Maria Grazia Roncarolo

STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25-effector T cells

Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.

In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells

Genetically engineered T memory stem cells preserve differentiation activity for decades after patient infusion. Sealing T cell fate Clinical trials are a relatively untapped source of experimental data that can be leveraged to explore both basic and pathological biology in humans. Now, Biasco et al. take advantage of two different gene therapy trials for inherited immunodeficiency to track in the long term T cell fate in humans. They find that the recently described T memory stem cells (TSCM) are able to persist and preserve their precursor potential in human recipients for up to 12 years after genetic correction and infusion into patients. The safety and long-term survival of these cells not only strengthen our knowledge of human immunology but also support the use of TSCM cells for adoptive immunotherapy. A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow–transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.

Functional type 1 regulatory T cells develop regardless of FOXP3 mutations in patients with IPEX syndrome

Mutations of forkhead box p3 (FOXP3), the master gene for naturally occurring regulatory T cells (nTregs), are responsible for the impaired function of nTregs, resulting in an autoimmune disease known as the immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) syndrome. The relevance of other peripheral tolerance mechanisms, such as the presence and function of type 1 regulatory T (Tr1) cells, the major adaptive IL‐10‐producing Treg subset, in patients with IPEX syndrome remains to be clarified. FOXP3mutated Tr1‐polarized cells, differentiated in vitro from CD4+ T cells of four IPEX patients, were enriched in IL‐10+IL‐4−IFN‐γ+ T cells, a cytokine production profile specific for Tr1 cells, and expressed low levels of FOXP3 and high levels of Granzyme‐B. IPEX Tr1 cells were hypoproliferative and suppressive, thus indicating that FOXP3 mutations did not impair their function. Furthermore, we isolated Tr1 cell clones from the peripheral blood of one FOXP3null patient, demonstrating that Tr1 cells are present in vivo and they can be expanded in vitro in the absence of WT FOXP3. Overall, our results (i) show that functional Tr1 cells differentiate independently of FOXP3, (ii) confirm that human Tr1 and nTregs are distinct T‐cell lineages, and (iii) suggest that under favorable conditions Tr1 cells could exert regulatory functions in IPEX patients.

Cord blood B cells are mature in their capacity to switch to IgE-producing cells in response to interleukin-4 in vitro.

Neonatal B cells have been considered immature because of their impaired capacity to produce immunoglobulins in response to polyclonal activators in vitro. Here we demonstrate that cord blood mononuclear cells (MNC) produce normal levels of IgE in vitro when cultured in the presence of interleukin‐4 (IL‐4), indicating that the B cells are mature in their capacity to switch to IgE‐producing cells. However, in contrast to adult peripheral blood T cells, cord blood T cells failed to produce detectable levels of IL‐4 upon activation by phytohaemagglutinin (PHA) concanavalin A (Con A) or combinations of PHA and the phorbol ester TPA. Interferon‐gamma (IFN‐γ) production by cord blood T cells following activation by Con A or PHA was also strongly reduced. However, high levels of IFN‐γ, significantly higher than those produced by adult T cells, were synthesized in response to combinations of PHA and TPA, indicating that IFN‐γ production by cord blood T cells is not intrinsically defective. In contrast, cord blood T cells produced levels of IL‐2 that were significantly higher than those obtained by adult T cells tested in parallel. Collectively, our data indicate that the minimal levels of IgE production measured in cord blood (< 1 U/ml) are not due to immaturity of the cord blood B cells, but may be associated with the failure of cord blood T cells to produce detectable levels of IL‐4, which has been shown to be responsible for induction of IgE synthesis both in vitro and in vivo.

Absence of VOD in paediatric thalassaemic HSCT recipients using defibrotide prophylaxis and intravenous Busulphan.

Hepatic veno‐occlusive disease (VOD) is a common complication of haematopoietic stem cell transplantation (HSCT), with reported incidences of 5–40% in children. Recently, defibrotide (DF) has been successfully used as prophylaxis and treatment of VOD. This study reports data on 63 human leucocyte antigen‐matched HSCT performed in 57 children affected by beta thalassemia at very high risk for developing VOD (liver fibrosis, iron overload, hepatitis C virus infections, busulphan‐based conditioning, methotraexate + ciclosporine). All patients received a busulphan‐based conditioning regimen, either orally (four HSCT) or intravenously (59 HSCT). All patients received oral DF (40 mg/kg per day, final dose) as VOD prophylaxis from median day −9 to median day +29. In order to overcome the lack of oral paediatric formulations, a galenic formulation was administered. DF was well tolerated. Only one patient fulfilled Seattle Criteria for VOD diagnosis. This patient had discontinued DF 6 d prior to VOD onset, due to high risk of haemorrhage. We concluded that oral defibrotide prophylaxis and i.v. busulphan safely abated VOD incidence in high‐risk patients who had undergone HSCT. A galenic preparation of oral DF also permits this treatment in low‐weight patients. Costs of DF prophylaxis are acceptable considering the reduced incidence of VOD.

Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation.

Marktel S, Napolitano S, Zino E, Cappelli B, Chiesa R, Poli F, Crocchiolo R, Ronchi P, Rossini S, Ciceri F, Roncarolo MG, Fleischhauer K. Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation.
Pediatr Transplantation 2010: 14:393–401.

The capacity of interleukin-4 to induce in vitro IgE synthesis by B cells of patients with common variable immunodeficiency

Interleukin‐4 (IL‐4) has been shown to induce IgE synthesis by peripheral blood mononuclear cells (PBMC) of normal donors in vitro. However, induction of PBMC of patients with common variable immunodeficiency (CVI) with IL‐4 resulted in IgE production in only two out of eight cases tested. PBMC of the first patient that produced IgE in response to IL‐4 also secreted normal levels of IL‐4 upon activation. PBMC of the second patient secreted very low levels of IL‐4 in vitro which may account for the very low serum IgE levels in this patient. Of the other six patients who had very low serum IgE levels and whose PBMC failed to produce IgE in response to IL‐4 in vitro, five did not secrete IL‐4 upon in vitro activation. The capacity of the T cells to produce IL‐4 was intact in the sixth patient. Collectively our data indicate the PBMC of the majority of patients with CVI are defective since they failed to respond appropriately to IL‐4 and they failed to produce IL‐4, contributing to the view that CVI is a heterogeneous disorder in which a variety of T and B cell defects occur.

T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity

Gangliosides form a component of the glycosphingolipid‐rich membrane microdomains recently shown to play an important role in receptor signal transduction. Specific gangliosides also serve as receptors for binding and internalization of bacterial toxins. In the course of characterizing the basis of the native tetanus toxin (TTx) reactivity of a human γ δ T cell clone, we observed that transfer of the TCR was required to impart TTx reactivity on a TCR‐negative recipient T cell. However, the reconstitution of toxin reactivity could be achieved regardless of the antigen specificity of the TCR chains. Further analysis showed that the T cell recognition of native TTx was dependent on the presence of its ganglioside receptor, GT1b, on the T cell surface. Incorporation of exogenous GT1b into plasma membrane conferred TTx reactivity on otherwise non‐reactive T cells provided these cells expressed the TCR. Finally, reconstitution of TCR‐negative Jurkat T cells with a CD8‐CD3ζ chain chimera demonstrated that the cytoplasmic region of the CD3ζ chain was sufficient to couple ganglioside‐mediated TTx binding to T cell activation. These data reveal a novel mode of TCR‐dependent reactivity to a bacterial toxin that could mobilize a large subset of T cells, thus representing a form of innate immunity. Given the possibility that endogenous ligands may bind to cell surface gangliosides, regulation of their levels and topology on the cell surface may constitute an immunoregulatory mechanism.

IPEX Syndrome: Clinical Profile, Biological Features, and Current Treatment

Children carrying mutations in the forkhead box p3 (FOXP3) gene are affected by the syndrome known as Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). Early onset severe enteropathy, Type-1 diabetes (T1D) and eczema with elevated IgE serum levels are the hallmarks of the disease. Mortality is generally high within the first year of life, although some patients can partially respond to conventional immunosuppression showing clinical improvement. Progress in understanding the pathogenesis of IPEX have been made possible by the recognition that FOXP3 is the driving force for the function of naturally occurring regulatory T cells, a T-cell subset specialized in controlling immune responses. However, many open questions concerning the disease mechanisms, the indications for genetic screening and appropriate treatment of IPEX syndrome remain unanswered. In addition, several patients presenting IPEX-like symptoms do not carry mutations in the FOXP3 gene. In the present chapter, we will (1) summarize the latest findings on the biologic function of FOXP3 and its role in immune regulation, (2) highlight the most relevant signs for a correct diagnosis of IPEX syndrome, and (3) provide indications for the development of more targeted therapeutic strategies for the treatment of this devastating pediatric autoimmune disease.

Erratum: Interleukin-10- secreting type 1 regulatory T cells in rodents and humans (Immunological Reviews (2006) 212, (28-50))

(p.34) These alloreactive anergic CD4þ T cells are incapable of inducing GVHD and suppress responses of naı̈ve T cells, after in vivo transfer into histo-incompatible recipients (93–95). (p.39) Furthermore, administration of vitamin D3 to glucocorticoid-resistant patients enhanced T-cell responsiveness to dexamethasone with an increase in IL-10 production (168). (p.40) Examples of pathogens encoding suppressive IL-10 homologues themselves include human cytomegalovirus and EpsteinBarr virus (EBV) (7, 10, 185). (p.41) For example, stimulation of DCs with peptidoglycan (which binds to NOD1) or b-glucan (which binds to dectin-1) increases IL-10 production (189, 190). (p.43) Furthermore, CD4þ T cells obtained from allogenic MLR in the presence of IL-10 reduce skin graft GVHD in skin explant assays (229).