Fig 1.
FOXP3+ CD4+ T cells in the steady-state nasal mucosa display a memory phenotype.
(A) Samples of enzymatically digested nasal mucosa contained readily identifiable and viable CD3+ T cells. Representative plots showing the gating strategy when comparing the CD45RO-expression on CD4+ T cells derived from PBMCs or released from nasal mucosal tissue samples. (B) Percentage of CD4+ T cells expressing the memory marker CD45RO derived from PBMC (n = 9) or nasal mucosa (n = 7). (C) Representative plots comparing FOXP3-staining in CD4+ T cells derived from PBMCs or nasal mucosa. (D) Percentage of CD4+ T cells expressing FOXP3 in PBMC (n = 9) and nasal mucosa (n = 7).
Fig 2.
The majority of mucosal CD127lo FOXP3+ Helios+ CD4+ T cells express CTLA-4.
(A) Representative plots gated on CD4+ T cells showing the expression of CD127 on FOXP3+ CD4+ T cells derived from nasal mucosa and in PBMC. (B) Representative plots (left) and compiled percentages of CD4+ T cells expressing FOXP3 and Helios in PBMC (n = 13) and in nasal mucosa (n = 11). (C) Representative plots (left) and percentages of intracellular CTLA-4 expression in CD4+ T cells subsets separated on FOXP3 and Helios expression in PBMC n = 5 and nasal mucosa (n = 4). Statistical difference between PMBC and nasal mucosa is indicated, *p<0.05.
Fig 3.
FOXP3hi and FOXP3lo CD4+ mucosal T-cells co-expressing Helios scarcely produce cytokines.
Representative plots of nasal mucosal CD4+ T cells stained for IFN-μ (top), IL-2 (middle) and IL-17 (bottom) together with FOXP3 and Helios following 4 h stimulation with PMA/ionomycin in the presence of secretion blockade. FOXP3hi and FOXP3lo cells were defined by dividing the total proportion of FOXP3+ cells equally into a high- and low-intensity fraction. Compiled percentages (right panels) of cells expressing either IFN-μ (n = 10), IL-2 (n = 3) or IL-17 (n = 7). *p<0.03.
Fig 4.
IL-10 production in mucosal FOXP3+ CD4+ T cells is most prominent in cells lacking Helios.
(A) Representative plots of gating strategy of CD4+ T cells from nasal mucosa and PBMCs when comparing IL-10 and Helios staining with increasing levels of FOXP3 expression, revealing that IL-10 is preferentially expressed by FOXP3+ cells negative for Helios. (B) Compiled percentages from mucosal samples (n = 6) and PBMC (n = 9) Statistical difference between PBMC and nasal mucosa is indicated, *p<0.01, **p<0.0004. (C) Compiled percentages of IL-10+ memory CD4+ T cell distribution within these subsets from nasal mucosa samples (n = 6) and PBMC (n = 9) reveal that the vast majority of IL-10+ cells are found within the FOXP3-Helios- population.
Fig 5.
IL-10 is preferentially coexpressed with IFN-μ by mucosal CD4+ T cells.
Representative plots showing the gating strategy of CD4+ T cells from nasal mucosa (A) and PBMCs (B) when comparing production of IL-10 and IFN-μ in cells expressing FOXP3 and Helios. (C) Compiled percentages from mucosal samples (n = 5) and PBMC (n = 9). Statistical difference between nasal mucosa and PBMC is indicated, *p<0.05.