Figure 3.

nBreg Cells Are Preferentially Infected by RSV

(A–C) 10⁵ cord blood nBreg cells were FACS sorted and left untreated (0 hr) or exposed to HRSV-A or to rHRSV-Ch (MOI = 2.5).

(A) Representative plot of IL10 gene expression, as measured by qRT-PCR at 0, 6, and 24 hr.

(B) 10⁵ B cell subsets were FACS sorted as nBreg cells, MN, or IMT cells and stimulated with rHRSV-Ch. mCherry expression was assessed by fluorescent microscopy at 48 hr after infection (left) or by monitoring the red object count (R.O.C) through live imaging (right). Results are representitative of 3–5 independent experiments.

(C) Representative FACS plot for intra-cellular IL-10 expression at 48 hr after infection as compared to untreated cells (No stimulus).

(D) The frequency of IL-10⁺ nBreg cells among rHRSV-Ch-positive or -negative nBreg cells (n = 3). Unpaired t test was used for comparison.

(E) IL-10 production after nBreg cell exposure to live or UV-treated HRSV-mCherry was measured at 48 hr by ELISA (n = 5). Paired t test was also used to compare the three conditions.

(F and G) HEp-2 cells were infected with rHRSV-Ch (MOI = 0.1) then cocultured with B cell subsets.

(F) The percentage of rHRSV-Ch⁺ B cells in co-culture with HEp-2 cells is shown by FACS at 48 hr after coculture (n = 3). ANOVA test was used to compare the three groups.

(G) IL-10 production was measured by ELISA (n = 3) and unpaired t test was used for comparison.

(H) nBreg cells were stimulated or not with HRSV-A for 24 hr. IL-10⁺ nBreg cells were enriched using IL-10 enrichement beads, then FACS sorted IL-10⁺ nBreg cells were used for fluorescent IgM ELISPOT. Left panel is a representative FACS plot after IL-10 enrichement and sorting purity. Right panel indicates the frequency of IgM⁺ cells.

Results are expressed as the means ± SD of triplicates. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.