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CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes.

Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM - J. Exp. Med. (2004)

Bottom Line: The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells.Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo.This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

ABSTRACT
In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet beta cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 10(5) polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.

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NOD DCs induce growth of CD25+ CD4+ T cells from NOD.BDC2.5 or NOD mice. (A) In vitro–derived NOD DCs were stained with antibodies specific for CD86 and MHC class II before (left) and after (right) magnetic bead enrichment of CD86+ cells. (B) CD25+ CD4+ or CD25− CD4+ T cells sorted from BDC2.5 TCR transgenic mice were cultured with CD86+ NOD DCs with and without 30 ng ml−1 BDC peptide and IL-2. In the same experiment, NOD spleen cells were used with IL-2, with and without BDC peptide (right). A 12-h [3H]thymidine pulse was given on day 3. (C) Same as B, but the dose of BDC peptide was 100 ng ml−1 and the fold increase in T cell numbers was monitored by counting on days 3, 5, and 7. (D) CD25+ or CD25− CD4+ T cells were isolated from NOD mice and cultured with NOD CD86+ DCs, with and without anti-CD3 and IL-2 as indicated. Proliferation was determined by [3H]thymidine incorporation on day 3. (E) As in D, but cells were counted on days 3, 5, and 7, and the fold increase in cell numbers was calculated. One result of at least three similar experiments is shown.
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fig1: NOD DCs induce growth of CD25+ CD4+ T cells from NOD.BDC2.5 or NOD mice. (A) In vitro–derived NOD DCs were stained with antibodies specific for CD86 and MHC class II before (left) and after (right) magnetic bead enrichment of CD86+ cells. (B) CD25+ CD4+ or CD25− CD4+ T cells sorted from BDC2.5 TCR transgenic mice were cultured with CD86+ NOD DCs with and without 30 ng ml−1 BDC peptide and IL-2. In the same experiment, NOD spleen cells were used with IL-2, with and without BDC peptide (right). A 12-h [3H]thymidine pulse was given on day 3. (C) Same as B, but the dose of BDC peptide was 100 ng ml−1 and the fold increase in T cell numbers was monitored by counting on days 3, 5, and 7. (D) CD25+ or CD25− CD4+ T cells were isolated from NOD mice and cultured with NOD CD86+ DCs, with and without anti-CD3 and IL-2 as indicated. Proliferation was determined by [3H]thymidine incorporation on day 3. (E) As in D, but cells were counted on days 3, 5, and 7, and the fold increase in cell numbers was calculated. One result of at least three similar experiments is shown.

Mentions: As previously described (26), spleen and lymph node cell suspensions were enriched for CD4+ cells by panning and were sorted on a FACS Vantage™ (BD Biosciences) into CD25+ CD4+ and CD25− CD4+ populations (>95 and >97% pure). 104 T cells from BDC2.5 or NOD mice were cultured for 3 d with the indicated number of DCs and a mimetope peptide (termed 1040–55; 30–100 ng ml−1) having the sequence RVRPLWVRME (38), or with purified anti-CD3 antibody (0.3–1 μg ml−1). RHu IL-2 (Chiron Corp.) was added where indicated in Fig. 1 and with cultured CD25+ cells in Fig. 2 at 100 U ml−1. All CD25+ CD4+ T cell expansions for in vivo injection were performed with IL-2 in the cultures. To assess suppression by CD25+ CD4+ T cells, 5 × 104 whole NOD spleen cells irradiated with 15 Gy were used to stimulate mixtures of 104 CD25− CD4+ and the indicated number of CD25+ CD4+ T cells from BDC2.5 or NOD mice. If DC-expanded CD25+ CD4+ T cells were used, CD11c+ cells were removed using magnetic microbeads (Miltenyi Biotec) after harvesting the cells on days 5–7. [3H]thymidine uptake (1 μCi/well; PerkinElmer) by proliferating lymphocytes was measured at 60–72 h. For in vivo proliferation, CD25+ CD4+ and CD25− CD4+ cells were purified by flow cytometry and labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes). 3.3 × 105 T cells were injected i.v. into NOD recipients. 1 d later, 2 × 105 BDC peptide–pulsed or –unpulsed, LPS-matured bone marrow DCs were injected s.c. in each paw. 3 d after DCs were injected, lymph nodes were collected and cells were stained with CD4 and BDC2.5 clonotype antibody, and the level of CFSE staining was determined by flow cytometry.


CD25+ CD4+ T cells, expanded with dendritic cells presenting a single autoantigenic peptide, suppress autoimmune diabetes.

Tarbell KV, Yamazaki S, Olson K, Toy P, Steinman RM - J. Exp. Med. (2004)

NOD DCs induce growth of CD25+ CD4+ T cells from NOD.BDC2.5 or NOD mice. (A) In vitro–derived NOD DCs were stained with antibodies specific for CD86 and MHC class II before (left) and after (right) magnetic bead enrichment of CD86+ cells. (B) CD25+ CD4+ or CD25− CD4+ T cells sorted from BDC2.5 TCR transgenic mice were cultured with CD86+ NOD DCs with and without 30 ng ml−1 BDC peptide and IL-2. In the same experiment, NOD spleen cells were used with IL-2, with and without BDC peptide (right). A 12-h [3H]thymidine pulse was given on day 3. (C) Same as B, but the dose of BDC peptide was 100 ng ml−1 and the fold increase in T cell numbers was monitored by counting on days 3, 5, and 7. (D) CD25+ or CD25− CD4+ T cells were isolated from NOD mice and cultured with NOD CD86+ DCs, with and without anti-CD3 and IL-2 as indicated. Proliferation was determined by [3H]thymidine incorporation on day 3. (E) As in D, but cells were counted on days 3, 5, and 7, and the fold increase in cell numbers was calculated. One result of at least three similar experiments is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211787&req=5

fig1: NOD DCs induce growth of CD25+ CD4+ T cells from NOD.BDC2.5 or NOD mice. (A) In vitro–derived NOD DCs were stained with antibodies specific for CD86 and MHC class II before (left) and after (right) magnetic bead enrichment of CD86+ cells. (B) CD25+ CD4+ or CD25− CD4+ T cells sorted from BDC2.5 TCR transgenic mice were cultured with CD86+ NOD DCs with and without 30 ng ml−1 BDC peptide and IL-2. In the same experiment, NOD spleen cells were used with IL-2, with and without BDC peptide (right). A 12-h [3H]thymidine pulse was given on day 3. (C) Same as B, but the dose of BDC peptide was 100 ng ml−1 and the fold increase in T cell numbers was monitored by counting on days 3, 5, and 7. (D) CD25+ or CD25− CD4+ T cells were isolated from NOD mice and cultured with NOD CD86+ DCs, with and without anti-CD3 and IL-2 as indicated. Proliferation was determined by [3H]thymidine incorporation on day 3. (E) As in D, but cells were counted on days 3, 5, and 7, and the fold increase in cell numbers was calculated. One result of at least three similar experiments is shown.
Mentions: As previously described (26), spleen and lymph node cell suspensions were enriched for CD4+ cells by panning and were sorted on a FACS Vantage™ (BD Biosciences) into CD25+ CD4+ and CD25− CD4+ populations (>95 and >97% pure). 104 T cells from BDC2.5 or NOD mice were cultured for 3 d with the indicated number of DCs and a mimetope peptide (termed 1040–55; 30–100 ng ml−1) having the sequence RVRPLWVRME (38), or with purified anti-CD3 antibody (0.3–1 μg ml−1). RHu IL-2 (Chiron Corp.) was added where indicated in Fig. 1 and with cultured CD25+ cells in Fig. 2 at 100 U ml−1. All CD25+ CD4+ T cell expansions for in vivo injection were performed with IL-2 in the cultures. To assess suppression by CD25+ CD4+ T cells, 5 × 104 whole NOD spleen cells irradiated with 15 Gy were used to stimulate mixtures of 104 CD25− CD4+ and the indicated number of CD25+ CD4+ T cells from BDC2.5 or NOD mice. If DC-expanded CD25+ CD4+ T cells were used, CD11c+ cells were removed using magnetic microbeads (Miltenyi Biotec) after harvesting the cells on days 5–7. [3H]thymidine uptake (1 μCi/well; PerkinElmer) by proliferating lymphocytes was measured at 60–72 h. For in vivo proliferation, CD25+ CD4+ and CD25− CD4+ cells were purified by flow cytometry and labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes). 3.3 × 105 T cells were injected i.v. into NOD recipients. 1 d later, 2 × 105 BDC peptide–pulsed or –unpulsed, LPS-matured bone marrow DCs were injected s.c. in each paw. 3 d after DCs were injected, lymph nodes were collected and cells were stained with CD4 and BDC2.5 clonotype antibody, and the level of CFSE staining was determined by flow cytometry.

Bottom Line: The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells.Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo.This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

ABSTRACT
In the nonobese diabetic (NOD) mouse model of type 1 diabetes, the immune system recognizes many autoantigens expressed in pancreatic islet beta cells. To silence autoimmunity, we used dendritic cells (DCs) from NOD mice to expand CD25+ CD4+ suppressor T cells from BDC2.5 mice, which are specific for a single islet autoantigen. The expanded T cells were more suppressive in vitro than their freshly isolated counterparts, indicating that DCs from autoimmune mice can increase the number and function of antigen-specific, CD25+ CD4+ regulatory T cells. Importantly, only 5,000 expanded CD25+ CD4+ BDC2.5 T cells could block autoimmunity caused by diabetogenic T cells in NOD mice, whereas 10(5) polyclonal, CD25+ CD4+ T cells from NOD mice were inactive. When islets were examined in treated mice, insulitis development was blocked at early (3 wk) but not later (11 wk) time points. The expanded CD25+ CD4+ BDC2.5 T cells were effective even if administered 14 d after the diabetogenic T cells. Our data indicate that DCs can generate CD25+ CD4+ T cells that suppress autoimmune disease in vivo. This might be harnessed as a new avenue for immunotherapy, especially because CD25+ CD4+ regulatory cells responsive to a single autoantigen can inhibit diabetes mediated by reactivity to multiple antigens.

Show MeSH
Related in: MedlinePlus