Delia Zanzi

Recognition of autologous dendritic cells by human NK cells.

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK‐target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40‐transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co‐stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL‐2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF‐α of the cultures stimulated the development of a more mature DC phenotype, while addition of IL‐10 resulted in a less mature phenotype, with lower expression of CD40 and other co‐stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.

Intestinal T cell responses to gluten peptides are largely heterogeneous: implications for a peptide-based therapy in celiac disease.

The identification of gluten peptides eliciting intestinal T cell responses is crucial for the design of a peptide-based immunotherapy in celiac disease (CD). To date, several gluten peptides have been identified to be active in CD. In the present study, we investigated the recognition profile of gluten immunogenic peptides in adult HLA-DQ2+ celiac patients. Polyclonal, gliadin-reactive T cell lines were generated from jejunal mucosa and assayed for both proliferation and IFN-γ production in response to 21 peptides from wheat glutenins and α-, γ-, and ω-gliadins. A magnitude analysis of the IFN-γ responses was performed to assess the hierarchy of peptide potency. Remarkably, 12 of the 14 patients recognized a different array of peptides. All α-gliadin stimulatory peptides mapped the 57–89 N-terminal region, thus confirming the relevance of the known polyepitope 33-mer, although it was recognized by only 50% of the patients. By contrast, γ-gliadin peptides were collectively recognized by the great majority (11 of 14, 78%) of CD volunteers. A 17-mer variant of 33-mer, QLQPFPQPQLPYPQPQP, containing only one copy of DQ2-α-I and DQ2-α-II epitopes, was as potent as 33-mer in stimulating intestinal T cell responses. A peptide from ω-gliadin, QPQQPFPQPQQPFPWQP, although structurally related to the α-gliadin 17-mer, is a distinct epitope and was active in 5 out of 14 patients. In conclusion, these results showed that there is a substantial heterogeneity in intestinal T cell responses to gluten and highlighted the relevance of γ- and ω-gliadin peptides for CD pathogenesis. Our findings indicated that α-gliadin (57–73), γ-gliadin (139–153), and ω-gliadin (102–118) are the most active gluten peptides in DQ2+ celiac patients.

Gliadin-Specific Type 1 Regulatory T Cells from the Intestinal Mucosa of Treated Celiac Patients Inhibit Pathogenic T Cells

Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-γ, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-γ, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.

IL-15 Interferes With Suppressive Activity of Intestinal Regulatory T Cells Expanded in Celiac Disease

OBJECTIVES:Celiac disease (CD) is a condition in which the regulation of the mucosal immune response to dietary gliadin might be altered. The transcription factor forkhead box P3 (Foxp3) has been identified as a marker of a subset of regulatory T cells (Treg). In this study, we have investigated the presence and the suppressive function of Treg cells in the celiac small intestinal mucosa, their correlation with the disease state, and the inducibility by gliadin in an organ culture system; moreover, we tried to define whether interleukin 15 (IL-15), overexpressed in CD, could influence the regulatory activity of such cells.METHODS:The expression of Foxp3, CD3, CD4, and CD8 were analyzed by immunohistochemistry and flow cytometry in duodenal biopsies taken from patients with untreated CD, treated CD, and from non-CD controls, as well as in vitro cultured biopsy samples from treated CD patients, upon challenge with gliadin. Furthermore, we analyzed the suppressive function of CD4+CD25+ T cells, isolated from untreated CD biopsy samples, on autologous responder CD4+CD25− T cells, in the presence of a polyclonal stimulus, with or without IL-15.RESULTS:Higher density of CD4+CD25+Foxp3+ T cells was seen in duodenal biopsy samples from active CD patients in comparison with treated CD and non-CD controls. In coculture, CD4+CD25+ T cells were functionally suppressive, but their activity was impaired by IL-15. Cells from CD subjects showed increased sensitivity to the IL-15 action, likely due to enhanced expression of IL-15 receptor. Finally, we demonstrated an expansion of Foxp3 in treated CD mucosa following in vitro challenge with gliadin.CONCLUSIONS:These data suggest that CD4+CD25+Foxp3+ T cells are induced in situ by gliadin. However, their suppressor capacity might be impaired in vivo by IL-15; this phenomenon contributes to maintain and expand the local inflammatory response in CD.

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

Background Celiac Disease (CD) is both a frequent disease (1∶100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called “toxic” A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. Methods/Principal Findings Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. Conclusions P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosin Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.

Gliadin Regulates the NK-Dendritic Cell Cross-Talk by HLA-E Surface Stabilization

We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-γ production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The α and ω fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin α increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.

Inhibition of Human NK Cell-Mediated Killing by CD1 Molecules

It is now well established that NK cells recognize classical and nonclassical MHC class I molecules and that such recognition typically results in the inhibition of target cell lysis. Given the known structural similarities between MHC class I and non-MHC-encoded CD1 molecules, we investigated the possibility that human CD1a, -b, and -c proteins might also function as specific target structures for NK cell receptors. Here we report that expression of CD1a, -b, or -c can partially inhibits target cell lysis by freshly isolated human NK cells and cultured NK lines. The inhibitory effects of CD1 molecules on NK cell could be shown upon expression of individual CD1 proteins in transfected NK-sensitive target cells, and these effects could be reversed by incubation of the target cells with mAbs specific for the expressed form of CD1. Inhibitory effects of CD1 expression on NK-mediated lysis could also be shown for cultured human dendritic cells, which represent a cell type that prominently expresses the various CD1 proteins in vivo. In addition, the bacterial glycolipid Ags known to be bound and presented by CD1 proteins could significantly augment the observed inhibitory effects on target cell lysis by NK cells.

Majority of Children With Type 1 Diabetes Produce and Deposit Anti-Tissue Transglutaminase Antibodies in the Small Intestine

OBJECTIVE Anti-tissue transglutaminase (TG2) antibodies are the serological marker of celiac disease. Given the close association between celiac disease and type 1 diabetes, we investigated the production and deposition of anti-TG2 antibodies in the jejunal mucosa of type 1 diabetic children. RESEARCH DESIGN AND METHODS Intestinal biopsies were performed in 33 type 1 diabetic patients with a normal mucosal architecture: 14 had high levels (potential celiac disease patients) and 19 had normal levels of serum anti-TG2 antibodies. All biopsy specimens were investigated for intestinal deposits of IgA anti-TG2 antibodies by double immunofluorescence. In addition, an antibody analysis using the phage display technique was performed on the intestinal biopsy specimens from seven type 1 diabetic patients, of whom four had elevated and three had normal levels of serum anti-TG2 antibodies. RESULTS Immunofluorescence studies showed that 11 of 14 type 1 diabetic children with elevated levels and 11 of 19 with normal serum levels of anti-TG2 antibodies presented with mucosal deposits of such autoantibodies. The phage display analysis technique confirmed the intestinal production of the anti-TG2 antibodies; however, whereas the serum-positive type 1 diabetic patients showed a preferential use of the VH5 antibody gene family, in the serum-negative patients the anti-TG2 antibodies belonged to the VH1 and VH3 families, with a preferential use of the latter. CONCLUSIONS Our findings demonstrate that there is intestinal production and deposition of anti-TG2 antibodies in the jejunal mucosa of the majority of type 1 diabetic patients. However, only those with elevated serum levels of anti-TG2 antibodies showed the VH usage that is typical of the anti-TG2 antibodies that are produced in patients with celiac disease.

Allelic distribution of human leucocyte antigen in historical and recently diagnosed tuberculosis patients in Southern Italy

This study addresses the analysis of the human leucocyte antigen (HLA) allele distribution in 54 historical and in 68 recently diagnosed tuberculosis (TB) patients. The historical cohort was characterized by the presence of large fibrocavernous lesions effectively treated with therapeutic pneumothorax during the period 1950–55. Patients and healthy controls enrolled in the study were from the Campania region of southern Italy. No significant association between HLA alleles and TB in the population of recently diagnosed TB patients was observed. On the contrary, among the historical TB patients there was a strong association with an increased frequency of the HLA‐DR4 allele alone and/or in the presence of the HLA‐B14 allele (P = 0·000004; Pc = 0·0008), as well as with a decreased frequency of the HLA‐A2+,‐B14−,DR4− allele association (P = 0·00005; Pc = 0·01). In order to exclude any interference from age‐related factors, these results were confirmed by comparing the historical cohort of TB patients with an age‐matched healthy control population of the same ethnic origin (P = 0·00004; Pc = 0·008; and P = 0·0001; and Pc = 0·02, respectively).