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Foxp3+ Treg expanded from patients with established diabetes reduce Helios expression while retaining normal function compared to healthy individuals.

Du W, Shen YW, Lee WH, Wang D, Paz S, Kandeel F, Liu CP - PLoS ONE (2013)

Bottom Line: After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity.Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8(+) effector T cells equally well.Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California, United States of America.

ABSTRACT
Foxp3(+) regulatory T cells (Treg) play a crucial role in regulating immune tolerance. The use of Treg to restore immune tolerance is considered an attractive novel approach to inhibit autoimmune disease, including type 1 diabetes (T1D), and to prevent rejection of organ transplants. In view of the goal of developing autologous Treg-based cell therapy for patients with long-term (>15 years) T1D, it will be necessary to expand a sufficient amount of functional Treg in vitro in order to study and compare Treg from T1D patients and healthy subjects. Our results have demonstrated that there is a comparable frequency of Treg in the peripheral blood lymphocytes (PBLs) of patients with long-term T1D relative to those in healthy subjects; however, Th1 cells, but not Th17 cells, were increased in the T1D patients. Further, more Treg in PBLs from T1D patients than from healthy subjects expressed the CD45RO(+) memory cell phenotype, suggesting they were antigen-experienced cells. After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity. Although there was no difference in Helios expression on Treg in PBLs, in vitro expansion led to fewer Helios-expressing Treg from T1D patients than healthy subjects. While more Th1-like Treg expressing IFN-γ or TNF-α were found in the PBLs of T1D patients than healthy controls, there was no such difference in the expanded Treg. Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8(+) effector T cells equally well. Our findings demonstrate that a large number of ex vivo expanded functional Treg can be obtained from long-term T1D patients, although fewer expanded Treg expressed a high level of Helios. Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

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Expression of IFN-γ, TNF-α, IL17 in Treg of PBLs or in expanded Treg.Cells from PBMCs and expanded Treg on Day 14 after anti-CD3/CD28 activation were stained with indicated antibodies following stimulation with PMA/ionomycin for 5 h and analyzed by FACS. (A–B) IFN-γ, TNF-α, and IL17 expression in Foxp3+Treg of (A) PBLs and (B) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4+ T cell population. (C–D) Collective analyses of the percentage of IFN-γ+, TNF-α+, or IL17+ in (C) Foxp3+ Treg of PBLs or (D) expanded Foxp3+ Treg.
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pone-0056209-g007: Expression of IFN-γ, TNF-α, IL17 in Treg of PBLs or in expanded Treg.Cells from PBMCs and expanded Treg on Day 14 after anti-CD3/CD28 activation were stained with indicated antibodies following stimulation with PMA/ionomycin for 5 h and analyzed by FACS. (A–B) IFN-γ, TNF-α, and IL17 expression in Foxp3+Treg of (A) PBLs and (B) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4+ T cell population. (C–D) Collective analyses of the percentage of IFN-γ+, TNF-α+, or IL17+ in (C) Foxp3+ Treg of PBLs or (D) expanded Foxp3+ Treg.

Mentions: Previous studies showed that some human Foxp3+ Treg might also express IFN-γ [25]. We first examined whether there would be differences in IFN-γ expression in Treg from CD4+ PBLs of T1D subjects versus those from healthy controls. In addition, we also examined whether Treg may also express another Th1 cytokine TNF-α. We found that there were more IFN-γ+ or TNF-α+ (p = 0.003) cells in the CD4+Foxp3+ Treg from PBLs of T1D subjects than in those of healthy controls (Fig. 7A and 7C). A small and comparable percentage of the CD4+Foxp3+ Treg in PBLs from both T1D and healthy subjects expressed IL17. Further analyses showed that the observed difference in cell phenotype is independent of the age of subjects in the two cohorts (data not shown).


Foxp3+ Treg expanded from patients with established diabetes reduce Helios expression while retaining normal function compared to healthy individuals.

Du W, Shen YW, Lee WH, Wang D, Paz S, Kandeel F, Liu CP - PLoS ONE (2013)

Expression of IFN-γ, TNF-α, IL17 in Treg of PBLs or in expanded Treg.Cells from PBMCs and expanded Treg on Day 14 after anti-CD3/CD28 activation were stained with indicated antibodies following stimulation with PMA/ionomycin for 5 h and analyzed by FACS. (A–B) IFN-γ, TNF-α, and IL17 expression in Foxp3+Treg of (A) PBLs and (B) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4+ T cell population. (C–D) Collective analyses of the percentage of IFN-γ+, TNF-α+, or IL17+ in (C) Foxp3+ Treg of PBLs or (D) expanded Foxp3+ Treg.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569420&req=5

pone-0056209-g007: Expression of IFN-γ, TNF-α, IL17 in Treg of PBLs or in expanded Treg.Cells from PBMCs and expanded Treg on Day 14 after anti-CD3/CD28 activation were stained with indicated antibodies following stimulation with PMA/ionomycin for 5 h and analyzed by FACS. (A–B) IFN-γ, TNF-α, and IL17 expression in Foxp3+Treg of (A) PBLs and (B) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4+ T cell population. (C–D) Collective analyses of the percentage of IFN-γ+, TNF-α+, or IL17+ in (C) Foxp3+ Treg of PBLs or (D) expanded Foxp3+ Treg.
Mentions: Previous studies showed that some human Foxp3+ Treg might also express IFN-γ [25]. We first examined whether there would be differences in IFN-γ expression in Treg from CD4+ PBLs of T1D subjects versus those from healthy controls. In addition, we also examined whether Treg may also express another Th1 cytokine TNF-α. We found that there were more IFN-γ+ or TNF-α+ (p = 0.003) cells in the CD4+Foxp3+ Treg from PBLs of T1D subjects than in those of healthy controls (Fig. 7A and 7C). A small and comparable percentage of the CD4+Foxp3+ Treg in PBLs from both T1D and healthy subjects expressed IL17. Further analyses showed that the observed difference in cell phenotype is independent of the age of subjects in the two cohorts (data not shown).

Bottom Line: After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity.Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8(+) effector T cells equally well.Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope, Duarte, California, United States of America.

ABSTRACT
Foxp3(+) regulatory T cells (Treg) play a crucial role in regulating immune tolerance. The use of Treg to restore immune tolerance is considered an attractive novel approach to inhibit autoimmune disease, including type 1 diabetes (T1D), and to prevent rejection of organ transplants. In view of the goal of developing autologous Treg-based cell therapy for patients with long-term (>15 years) T1D, it will be necessary to expand a sufficient amount of functional Treg in vitro in order to study and compare Treg from T1D patients and healthy subjects. Our results have demonstrated that there is a comparable frequency of Treg in the peripheral blood lymphocytes (PBLs) of patients with long-term T1D relative to those in healthy subjects; however, Th1 cells, but not Th17 cells, were increased in the T1D patients. Further, more Treg in PBLs from T1D patients than from healthy subjects expressed the CD45RO(+) memory cell phenotype, suggesting they were antigen-experienced cells. After isolation, Treg from both T1D patients and healthy subjects were successfully expanded with high purity. Although there was no difference in Helios expression on Treg in PBLs, in vitro expansion led to fewer Helios-expressing Treg from T1D patients than healthy subjects. While more Th1-like Treg expressing IFN-γ or TNF-α were found in the PBLs of T1D patients than healthy controls, there was no such difference in the expanded Treg. Importantly, expanded Treg from both subject groups were able to suppress autologous or allogeneic CD8(+) effector T cells equally well. Our findings demonstrate that a large number of ex vivo expanded functional Treg can be obtained from long-term T1D patients, although fewer expanded Treg expressed a high level of Helios. Thus, based on the positive outcomes, these potent expanded Treg from diabetic human patients may be useful in treating T1D or preventing islet graft rejection.

Show MeSH
Related in: MedlinePlus