Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells.

T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti-PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

Approaches to Detect microRNA Expression in T Cell Subsets and T Cell Differentiation.

MicroRNAs (miRNAs) are crucial components of the molecular networks regulating differentiation and responses of T lymphocytes in health and disease. It is therefore essential to rely on robust methods of qualitative and quantitative investigation of miRNA expression in T cell subsets, and during T cell activation and differentiation. Here, we focus on different methods for miRNA analysis, including Northern blots, quantitative RT-PCR, and next-generation sequencing, and we discuss advantages and disadvantages of each method. While we mainly focus on the study of miRNA expression in human T lymphocytes, these methods can also be applied to other species and/or cell types.

Dynamic Changes in Chromatin Accessibility Occur in CD8+ T Cells Responding to Viral Infection.

In response to acute infection, naive CD8+ T cells expand, differentiate into effector cells, and then contract to a long-lived pool of memory cells after pathogen clearance. During chronic infections or in tumors, CD8+ T cells acquire an "exhausted" phenotype. Here we present genome-wide comparisons of chromatin accessibility and gene expression from endogenous CD8+ T cells responding to acute and chronic viral infection using ATAC-seq and RNA-seq techniques. Acquisition of effector, memory, or exhausted phenotypes was associated with stable changes in chromatin accessibility away from the naive T cell state. Regions differentially accessible between functional subsets in vivo were enriched for binding sites of transcription factors known to regulate these subsets, including E2A, BATF, IRF4, T-bet, and TCF1. Exhaustion-specific accessible regions were enriched for consensus binding sites for NFAT and Nr4a family members, indicating that chronic stimulation confers a unique accessibility profile on exhausted cells.

Control of Foxp3 stability through modulation of TET activity.

Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine and other oxidized methylcytosines, intermediates in DNA demethylation. In this study, we examine the role of TET proteins in regulating Foxp3, a transcription factor essential for the development and function of regulatory T cells (T reg cells), a distinct lineage of CD4(+) T cells that prevent autoimmunity and maintain immune homeostasis. We show that during T reg cell development in the thymus, TET proteins mediate the loss of 5mC in T reg cell-specific hypomethylated regions, including CNS1 and CNS2, intronic cis-regulatory elements in the Foxp3 locus. Similar to CNS2-deficient T reg cells, the stability of Foxp3 expression is markedly compromised in T reg cells from Tet2/Tet3 double-deficient mice. Vitamin C potentiates TET activity and acts through Tet2/Tet3 to increase the stability of Foxp3 expression in TGF-ß-induced T reg cells. Our data suggest that targeting TET enzymes with small molecule activators such as vitamin C might increase induced T reg cell efficacy.

MicroRNA-directed program of cytotoxic CD8+ T-cell differentiation.

Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTLs, and that deletion or depletion of Dicer in mouse or human activated CD8(+) T cells causes up-regulation of perforin, granzymes, and effector cytokines. Genome-wide analysis of microRNA (miR, miRNA) changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of an miRNA network that controls perforin, eomesodermin, and IL-2Rα expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation, and antigenic stimulation. Overall, our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation.

Functional studies on the IBD susceptibility gene IL23R implicate reduced receptor function in the protective genetic variant R381Q.

Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23R(R381) and IL23R(Q381) haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23R(Q381) was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23R(Q381) positive donors. Our study shows conclusively that IL23R(Q381) is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD.

Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161.

Innate lymphoid cells (ILCs) are emerging as a family of effectors and regulators of innate immunity and tissue remodeling. Interleukin 22 (IL-22)- and IL-17-producing ILCs, which depend on the transcription factor RORγt, express CD127 (IL-7 receptor α-chain) and the natural killer cell marker CD161. Here we describe another lineage-negative CD127(+)CD161(+) ILC population found in humans that expressed the chemoattractant receptor CRTH2. These cells responded in vitro to IL-2 plus IL-25 and IL-33 by producing IL-13. CRTH2(+) ILCs were present in fetal and adult lung and gut. In fetal gut, these cells expressed IL-13 but not IL-17 or IL-22. There was enrichment for CRTH2(+) ILCs in nasal polyps of chronic rhinosinusitis, a typical type 2 inflammatory disease. Our data identify a unique type of human ILC that provides an innate source of T helper type 2 (T(H)2) cytokines.

Regulation of cytokine secretion in human CD127(+) LTi-like innate lymphoid cells by Toll-like receptor 2.

Lymphoid tissue inducer cells are members of an emerging family of innate lymphoid cells (ILC). Although these cells were originally reported to produce cytokines such as interleukin-17 (IL-17) and IL-22, we demonstrate here that human CD127(+)RORC(+) and CD56(+)CD127(+) LTi-like ILC also express IL-2, IL-5, and IL-13 after activation with physiologic stimuli such as common γ-chain cytokines, Toll-like receptor (TLR) 2 ligands, or IL-23. Whereas TLR2 signaling induced IL-5, IL-13, and IL-22 expression in a nuclear factor κB (NF-κB)-dependent manner, IL-23 costimulation induced only IL-22 production. CD127(+) LTi-like ILC displayed clonal heterogeneity for IL-13 and IL-5 production, suggesting in vivo polarization. Finally, we identified a role for autocrine IL-2 signaling in mediating the effects of TLR2 stimulation on CD56(+)CD127(+) and CD127(+) LTi-like ILC. These results indicate that human LTi-like ILC can directly sense bacterial components and unravel a previously unrecognized functional heterogeneity among this important population of innate lymphoid cells.

IL-22-producing CD4+ T cells: middle-men between the immune system and its environment.

CD4(+) Th cell populations such as Th1, Th2, Th17 and regulatory T cells regulate immune responses by inducing (or inhibiting) proliferation, differentiation and activation of other immune cells. Recent findings have expanded the universe of CD4(+) T-cell subsets by identifying a cell population dedicated to the production of the cytokine IL-22. These so-called Th22 cells may mediate interactions of the immune system with stromal cells. Th22 cells have so far only been observed in humans and are present in inflamed tissues in some skin diseases and evidence suggests that these cells may play a role in the disease process. It is therefore of importance to learn the mechanisms of regulation of development and function of these cells. A paper in the current issue of the European Journal of Immunology indicates that the ligand-dependent transcription factor aryl hydrocarbon receptor is important for regulating IL-22 production of this new class of human Th cells.

Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells.

Lymphoid tissue inducer (LTi) cells are required for lymph node formation during fetal development, and recent evidence implies a role in mucosal immunity in the adult. LTi cells share some phenotypic features of conventional natural killer (NK; cNK) cells; however, little is known to date about the relationship between these two cell types. We show that lineage(-) (Lin(-)) CD127(+)RORC(+) LTi-like cells in human tonsil are precursors to CD56(+)CD127(+)RORC(+)NKp46(+) cells, which together comprise a stable RORC(+) lineage. We find that LTi-like cells and their CD56(+) progeny can be expanded and cloned ex vivo without loss of function and without conversion into cNK cells. Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population. Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells. Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: analysis of function and distribution in lymphoid organs.

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE: The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS: A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS: The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION: Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.

Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells.

Interleukin 22 (IL-22) is a member of the IL-10 cytokine family that is involved in inflammatory and wound healing processes. Originally considered a T helper type 1 (T(H)1)-associated cytokine, IL-22 has since been shown to be produced mainly by IL-17-producing helper T cells (T(H)-17 cells). Here we describe a previously uncharacterized IL-22-producing human helper T cell population that coexpressed the chemokine receptor CCR6 and the skin-homing receptors CCR4 and CCR10. These cells were distinct from both T(H)-17 cells and T(H)1 cells. Downregulation of either the aryl hydrocarbon receptor (AHR) or the transcription factor RORC by RNA-mediated interference affected IL-22 production, whereas IL-17 production was affected only by downregulation of RORC by RNA-mediated interference. AHR agonists substantially altered the balance of IL-22- versus IL-17-producing cells. This subset of IL-22-producing cells may be important in skin homeostasis and pathology.

WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells.

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

Current understanding of the Wiskott-Aldrich syndrome and prospects for gene therapy.

Gene therapy, based on the transplantation of genetically corrected autologous hematopoietic stem cells (HSCs), has proven to be an effective therapeutic approach as an alternative to allogenic HSC transplantation for the cure of severe combined immunodeficiencies (SCID). In this article, the recent preclinical studies aiming towards gene therapy trials for the Wiskott-Aldrich syndrome (WAS), a life-threatening immunodeficiency characterized by infections, hemorrhages, autoimmune disorders and lymphomas, will be reviewed. An update of the safety and efficacy data obtained in studies performed in murine disease models and in cells from WAS patients will be presented. Based on these data and on the clinical results of the recent trials for SCID, the most critical issues regarding the implementation of a gene therapy approach for WAS will be discussed.

Defective Th1 cytokine gene transcription in CD4+ and CD8+ T cells from Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) protein (WASP) plays a key role in TCR-mediated activation and immunological synapse formation. However, the effects of WASP deficiency on effector functions of human CD4+ and CD8+ T cells remain to be determined. In this study, we report that TCR/CD28-driven proliferation and secretion of IL-2, IFN-gamma, and TNF-alpha are strongly reduced in CD8+ T cells from WAS patients, compared with healthy donor CD8+ T cells. Furthermore, WAS CD4+ T cells secrete low levels of IL-2 and fail to produce IFN-gamma and TNF-alpha, while the production of IL-4, IL-5, and IL-10 is only minimally affected. Defective IL-2 and IFN-gamma production persists after culture of naive WAS CD4+ T cells in Th1-polarizing conditions. The defect in Th1 cytokine production by WAS CD4+ and CD8+ T cells is also present at the transcriptional level, as shown by reduced IL-2 and IFN-gamma mRNA transcripts after TCR/CD28 triggering. The reduced transcription of Th1 cytokine genes in WAS CD4+ T cells is associated with a defective induction of T-bet mRNA and a reduction in the early nuclear recruitment of NFAT-1, while the defective activation of WAS CD8+ T cells correlates with reduced nuclear recruitment of both NFAT-1 and NFAT-2. Together, our data indicate that WASP regulates the transcriptional activation of T cells and is required specifically for Th1 cytokine production.

Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/cDNA-containing lentiviral vector and nonlethal irradiation.

Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked primary immunodeficiency characterized by infections, hemorrhages, autoimmune disorders, and lymphomas. Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could represent an alternative treatment to allogeneic HSC transplantation, the latter being often associated with severe complications. We used WAS-/- mice to test the efficacy of a gene therapy approach based on nonlethal irradiation followed by transplantation of WAS-/- HSCs transduced with lentiviral vectors encoding the WAS protein (WASP) from either the ubiquitous PGK promoter or the tissue- specific WAS promoter. The procedure resulted in significant levels of engraftment of WASP-expressing T cells, B cells, platelets, and myeloid cells. T cells harbored one or two vector copies and displayed partial to full correction of T cell receptor-driven interleukin-2 production and proliferation. In addition, polymerization of F-actin and localization of WASP at the site of the immunological synapse were restored. The treatment was well tolerated and no pathology was detected by systematic blood analysis and autopsy. The efficacy of WAS gene transfer into HSCs, using the WAS promoter-containing lentiviral vector, combined with nonlethal irradiation provides a strong rationale for the development of gene therapy for WAS patients.

Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.

Wiskott-Aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation.

Immunological synapse assembly relies on the clustering of lipid rafts and is required for optimal T cell activation. We demonstrate that the Wiskott-Aldrich syndrome protein (WASP) is recruited to lipid rafts immediately after TCR and CD28 triggering and is required for the movements of lipid rafts. T cells from Wiskott-Aldrich syndrome (WAS) patients, lacking WASP, proliferate poorly after TCR/CD28 activation and have impaired capacities to cluster the lipid raft marker GM1 and to upregulate GM1 cell surface expression. T cell proliferation and lipid raft clustering are restored by retroviral transfer of the WASP gene. These results demonstrate that WASP plays a central role in the movements of lipid rafts and identify a potential mechanism underlying the T cell defect affecting WAS patients.