Limits...
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.

Show MeSH

Related in: MedlinePlus

In vitro expansion of Tregs. (A) Representative flow cytometry plots of CD25 and CD62L expression on CD4 cells from NOD (left), BDC2.5 (middle), and GAD286 (right) mice. FACS®-purified Tregs (•) and CD4+ CD62L+ CD25− cells (○) from NOD (B), BDC2.5 TCR Tg (C), or GAD286 TCR Tg (D) mice were stimulated in vitro with anti-CD3– and anti-CD28–coated beads along with IL-2. (E) T cells from BDC2.5 TCR Tg mice were expanded as described above with p31-linked IAg7-mIgG2a immobilized on latex beads. All cultures were quantitated by viable cell counting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211775&req=5

fig1: In vitro expansion of Tregs. (A) Representative flow cytometry plots of CD25 and CD62L expression on CD4 cells from NOD (left), BDC2.5 (middle), and GAD286 (right) mice. FACS®-purified Tregs (•) and CD4+ CD62L+ CD25− cells (○) from NOD (B), BDC2.5 TCR Tg (C), or GAD286 TCR Tg (D) mice were stimulated in vitro with anti-CD3– and anti-CD28–coated beads along with IL-2. (E) T cells from BDC2.5 TCR Tg mice were expanded as described above with p31-linked IAg7-mIgG2a immobilized on latex beads. All cultures were quantitated by viable cell counting.

Mentions: Previous studies have shown that the number and function of Tregs in NOD mice decrease over time correlating with clinical disease onset between 16 and 24 wk of age (9, 40). These observations support the use of Tregs to prevent or treat diabetes even after disease onset. However, the ability to use these cells therapeutically is severely limited by the small numbers of cells resident in the circulation or lymphoid organs (<5% of CD4+ T cells in NOD mice and <2% of CD4+ T cells in humans with T1D; references 8 and 39). Moreover, a large number of cells are required for therapeutic efficacy due to an inability at present to select the cells based on antigen specificity. Therefore, we developed a technique for rapid and efficient expansion of autoantigen-specific Tregs based on observations that these cells, present in TCR Tg mice, can be driven into cell cycle with coimmobilized anti-CD3 and anti-CD28 antibodies plus exogenous IL-2. As shown in Fig. 1, A and B, FACS®-purified NOD Tregs cultured with anti-CD3/anti-CD28–coated beads in the presence of IL-2 expanded 150–225-fold in 11 d (Fig. 1 B). In general, the CD4+ CD25− T cells expanded more vigorously (ranging from 300–800-fold in multiple experiments). A purity of >98% CD4+ CD25+ CD62+ T cells was essential to enable successful Treg expansion as a small contamination of either CD25− CD4+ or CD8+ T cells significantly impacted the ability to specifically expand the Tregs (unpublished data). It should be noted that the Treg expansion is dependent on the high level of IL-2 (2,000 IU/ml). No Treg expansion was observed when 200 IU/ml IL-2 was used (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 vitro expansion of Tregs. (A) Representative flow cytometry plots of CD25 and CD62L expression on CD4 cells from NOD (left), BDC2.5 (middle), and GAD286 (right) mice. FACS®-purified Tregs (•) and CD4+ CD62L+ CD25− cells (○) from NOD (B), BDC2.5 TCR Tg (C), or GAD286 TCR Tg (D) mice were stimulated in vitro with anti-CD3– and anti-CD28–coated beads along with IL-2. (E) T cells from BDC2.5 TCR Tg mice were expanded as described above with p31-linked IAg7-mIgG2a immobilized on latex beads. All cultures were quantitated by viable cell counting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211775&req=5

fig1: In vitro expansion of Tregs. (A) Representative flow cytometry plots of CD25 and CD62L expression on CD4 cells from NOD (left), BDC2.5 (middle), and GAD286 (right) mice. FACS®-purified Tregs (•) and CD4+ CD62L+ CD25− cells (○) from NOD (B), BDC2.5 TCR Tg (C), or GAD286 TCR Tg (D) mice were stimulated in vitro with anti-CD3– and anti-CD28–coated beads along with IL-2. (E) T cells from BDC2.5 TCR Tg mice were expanded as described above with p31-linked IAg7-mIgG2a immobilized on latex beads. All cultures were quantitated by viable cell counting.
Mentions: Previous studies have shown that the number and function of Tregs in NOD mice decrease over time correlating with clinical disease onset between 16 and 24 wk of age (9, 40). These observations support the use of Tregs to prevent or treat diabetes even after disease onset. However, the ability to use these cells therapeutically is severely limited by the small numbers of cells resident in the circulation or lymphoid organs (<5% of CD4+ T cells in NOD mice and <2% of CD4+ T cells in humans with T1D; references 8 and 39). Moreover, a large number of cells are required for therapeutic efficacy due to an inability at present to select the cells based on antigen specificity. Therefore, we developed a technique for rapid and efficient expansion of autoantigen-specific Tregs based on observations that these cells, present in TCR Tg mice, can be driven into cell cycle with coimmobilized anti-CD3 and anti-CD28 antibodies plus exogenous IL-2. As shown in Fig. 1, A and B, FACS®-purified NOD Tregs cultured with anti-CD3/anti-CD28–coated beads in the presence of IL-2 expanded 150–225-fold in 11 d (Fig. 1 B). In general, the CD4+ CD25− T cells expanded more vigorously (ranging from 300–800-fold in multiple experiments). A purity of >98% CD4+ CD25+ CD62+ T cells was essential to enable successful Treg expansion as a small contamination of either CD25− CD4+ or CD8+ T cells significantly impacted the ability to specifically expand the Tregs (unpublished data). It should be noted that the Treg expansion is dependent on the high level of IL-2 (2,000 IU/ml). No Treg expansion was observed when 200 IU/ml IL-2 was used (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