Pathological Role of Regulatory T Cells in the Initiation and Maintenance of Eczema Herpeticum Lesions

Expansion of skin-homing Foxp3⁺CD25⁺CTLA-4⁺CD127 ^dim/−CD39⁺HELIOS⁺ Tregs during the acute stage of ADEH. (A) Representative flow cytometry dot plots showing the expression of Foxp3 versus CD25, Foxp3 versus CLA, and Foxp3 versus CCR4 in CD4⁺ T cells from ADEH, ADEH⁻, and healthy controls. Numbers in each quadrant indicate the frequency of each fraction. The mean frequency of Foxp3⁺CD25⁺, Foxp3⁺CLA⁺, and Foxp3⁺CCR4⁺ Tregs in CD4⁺ T cells in the patients and healthy controls is shown (right panels). ADEH acute stage (n = 19), ADEH resolution stage (RESO; n = 23), ADEH⁻ (n = 16), and healthy controls (n = 34). Data are mean ± SEM. (B) Phenotypic analysis of CD4⁺Foxp3⁺ Tregs obtained from the patients and healthy controls. Representative flow cytometry dot plots showing the expression of Foxp3 versus CTLA-4, Foxp3 versus CD127, Foxp3 versus CD39, Foxp3 versus HELIOS, and Foxp3 versus Ki67 in CD4⁺ T cells. The mean frequency of Foxp3⁺CTLA-4⁺, Foxp3⁺CD127^dim/−, Foxp3⁺CD39⁺, Foxp3⁺HELIOS⁺, and Foxp3⁺Ki67⁺ Tregs in CD4⁺ T cells from the patients and healthy controls is shown (right panels). (C) CD4⁺Foxp3⁺ cells can be divided into three functionally distinct subpopulations defined by Foxp3 and CD45RA expression: CD45RA⁺Foxp3⁺ rTreg, CD45RA⁻Foxp3⁺⁺ iTregs, and CD45RA⁻Foxp3⁺ non-Tregs. (D) Increased CD4⁺CD45RA⁻Foxp3⁺⁺ iTregs are seen during the acute stage of ADEH but not during the resolution stage or in ADEH⁻ patients. Representative flow cytometry dot plots showing the expression of CD45RA versus Foxp3 in CD4⁺ T cells. Fraction II is indicated by red boxes. The mean frequency of each fraction in CD4⁺ T cells from the patients and healthy controls is shown (lower panels). In (B) and (D): ADEH (acute stage, n = 9; resolution stage [RESO], n = 12), ADEH⁻ (n = 6), and healthy controls (n = 8). The p values were determined using the Student t test (versus healthy controls). Fr., Fraction.

A sequential analysis of Treg frequency, serum HSV IgG level, and clinical course in a single AD patient with EH induced by a primary infection with HSV. (A) Flow cytometry dot plots showing the expression of Foxp3 versus CD25 in CD4⁺ T cells. (B) Clinical course of symptoms in relation to treatment, Treg frequency, and serum HSV IgG titers.

Functional analysis of Tregs at the different disease stages of ADEH, ADEH⁻, and healthy controls. Highly purified CD4⁺CD25⁺⁺ Treg populations from patients with EH at either the acute or resolution stage, ADEH⁻, or healthy controls were cocultured at different ratios with highly purified CD4⁺CD25⁻ effector T cell populations from the same stage or from healthy controls in the presence of mitomycin C–treated allogeneic APCs and anti-CD3 and anti-CD28 Abs. Proliferation was assessed by a [³H]thymidine-incorporation assay. The results are expressed as the percentage proliferation of CD4⁺CD25⁻ effector T cells in the absence of CD4⁺CD25⁺⁺ Tregs. Mean (n = 3) and SEM are shown.

Intracellular expression of IFN-γ, TNF-α, IL-17, and IL-4 by CD8⁺ T, CD4⁺ T, CD56⁺ NK, and TCR-γ/δ⁺ cells from ADEH patients, ADEH⁻ patients, and healthy controls. (A) Representative experiment showing intracellular IFN-γ versus TNF-α dot plots in CD8⁺ T cells from ADEH and ADEH⁻ patients and healthy controls. (B) Mean percentage of IFN-γ⁺, TNF-α⁺, IL-17⁺, or IL-4⁺ cells ± SEM in each subset. ADEH (acute stage, n = 9–17; resolution [RESO] stage, n = 9–21), ADEH⁻ (n = 7), and healthy controls (n = 14–35). *p < 0.001, ^†p < 0.01, ^‡p < 0.05 versus healthy controls, Student t test.

Restoration of IFN-γ production by HSV-1–specific CD8⁺ and CD4⁺ T cells obtained during the acute stage of disease from ADEH patients by Treg depletion. (A) PBMCs were obtained from ADEH patients, ADEH⁻ patients, and healthy controls, and CD4⁺CD25⁺CD127^dim/− Tregs were depleted from the PBMCs (Treg⁻) as described in Materials and Methods. PBMCs and Treg⁻, respectively, were stimulated with 25 μg/ml HSV-1gD protein or medium for 24 h with CD28/CD49d Abs. After 24 h of culture, brefeldin A was added, and culture continued for an additional 4 h. Intracellular cytokine was detected by FACS. The results are expressed as the average (± SEM) percentage of IFN-γ⁺ cells in CD8⁺ and CD4⁺ T cells from PBMCs and Treg-depleted PBMCs, respectively. ADEH (n = 16), ADEH⁻ (n = 15), and healthy controls (n = 16). (B) Restoration of IFN-γ production from CD8⁺ T cells by anti–IL-10 Ab. Representative flow cytometry dot plots showing the expression of granzyme B (GraB) versus IFN-γ in CD8⁺ T cells from the acute stage of ADEH. (C) Average (± SEM) percentage of GraB⁺IFN-γ⁺ cells in CD8⁺ T cells from PBMCs (n = 8). The p values were determined using the Student t test.

Expansion of CD14^dimCD16⁺ pMOs with the potent ability to produce IL-10 upon TLR-2 ligand stimulation in ADEH patients at onset of EH. (A) PBMCs were stained by anti-CD14 Ab and anti-CD16 Ab. The result of flow cytometric analysis of cell surface expression of CD14 and CD16 is shown. Monocytes can be classified into distinct populations based on the CD14 and CD16 expression, CD14^dimCD16⁺ pMOs and CD14⁺CD16^– cMOs in healthy controls. (B) Representative dot plots showing these subsets in ADEH patients, ADEH⁻ patients, and healthy controls. (C) Representative graph showing the expression of FcεRI on CD14^dimCD16⁺ pMOs in healthy controls. The vast majority of CD14^dimCD16⁺ pMOs are negative for FcεRI. (D) The percentages of monocytes and pMOs in PBMCs from ADEH patients, ADEH⁻ patients, and healthy controls. (E) Mean percentage of TNF-α⁺, IL-1β⁺, IL-6⁺, or IL-10⁺ cells ± SEM in pMOs. ADEH (acute stage, n = 8–19; resolution [RESO] stage, n = 7–15), ADEH⁻ (n = 5–6), and healthy controls (n = 8–15). *p < 0.001, ^†p < 0.01, ^‡p < 0.05 versus healthy controls, Student t test.

Expansion of iTregs by pMOs from the acute stage of disease in ADEH patients. Purified CD14^dim monocytes from ADEH patients at the acute stage of disease or healthy controls were cocultured with allogeneic CD3⁺ T cells from a healthy volunteer in the presence of anti-CD3 Ab + CD28 Ab for 7 d. (A) CD45RA⁻Foxp3⁺⁺ activated/iTregs (fraction II) were analyzed. Representative flow cytometry dot plots showing the expansion of iTregs induced by pMOs from ADEH patients at onset of disease and from healthy control. Numbers indicate the frequency of CD45RA⁻Foxp3⁺⁺ iTregs (Fr. II). (B) Average percentage and absolute cell numbers per culture. Mean and SEM are shown. ADEH (acute stage, n = 4) and healthy controls (n = 5). Student t test. *p < 0.05 versus healthy controls. Fr., Fraction.