Expansion of regulatory T cells via IL-2/anti-IL-2 mAb complexes suppresses experimental myasthenia

Abstract

Human autoimmune diseases are often characterized by a relative deficiency in CD4(+)CD25(+) regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B-cell-mediated disease characterized by auto-Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL-2 and anti-IL-2 mAb (JES6-1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4(+)CD25(+)Foxp3(+) cells and peripheral conversion of CD4(+)CD25(-)Foxp3(-) cells. The expanded Treg potently suppressed autoreactive T- and B-cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL-2/anti-IL-2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.

Figure 1

Homeostasis of CD4⁺CD25^high Treg in AChR-primed mice treated with IL-2 complexes. Splenocytes from AChR-immunized B6 mice treated with isotype control IgG or IL-2 complexes were prepared on the indicated days after immunization, and stained with anti-CD4 and anti-CD25 mAb as described in the Materials and methods section. Dot plots were gated on lymphocytes. Results of frequencies of individual cell populations were pooled from three independent experiments (n=2 or 3 mice/group for each experiment). *p<0.05, **p<0.01, ANOVA. (A) CD4⁺ CD25^high Treg frequencies; (B) CD4⁺CD25^high Treg numbers; (C) kinetics of the CD4⁺CD25^high population during EAMG ; (D) Foxp3⁺ Treg frequencies among CD4⁺CD25^high cells; (E) Foxp3⁺ Treg numbers among CD4⁺CD25^high cells; and (F) kinetics of the Foxp3⁺ cell population of CD4⁺CD25^high cells during EAMG.

Figure 2

The profile of IL-2 complex-expanded CD4⁺CD25^high Treg in Foxp3^gfp mice. Lymphocytes from AChR-immunized Foxp3^gfp mouse draining lymph nodes treated with isotype control IgG, IL-2 alone, anti-IL-2 mAb alone or IL-2 complexes were prepared on day 35 after immunization and were stained with anti-CD4 and anti-CD25 mAb. Representative dot plots were gated on lymphocytes. Results of frequencies of individual cell populations were pooled from three independent experiments (n=2 or 3 mice/group for each experiment). *p<0.05, **p<0.01; ANOVA. (A) CD4⁺CD25⁺ Treg; (B) CD4⁺Foxp3^gfp+ cells; and (C) CD25⁺Foxp3^gfp+ cells.

Figure 3

IL-2 complexes expand Treg via proliferation of peripheral CD4⁺CD25⁺ T cells and conversion of extrathymic CD4⁺CD25⁻ T cells. On day 35 after immunization, thymocytes from the AChR-immunized Foxp3^gfp mice treated with isotype control IgG or IL-2 complexes were isolated from thymus by passing through a 40-µM cell strainer and stained with anti-CD4, and anti-CD25 Ab. Representative dot plots were gated on lymphocytes. Results of frequencies of individual cell populations were pooled from three independent experiments (n=2 or 3 mice/group for each experiment). (A) CD4⁺CD25⁺ Treg; (B) CD41Foxp3^gfp+ cells; and (C) CD25⁺Foxp3^gfp+ cells. Splenocytes from Foxp3^gfp mice were separated into CD4⁺CD25⁺Foxp3^gfp+ or CD4⁺CD25⁻Foxp3^gfp− T-cell subsets as described in the Materials and methods. CD4⁺CD25⁺Foxp3^gfp+ and CD4⁺ CD25⁻Foxp3^gfp− T cells (1×10⁶ cells/mL) were cultured with IL-2 complexes for 4 days, and then harvested for staining with anti-CD4 and anti- CD25 Ab. Representative dot plots were gated on lymphocytes. Frequencies of the indicated cell populations are shown from one of three individual experiments with similar results. (D) Proliferation of T cells labeled in vitro with BrdU. (E) Bar plots of Foxp3⁺ cell frequencies and numbers. **p<0.01, Student’s t-test. (F) Dot plots of the indicated T-cell frequencies.

Figure 4

IL-2 complexes expand Treg in thymectomized Foxp3^gfp mice. Foxp3^gfp mice were thymectomized and then immunized with AChR/CFA, treated with isotype control IgG or IL-2 complexes. On day 35 after immunization, lymphocytes were prepared from these Foxp3^gfp mouse draining lymph nodes and stained with anti-CD4 and anti-CD25 mAb. Representative dot plots were gated on lymphocytes. Results of frequencies of individual cell populations were pooled from two independent experiments (n=2 or 3 mice/group for each experiment). *p<0.05, **p<0.01; Student’s t-test. (A) CD4⁺CD25⁺ Treg; (B) CD4⁺ Foxp3^gfp+ cells; and (C) CD25⁺Foxp3^gfp+ cells. Data are summarized in (D).

Figure 5

CD4⁺CD25^high Treg expanded by IL-2 complexes display suppressor activity. Purified CD4⁺CD25⁺ Treg and purified responder CD41CD25⁻ T cells (1×10⁵) from AChR-primed B6 mice treated with isotype control IgG or IL-2 complexes were prepared on day 35 after immunization and cultured at the indicated ratios and stimulated with (A) AChR (10 µg/mL), (B) anti-CD3 (0.2 µg/mL, clone 145.2C11), or (C) Con A (1µg/mL), in the presence of irradiated (2000 rad) splenocytes (50×10³ cells/well) for 72h, and then pulsed with 1 µCi/well of [³H] thymidine for the last 18 h of the culture. Data are shown as mean [³H] thymidine incorporation in triplicate cultures. The data are representative of three separate experiments with similar outcomes. In control experiments, with responder cells only, levels of proliferation were similar to those with Treg:T effector cell ratios of 0.05:1 (data not shown). **p<0.05, **p<0.01; Student’s t-test.

Figure 6

Impact of IL-2 complexes on EAMG. Groups of B6 mice treated with isotype control IgG or IL-2 complexes were immunized with AChR and monitored for clinical scores of muscle weakness as described in the Materials and methods section. (A) Treatment started on the same day as AChR immunization; (B) treatment started on day 35 after immunization (the arrows indicate the start of injection). Results are pooled from three independent experiments (n=4–9 mice/group in each experiment) and are expressed as daily mean clinical scores. Bars indicate standard errors. (C) Groups of B6 mice (n=3–5) received CD4⁺ CD25⁺Foxp3⁺ cells from Foxp3^gfp mice treated with isotype control IgG or IL-2 complexes (the arrow indicates the day of cell transfer). This experiment was repeated one time with similar results. *p<0.05, **p<0.01; Mann–Whitney U test.

Figure 7

Effect of IL-2 complexes on AChR-specific T-cell proliferation and cytokine production. Splenocytes from unmanipulated B6 mice or AChR-immunized B6 mice treated with or without IL-2 complexes were harvested on day 35~40 post-immunization or on the indicated days and analyzed for proliferative responses to (A) AChR (0.1, 1, 10 and 100 µg/mL) and (B) Con A (5 µg/mL) by [³H] thymidine incorporation in vitro. The results are presented as cpm and are expressed as means ± SD (pooled from three independent experiments, n=2 or 3 mice/group for each experiment). Splenocytes from AChR-immunized B6 mice treated with or without IL-2 complexes were collected between weeks 5 and 6 p.i. and cultured in the presence of AChR (10 µg/mL) for 72 h. Intracellular cytokines were examined by flow cytometry. Dot plots were gated on lymphocytes as shown in representative results from three separate experiments (n=2 or 3 mice/group in each experiment). The bars present percentages of the indicated cytokine-producing cells and are expressed as means ± SD. (C) AChR-induced IFN-g and IL-17 production by CD4⁺ T cells. (D) AChR-induced IL-4 production by CD4⁺ T cells; each symbol represents one mouse. (E) Serum TGF-β profile. (F) Serum IL-6 profile. *p<0.05, **p<0.01; Student’s t-test.

Figure 8

Effect of IL-2 complexes on humoral and AChR-reactive B-cell responses. (A) Serum samples were collected at 6 wk (day 35~40) post-immunization from B6 mice treated with isotype control mAb (n=8) or IL-2 complexes (n=16). Serum levels of anti-AChR-specific total IgG, IgG1, IgG2a, IgG2b, IgG3 and IgM were determined by ELISA on AChR-coated plates as described in the Materials and methods section. Serum dilution for total IgG, IgG1, IgG3 and IgM was 1/400, for IgG2b 1/200, and for IgG2a 1/40. The pooled data are expressed as mean OD values± SE. (B) Serum anti-AChR Ab concentrations were measured by RIA and expressed as moles of toxin-binding sites bound per liter of serum (n=4/group). Splenocytes from AChR-immunized mice treated with isotype control IgG or IL-2 complexes were prepared on day 35~40 after immunization, and stained with surface mAb. Dot plots were gated on lymphocytes in three independent experiments (n=2 or 3 mice/group per experiment); each symbol represents one mouse. (C) CD19⁺ cell frequency; (D) IgM⁺IgD⁺ cell frequency among CD19⁺ cells; (E) CD138⁺ cell frequency among CD19⁺ cells. The values between groups were compared; *p<0.05, p<0.01; NS indicates not statistically significant; Student’s t-test.