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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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Both diabetic and healthy Treg can be expanded with comparable purity and yield in vitro.CD4+CD25+CD127−/lo Treg, electronically sorted using FACS from diabetic patients and healthy subjects, were activated in vitro using anti-CD3/anti-CD28-coated beads plus recombinant human IL-2. Expanded Treg were stained with CD4, CD25 and Foxp3 antibodies and analyzed by FACS on day 7 (D7) and day 14 (D14) following activation. (A) FACS analyses of CD4 and Foxp3 expression of expanded Treg on D7 and D14. The representative results were from cells obtained from one individual of each subject group. (B) Collective analyses of the purity and expansion fold of expanded Treg cells in each group on D7 and D14.
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pone-0056209-g003: Both diabetic and healthy Treg can be expanded with comparable purity and yield in vitro.CD4+CD25+CD127−/lo Treg, electronically sorted using FACS from diabetic patients and healthy subjects, were activated in vitro using anti-CD3/anti-CD28-coated beads plus recombinant human IL-2. Expanded Treg were stained with CD4, CD25 and Foxp3 antibodies and analyzed by FACS on day 7 (D7) and day 14 (D14) following activation. (A) FACS analyses of CD4 and Foxp3 expression of expanded Treg on D7 and D14. The representative results were from cells obtained from one individual of each subject group. (B) Collective analyses of the purity and expansion fold of expanded Treg cells in each group on D7 and D14.

Mentions: Next, we determined, compared to healthy controls, how well Treg isolated from PBLs of T1D patients with established long-term disease can be expanded in vitro. Previous studies showed that the CD4+CD25+CD127−/lo T cells contain the greatest population of Foxp3+ Treg [21]. Therefore, we isolated CD4+CD25+CD127−/lo T cells from PBLs of T1D or healthy subjects to obtain a highly purified Foxp3+ Treg population. Similar purity and cell yield of sorted Treg were obtained from both subject groups on day 0 (mean: 97.6±1.8%, range: 93.3%–99.9%)(Table 2). The purified Treg were then activated and expanded in vitro. During the culture period, there was no significant difference in the purity of cultured Treg isolated from T1D or healthy subjects on day 7 and day 14 (Fig. 3A–B). Furthermore, the purified Treg from both groups could be expanded equally well in vitro (Fig. 3B).


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)

Both diabetic and healthy Treg can be expanded with comparable purity and yield in vitro.CD4+CD25+CD127−/lo Treg, electronically sorted using FACS from diabetic patients and healthy subjects, were activated in vitro using anti-CD3/anti-CD28-coated beads plus recombinant human IL-2. Expanded Treg were stained with CD4, CD25 and Foxp3 antibodies and analyzed by FACS on day 7 (D7) and day 14 (D14) following activation. (A) FACS analyses of CD4 and Foxp3 expression of expanded Treg on D7 and D14. The representative results were from cells obtained from one individual of each subject group. (B) Collective analyses of the purity and expansion fold of expanded Treg cells in each group on D7 and D14.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056209-g003: Both diabetic and healthy Treg can be expanded with comparable purity and yield in vitro.CD4+CD25+CD127−/lo Treg, electronically sorted using FACS from diabetic patients and healthy subjects, were activated in vitro using anti-CD3/anti-CD28-coated beads plus recombinant human IL-2. Expanded Treg were stained with CD4, CD25 and Foxp3 antibodies and analyzed by FACS on day 7 (D7) and day 14 (D14) following activation. (A) FACS analyses of CD4 and Foxp3 expression of expanded Treg on D7 and D14. The representative results were from cells obtained from one individual of each subject group. (B) Collective analyses of the purity and expansion fold of expanded Treg cells in each group on D7 and D14.
Mentions: Next, we determined, compared to healthy controls, how well Treg isolated from PBLs of T1D patients with established long-term disease can be expanded in vitro. Previous studies showed that the CD4+CD25+CD127−/lo T cells contain the greatest population of Foxp3+ Treg [21]. Therefore, we isolated CD4+CD25+CD127−/lo T cells from PBLs of T1D or healthy subjects to obtain a highly purified Foxp3+ Treg population. Similar purity and cell yield of sorted Treg were obtained from both subject groups on day 0 (mean: 97.6±1.8%, range: 93.3%–99.9%)(Table 2). The purified Treg were then activated and expanded in vitro. During the culture period, there was no significant difference in the purity of cultured Treg isolated from T1D or healthy subjects on day 7 and day 14 (Fig. 3A–B). Furthermore, the purified Treg from both groups could be expanded equally well in vitro (Fig. 3B).

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