involved in the differentiation and function of Th17 and Th1 cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Th17 and Th1 genes, suggesting that these SNPs may contribute to SpA pathogenesis through distinct cellular mechanisms.
Our results show that genetic variation at genes associated with the IL-23 signaling pathway affects the effector functions of Th17 and Th1 cells in SpA patients and provide a framework to delineate the mechanisms by which genetic variants contribute to pathology.
This project is performed in collaboration the team of Pr. M. Dougados, Director of the National Reference Center for ankylosing spondylitis in France and of the Service de Rhumatologie B at Hôpital Cochin and with the Center for Human Immunology (CIH). Reference: (Coffre et al., Arthritis Rheum. 2013 doi: 10.1002/art.37936. [Epub ahead of print])
Supplementary data for this article can be downloaded here 
 
Epigenetic mechanisms controlling the stability and plasticity of CD4+ T cell subsets
Project leader: Elisabetta Bianchi

Scientific Background
Differentiation of naïve CD4+ T lymphocytes into functionally distinct T helper cell subsets is essential for efficient immune responses against different types of pathogens. Effector T cell lineages selectively produce specific sets of cytokines, which modulate the responses of other cells involved in host defense. T helper type 1 (Th1) cells promote cell-mediated immunity and clear intracellular bacteria whereas Th2 responses are essential to fight extracellular helminthes. The recently identified Th17 subset is required for mucosal immunity and protection against extracellular bacteria and fungi. On the other hand, uncontrolled T helper cell responses play a critical role in the pathogenesis of various immunopathologies, such as allergies, asthma, chronic inflammatory and autoimmune diseases. Th1, Th2, and Th17 cells are primarily characterized by the secretion of “signature” cytokines, with Th1 cells producing interferon-g (IFN-g) and Th2 cells producing cytokines encoded by the “Th2 cytokine locus”, interleukin-4 (IL-4), IL-5, and IL-13. Th17 cells are characterized by the secretion of IL-17A and IL-17F, but may also produce additional cytokines such as IL-21, IL-22 and IL-26. Differentiation of T helper cells is regulated by “key” transcription factors (STAT4 and T-bet for Th1; GATA-3 and STAT6 for Th2 cell differentiation). The orphan nuclear receptor RORgt (RORC) is involved in the differentiation of Th17 cells. RORC is expressed during thymic development, but its expression is silenced in naive CD4+ T cells.
Studies from several laboratories, including ours, have provided clear evidence that the process of Th1 and Th2 cell differentiation from naïve CD4+ T cells is accompanied by extensive modifications of the chromatin structure at the loci of the Th1 and Th2 cytokine genes. These modifications occur early during the differentiation process, are stably inherited in fully differentiated Th1 and Th2 effector subsets, and are the basis for the maintenance of the differentiated T helper phenotype. On the other hand, the epigenetic modifications at the basis of human Th17 differentiation are still largely unknown.
 
Project Aim
The aim of this project is to study how differentiation of naïve CD4+ T cells into Th1 and Th17 effector cells is coordinated at the level of the chromatin structure.
To this aim, we will analyze the levels of permissive or repressive chromatin modifications at the signature cytokine loci and at the master transcription factors loci of Th1 and Th17 cells.
These studies will be performed on T cell populations (CD4+, CD4+CD8+, and double-negative) isolated from human thymic specimens, on in vitro differentiated Th1 and Th17 cells, and on IFN-g or IL-17-secreting T cells isolated from the peripheral blood of healthy individuals (buffy coats), or from peripheral lymphatic structures (tonsils).
 
 

Analysis of human CD4+FOXP3+ regulatory T cell populations in homeostatic conditions and during graft-versus-host disease


The perspective to harness the potent immunosuppressive activity of CD4+FOXP3+ regulatory T cells (Treg) for clinical applications has sparked enormous interest in this cell population. Recent studies, however, have shown that the human Treg landscape may be more complicated than previously thought: not only do several distinct Treg subsets exist, but also the possibility has been raised that Treg may represent a “plastic” lineage with propensity to convert into an inflammatory cell phenotype. Understanding the heterogeneity of human Treg and their potential for lineage reprogramming to pro-inflammatory effector T cells is key for moving Treg therapy into the clinics.
In a collaboration with Pr. Gérard Socié and his teal at Hôpital St. Louis in Paris, we have explored the heterogeneity and functional diversity of human Treg using multi-parameter single-cell analysis techniques in healthy donors and in the severely inflammatory and lymphopenic environment of patients after allogeneic hematopoietic stem cell transplantation (alloHSCT). We found that the human regulatory T cell compartment displayed a similar level of complexity with respect to the expression of transcription factors, homing receptors and inflammatory cytokines as conventional CD4+ effector T cells. Single-cell gene profiling analysis of very small populations of IFN-γ and IL-17A-producing Treg revealed that cytokine-secreting Treg are characterized by an overlap of gene expression signatures of Th1 or Th17 cells and of Treg within the same cell. Analysis of Treg homeostasis at the time of engraftment 2-3 weeks after alloHSCT revealed a revealed a marked depletion of Treg with a naïve phenotype and increased Treg proliferation in patients developing acute graft-versus-host disease, compared to tolerant patients. However, the overall frequency of CD4+FOXP3+ T cells and their suppressive activity were preserved.
Our work suggests that heterogeneity at the single-cell level, rather than reprogramming of CD4+FOXP3+ T cells, explains the remarkable complexity and functional diversity of human Treg. Moreover, we demonstrate that the use of single-cell analyses offers the unique opportunity to investigate the early events that shape immune reconstitution after alloHSCT, at a time point when it may still be possible to tilt the balance between excessive inflammation and tolerance induction.