Cord Blood Treg Research Articles

Comparative study of regulatory T cells expanded ex vivo from cord blood and adult peripheral blood

In this study, we expanded regulatory T cells (Tregs) ex vivo from CD4(+) CD25(+) T cells from cord blood (CB) and CD4(+) CD25(+) CD127(-) T cells from adult peripheral blood (APB) and compared the suppressive functions of the newly generated Tregs. The Tregs from CB and APB were expanded either in two cycles with a polyclonal stimulus or in two cycles with an alloantigen stimulus in the first cycle and a polyclonal stimulus in the second cycle. Cell yield after Treg expansion with polyclonal stimulation was greater than that of Tregs expanded with combined alloantigen and polyclonal stimulation. The expanded Tregs expressed high levels of Foxp3, CD39 and cytotoxic T-lymphocyte antigen-4 and low levels of CD127, interleukin-2 and interferon-γ. After two cycles of expansion, the CB Tregs maintained expression of the GARP gene and showed greater suppressive function than APB Tregs. The CB Tregs that were expanded with two cycles of polyclonal stimulation suppressed not only the polyclonal antigen-driven responder T (T(resp)) cell proliferation but also the HLA mismatched dendritic cell-driven T(resp) cell proliferation. When CB and APB Tregs were expanded with a primary alloantigen stimulus followed by a secondary polyclonal stimulus, the Tregs showed a potent, antigen-specific suppressive capacity. The Tregs expanded with two cycles of polyclonal stimulation from both CB and APB alleviated acute graft-versus-host disease symptoms and prolonged survival in a murine model of graft-versus-host disease. In conclusion, CB Tregs expanded with two cycles of polyclonal stimulation had a stronger immunosuppressive function than APB Tregs. It is feasible to obtain human functional alloantigen-specific Tregs expanded ex vivo from CB and APB in large numbers.

Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year

Regulatory T cells (Tregs) with stable FOXP3 expression are characterized by a specific demethylated region in the FOXP3 gene (Treg-specific demethylated region, TSDR). The aim of this study was to analyse the influence of prenatal factors on cord blood Treg numbers, as detected by changes in the TSDR demethylation, and the subsequent risk for allergic diseases. Analyses were performed within the LINA study in blood samples from pregnant women (34th gestational week) and in cord blood (n = 346 mother-child pairs). Treg numbers were detected via DNA demethylation in the FOXP3 TSDR. At age 1, total and specific IgE was measured in children's blood. In addition, maternal cytokine production (Th1/Th2/Th17) was analysed. Exposure and disease outcomes were assessed by questionnaires. Boys had lower Treg numbers compared with girls (P < 0.001). Parental atopy history, particularly maternal hay fever and paternal asthma were related to lower Treg numbers in cord blood (adj. MR = 0.81, 95% CI = 0.68-0.97; adj. MR = 0.60, 95% CI = 0.45-0.81). Maternal cytokines (IL-13, IL-17E and IFN-γ) and maternal smoking/exposure to tobacco smoke during pregnancy were also associated with decreased cord blood Treg numbers (adj. MR = 0.89, 95% CI = 0.97-1.00). Children with lower Treg numbers at birth had a higher risk to develop atopic dermatitis (adj. OR = 1.55, 95% CI = 1.00-2.41) and sensitization to food allergens (adj. OR = 1.55, 95% CI = 1.06-2.25) during the first year of life. These results indicate that both genetic and environmental factors presumably influence the development of foetal Tregs. Low cord blood Treg numbers may predict early atopic dermatitis.

Functional analysis and gene expression profile of umbilical cord blood regulatory T cells

The aim of the study was to analyze and compare the functional properties and the gene expression profile of regulatory T cells (Tregs) isolated from cord blood (CB) units (n = 23) and from the peripheral blood (PB) of adult normal donors (n = 13). Tregs were purified from mononuclear cells and expanded for 6days with anti-CD3, anti-CD28, and IL-2. CB and PB Tregs presented similar immunophenotypic features. However, Tregs isolated from CB presented a much higher expansion capacity; this was confirmed by the genomic characterization that showed in CB-derived Tregs significant enrichments of genes involved in cell proliferation, chromatin modification, and regulation of gene expression. All samples were positive for the FoxP3 gene and protein after expansion. CB and PB expanded Tregs exerted a comparable and potent suppressive function on the proliferative reaction of autologous T cells stimulated by allogeneic dendritic cells and presented a high in vitro IL-10 production capacity. Gene profile analysis also revealed for PB Tregs significant enrichments of genes involved in the adaptive immune response. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T cell response.

The Effect of Mode of Delivery on T Regulatory (Treg) Cells of Cord Blood

To evaluate whether the mode of delivery (vaginal versus C-section) influences the levels of CD4+CD25+FOXP3+ Treg cells in cord blood and maternal peripheral blood and also to examine its relationship with plasma cortisol levels. Newborns either born vaginally (n = 19) or via elective C- section (n = 20) and their mothers, as well as 20 healthy but not pregnant women, were included in the study. CD4+CD25+FOXP3 (Treg) cells were examined by flow cytometry. Total lymphocyte counts (TLC) and serum cortisol levels were also determined for all the groups. The percentages of CD4+CD25+FOXP3 cells and the serum cortisol levels of infants born vaginally (p < 0.004 and p < 0.0001) and their mothers (p < 0.0001 for both) were found to be significantly higher than those of newborns born by C-section and their mothers. Positive correlations were seen between CD4+CD25+FOXP3+ cells (r = 0.741) and serum cortisol levels (r = 0.468). No relationship was observed between newborns delivered by C-section and their mothers (r = 0.022 for both). This study suggests that mode of delivery affects cord blood Treg cells. Higher CD4+CD25+FOXP3+ Treg cells of newborns and their mothers in vaginal delivery group and their relationship with serum cortisol levels suggest a stress phenomenon related to vaginal delivery.

Genome-Wide Identification of Human FOXP3 Target Genes in Natural Regulatory T Cells

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3(+) Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25(+)FOXP3(+) Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

Reduced maternal regulatory T cell numbers and increased T helper type 2 cytokine production are associated with elevated levels of immunoglobulin E in cord blood

There is evidence that the basis of an atopic-skewed immune response is acquired early in life, perhaps at the fetal stage. Thus, we hypothesized that the development of the fetal immune system might be influenced by maternal regulatory T cells (Treg) and maternal T cell cytokine production during pregnancy. The aim of the present study was to assess the influence of maternal Treg and cytokine production during pregnancy on Treg and atopy at birth. Within the mother-child study LINA (Lifestyle and Environmental factors and their Influence on Newborns Allergy risk), we determined the frequency and function of Treg and the total IgE concentration in pregnant women in the 34th week of gestation and in corresponding cord bloods at birth (n=24). Furthermore, we assessed how maternal mitogen-induced T-helper type 1/T-helper type 2 and inflammatory cytokines influence the level of cord blood Treg and IgE. Frequencies of CD4(+)CD25(high) T cells were higher (P=0.001), whereas percentages of FOXP3+ T cells were lower (P<0.001) in cord blood cells compared with maternal blood. Reduced maternal CD4(+)CD25(high) Treg frequencies correlated with increased total IgE concentrations at the 34th week of gestation (r=-0.32, P=0.028) and with increased IgE concentrations in cord blood (r=-0.50, P<0.001). Elevated maternal mitogen-induced Th2 cytokine production was related to increased total IgE levels in the serum of corresponding cord bloods (IL-4, r=0.53; IL-5, r=0.43; IL-13, r=0.52). Because cord blood IgE has been shown to be predictive for allergic diseases in early childhood, our results indicate that reduced maternal Treg numbers and increased Th2 cytokine production during pregnancy might influence the allergy risk of the child.

Functional Analysis and Gene Expression Profile of Umbilical Cord Blood Regulatory T Cells.

Abstract 4085Poster Board III-1020 IntroductionUmbilical cord blood (CB) stem cells are now broadly used in the unrelated stem cell transplant setting and comparative studies with different stem cell sources have shown that CB transplant is characterized by a lower risk of graft-versus-host disease (GVHD). The immaturity of CB T cells has been generally regarded as the main contributing factor accounting for this phenomenon; the possible role played by CB regulatory T cells (Tregs) for the suppression of the allogeneic T-cell response is now under investigation, but very scare data are so far available. Aim of this study was to analyze and compare the functional properties and the gene expression profile of Tregs expanded from CB units with those expanded from the peripheral blood (PB) of adult normal donors. MethodsTregs were purified from mononuclear cells obtained from 23 CB units and from the PB of 13 adult normal donors using the CD4+CD25+ regulatory T-cell isolation kit (Miltenyi Biotec) and expanded for 6 days in 96-well U-Bottom plates coated with the anti-CD3 (5 ug/ml) and anti-CD28 (5 ug/ml) MoAbs in the presence of IL-2 (100 U/ml). Immunophenotypic analyses were performed before and after expansion. To assess their suppressive functions, expanded Tregs were seeded with autologous effector T cells stimulated with allogeneic dendritic cells (DC) pulsed with apoptotic leukemic blasts, then incubated with [3H]-thymidine and counted in a beta-counter. Suppressor activity was measured as [3H]-thymidine incorporation in the presence or absence of Tregs. The IL-10 production capacity of expanded Tregs was tested using an ELISA assay. The two-sided student t test was used to evaluate the significance of differences between groups. Gene expression profile experiments were performed using the HGU133 Plus 2.0 arrays (Affymetrix); statistical analyses were carried out using the dChip software; a t test was used to evaluate the presence of specifically expressed classes of genes. Functional annotation analysis was performed using the DAVID software. ResultsCB and PB Tregs presented similar immunophenotypic appearances before and after expansion. Im particular, after expansion they presented a comparable expression of surface CD4, CD25, CD62L, CCR5 and CD45RO, and of cytoplasmic CTLA-4 and Foxp3, while they both were negative for the CD45RA antigen, thus indicating the loss of their naïve features. On the contrary, Tregs obtained from CB (n=23) presented a much higher expansion capacity compared to those obtained from PB (n=13): mean fold increase (range), CB 10.3 (1.6-24), PB 3.9 (1.5-10), p 0.003. CB expanded Tregs (n=6) exerted a potent suppressive function on the proliferative reaction of T cells stimulated by allogeneic DC, that resulted inferior even though not significantly compared to that exerted by PB expanded Tregs (n=5): mean fold reduction (range), CB 7.8 (2.5-15.1), PB 14.3 (1.5-23.7), p 0.14. Tregs expanded from CB (n=4) and PB (n=1) presented a high and comparable in vitro IL-10 production capacity: mean pg/ml (range), CB 326.5 (226-426), PB 382. Gene expression profile analysis showed a higher number of upregulated genes in Tregs expanded from CB (n=2) compared to Tregs expanded from PB (n=3); among them, a significant enrichment of genes involved in cell proliferation, cell cycle checkpoints, signal transduction, cell differentiation, apoptosis, TGF-β receptor pathway and the GrNH pathway was observed. This suggests that CB Tregs retain a more undifferentiated program and are characterized by the high expression of genes which might provide an advantage in cell expansion. Finally, when looking at the Foxp3 gene expression levels, no difference was observed between the two populations. ConclusionsThese results demonstrate that Tregs contained in CB retain an expansion potential superior to that of Tregs isolated from the PB of normal donors, as confirmed by functional analyses and gene profile. Tregs expanded from CB and PB seem to exert a potent and comparable suppressive function of the proliferative effect in mixed lymphocyte reaction assays. The maintaining of the modulatory properties after expansion is confirmed by the expression of the Foxp3 gene and protein, and by the production of IL-10. These data offer further insights into the understanding of the biology of CB transplantation indicating a possible role played by CB Tregs in the suppression of the allogeneic T-cell response. Disclosures:No relevant conflicts of interest to declare.

Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells

Cross-sectional studies suggest that maternal exposure to farming decreases the risk of allergic diseases in offspring. The potential underlying immunologic mechanisms are not understood. We sought to assess whether maternal farm exposure activates regulatory T (Treg) cells in cord blood, exerting T(H)2-suppressive effects after microbial stimulation. Eighty-four pregnant mothers were recruited before delivery. Detailed questionnaires (60 nonfarming and 22 farming mothers with 2 exclusions) assessed the farming exposures. Cord blood was stimulated with the microbial stimulus peptidoglycan (Ppg), the mitogen PHA, house dust mite extracts (Der p 1), and combinations. Treg cells (CD4+CD25(high) cells; intracellular forkhead/winged-helix family transcriptional repressor p3 [FOXP3] expression, FOXP3 levels, lymphocyte activation gene 3 mRNA expression, functional studies, and DNA methylation of the FOXP3 locus), proliferation, and T(H)2/T(H)1/T(H)17 cytokines were examined. Cord blood Treg cell counts (both unstimulated and PHA stimulated) were increased with maternal farming exposures and associated with higher FOXP3 (Der p 1 + Ppg stimulation) and trendwise higher lymphocyte activation gene 3 (Ppg) expression. Furthermore, Treg cell function was more efficient with farming exposure (effector cell suppression, P = .004). In parallel, T(H)2 cytokine (IL-5) levels were decreased and associated with decreased lymphoproliferation and increased IL-6 levels (Ppg stimulation, Der p 1 + Ppg stimulation, or both; P < .05). Maternal exposure to increasing numbers of farm animals and stables was discovered to exert distinct effects on Treg cells, T(H)1/T(H)2 cells, or both. Additionally, FOXP3 demethylation in offspring of mothers with farm milk exposure was increased (P = .02). Farm exposures during pregnancy increase the number and function of cord blood Treg cells associated with lower T(H)2 cytokine secretion and lymphocyte proliferation on innate exposure. One fascinating speculation is that maternal farm exposure might reflect a natural model of immunotherapy, potentially including a selection of innate stimuli in addition to allergen, shaping a child's immune system at an early stage.

Children with egg allergy have evidence of reduced neonatal CD4 +CD25 +CD127 lo/− regulatory T cell function

The role of regulatory T (Treg) cells in allergic predisposition is not known. This study compared the frequency and function of cord blood Treg cells from nonallergic children (n = 18) with those from children who have egg allergy (n = 15) in the first year of life. CD4(+) effector T cells and autologous antigen-presenting cells isolated from cord blood mononuclear cells were cocultured with or without CD4(+)CD25(+)CD127(lo/-) Treg cells, and cytokine responses to staphylococcal endotoxin B were assessed after 48 hours. The addition of Treg cell populations to cord blood mononuclear cell cultures resulted in significant reduction in IL-10 (P = .002), IL-13 (P = .012), and IFN-gamma (P < .001) production. Consistent with other reports, effector CD4(+) T-cell responses (IFN-gamma and IL-13) tended to be lower in the allergic group. These neonates showed less significant Treg cell-associated suppression of IFN-gamma (P = .015) compared with that seen in the nonallergic group (P = .001). The allergic group was also less likely (44%) to show Treg cell-associated suppression of IFN-gamma effector responses compared with that seen in the nonallergic group (78%, P = .015). The magnitude of suppression (change in IFN-gamma level when CD4(+)CD25(+)CD127(lo/-) Treg cells were added to responding effector T-cell cultures) was significantly lower in the allergic group (P = .004). There were no between-group differences in the circulating CD4(+)CD25(+)CD127(lo/-) Treg cells (as a percentage of cord blood T cells) or in the FOXP3 expression of these cells. This study confirms the presence and activity of Treg cells in cord blood and provides preliminary evidence of differences in neonates who progress to allergic disease in the first year of life.

Naive regulatory T cells: a novel subpopulation defined by resistance toward CD95L-mediated cell death

AbstractMost CD4+CD25hiFOXP3+ regulatory T cells (Tregs) from adult peripheral blood express high levels of CD45RO and CD95 and are prone to CD95L-mediated apoptosis in contrast to conventional T cells (Tconvs). However, a Treg subpopulation remained consistently apoptosis resistant. Gene microarray and 6-color flow cytometry analysis including FOXP3 revealed an increase in naive T-cell markers on the CD95L-resistant Tregs compared with most Tregs. In contrast to Tregs found in adult humans, most CD4+CD25+FOXP3+ T cells found in cord blood are naive and exhibit low CD95 expression. Furthermore, most of these newborn Tregs are not sensitive toward CD95L similar to naive Tregs from adult individuals. After short stimulation with anti-CD3/CD28 monoclonal antibodies (mAbs), cord blood Tregs strongly up-regulated CD95 and were sensitized toward CD95L. This functional change was paralleled by a rapid up-regulation of memory T-cell markers on cord blood Tregs that are frequently found on adult memory Tregs. In summary, we show a clear functional difference between naive and memory Tregs that could result in different survival rates of those 2 cell populations in vivo. This new observation could be crucial for the planning of therapeutic application of Tregs.