Erica Valencic

Regulatory T-Cell Function Is Impaired in Celiac Disease

Celiac disease (CD) is characterized by intolerance to gluten and high risk of developing autoimmune phenomena. Possible defects in immune tolerance could have a role in the pathogenesis of the disease. As regulatory T-cells (Tregs) are the main population involved in maintaining peripheral tolerance, we investigated the number of these cells in celiac patients as compared with healthy donors. Moreover, we analyzed the suppressive function of CD4+CD25+ T-cells from celiac disease patients and controls on autologous responder T-cells (CD4+CD25−). The percentage of CD4+CD25+FOXP3+ cells was not different in celiacs and in healthy controls, and among positive cells the level of expression of the two regulatory markers was comparable. However, the suppressor activity of Tregs was significantly impaired in CD patients. These results suggest that a defect in Tregs function could play a role in the pathogenesis of CD and in CD-associated autoimmunity.

The immunosuppressive effect of Wharton's jelly stromal cells depends on the timing of their licensing and on lymphocyte activation

BACKGROUND Mesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However, in most models, MSC were added at the same time of lymphocyte stimulation, which is quite different from what occurs in vivo. AIMS To investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Whartons jelly stromal cells (WJSC). METHODS WJSC, licensed or not with activation-related cytokines, were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture. RESULTS Lymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines, in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan, suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect, in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h, and added afterwards to non-licensed WJSC, showed normal or even increased proliferation. On the other hand, their proliferation was strongly inhibited by licensed WJSC. CONCLUSIONS WJSC have a potent immunosuppressive function best realized with direct contact, and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders.

Wild-type FOXP3 is selectively active in CD4+CD25hi regulatory T cells of healthy female carriers of different FOXP3 mutations

Forkhead box P3 (FOXP3) is constitutively expressed by CD4(+)CD25(hi) regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)-FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4(+)T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.

Wharton's jelly derived mesenchymal stromal cells: Biological properties, induction of neuronal phenotype and current applications in neurodegeneration research

Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from bone marrow or other tissues, including fat, muscle and umbilical cord. It has been shown that MSC behave in vitro as stem cells: they self-renew and are able to differentiate into mature cells typical of several mesenchymal tissues. Moreover, the differentiation toward non-mesenchymal cell lineages (e.g. neurons) has been reported as well. The clinical relevance of these cells is mainly related to their ability to spontaneously migrate to the site of inflammation/damage, to their safety profile thanks to their low immunogenicity and to their immunomodulation capacities. To date, MSCs isolated from the post-natal bone marrow have represented the most extensively studied population of adult MSCs, in view of their possible use in various therapeutical applications. However, the bone marrow-derived MSCs exhibit a series of limitations, mainly related to their problematic isolation, culturing and use. In recent years, umbilical cord (UC) matrix (i.e. Whartons jelly, WJ) stromal cells have therefore emerged as a more suitable alternative source of MSCs, thanks to their primitive nature and the easy isolation without relevant ethical concerns. This review seeks to provide an overview of the main biological properties of WJ-derived MSCs. Moreover, the potential application of these cells for the treatment of some known dysfunctions in the central and peripheral nervous system will also be discussed.

Medium-term survival without haematopoietic stem cell transplantation in a case of IPEX: insights into nutritional and immunosuppressive therapy

Immunodysregulation, polyendocrinopathy, enteropathy, Xlinked (IPEX; OMIM 304930) is a severe disorder of immune development characterised by a usually fatal course in early childhood [6]. It is due to mutations in the FOXP3 gene (locus Xp11.23-q13.3) [2, 4, 9], leading to failure in immune tolerance. Haematopoietic stem cell transplantation (HSCT) is the only definitive therapy and, even if its success is inconstant, it is considered to be the treatment of choice for the disease [6, 8, 10]. On the other hand, just two cases have been reported as surviving the first decade with a severe form of the disease without HSCT [5, 7]. A 1-month-old male was referred to our department for a severe eczema (Fig. 1), intractable mucous diarrhoea and failure to thrive. Extensive investigations ruled out a severe combined immunodeficiency (SCID). Symptoms were controlled with intravenous immunoglobulin, cyclosporin A, steroids and total parenteral nutrition (TPN) (Fig. 2). During his second year of life, he developed autoimmune diabetes, autoimmune haemolytic anaemia (AHA), inflammatory interstitial pneumonia, alopecia and thyroiditis. Due to the unsatisfactory response to prednisone, oral betamethasone was prescribed, with a dramatically greater efficacy at an equipotent dosage. A bone marrow transplantation was refused by the parents. Flares of dermatitis, AHA and pneumonia occurred after every cold and required increasing doses of steroids, antibiotics and oxygen administration. Cyclosporin A (10 mg/kg/day) did not allow a reduction of steroids and the child developed a Cushing-like aspect. An experimental treatment with fludarabine followed by the infusion of autologous lymphocytes treated in vitro with vincristine and prednisone was repeated twice, allowing a prolonged reduction of steroid dosage (0.5–0.25 mg/day of betamethasone) and fairly good clinical improvement with less frequent and less intense reactivation of symptoms. At Eur J Pediatr (2007) 166:1195–1197 DOI 10.1007/s00431-006-0395-6

Block of the Mevalonate Pathway Triggers Oxidative and Inflammatory Molecular Mechanisms Modulated by Exogenous Isoprenoid Compounds

Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD). One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3), cytokines and nitric oxide (NO)]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD.

Failure of interferon-γ pre-treated mesenchymal stem cell treatment in a patient with Crohn's disease.

Mesenchymal stem cells (MSC) are cells of stromal origin which exhibit unlimited self-renewal capacity and pluripotency in vitro. It has recently been observed that MSC may also exert a profound immunosuppressive and anti-inflammatory effect both in vitro and in vivo with consequent potential use in autoimmune disorders. We present the case of a patient suffering from childhood-onset, multidrug resistant and steroid-dependent Crohns disease who underwent systemic infusions of MSC, which led to a temporary reduction in CCR4, CCR7 and CXCR4 expression by T-cells, and a temporary decrease in switched memory B-cells, In addition, following MSC infusion, lower doses of steroids were needed to inhibit proliferation of the patients peripheral blood mononuclear cells. Despite these changes, no significant clinical benefit was observed, and the patient required rescue therapy with infliximab and subsequent autologous hematopoietic stem cell transplantation. The results of biological and in vitro observations after MSC use and the clinical effects of infusion are discussed, and a brief description is provided of previous data on MSC-based therapy in autoimmune disorders.

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media

IntroductionDespite having a proven immunosuppressive potential in vitro, human mesenchymal stromal cells (MSCs) are reported to display variable efficacy in vivo and, in fact, their proven benefit in the clinical practice is still limited and controversial.MethodsThe interplay between clinical grade MSCs and pre-activated donor lymphocytes or selected lymphocyte subsets was studied in vitro. The kinetics of MSC growth and viability was evaluated by adhesion-dependent changes of culture plate impedance and biochemically by a colorimetric assay. Activation of natural killer (NK) cells was assessed as well, using a flow cytometry assay.ResultsA strong inhibition of MSC growth was rapidly induced by the addition of pre-activated lymphocytes but not of resting lymphocytes. Inhibition seems not to be attributable to a single cell population, as similar results can be obtained by depleting NK cells or by using either selected CD4+ or CD8+ lymphocytes. In addition, conditioned medium (CM) from activated lymphocytes was able to inhibit MSC growth in a dose-dependent manner. Furthermore, licensing with IFN-γ partially protected MSCs from pre-activated lymphocytes but not from their CM. These results suggest an inhibitory role of lymphocyte-activation-derived substances. However, the identification of a single molecule responsible for MSC inhibition remained elusive, even if preliminary experiments showed that ATP and, to a lesser extent, TNF-α might play a role.ConclusionsThese results suggest that survival of MSCs can be affected by soluble mediators released by activated lymphocytes. Thus it can be hypothesized that MSC immunosuppressive action in vivo could be impaired by ongoing immune activation through the release of inflammatory mediators.

T cells stimulated in vitro have a suppressive function but do not contain only regulatory T cells

The generation of regulatory T cells (Tregs) in vitro represents an attractive possibility to set up cellular therapies that could prevent and cure autoimmune disorders. Different methods have been proposed to generate Tregs in vitro and to evaluate their phenotype and function. Moreover, the overlap between generation of activated and regulatory cells could often be underestimated. We showed that in vitro treatment of CD4+ CD25– lymphocytes with different stimuli leads to a good expression of CD25 and forkhead box P3 (FoxP3) on most cells, but to a full Treg phenotype (including CD127 negativity) in only a minor percentage of cells, ranging from 17·38% of cells treated with phytohaemagglutinin (PHA) to 50·91% of cells treated with T cell receptor (TCR) stimulation in association with transforming growth factor (TGF)‐β. Some suppressive activity was demonstrated for T cells activated with all the different stimuli. However, while suppression mediated by TCR/TGF‐β treated T cells was associated with an inhibition of both interleukin (IL)‐2 and interferon (IFN)‐γ in the co‐culture supernatant, the suppression observed for PHA‐activated cells occurred in the presence of large amounts of these cytokines. In conclusion, also taking into account other recent publications, caution should be taken in interpretation of data in the field of regulatory T cells.

In vivo detection of polyomaviruses JCV and SV40 in mesenchymal stem cells from human umbilical cords

Multipotent stromal cells are present in the Whartons jelly matrix (WJSC) of the umbilical cord and can be used as an allogeneic source of cells to treat immunological disorders. Recently it was demonstrated that adult bone marrow (BM)‐derived mesenchimal stromal cells (MSC) are susceptible to infection with viruses showing potential oncogenic properties, such as the polyomavirus JC (JCV). The aim of this study was to investigate the presence of human polyomaviruses (JCV, BK Virus‐BKV, SV40, and Merkel cell polyomavirus‐MCPyV) in WJSC, and explore the risk of infection.