Chemokine responsiveness of CD4+ CD25+ regulatory and CD4+ CD25- T cells from atopic and nonatopic donors

Abstract

Background: Allergic inflammation is associated with Th2-type T cells, which can be suppressed by CD4+ CD25+ regulatory T cells (Tregs). Both express chemokine receptors (CCR) 4 and CCR8, but the dynamics of expression and effect of atopic status are unknown.

Objective: To examine the expression of chemokine receptors by CD4+ CD25+ and CD4+ CD25- T cells from atopic and nonatopic donors, and document response to allergen stimulation in vitro.

Methods: Chemokine receptor expression was examined by flow cytometry and quantitative PCR of CD4+ CD25hi and CD4+ CD25- T cells from atopics and nonatopics. Responsiveness to chemokines was by actin polymerization. Dynamics of chemokine receptor expression in 6-day allergen-stimulated cultures was analysed by carboxyfluoroscein succinimidyl ester labelling.

Results: CD4+ CD25hi Tregs preferentially expressed CCR3, CCR4, CCR5, CCR6 and CCR8. CD4+ CD25hi Tregs responded to the chemokine ligands for CCR4, CCR6 and CCR8 (CCL17, 22, 20 and 1 respectively), with no differences between atopic and nonatopic donors. Over 6-day allergen stimulation, CD4+ CD25+ T-cells downregulated CCR4 and upregulated CCR7, in contrast to CD4+ CD25- effector cells, which downregulated CCR7 and upregulated CCR4.

Conclusions: CCR4, CCR6 and CCR8 have potential roles in localization of both CD4+ CD25+ regulatory and CD4+ CD25- effector T cells to sites of allergic inflammation. Upregulation of CCR7 and downregulation of CCR4 upon allergen stimulation of Tregs may allow their recirculation from sites of inflammation, in contrast to retention of effector T cells.

Figure 1

Differential chemokine receptor expression on CD4+ CD25hi and CD4+ CD25− T cells. Chemokine receptor expression by CD4+ CD25hi, FoxP3+, CD127lo and CD4+ CD25− human peripheral blood T cells. Gated CD4+ CD25hi T cells were confirmed to be FoxP3+ and CD127−, whilst CD4+ CD25− T cells were FoxP3− and CD127+ (A–C). These gates were then used to determine chemokine receptor expression by CD4+ CD25hi (black bars) and CD4+ CD25− T cells (white bars) using flow cytometry. Results shown are from n = 7 donors. *P < 0.05. Chemokine receptor expression is shown as percent positive for CD4⁺ CD25^hi and CD4⁺ CD25^lo T cells (D).

Figure 2

Chemokine responsiveness of CD4+ CD25+, CD4+ CD25− and CD4+ CD25high T cells. The percentage increase in actin polymerization over baseline was measured for chemokine-stimulated CD4+ T cells at 15-s intervals up to 1 min. The chemokine responsiveness of CD4+ CD25− (solid line), CD4+ CD25+ (dashed line) and CD4+ CD25high T cells (dotted line) to the chemokines CCL17 (A), CCL22 (B) and CCL20 (C) is shown for n = 13 donors.

Figure 3

CD4+ CD25+ T cells express CCR8 mRNA and respond to its ligand CCL1. mRNA expression of CCR8 (A), FoxP3 (B), GATA3 (C) and Tbet (D) was measured in freshly isolated CD4+ CD25+ and CD4+ CD25) T cells and in anti-CD3 stimulated T cells from n = 6 donors. mRNA expression in Th1 and Th2 polarized cell lines is also shown. The chemokine responsiveness of CD4+ CD25-, CD4+ CD25+ and CD4+ CD25high T cells to stimulation with CCL1 in the actin polymerization assay is shown for n = 13 donors (E). This response was blocked in the presence of MC148 (10 μg/ml) (F). *P < 0.05; St, anti-CD3 stimulated cells.

Figure 4

Modulation of CD4+ CD25+ and CD4+ CD25− T cell chemokine receptor expression during allergen-driven cell culture. Graphs A–I showing expression of CCR4 (A–C), CCR7 (D–F) and CXCR4 (G–I) on CD4+ CD25+ (A,D,G), nonproliferating CD4+ CD25− (B,E,H) and proliferating CD4+ CD25− T cells (C,F,I) on days 0, 2, 4 and 6 of allergen-stimulated cell culture. Due to the very low numbers of proliferating cells in culture on days 2 and 4, only the expression on days 0 and 6 is shown for proliferating CD4+ CD25− T cells (C,F,I). Results are shown for n = 8 different donors. *P < 0.05; **P < 0.01.