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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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Prevention of diabetes transfer by expanded Tregs. (A) Activated diabetogenic BDC2.5 CD4+ CD62L+ CD25− cells (3.5 × 105) were cotransferred with BDC2.5-expanded Tregs to 8-wk-old NOD. RAG−/− recipients at the indicated ratio. The blood glucose for individual recipient mouse was monitored and plotted to access diabetes. n = 3 for no Tregs and 1:9 groups; n = 4 for 1:1 and 1:3 groups. Results are representative of three independent experiments. (B) Diabetes was induced in 6-wk-old NOD.RAG−/− mice in the same manner as described in A, except that the number of transferred expanded Tregs from GAD286 TCR Tg mice and BDC2.5 TCR Tg mice equaled the number of transferred Teffs (n = 3 mice/group). (C) Diabetes was induced in 5–8-wk-old NOD.RAG−/− or NOD.TCR-α−/− recipients by injection of 25 × 106 pooled spleen and LN cells from diabetic donors (n = 8). Some recipient mice were coinjected with expanded Tregs from NOD (2 × 106, n = 3; 5 × 106, n = 3; 8 × 106, n = 4) or BDC2.5 TCR Tg mice (2 × 106, n = 4). Results represent two independent experiments.
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fig5: Prevention of diabetes transfer by expanded Tregs. (A) Activated diabetogenic BDC2.5 CD4+ CD62L+ CD25− cells (3.5 × 105) were cotransferred with BDC2.5-expanded Tregs to 8-wk-old NOD. RAG−/− recipients at the indicated ratio. The blood glucose for individual recipient mouse was monitored and plotted to access diabetes. n = 3 for no Tregs and 1:9 groups; n = 4 for 1:1 and 1:3 groups. Results are representative of three independent experiments. (B) Diabetes was induced in 6-wk-old NOD.RAG−/− mice in the same manner as described in A, except that the number of transferred expanded Tregs from GAD286 TCR Tg mice and BDC2.5 TCR Tg mice equaled the number of transferred Teffs (n = 3 mice/group). (C) Diabetes was induced in 5–8-wk-old NOD.RAG−/− or NOD.TCR-α−/− recipients by injection of 25 × 106 pooled spleen and LN cells from diabetic donors (n = 8). Some recipient mice were coinjected with expanded Tregs from NOD (2 × 106, n = 3; 5 × 106, n = 3; 8 × 106, n = 4) or BDC2.5 TCR Tg mice (2 × 106, n = 4). Results represent two independent experiments.

Mentions: Next, we examined the ability of the expanded BDC2.5 Tregs to suppress diabetes after in vivo cotransfer of activated BDC2.5 T cells into NOD.RAG mice. The Tregs were effective in blocking the transfer of diabetes, functioning at as low as a 1:9 ratio of Treg/Teff (Fig. 5 A), whereas the GAD286 Tregs did not protect even at a Treg/Teff ratio of 1:1 (Fig. 5 B). In fact, the expanded BDC2.5 Tregs suppressed polyclonal T cell–mediated disease. As few as 2 × 106 expanded BDC2.5 Tregs blocked the ability of 25 × 106 diabetogenic NOD spleen and LN cells to transfer disease (Fig. 5 C). The expanded antigen-specific Tregs from the BDC2.5 mice were far more efficient than expanded polyclonal NOD Tregs in preventing the onset of diabetes. The same number (2 × 106) as well as 5 × 106 expanded NOD Tregs did not confer any protection under the same conditions (Fig. 5 C). In fact, even the transfer of four times as many expanded NOD Tregs (8 × 106) only slightly delayed diabetes onset and prevented diabetes in only one diabetogenic cell recipient (Fig. 5 C). This result is consistent with previous findings suggesting that a high ratio of polyclonal Tregs to Teffs is necessary to efficiently suppress disease transfer in this setting (8, 16, 17). Importantly, these data suggest that in vitro activity of the Tregs does not predict in vivo function in this disease setting.


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)

Prevention of diabetes transfer by expanded Tregs. (A) Activated diabetogenic BDC2.5 CD4+ CD62L+ CD25− cells (3.5 × 105) were cotransferred with BDC2.5-expanded Tregs to 8-wk-old NOD. RAG−/− recipients at the indicated ratio. The blood glucose for individual recipient mouse was monitored and plotted to access diabetes. n = 3 for no Tregs and 1:9 groups; n = 4 for 1:1 and 1:3 groups. Results are representative of three independent experiments. (B) Diabetes was induced in 6-wk-old NOD.RAG−/− mice in the same manner as described in A, except that the number of transferred expanded Tregs from GAD286 TCR Tg mice and BDC2.5 TCR Tg mice equaled the number of transferred Teffs (n = 3 mice/group). (C) Diabetes was induced in 5–8-wk-old NOD.RAG−/− or NOD.TCR-α−/− recipients by injection of 25 × 106 pooled spleen and LN cells from diabetic donors (n = 8). Some recipient mice were coinjected with expanded Tregs from NOD (2 × 106, n = 3; 5 × 106, n = 3; 8 × 106, n = 4) or BDC2.5 TCR Tg mice (2 × 106, n = 4). Results represent two independent experiments.
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Related In: Results  -  Collection

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fig5: Prevention of diabetes transfer by expanded Tregs. (A) Activated diabetogenic BDC2.5 CD4+ CD62L+ CD25− cells (3.5 × 105) were cotransferred with BDC2.5-expanded Tregs to 8-wk-old NOD. RAG−/− recipients at the indicated ratio. The blood glucose for individual recipient mouse was monitored and plotted to access diabetes. n = 3 for no Tregs and 1:9 groups; n = 4 for 1:1 and 1:3 groups. Results are representative of three independent experiments. (B) Diabetes was induced in 6-wk-old NOD.RAG−/− mice in the same manner as described in A, except that the number of transferred expanded Tregs from GAD286 TCR Tg mice and BDC2.5 TCR Tg mice equaled the number of transferred Teffs (n = 3 mice/group). (C) Diabetes was induced in 5–8-wk-old NOD.RAG−/− or NOD.TCR-α−/− recipients by injection of 25 × 106 pooled spleen and LN cells from diabetic donors (n = 8). Some recipient mice were coinjected with expanded Tregs from NOD (2 × 106, n = 3; 5 × 106, n = 3; 8 × 106, n = 4) or BDC2.5 TCR Tg mice (2 × 106, n = 4). Results represent two independent experiments.
Mentions: Next, we examined the ability of the expanded BDC2.5 Tregs to suppress diabetes after in vivo cotransfer of activated BDC2.5 T cells into NOD.RAG mice. The Tregs were effective in blocking the transfer of diabetes, functioning at as low as a 1:9 ratio of Treg/Teff (Fig. 5 A), whereas the GAD286 Tregs did not protect even at a Treg/Teff ratio of 1:1 (Fig. 5 B). In fact, the expanded BDC2.5 Tregs suppressed polyclonal T cell–mediated disease. As few as 2 × 106 expanded BDC2.5 Tregs blocked the ability of 25 × 106 diabetogenic NOD spleen and LN cells to transfer disease (Fig. 5 C). The expanded antigen-specific Tregs from the BDC2.5 mice were far more efficient than expanded polyclonal NOD Tregs in preventing the onset of diabetes. The same number (2 × 106) as well as 5 × 106 expanded NOD Tregs did not confer any protection under the same conditions (Fig. 5 C). In fact, even the transfer of four times as many expanded NOD Tregs (8 × 106) only slightly delayed diabetes onset and prevented diabetes in only one diabetogenic cell recipient (Fig. 5 C). This result is consistent with previous findings suggesting that a high ratio of polyclonal Tregs to Teffs is necessary to efficiently suppress disease transfer in this setting (8, 16, 17). Importantly, these data suggest that in vitro activity of the Tregs does not predict in vivo function in this disease setting.

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