In vitro Treg expansion favors the full-length splicing isoform of CTLA4.

AIM: We compared fresh and in vitro expanded human Tregs for their CTLA4 splicing isoform expression. METHODS: The CD4(+)CD25(+)CD127(low/-)phenotype was used for sorting Tregs and mRNA levels were measured with relative qRT-PCR. RESULTS: In fresh Tregs the level of soluble CTLA4 (sCTLA4) was half of that of full-length CTLA4, whereas in expanded cells sCTLA4 level was tenfold lower. The most striking change took place early on: sCTLA4 expression decreased significantly when cells were simply kept in culture. CONCLUSION: In the in vitro expanded Tregs, the splicing of CTLA4 is affected. Our findings can be significant for clinical cell manufacturing. First, even minimal processing of cells may impact the functional molecules. Second, Treg expansion yields more potent CTLA4 receptor bearing cells.

Interaction with intestinal epithelial cells promotes an immunosuppressive phenotype in Lactobacillus casei.

Maintenance of the immunological tolerance and homeostasis in the gut is associated with the composition of the intestinal microbiota. We here report that cultivation of Lactobacillus casei ATCC 334 in the presence of human intestinal epithelial cells promotes functional changes in bacteria. In particular, the interaction enhanced the immunosuppressive phenotype of L. casei as demonstrated by the ability of L. casei to generate functional regulatory T cells (CD4+CD25+FoxP3+) and production of the anti-inflammatory cytokine interleukin-10 by human peripheral blood mononuclear cells. The results indicate microbe-host cross-talk that changes features of microbes, and suggest that in vitro simulation of epithelial cell interaction can reveal functional properties of gut microbes more accurately than conventional cultivation.

Lectin from Erythrina cristagalli supports undifferentiated growth and differentiation of human pluripotent stem cells.

Lectins are carbohydrate-binding proteins, which occur ubiquitously in nature and are abundant in all living organisms from bacteria to mammals. They have several biological functions among which cell adhesion is well known and characterized. Based on the characterization of the glycome of human embryonic stem cells (hESCs), we have investigated the properties of glycan-binding lectins as a novel class of culture support matrices supporting hESC culture. We report that an Erythrina cristagalli lectin (agglutinin) (ECA) matrix supported the undifferentiated growth and significantly increased the plating efficiency of both hESC and human induced pluripotent stem cells when used in conjunction with pinacidil, an antihypertensive drug with ROCK inhibition activity. As a matrix, ECA maintained pluripotency, robust proliferation with a normal karyotype, and the ability to differentiate both in vitro and in vivo. Therefore, our findings indicate that lectins are potential candidates for design of culture and differentiation methods, and that ECA is a potent simple defined matrix for human pluripotent stem cells.

Infiltration of Foxp3- and Toll-like receptor-4-positive cells in the intestines of children with food allergy.

OBJECTIVES: Regulatory T (Treg) cells together with intestinal microflora play a central role in controlling allergic inflammation. We examined the markers related to Treg cells, and bacterial signaling, such as Toll-like receptors (TLR)-2 and -4, in the duodenal mucosa of patients with food allergy (FA). PATIENTS AND METHODS: Small intestinal samples were collected from patients with FA on a normal or an elimination diet, from healthy controls and patients with untreated celiac disease. Single and double immunohistochemistry were used to enumerate the densities of Foxp3-positive cells and TLR2- and TLR4-positive cells in the mucosa and evaluate the colocalization of Foxp3 expression in CD4, CD25, and CTLA-4 cells. The mRNA expression of CD25, Foxp3, TLR2, and TLR4 was measured by reverse transcriptase-polymerase chain reaction. RESULTS: The densities of Foxp3 and TLR4 cells were significantly increased in patients with untreated FA compared with healthy controls (P = 0.003, P = 0.033), and the Foxp3 cells were higher in untreated than in treated allergic patients (P < 0.001). The immense majority of Foxp3 cells were CD4 (median 100%), CTLA-4 (100%), or CD25 (81%). The ratio of Foxp3 mRNA to Foxp3 cells was decreased in patients with FA and in patients with celiac disease compared with controls (P = 0.036, P = 0.035). CONCLUSIONS: Foxp3 cells are increased in the duodenum of patients with untreated FA, but these cells are not able to suppress the harmful immune response, indicated by the low expression of Foxp3 transcripts. The increase of TLR4 cells and their correlation with TCRgammadelta intraepithelial lymphocytes suggest a role for the innate immunity and intestinal microbiota in FA.

The N-glycome of human embryonic stem cells.

BACKGROUND: Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. RESULTS: The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. CONCLUSION: The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

Insulin treatment in patients with type 1 diabetes induces upregulation of regulatory T-cell markers in peripheral blood mononuclear cells stimulated with insulin in vitro.

Patients with type 1 diabetes are treated with daily injections of human insulin, an autoantigen expressed in thymus. Natural CD4(+)CD25(high) regulatory T-cells are derived from thymus, and accordingly human insulin-specific regulatory T-cells should exist. We had a chance to study peripheral blood mononuclear cells (PBMCs) from children with type 1 diabetes both before and after starting insulin treatment, and thus we could analyze the effects of insulin treatment on regulatory T-cells in children with type 1 diabetes. PBMCs were stimulated for 72 h with bovine/human insulin. The mRNA expression of regulatory T-cell markers (transforming growth factor-beta, Foxp3, cytotoxic T-lymphocyte antigen-4 [CTLA-4], and inducible co-stimulator [ICOS]) or cytokines (gamma-interferon [IFN-gamma], interleukin [IL]-5, IL-4) was measured by quantitative RT-PCR. The secretion of IFN-gamma, IL-2, IL-4, IL-5, and IL-10 was also studied. The expression of Foxp3, CTLA-4, and ICOS mRNAs in PBMCs stimulated with bovine or human insulin was higher in patients on insulin treatment than in patients studied before starting insulin treatment. The insulin-induced Foxp3 protein expression in CD4(+)CD25(high) cells was detectable in flow cytometry. No differences were seen in cytokine activation between the patient groups. Insulin stimulation in vitro induced increased expression of regulatory T-cell markers, Foxp3, CTLA-4, and ICOS only in patients treated with insulin, suggesting that treatment with human insulin activates insulin-specific regulatory T-cells in children with newly diagnosed type 1 diabetes. This effect of the exogenous autoantigen could explain the difficulties to detect in vitro T-cell proliferation responses to insulin in newly diagnosed patients. Furthermore, autoantigen treatment-induced activation of regulatory T-cells may contribute to the clinical remission of the disease.

Dietary insulin as an immunogen and tolerogen.

We have shown that exposure to bovine insulin (BI) in cow's milk (CM) formula induces an insulin-specific immune response in infants. Here we studied the role of human insulin (HI) in breast milk as a modulator of the immune response to insulin. In a group of 128 children participating in the TRIGR pilot study, maternal breast milk samples were collected 3-7 days and/or 3 months after delivery. After exclusive breast-feeding, the children received either CM formula or casein hydrolysate during the first 6-8 months of life. Insulin concentration in breast milk and immunoglobulin G (IgG) antibodies to BI in plasma samples were measured by EIA. The levels of insulin in breast milk samples were higher in mothers affected by type 1 diabetes than in non-diabetic mothers (p = 0.007 and p < 0.001). The concentration of insulin in breast milk correlated inversely with the plasma levels of IgG antibodies to BI at 6 months of age in children who received CM formula (r = -0.39, p = 0.013), and at 12 months of age in all children (r = -0.25, p = 0.029). The levels of breast milk insulin were higher in the mothers of nine children who developed beta-cell autoimmunity when compared with autoantibody-negative children (p = 0.030); this holds true also when only children of diabetic mothers were included (p = 0.045). BI in CM induces higher levels of IgG to insulin in infants than does HI in breast-fed children. Instead, HI in breast milk seems to be tolerogenic and may downregulate the IgG response to dietary BI. However, our results in infants who developed beta-cell autoimmunity suggest that in this subgroup of children breast milk insulin does not promote tolerance.

Lactobacillus GG effect in increasing IFN-gamma production in infants with cow's milk allergy.

BACKGROUND: Probiotic bacteria are potentially beneficial to maturation of the infant's immune system. OBJECTIVE: To examine the role of probiotic bacteria in treatment of cow's milk allergy (CMA) and IgE-associated dermatitis, we investigated the immunologic effects of Lactobacillus rhamnosus GG (LGG) and a mixture of 4 bacterial species (MIX). METHODS: In a randomized, double-blind study design, concomitantly with elimination diet and skin treatment, LGG, MIX, or placebo was given for 4 weeks to infants with suspected CMA. After anti-CD3 (OKT3) and anti-CD28 stimulation of PBMCs, IFN-gamma, IL-4, IL-5, and IL-12 levels were measured in culture supernatants by ELISA. Intracellular IFN-gamma, IL-4, and IL-5 production on CD4 lymphocytes was analyzed with fluorescence-activated cell sorting. RESULTS: Secretion of IFN-gamma by PBMCs before the treatment was significantly lower in infants with CMA (P=.016) and in infants with IgE-associated CMA (P=.003) than in non-CMA infants. Among the infants who received LGG, the level of secreted IFN-gamma increased in those with CMA (P=.006) and in those with IgE-associated dermatitis (P=.017) when compared with the placebo group. Secretion of IL-4 increased significantly in infants with CMA in the MIX (P=.034) but not in the LGG group. CONCLUSION: Deficiency in IFN-gamma response appears to be related to CMA. LGG raises IFN-gamma production of PBMC in infants with CMA and in infants with IgE-associated dermatitis and may thus provide beneficial TH1 immunomodulatory signals. MIX, although containing LGG, appears to modulate the immune responses differently.

Anti-insulin activity in IgG-fractions from children with newly-diagnosed type 1 diabetes and negative for insulin autoantibodies.

Insulin autoantibodies (IAA) are often detected as the first humoral sign of beta-cell autoimmunity in prospective studies in young children with increased genetic risk of type 1 diabetes. After the appearance of IAA their level typically rise but seems to decline in many cases before the clinical presentation of type 1 diabetes. We hypothesized that the reason for the sudden drops in the levels of IAA could be the formation of immune complexes caused by binding of antibodies to free insulin in plasma. We studied whether isolation of the IgG-fraction and dissociation of immune complexes by acid treatment using protein A column results in the appearance of detectable IAA in those children with newly-diagnosed type 1 diabetes whose plasma samples test negative for IAA. IAA assay was performed in IgG-fractions and corresponding plasma samples from 17 children with type 1 diabetes and 23 unaffected children all testing negative for plasma IAA. The levels of IAA measured from IgG-fractions of diabetic children were higher than the levels of IAA measured from IgG-fractions in the control children (p = 0.004 in Mann-Whitney U-test). Forty-seven percent (8 out of 17) of newly-diagnosed patients negative for plasma IAA before IgG separation had increased levels of IAA in IgG-fractions and only 13% (3 out of 23) of controls. The levels of glutamate decarboxylase autoantibodies (GADA) did not differ between patients (n = 14) and controls (n = 21) negative for plasma GADA when measured in IgG-fractions. Our results suggest that formation of immune complexes results in false negative results in tests for IAA but not for GADA.

CCR3, CCR5, interleukin 4, and interferon-gamma expression on synovial and peripheral T cells and monocytes in patients with rheumatoid arthritis.

OBJECTIVE: To characterize cytokine and chemokine receptor profiles of T cells and monocytes in inflamed synovium and peripheral blood (PB) in patients with rheumatoid arthritis (RA) and other arthritides. METHODS: We studied PB and synovial fluid (SF) samples taken from 20 patients with RA and 9 patients with other arthritides. PB cells from 8 healthy adults were used as controls. CCR3, CCR5, and intracellular interferon-g (IFN-g) and interleukin 4 (IL-4) expression in CD8+ and CD8- T cell populations and in CD14+ cells were determined with flow cytometry. RESULTS: Expression of CCR5 and CCR3 by CD8-, CD8+ T cells and CD14+ monocytes was increased in SF compared to PB cells in patients with RA and other arthritides. The number of CD8+ T cells spontaneously expressing IL-4 and IFN-g was higher in SF than in PB in RA patients. Spontaneous CCR5 expression was associated with intracellular IFN-g expression in CD8+ T cells derived from SF in RA. In CD8- T cells the ratio of CCR5+/CCR3+ cells was increased in patients with RA compared to patients with other arthritides. The number of PB CD8- T cells expressing IFN-g after mitogen stimulation was higher in controls than in patients. In PB monocytes the ratio of CCR5+/CCR3+ cells was increased in patients with RA compared to patients with other arthritides and controls. CONCLUSION: T cells and monocytes infiltrating joints in RA and in other arthritides showed increased activation of both type 1 and type 2 immune markers. More pronounced type 1 immune response in joints, shown as an increased CCR5/CCR3 ratio, was found in the patients with RA versus those with other arthritides. Monocytes but not T cells in PB showed increased activation in RA.