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In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.

Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA - J. Exp. Med. (2004)

Bottom Line: Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2.The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions.Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: UCSF Diabetes Center, University of California San Francisco, 94143, USA.

ABSTRACT
The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

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In vivo survival and activation of expanded Tregs. (A) Freshly isolated and expanded BALB/c Tregs were labeled with CFSE and then injected into normal BALB/c mice (106/mouse). All recipient mice were killed on day 30 after injection and the numbers of CFSE+ cells in the peripheral LN and spleen (not shown) were determined by flow cytometry. The data are presented as the number of CD4+ CFSE+ cells/106 endogenous CD4+ T cells. Each circle represents the value from one mouse and the black bar represents the mean of the group. (B) Expanded Tregs from NOD.Thy1.2 (top) and GAD286 TCR Tg Thy1.2 (middle) were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.1 recipients. Expanded BDC2.5 TCR Tg Thy1.1 were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.2 recipients. The presence of transferred cells and their activation status in spleens, LNs, and pancreatic LNs were determined by flow cytometry on day 7 after cell transfer. The dot plots shown are gated on the Thy1.2 for NOD and GAD286 cells and Thy1.1 for BDC2.5 cells. The percentages of cells with CFSE dilution are shown on the plots. Results are representative of at least three recipient mice in two separate experiments.
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fig4: In vivo survival and activation of expanded Tregs. (A) Freshly isolated and expanded BALB/c Tregs were labeled with CFSE and then injected into normal BALB/c mice (106/mouse). All recipient mice were killed on day 30 after injection and the numbers of CFSE+ cells in the peripheral LN and spleen (not shown) were determined by flow cytometry. The data are presented as the number of CD4+ CFSE+ cells/106 endogenous CD4+ T cells. Each circle represents the value from one mouse and the black bar represents the mean of the group. (B) Expanded Tregs from NOD.Thy1.2 (top) and GAD286 TCR Tg Thy1.2 (middle) were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.1 recipients. Expanded BDC2.5 TCR Tg Thy1.1 were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.2 recipients. The presence of transferred cells and their activation status in spleens, LNs, and pancreatic LNs were determined by flow cytometry on day 7 after cell transfer. The dot plots shown are gated on the Thy1.2 for NOD and GAD286 cells and Thy1.1 for BDC2.5 cells. The percentages of cells with CFSE dilution are shown on the plots. Results are representative of at least three recipient mice in two separate experiments.

Mentions: Effective suppression of immune responses in vivo by Tregs requires that the cells migrate to appropriate sites, respond to antigen, and survive long-term. We have observed recently that blockade of the CD28/B7 pathway resulted in rapid loss of Tregs in vivo and subsequent loss of critical immune regulation (8, 46). Thus, we examined the ability of expanded Tregs to survive and proliferate in vivo. Expanded Tregs were labeled with CFSE and transferred into normal nonlymphopenic syngeneic mice. At 30 d after transfer, the mice were killed and examined for the number of CFSE+ cells as an indication of cell survival. As seen in Fig. 4 A, a significant number of CFSE+ cells were recovered from mice transferred with expanded Tregs. The number of CFSE+ Tregs was equal to that observed with fresh Tregs transferred in the same manner (Fig. 4 A). In fact, Thy1.1-marked expanded Tregs were observed at least 50 d after transfer (unpublished data).


In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.

Tang Q, Henriksen KJ, Bi M, Finger EB, Szot G, Ye J, Masteller EL, McDevitt H, Bonyhadi M, Bluestone JA - J. Exp. Med. (2004)

In vivo survival and activation of expanded Tregs. (A) Freshly isolated and expanded BALB/c Tregs were labeled with CFSE and then injected into normal BALB/c mice (106/mouse). All recipient mice were killed on day 30 after injection and the numbers of CFSE+ cells in the peripheral LN and spleen (not shown) were determined by flow cytometry. The data are presented as the number of CD4+ CFSE+ cells/106 endogenous CD4+ T cells. Each circle represents the value from one mouse and the black bar represents the mean of the group. (B) Expanded Tregs from NOD.Thy1.2 (top) and GAD286 TCR Tg Thy1.2 (middle) were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.1 recipients. Expanded BDC2.5 TCR Tg Thy1.1 were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.2 recipients. The presence of transferred cells and their activation status in spleens, LNs, and pancreatic LNs were determined by flow cytometry on day 7 after cell transfer. The dot plots shown are gated on the Thy1.2 for NOD and GAD286 cells and Thy1.1 for BDC2.5 cells. The percentages of cells with CFSE dilution are shown on the plots. Results are representative of at least three recipient mice in two separate experiments.
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Related In: Results  -  Collection

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fig4: In vivo survival and activation of expanded Tregs. (A) Freshly isolated and expanded BALB/c Tregs were labeled with CFSE and then injected into normal BALB/c mice (106/mouse). All recipient mice were killed on day 30 after injection and the numbers of CFSE+ cells in the peripheral LN and spleen (not shown) were determined by flow cytometry. The data are presented as the number of CD4+ CFSE+ cells/106 endogenous CD4+ T cells. Each circle represents the value from one mouse and the black bar represents the mean of the group. (B) Expanded Tregs from NOD.Thy1.2 (top) and GAD286 TCR Tg Thy1.2 (middle) were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.1 recipients. Expanded BDC2.5 TCR Tg Thy1.1 were labeled with CFSE and 3 × 106 were transferred to normal 8–12-wk-old NOD.Thy1.2 recipients. The presence of transferred cells and their activation status in spleens, LNs, and pancreatic LNs were determined by flow cytometry on day 7 after cell transfer. The dot plots shown are gated on the Thy1.2 for NOD and GAD286 cells and Thy1.1 for BDC2.5 cells. The percentages of cells with CFSE dilution are shown on the plots. Results are representative of at least three recipient mice in two separate experiments.
Mentions: Effective suppression of immune responses in vivo by Tregs requires that the cells migrate to appropriate sites, respond to antigen, and survive long-term. We have observed recently that blockade of the CD28/B7 pathway resulted in rapid loss of Tregs in vivo and subsequent loss of critical immune regulation (8, 46). Thus, we examined the ability of expanded Tregs to survive and proliferate in vivo. Expanded Tregs were labeled with CFSE and transferred into normal nonlymphopenic syngeneic mice. At 30 d after transfer, the mice were killed and examined for the number of CFSE+ cells as an indication of cell survival. As seen in Fig. 4 A, a significant number of CFSE+ cells were recovered from mice transferred with expanded Tregs. The number of CFSE+ Tregs was equal to that observed with fresh Tregs transferred in the same manner (Fig. 4 A). In fact, Thy1.1-marked expanded Tregs were observed at least 50 d after transfer (unpublished data).

Bottom Line: Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2.The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions.Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: UCSF Diabetes Center, University of California San Francisco, 94143, USA.

ABSTRACT
The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.

Show MeSH
Related in: MedlinePlus