Figure 5.
iNKT Cell Number Is Recovered in SLE Patients Responding to Rituximab after B Cell Repopulation
Ex vivo PBMCs from SLE patients before, during, and after rituximab treatment stained for expression of CD19, CD3, and iTCR.
(A) Representative flow cytometry plots showing frequency of iNKT and B cells from SLE patients, B cell-depleted SLE patients (BCD), repopulated nonresponding SLE patients (RBRnr), and repopulated responding patients (RBRr).
(B and C) Scatter plots show mean percentage of B cells (B) and percentage and absolute number of iNKT cells (C) in PB of 254 SLE, 92 BCD, 32 RBRnr, and 113 RBRr patients. ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA.
(D and E) PBMCs isolated from SLE patients before, during, and after rituximab treatment were cultured with αGalCer and IL-2 for 7 days. Supernatants were collected and cells were stained with CD3 and iTCR mAbs. Representative flow cytometry plots at day 7 (D) and bar charts showing the cumulative data of iNKT cells (E) before and after the 7 day culture from 10 SLE, 10 BCD, 13 RBRnr, and 13 RBRr patients. ∗∗p < 0.01; paired t test.
(F) Cytokines from supernatants collected in experiment (D) were measured by CBA. Bar charts of cumulative results from three independent experiments with 10 SLE, 10 BCD, 13 RBRnr, and 13 RBRr patients, mean ± SE. ∗p < 0.05, ∗∗p < 0.01; one-way ANOVA.
(G) Ex vivo PBMCs from SLE patients before, during, and after rituximab treatment stained for expression of CD19 and CD1d. Scatter plots showing CD1d MFI on B cells in PB of 42 healthy donors, 33 SLE patients, 21 RBRnr, and 66 RBRr patients. ∗p < 0.05; one-way ANOVA.
(H) Bar chart showing CD1d internalization on B cells from three healthy donors, three SLE patients, three RBRnr, and three RBRr patients after 5 min. One of three experiments is shown. Mean percentage ± SE. ∗∗∗p < 0.001; one-way ANOVA.
(I) Confocal microscopy analysis depicting CD1d localization on B cells from one RBRnr and one RBRr patient before (0) and 5 min after CD1d endocytosis. Cells were stained for CD1d (green) and DAPI (blue). Scale bars represent 1 μm. Arrows indicate areas of CD1d internalization.