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

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.

<p>PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (<b>A</b>) Percentage of CD45RA⁺ or CD45RO⁺ cells detected in the CD4⁺ or CD8⁺ T cells. (<b>B</b>) Percentage of CD45RA⁺ or CD45RO⁺ cells in CD4⁺Foxp3⁺ Treg. (<b>C</b>) 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. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Expanded CD4⁺CD25⁺CD127^lo/− T cells from healthy controls and T1D patients are highly enriched Foxp3⁺ Treg^*.

*<p>Subject 1–10 were healthy controls and subject 11–20 were T1D patients.</p

Treg expanded from diabetic patients or healthy subjects have comparable potent suppressive function.

<p>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. (<b>A</b>–<b>B</b>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056209#s2" target="_blank">Results</a> from functional analyses of co-culture of expanded Treg with autologous PBMCs from two representative healthy subjects (<b>A</b>) or diabetic patients (<b>B</b>) from at least 8 different subjects for each group. (<b>C</b>–<b>D</b>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056209#s2" target="_blank">Results</a> 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 (<b>C</b>) or T1D patient (<b>D</b>). (<b>E–F</b>) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (<b>E</b>) or diabetic patients (<b>F</b>) using autologous Teff. (<b>G–H</b>) The % suppression as a function of Teff/Treg ratio for Treg from either healthy controls (<b>G</b>) or diabetic patients (<b>H</b>) using allogeneic Teff from either healthy controls or diabetic patients.</p

T cell subset distribution in peripheral blood of healthy and diabetic subjects^*.

*<p>Subjects 1–10 were healthy controls and subjects 11–20 were T1D patients.</p

Comparable frequency of CD4⁺, CD8⁺ or Treg in PBLs of T1D patients and healthy subjects.

<p>PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. (<b>A</b>) Percentage of CD4⁺ and CD8⁺ T cells in PBLs. (<b>B</b>) Ratios of CD4⁺ and CD8⁺ T cells (left) and the frequency of CD4⁺Foxp3⁺ Treg (right). The horizontal lines represent the median of all analyzed samples in each group.</p

Expression of IFN-γ, TNF-α, IL17 in Treg of PBLs or in expanded Treg.

<p>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. (<b>A</b>–<b>B</b>) IFN-γ, TNF-α, and IL17 expression in Foxp3⁺Treg of (<b>A</b>) PBLs and (<b>B</b>) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4⁺ T cell population. <b>(C</b>–<b>D</b>) Collective analyses of the percentage of IFN-γ⁺, TNF-α⁺, or IL17⁺ in (<b>C</b>) Foxp3⁺ Treg of PBLs or (<b>D</b>) expanded Foxp3⁺ Treg.</p

<i>In vitro</i> expansion leads to decreased expression of Helios on T1D patient but not healthy subject Treg.

<p>PBMCs and expanded Treg were stained with indicated antibodies and analyzed by FACS. (<b>A</b>) Percentage of Helios⁺ cells in total CD4⁺ T cells (CD4⁺Helios⁺) or in Foxp3⁺CD4⁺ T cells (CD4⁺Foxp3⁺Helios⁺) in PBLs of T1D patients or healthy controls. (<b>B</b>) FACS analyses of Helios and Foxp3 expression in PBLs and expanded Treg on D14 from one representative individual of each subject group. The cells were electronically gated on CD4⁺ T cell population. (<b>C</b>) Collective analyses of the percentage of Helios⁺ cell frequency in PBLs or expanded Foxp3⁺ Treg on D14 in each subject group. (<b>D</b>) Paired comparison analyses of Helios expression on Foxp3⁺ Treg in PBLs vs. its expression on expanded Foxp3⁺ Treg in each subject group.</p

TNFRII and GARP expression on Foxp3⁺ Treg.

<p>PBMCs and expanded Treg were stained with indicated antibodies and analyzed by FACS. FACS analyses of (<b>A</b>) TNFRII, (<b>C</b>) GARP expression on Foxp3⁺Treg in PBLs and expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4⁺ T cell population. Collective analyses of the percentage of (<b>B</b>) TNFRII⁺ or (<b>D</b>) GARP⁺ cell frequency in PBLs or expanded Foxp3⁺ Treg.</p