Adaptive TGF-beta-dependent regulatory T cells control autoimmune diabetes and are a privileged target of anti-CD3 antibody treatment

Abstract

Previous results have shown that CD4(+)CD25(+) regulatory T cells (Tregs) control autoimmunity in a spontaneous model of type 1 diabetes, the nonobese diabetic (NOD) mouse. Moreover, anti-CD3 reverses diabetes in this setting by promoting Tregs that function in a TGF-beta-dependent manner. This finding contrasts with a large body of work suggesting that CD4(+)CD25(high) Tregs act in a cytokine-independent manner, thus suggesting that another type of Treg is operational in this setting. We sought to determine the basis of suppression both in untreated NOD mice and in those treated with anti-CD3. Our present results show that a subset of foxP3(+) cells present within a CD4(+)CD25(low) lymphocyte subset suppresses T cell immunity in spontaneously diabetic NOD mice in a TGF-beta-dependent manner, a functional property typical of "adaptive" regulatory T cells. This distinct Treg subset is evident in NOD, but not normal, mice, suggesting that the NOD mice may generate these adaptive Tregs in an attempt to regulate ongoing autoimmunity. Importantly, in two distinct in vivo models, these TGF-beta-dependent adaptive CD4(+)CD25(low) T cells can be induced from peripheral CD4(+)CD25(-) T lymphocytes by anti-CD3 immunotherapy which correlates with the restoration of self-tolerance.

Fig. 1.

Regulatory capacities of thymic CD4⁺CD25⁺ T cells. Diabetes was monitored in NOD–SCID recipients injected with diabetogenic cells alone (spleen cells from diabetic NOD mice, 5 × 10⁶, Diab) or with 1 × 10⁶ CD4⁺CD25⁺ thymocytes (A) or CD4⁺CD25⁺ splenocytes (B) isolated from 6-week-old NOD mice. A significant protection was observed (P < 0.0001) with both regulatory populations. Administration of anti-TGF-β antibody abrogated diabetes protection afforded only by CD4⁺CD25⁺ T cells from the spleen but not from the thymus (P < 0.016). (C) Suppression of CD4⁺CD25⁻ T cell proliferation by CD4⁺CD25⁺ thymocytes (n = 10). Antibodies to IL-10 receptor (50 μg/ml) or TGF-β (10 or 50 μg/ml) were added in the culture. Data were expressed as the percent inhibition.

Fig. 2.

In vivo suppressive capacities of CD4⁺CD25^high and CD4⁺CD25^low T cells from NOD mice. (A) Splenic CD4⁺ T cells from 6-week-old NOD mice were labeled with CD4 and CD25 antibodies and sorted into CD4⁺CD25^high (1.9 ± 0.1%) and CD4⁺CD25^low (2.3 ± 0.1%) T cell populations by FACS. (B) NOD–SCID mice were adoptively transferred with diabetogenic cells (1 × 10⁶, Diab) alone or in combination with CD25^high or CD25^low T cells (2.5 × 10⁵ per recipient) recovered from 6-week-old NOD mice. Both populations significantly delay diabetes onset (P < 0.002).

Fig. 3.

In vitro suppressive activity of CD4⁺CD25^high and CD4⁺CD25^low T cells from NOD mice. (A) Expression of foxP3 by FACS-sorted CD4⁺CD25⁻, CD4⁺CD25^high, and CD4⁺CD25^low T cells recovered from the spleen of prediabetic NOD mice. The numbers in each histogram represent the percentage of positively stained cells. (B) CD4⁺CD25⁻ T cells from NOD mice were incubated with CD25^high or CD25^low T cells and stimulated with antigen-presenting cells (APCs) and anti-CD3 antibody for 72 h with or without antibodies to TGF-β or IL10 receptor (50 μg/ml) (n = 6). Data were expressed as the percent inhibition of proliferation. (C) The proliferation of carboxyfluorescein-diacetate-succinimidyl-ester-labeled CD4⁺CD25⁻ T cells was analyzed by FACS after being incubated for 4 days with CD25^low or CD25^high T cells separated or not separated by a transwell membrane.

Fig. 4.

IL-2 production. CD4⁺CD25^high, CD4⁺CD25^low, and CD4⁺CD25⁻ T cells T cells from 6-week-old NOD (A), BALB/c (B), or C57BL/6 (C) mice were stimulated with PMA/ionomycin for 4 h, fixed, and stained with anti-IL-2 antibodies. The numbers in each histogram represent the percentage of positively stained cells.

Fig. 5.

In vitro suppressive activity of CD4⁺CD25^high and CD4⁺CD25^low T cells from nonautoimmune-prone mice. (A) Expression of foxP3 by FACS-sorted CD4⁺CD25⁻, CD4⁺CD25^high, and CD4⁺CD25^low T cells recovered from the spleen of BALB/c or C57BL/6 mice. (B) Suppression of CD4⁺CD25⁻ T cell proliferation to CD3 antibody by CD4⁺CD25^high or CD4⁺CD25^low T cells issued from BALB/c or C57BL/6 mice (n = 5). Each coculture was performed with or without anti-TGF-β or anti-IL10R antibodies (50 μg/ml). Data were expressed as the percent inhibition.

Fig. 6.

Immunoregulatory cytokine production pattern by CD4⁺CD25^high and CD4⁺CD25^low T cells. CD4⁺CD25^high and CD4⁺CD25^low T cells recovered from the spleen of NOD or C57BL/6 mice were stimulated with coated anti-CD3 and soluble anti-CD28 antibodies for 48 or 72 h. Supernatants were harvested and the amount of IL-4, IL-10, and TGF-β secreted by each population was determined ELISA.

Fig. 7.

Treatment of NOD CD28^−/− mice with anti-CD3 antibody restores the number and the suppressive capacities of the CD4⁺CD25⁺ T cells. Diabetic NOD CD28^−/− mice treated for 5 d with CD3-specific F(ab′)₂ fragments enter long-term remission. (A) Expression of CD25 in the CD4⁺ T cell subset recovered from the spleen of NOD CD28^−/− mice 3 weeks after the end of the treatment. (B) Analysis of the suppressive capacities of the CD4⁺CD25⁺ T cells from treated NOD CD28^−/− mice on the anti-CD3-induced proliferation of CD4⁺CD25⁻ T cells in the presence or absence of anti-TGF-β antibody. (C) Intracellular staining of foxP3 protein expressed by various T cell subsets isolated from the spleen or the thymus of treated NOD CD28^−/− mice.

Fig. 8.

Anti-CD3 antibody treatment preferentially induces CD4⁺CD25^low T cells. (A) NOD–SCID mice were injected with CD25⁻ T cells (2 × 10⁶ per recipient), became diabetic and were treated with CD3-specific F(ab′)₂ fragments. Long-term remission was induced in the majority of the mice. A small fraction of mice entered short-lasting remission (2 weeks). (B) Proportion of CD25^low T cells within the CD4⁺ T cell population recovered from recipient NOD–SCID mice treated with anti-CD3 antibodies and that entered long-lasting remission (>6 weeks, black bars) or short-lasting remission (dotted bars). (C and D) TGF-β and IL-10 production by CD4⁺ T cells were measured in anti-CD3 antibody-treated recipient mice.