Eosinophils affect functions of in vitro-activated human CD3-CD4+ T cells

Abstract

Background: The recent development of eosinophil-targeting agents has raised enthusiasm for management of patients with hypereosinophilic syndromes. Roughly half of anti-IL-5-treated patients with corticosteroid-responsive lymphocytic (L-HES) and idiopathic disease variants can be tapered off corticosteroids. Potential consequences of corticosteroid-withdrawal on clonal expansion of pre-malignant CD3⁻CD4⁺ T-cells associated with L-HES are a subject of concern. Indeed, corticosteroid treatment inhibits T-cell activation and may lower blood CD3⁻CD4⁺ cell counts. On the other hand, previous studies have shown that eosinophils support CD4 T-cell activation, suggesting that targeted eosinophil depletion may negatively regulate these cells.

Objectives: Effects of eosinophils on CD4 T-cell activation in vitro were investigated as an indirect means of exploring whether treatment-induced eosinophil depletion may affect pathogenic T-cells driving L-HES.

Methods: Helper (CD4) T-cells and CD3⁻CD4⁺ cells from healthy controls and L-HES patients, respectively, were cultured in vitro in presence of anti-CD3/CD28 or dendritic cells. Effects of eosinophils on T-cell proliferation and cytokine production were investigated.

Results: Eosinophils enhanced CD3-driven proliferation of CD4 T-cells from healthy subjects in vitro, while inhibiting TCR-independent proliferation and IL-5 production by CD3⁻CD4⁺ T-cells.

Conclusions: While this study confirms previous work showing that eosinophils support activation of normal helper T-cells, our in vitro findings with CD3⁻CD4⁺ T-cells suggest that eosinophil-depletion may favor activation and expansion of this pathogenic lymphocyte subset. With the ongoing development of eosinophil-targeted therapy for various eosinophilic conditions, the indirect consequences of treatment on the underlying immune mechanisms of disease should be investigated in detail in the setting of translational research programs.

Figure 1

L-HES-associated CD3^-CD4⁺ T-cells do not express IL-5Rα. CD3^-CD4⁺ T-cells from patient L-HES₁ stain negatively for CD125w (grey histogram), similar to normal CD4 T-cells (white histogram) (1a), whereas basophils stain positively for this receptor (grey histogram, 1b), as expected. Identical results were obtained for CD3^-CD4⁺ T-cells from patient L-HES₂ and a third patient with L-HES.

Figure 2

Eosinophils enhance proliferation and CD25 expression of in vitro activated CD4 T-cells from healthy controls. Purified CD4⁺ T-cells from healthy subjects were activated with coated anti-CD3 and soluble anti-CD28 (1 μg/ml) antibodies for 48 hours, in absence and in presence of autologous IgA/anti-IgA activated eosinophils. Cells were then cultured for an additional 18 hours with H³-thymidine to assess proliferation (n=8) (2a), washed and stained with fluoro-conjugated antibodies for assessment of membrane CD25 expression by flow cytometry (n=6) (2b), or underwent brief re-stimulation with PMA and A23187 to assess intracellular cytokine expression (n=10) (2c).

Figure 3

Eosinophils inhibit dendritic cell-induced activation of CD3^-CD4⁺ T cells in vitro. Purified CD3^-CD4⁺ T cells were cultured in presence of LPS-matured dendritic cells from healthy subjects for 5 days, in absence or in presence of IgA/anti-IgA-activated eosinophils. Proliferation was assessed on the basis of H³-thymidine incorporation (3a), IL-5 was measured in culture supernatants by ELISA (3b), and expression of activation markers CD25 and HLA-DR was assessed by flow cytometry (3c). Histograms represent mean proliferation (cpm) (3a, n=8) and IL-5 concentrations (pg/ml) (3b, n=6), and bars show the standard error of the mean for each condition. For cytometry studies (3c), histograms show CD25 and HLA-DR expression on CD3^-CD4⁺ T cells cultured alone (continuous fine line), with dendritic cells (dashed bold line), and with both dendritic cells and eosinophils (continuous bold line); results are representative of 3 independent experiments.