The role of Wiskott-Aldrich Syndrome protein in activation and function of human T cells

Wiskott-Aldrich Syndrome (WAS) is a X-linked genetic disease caused by mutation in the gene encoding for the Wiskott-Aldrich Syndrome protein (WASP). WASP is specifically expressed in hematopoietic cells, where it regulates the reorganization of actin cytoskeleton in response to extracellular stimuli. WAS is characterized by micro-throm bocytopenia, eczema, immunodeficiency and high susceptibility to autoimmune disorders and haematological malignancies. Although WASP deficiency affects the function of several haematopoietic cells, T cell dysfunction plays a predominant role in WAS pathogenesis. Our study focused on the functional and molecular analysis of different effector T cell subsets from WAS patients and on a possible defect in peripheral tolerance. We found that WASP lowers the threshold for T cell proliferation, induced by TCR/CD28 triggering. Restoration of WASP expression mediated by lentiviral vectors gene transfer fully reconstitutes TCR-driven proliferation. Furthermore, we showed that WASP regulates the levels of the ganglioside GM1 upon TCR/CD28 triggering. In addition, WASP deficiency selectively impairs the production of Th1 cytokines, after TCR/CD28 triggering in CD4+ and CD8+ T cells from WAS patients, as a consequence of reduced Th1 cytokine gene transcription. Reduced expression of IL-2 and IFN-ɣ mRNA in WAS T cells is associated with reduced nuclear levels of NFAT, and in WAS CD4+ T cells also with defective induction of T-bet transcription factor. The function of natural regulatory T cells (nTreg) from WAS patients was also investigated, showing that WASP deficiency impairs the suppressive activity of nTreg cells. This study clarifies the role of WASP in T cell activation and effector functions, and suggests that dysfunctions in regulatory T cells can be involved in the pathogenesis of WAS

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins

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

65. Long-Term Effects of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome: Persistence of Functional Correction of T Cells and Lack of Malignant Trasformation

Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS|[minus]|/|[minus]|), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupr|[eacute]|, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS|[minus]|/|[minus]| mice

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

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 β 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

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 IL23RR381 and IL23RQ381 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. IL23RQ381 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 IL23RQ381 positive donors. Our study shows conclusively that IL23RQ381 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

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 cell

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 patholog