Table 1.
Baseline characteristics.
Table 2.
Disease activity and laboratory variables in both groups before and two weeks after IVMP treatment.
Figure 1.
CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ Treg cells before and after IVMP pulse therapy.
(a) PBMCs from LN patients during IVMP pulse therapy were stimulated with anti-CD3 mAb stimulation (5 ug/ml) and IL-2 (10 U/ml) for five days, and cells with intracellular expression of FoxP3 were analyzed for CD4+CD25+ and CD8+CD25+ T cells, representative figures shown. Analysis of CD4+CD25+FoxP3 Treg cells (b) and CD8+CD25+FoxP3 Treg cells (c) in PBMCs before and after IVMP pulse therapy by flow cytometry. Bars represent mean ± SD.
Table 3.
CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ Treg cell numbers following anti-CD3 mAb stimulated PBMCs in both groups before (Day 0) and after IVMP treatment (Day 6).
Figure 2.
Confocal microscopic analysis of renal biopsy in active Class IV LN patient before IVMP pulse therapy.
(a) Specimen was stained for CD8, FoxP3, and 4′, 6-diamidino-2-phenylindole (DAPI) (nuclear stain). White arrows indicate CD8+FoxP3+ cells. (b) (c) right CD4+FoxP3+ and CD8+FoxP3+Treg cell expression significantly decreased before IVMP pulse therapy in renal tissue of Class IV LN (n = 50) FoxP3+ (brown); CD4+ or CD8+ (pink). (b) (c) left CD4+FoxP3+ and CD8+FoxP3+ Treg cell expression significantly increased after IVMP pulse therapy in renal tissue of follow-up biopsies in Fig. 2a patient.
Table 4.
Number of interstitial CD3+, FoxP3+, CD4FoxP3+ and CD8+FoxP3+ cells in renal biopsy.
Figure 3.
Intracellular IL-10 and granzyme B levels in CD8+CD25+ Treg cells before and after IVMP.
PBMCs were stimulated with anti-CD3 mAb for five days, followed by stimulation of PMA (10 ng/ml) plus ionomycin (1 µg/ml) for the last five hours, with addition of brefeldin A (10 µg/ml) for the final hour. Intracellular expression of IL-10 and granzyme B was measured by gating in CD8+CD25+ T cells, using flow cytometry. Isotype control (dotted line). (a) Results of 30 paired experiments for IL-10 (b) and granzyme B (c) production by PBMCs (* p<0.05).
Figure 4.
CD4+ cell proliferation in the presence of CD8+CD25+ Treg cells during IVMP.
(a) CFSE-labeled cells (Bulk PBMCs and CD8+-depleted PBMCs) were pretreated with anti-CD3 mAb for five days, CD8+-depleted PBMCs incubated with purified CD8+CD25+ T cells at a ratio of 10:1, proliferation of CD4+ T cells analyzed by flow cytometry. (b) There was significant suppression (*) of CD+ cells proliferation in the presence of CD8+CD25+ regulatory T cells compared to CD8+ depleted PMNCs alone. There was significant suppression (#) of CD4+ T cell proliferation after IVMP during SLE, data calculated from 20 paired experiments. (*# indicates p<0.05). (c) Th1 type IFN-r response to critical peptide epitopes (H3: 115–135, H4: 16–39) in PBMCs of LN patients before and after IVMP pulse therapy. CD8+ T cells significantly suppressed IFN-r response after IVMP pulse therapy. Data were calculated from 20 paired experiments; bars represent mean ± SD.
Figure 5.
Double fluorescence study by flow cytometry of CD45RO lymphocytes subpopulations and apoptosis by CD8+CD25+ regulatory T cells during IVMP.
(a). CD25+-depleted PBMCs were co-cultured with CD8+CD25+ Treg cells from control subjects or CD8+CD25+ Treg cells after IVMP during SLE. Apoptosis was simultaneously determined by Annexin V labeling and negative PI gating, representative histograms shown. (b). Percentage of Annexin V-positive CD4+CD45RO+ cells rose sharply after addition of Treg cells during IVMP, data calculated from 20 paired experiments. (*#p<0.05). (c). siRNA of FoxP3 decreased granzyme B protein expression in CD8+CD25+ Treg cells if pretreated with nucleosomal histone peptide epitope (H3:115–135) and dexamethasme (50 nM). Right column shows control without dexamethasone, nucleosomal histone peptide epitope and siRNA treatment; left column second control pretreated with nucleosomal histone peptide epitope with IL-2 (10 U/ml) for 3 days and third day treated with dexamethasone for 24 hours. Middle left column was a control RNA transfection for FoxP3 siRNA. Middle right column was pretreated with nucleosomal histone peptide for 3 days and third day treated with dexamethasone for 24 hours their FoxP3 siRNA treatment for 48 hours. Data were derived from three independent experiments; bars represent mean±SD.