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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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Phenotype of in vitro–expanded Tregs. (A) Expression of CD25 and CD62L on expanded Tregs and CD4+ CD62L+ CD25− cells was determined by flow cytometry on day 8 after the culture initiation. Results are representative of more than 20 independent experiments. (B) Levels of mRNA for the indicated genes in expanded NOD (filled symbols) or BDC2.5 TCR Tg T cells (open symbols) were determined by real time PCR analysis on day 10 after the initiation of the cultures. The relative expression ratio (Treg/TCD25−) for each pair of cultures was calculated from Ct values as described in Materials and Methods. The dashed line represents the ratio of 1 (i.e., identical level of gene expression in Treg and CD4+ CD62L+ CD25− cultures). (C) Western blot analysis of FoxP3 protein expression in fresh and expanded T cells. The level of tubulin expression was included as a loading control. Results are representative of three independent experiments. (D) Cytokine secretion by expanded BDC2.5 T cells 48 h after restimulation with antigenic peptide and splenic APC. Results are representative of two independent experiments.
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fig2: Phenotype of in vitro–expanded Tregs. (A) Expression of CD25 and CD62L on expanded Tregs and CD4+ CD62L+ CD25− cells was determined by flow cytometry on day 8 after the culture initiation. Results are representative of more than 20 independent experiments. (B) Levels of mRNA for the indicated genes in expanded NOD (filled symbols) or BDC2.5 TCR Tg T cells (open symbols) were determined by real time PCR analysis on day 10 after the initiation of the cultures. The relative expression ratio (Treg/TCD25−) for each pair of cultures was calculated from Ct values as described in Materials and Methods. The dashed line represents the ratio of 1 (i.e., identical level of gene expression in Treg and CD4+ CD62L+ CD25− cultures). (C) Western blot analysis of FoxP3 protein expression in fresh and expanded T cells. The level of tubulin expression was included as a loading control. Results are representative of three independent experiments. (D) Cytokine secretion by expanded BDC2.5 T cells 48 h after restimulation with antigenic peptide and splenic APC. Results are representative of two independent experiments.

Mentions: Next, we examined the phenotype of the expanded Tregs by flow cytometry, Western blot, and real time PCR. As can be seen in Fig. 2 A, the expanded Tregs maintained high levels of expression of CD25 as compared with expanded CD25− T cells, whereas the expression of CD62L remained high in both cell types. In addition, quantitative PCR showed that all of the Tregs expressed high levels of SOCS2, PD-1, and CTLA-4 as compared with similarly expanded CD25− T cells. Moreover, the recently identified markers neuropilin and TRAIL (20, 45) were also highly expressed on the expanded Tregs (Fig. 2 B). A high level of cell surface GITR expression was observed on the expanded Tregs. However, this previously identified Treg marker was also induced on the expanded CD25− T cells (19, 20, and unpublished data). It should be noted that the quantitative PCR studies were performed on five separate expanded Treg populations (including both polyclonal and BDC2.5 TCR Tg Tregs) and the relative expression of the Treg-specific genes was highly reproducible. Finally, we examined the recently identified lineage/differentiation marker for Tregs, FoxP3 (Fig. 2, B and C). As noted by both real time PCR and Western blot analyses, the expanded Tregs expressed levels of FoxP3 similar to those observed in fresh Tregs and significantly higher than those in fresh or expanded CD25− T cells. The RNA expression (10-fold) and protein amounts (20-fold) were consistent with previous studies of fresh Tregs, although there was clearly some increase in FoxP3 in CD25− Teffs, suggesting that the culture conditions may induce some Tregs within the CD25− subset, or FoxP3 is expressed at a low level in activated Teffs.


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)

Phenotype of in vitro–expanded Tregs. (A) Expression of CD25 and CD62L on expanded Tregs and CD4+ CD62L+ CD25− cells was determined by flow cytometry on day 8 after the culture initiation. Results are representative of more than 20 independent experiments. (B) Levels of mRNA for the indicated genes in expanded NOD (filled symbols) or BDC2.5 TCR Tg T cells (open symbols) were determined by real time PCR analysis on day 10 after the initiation of the cultures. The relative expression ratio (Treg/TCD25−) for each pair of cultures was calculated from Ct values as described in Materials and Methods. The dashed line represents the ratio of 1 (i.e., identical level of gene expression in Treg and CD4+ CD62L+ CD25− cultures). (C) Western blot analysis of FoxP3 protein expression in fresh and expanded T cells. The level of tubulin expression was included as a loading control. Results are representative of three independent experiments. (D) Cytokine secretion by expanded BDC2.5 T cells 48 h after restimulation with antigenic peptide and splenic APC. Results are representative of two independent experiments.
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fig2: Phenotype of in vitro–expanded Tregs. (A) Expression of CD25 and CD62L on expanded Tregs and CD4+ CD62L+ CD25− cells was determined by flow cytometry on day 8 after the culture initiation. Results are representative of more than 20 independent experiments. (B) Levels of mRNA for the indicated genes in expanded NOD (filled symbols) or BDC2.5 TCR Tg T cells (open symbols) were determined by real time PCR analysis on day 10 after the initiation of the cultures. The relative expression ratio (Treg/TCD25−) for each pair of cultures was calculated from Ct values as described in Materials and Methods. The dashed line represents the ratio of 1 (i.e., identical level of gene expression in Treg and CD4+ CD62L+ CD25− cultures). (C) Western blot analysis of FoxP3 protein expression in fresh and expanded T cells. The level of tubulin expression was included as a loading control. Results are representative of three independent experiments. (D) Cytokine secretion by expanded BDC2.5 T cells 48 h after restimulation with antigenic peptide and splenic APC. Results are representative of two independent experiments.
Mentions: Next, we examined the phenotype of the expanded Tregs by flow cytometry, Western blot, and real time PCR. As can be seen in Fig. 2 A, the expanded Tregs maintained high levels of expression of CD25 as compared with expanded CD25− T cells, whereas the expression of CD62L remained high in both cell types. In addition, quantitative PCR showed that all of the Tregs expressed high levels of SOCS2, PD-1, and CTLA-4 as compared with similarly expanded CD25− T cells. Moreover, the recently identified markers neuropilin and TRAIL (20, 45) were also highly expressed on the expanded Tregs (Fig. 2 B). A high level of cell surface GITR expression was observed on the expanded Tregs. However, this previously identified Treg marker was also induced on the expanded CD25− T cells (19, 20, and unpublished data). It should be noted that the quantitative PCR studies were performed on five separate expanded Treg populations (including both polyclonal and BDC2.5 TCR Tg Tregs) and the relative expression of the Treg-specific genes was highly reproducible. Finally, we examined the recently identified lineage/differentiation marker for Tregs, FoxP3 (Fig. 2, B and C). As noted by both real time PCR and Western blot analyses, the expanded Tregs expressed levels of FoxP3 similar to those observed in fresh Tregs and significantly higher than those in fresh or expanded CD25− T cells. The RNA expression (10-fold) and protein amounts (20-fold) were consistent with previous studies of fresh Tregs, although there was clearly some increase in FoxP3 in CD25− Teffs, suggesting that the culture conditions may induce some Tregs within the CD25− subset, or FoxP3 is expressed at a low level in activated Teffs.

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