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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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Altered populations of CD45RA or CD45RO-expressing Treg in T1D patients.PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (A) Percentage of CD45RA+ or CD45RO+ cells detected in the CD4+ or CD8+ T cells. (B) Percentage of CD45RA+ or CD45RO+ cells in CD4+Foxp3+ Treg. (C) Representative results from FACS analyses of CD45RA, CD45RO and Foxp3 expression in the CD4+ T cells in PBLs. The cells were electronically gated for FACS analyses. *p<0.05, **p<0.01, ***p<0.001.
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pone-0056209-g002: Altered populations of CD45RA or CD45RO-expressing Treg in T1D patients.PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (A) Percentage of CD45RA+ or CD45RO+ cells detected in the CD4+ or CD8+ T cells. (B) Percentage of CD45RA+ or CD45RO+ cells in CD4+Foxp3+ Treg. (C) Representative results from FACS analyses of CD45RA, CD45RO and Foxp3 expression in the CD4+ T cells in PBLs. The cells were electronically gated for FACS analyses. *p<0.05, **p<0.01, ***p<0.001.

Mentions: Within the CD4+ T cells in PBLs, we further analyzed the expression of CD45RA and CD45RO on CD4+Foxp3+ Treg. Similar to CD4+ T cells, less Treg in the PBLs of T1D subjects express CD45RA than healthy controls (p = 0.002)(Fig. 2B–C). In addition, more Foxp3+ Treg in the CD4+ T cells of T1D subjects express CD45RO than in healthy controls (p = 0.0011)(Fig. 2B–C). Further analyses showed that the observed difference in cell phenotype is independent of the age of subjects in the two cohorts (data not shown). Overall, these data support the conclusion that a larger percentage of not only CD4+ T cells but also CD4+Foxp3+ Treg in PBLs from T1D subjects than those from healthy controls express the CD45RO+ memory cell phenotype, and thus are likely antigen-experienced cells.


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)

Altered populations of CD45RA or CD45RO-expressing Treg in T1D patients.PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (A) Percentage of CD45RA+ or CD45RO+ cells detected in the CD4+ or CD8+ T cells. (B) Percentage of CD45RA+ or CD45RO+ cells in CD4+Foxp3+ Treg. (C) Representative results from FACS analyses of CD45RA, CD45RO and Foxp3 expression in the CD4+ T cells in PBLs. The cells were electronically gated for FACS analyses. *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056209-g002: Altered populations of CD45RA or CD45RO-expressing Treg in T1D patients.PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (A) Percentage of CD45RA+ or CD45RO+ cells detected in the CD4+ or CD8+ T cells. (B) Percentage of CD45RA+ or CD45RO+ cells in CD4+Foxp3+ Treg. (C) Representative results from FACS analyses of CD45RA, CD45RO and Foxp3 expression in the CD4+ T cells in PBLs. The cells were electronically gated for FACS analyses. *p<0.05, **p<0.01, ***p<0.001.
Mentions: Within the CD4+ T cells in PBLs, we further analyzed the expression of CD45RA and CD45RO on CD4+Foxp3+ Treg. Similar to CD4+ T cells, less Treg in the PBLs of T1D subjects express CD45RA than healthy controls (p = 0.002)(Fig. 2B–C). In addition, more Foxp3+ Treg in the CD4+ T cells of T1D subjects express CD45RO than in healthy controls (p = 0.0011)(Fig. 2B–C). Further analyses showed that the observed difference in cell phenotype is independent of the age of subjects in the two cohorts (data not shown). Overall, these data support the conclusion that a larger percentage of not only CD4+ T cells but also CD4+Foxp3+ Treg in PBLs from T1D subjects than those from healthy controls express the CD45RO+ memory cell phenotype, and thus are likely antigen-experienced cells.

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