Limits...
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.

Show MeSH

Related in: MedlinePlus

Treg expanded from diabetic patients or healthy subjects have comparable potent suppressive function.CFSE-labeled PBMCs were co-cultured with expanded Treg from the same donor or allogeneic donors at various ratios (Teff/Treg = 1∶0, 1∶1, 1∶0.5, 1∶0.25 or 1∶0.125). The cells were stimulated with soluble anti-CD3/anti-CD28 antibodies for 3 days, and the proliferation of CFSE-labeled CD8+ T cells in PBMCs was analyzed by FACS. (A–B) Results from functional analyses of co-culture of expanded Treg with autologous PBMCs from two representative healthy subjects (A) or diabetic patients (B) from at least 8 different subjects for each group. (C–D) Results from functional analyses of co-culture of expanded Treg with allogeneic PBMCs from either healthy subjects or diabetic patients. The Treg were from one representative healthy subject (C) or T1D patient (D). (E–F) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (E) or diabetic patients (F) using autologous Teff. (G–H) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (G) or diabetic patients (H) using allogeneic Teff from either healthy controls or diabetic patients.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569420&req=5

pone-0056209-g008: Treg expanded from diabetic patients or healthy subjects have comparable potent suppressive function.CFSE-labeled PBMCs were co-cultured with expanded Treg from the same donor or allogeneic donors at various ratios (Teff/Treg = 1∶0, 1∶1, 1∶0.5, 1∶0.25 or 1∶0.125). The cells were stimulated with soluble anti-CD3/anti-CD28 antibodies for 3 days, and the proliferation of CFSE-labeled CD8+ T cells in PBMCs was analyzed by FACS. (A–B) Results from functional analyses of co-culture of expanded Treg with autologous PBMCs from two representative healthy subjects (A) or diabetic patients (B) from at least 8 different subjects for each group. (C–D) Results from functional analyses of co-culture of expanded Treg with allogeneic PBMCs from either healthy subjects or diabetic patients. The Treg were from one representative healthy subject (C) or T1D patient (D). (E–F) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (E) or diabetic patients (F) using autologous Teff. (G–H) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (G) or diabetic patients (H) using allogeneic Teff from either healthy controls or diabetic patients.

Mentions: To compare the function of in vitro expanded Treg from the two subject groups, we first examined whether the expanded Treg may suppress the proliferation of autologous CD8+ Teff in PBLs from the same subject. Results from two representative subjects of either T1D patient or healthy controls are shown in Fig. 8A–B. Following a 3-day incubation of autologous PBLs with expanded Treg plus anti-CD3/CD28, the proliferation of CFSE-labeled CD8+ Teff cells in PBLs was analyzed at the end of the assay. The results demonstrated that the suppressive function of the expanded Treg was dose-dependent. When compared at varied Teff/Treg ratios, the expanded Treg from both T1D and healthy subjects were able to suppress autologous Teff equally well (Fig. A and E, B and F). In addition, the expanded Treg from both subject groups were still able to suppress Teff proliferation at a 1∶0.125 Teff∶Treg ratio. Therefore, expanded Treg from T1D patients or healthy subjects have comparable function in suppressing autologous Teff 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)

Treg expanded from diabetic patients or healthy subjects have comparable potent suppressive function.CFSE-labeled PBMCs were co-cultured with expanded Treg from the same donor or allogeneic donors at various ratios (Teff/Treg = 1∶0, 1∶1, 1∶0.5, 1∶0.25 or 1∶0.125). The cells were stimulated with soluble anti-CD3/anti-CD28 antibodies for 3 days, and the proliferation of CFSE-labeled CD8+ T cells in PBMCs was analyzed by FACS. (A–B) Results from functional analyses of co-culture of expanded Treg with autologous PBMCs from two representative healthy subjects (A) or diabetic patients (B) from at least 8 different subjects for each group. (C–D) Results from functional analyses of co-culture of expanded Treg with allogeneic PBMCs from either healthy subjects or diabetic patients. The Treg were from one representative healthy subject (C) or T1D patient (D). (E–F) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (E) or diabetic patients (F) using autologous Teff. (G–H) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (G) or diabetic patients (H) using allogeneic Teff from either healthy controls or diabetic patients.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056209-g008: Treg expanded from diabetic patients or healthy subjects have comparable potent suppressive function.CFSE-labeled PBMCs were co-cultured with expanded Treg from the same donor or allogeneic donors at various ratios (Teff/Treg = 1∶0, 1∶1, 1∶0.5, 1∶0.25 or 1∶0.125). The cells were stimulated with soluble anti-CD3/anti-CD28 antibodies for 3 days, and the proliferation of CFSE-labeled CD8+ T cells in PBMCs was analyzed by FACS. (A–B) Results from functional analyses of co-culture of expanded Treg with autologous PBMCs from two representative healthy subjects (A) or diabetic patients (B) from at least 8 different subjects for each group. (C–D) Results from functional analyses of co-culture of expanded Treg with allogeneic PBMCs from either healthy subjects or diabetic patients. The Treg were from one representative healthy subject (C) or T1D patient (D). (E–F) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (E) or diabetic patients (F) using autologous Teff. (G–H) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (G) or diabetic patients (H) using allogeneic Teff from either healthy controls or diabetic patients.
Mentions: To compare the function of in vitro expanded Treg from the two subject groups, we first examined whether the expanded Treg may suppress the proliferation of autologous CD8+ Teff in PBLs from the same subject. Results from two representative subjects of either T1D patient or healthy controls are shown in Fig. 8A–B. Following a 3-day incubation of autologous PBLs with expanded Treg plus anti-CD3/CD28, the proliferation of CFSE-labeled CD8+ Teff cells in PBLs was analyzed at the end of the assay. The results demonstrated that the suppressive function of the expanded Treg was dose-dependent. When compared at varied Teff/Treg ratios, the expanded Treg from both T1D and healthy subjects were able to suppress autologous Teff equally well (Fig. A and E, B and F). In addition, the expanded Treg from both subject groups were still able to suppress Teff proliferation at a 1∶0.125 Teff∶Treg ratio. Therefore, expanded Treg from T1D patients or healthy subjects have comparable function in suppressing autologous Teff 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