Patients treated with high-dose intravenous immunoglobulin show selective activation of regulatory T cells

Abstract

Intravenous immunoglobulin (IVIg) is used to treat autoimmune and systemic inflammatory diseases caused by derailment of humoral and cellular immunity. In this study we investigated whether IVIg treatment can modulate regulatory T cells (Tregs ) in humans in vivo. Blood was collected from IVIg-treated patients with immunodeficiency or autoimmune disease who were treated with low-dose (n = 12) or high-dose (n = 15) IVIg before, immediately after and at 7 days after treatment. Percentages and activation status of circulating CD4(+) CD25(+) forkhead box protein 3 (FoxP3(+)) Tregs and of conventional CD4(+) FoxP3(-) T-helper cells (Tconv) were measured. The suppressive capacity of Tregs purified from blood collected at the time-points indicated was determined in an ex-vivo assay. High-dose, but not low-dose, IVIg treatment enhanced the activation status of circulating Tregs , as shown by increased FoxP3 and human leucocyte antigen D-related (HLA-DR) expression, while numbers of circulating Tregs remained unchanged. The enhanced activation was sustained for at least 7 days after infusion, and the suppressive capacity of purified Tregs was increased from 41 to 70% at day 7 after IVIg treatment. The activation status of Tconv was not affected by IVIg. We conclude that high-dose IVIg treatment activates Tregs selectively and enhances their suppressive function in humans in vivo. This effect may be one of the mechanisms by which IVIg restores imbalanced immune homeostasis in patients with autoimmune and systemic inflammatory disorders.

Keywords: IVIg; human studies; intravenous immunoglobulin; regulatory T cells.

Fig. 1

Intravenous immunoglobulin (IVIg) treatment selectively activates circulating regulatory T cells (T_regs), but not conventional T helper cells (T_conv). (a) Percentages of CD3⁺CD4⁺ and CD3⁺CD8⁺ in peripheral blood mononuclear cells (PBMCs) did not alter after IVIg treatment. (b) Density plot shows the gating strategy of T_regs and T_conv after gating on CD4⁺ cells. Histograms show intracellular forkhead box protein 3 (FoxP3) expression in T_regs and surface expression of human leucocyte antigen D-related (HLA-DR) on T_regs and T_conv obtained before, immediately after and at day 7 after IVIg treatment of a representative patient. (c) Baseline percentages of CD25⁺FoxP3⁺ T_regs within circulating CD4⁺ T cells, (d) mean fluorescence intensity (MFI) of FoxP3 in T_regs and (e) percentages of T_regs expressing HLA-DR from healthy blood donors (n = 10) and patients treated with low-dose IVIg (n = 12) or high-dose IVIg (n = 15). (f) Percentages of CD25⁺FoxP3⁺ T_regs within the CD4⁺ population before, immediately after and 7 days after IVIg treatment in patients who received low-dose (n = 12) or high-dose IVIg (n = 15). (g) MFI of FoxP3 in T_regs, (h) percentages of T_regs and (i) percentages of T_conv expressing HLA-DR before, immediately after and 7 days after IVIg treatment in patients who received low-dose (n = 12) or high-dose IVIg treatment (n = 15). Horizontal lines represent median. *P < 0·05.

Fig. 2

Enhanced suppressive activity of regulatory T cells (T_regs) purified from blood after intravenous immunoglobulin (IVIg) treatment. (a) Regulatory T cells (T_regs) were obtained by flow cytometric sorting on 7-aminoactinomycin D (7-AAD)^–CD3⁺CD4⁺CD127^–CD25⁺ cells (T_regs) from peripheral blood mononuclear cells (PBMC) collected from seven high-dose IVIg-treated patients before treatment, immediately after treatment and at day 7 after treatment, and T responder cells (T_resp) were obtained by sorting both 7-AAD^–CD3⁺CD4^– (T_resp I) and 7-AAD^–CD3⁺CD4⁺CD127⁺CD25^– (T_resp II) cells from autologous PBMCs collected before IVIg-treatment. (b) T_regs purified from the patients before, after and at day 7 after treatment were co-cultured with T_resp (5 × 10⁴) obtained before treatment that were stimulated with 1 μg/ml phytohaemagglutinin (PHA) in the presence of 40 Gy-irradiated autologous PBMCs (5 × 10⁴) at a T_reg : T_resp ratio of 1:4 in culture medium in round-bottomed 96-well plates. After 5 days, supernatants were collected and the cumulative interferon (IFN)-γ production by T_resp was quantified by enzyme-linked immunosorbent assay. Each condition was tested at least in triplicate from which means were calculated. From each patient, these means were used to calculate the percentage of suppression using the formula: (IFN-γ level_Tresp–IFN-γ level_Tresp–Treg)/IFN-γ level_Tresp × 100%. Bar graphs represent mean ± standard error of the mean. **P < 0·01, compared to T_resp alone.