Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: (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.

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques: Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: 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).

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques:

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: (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 patient.

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: Number of interstitial CD3 + , FoxP3 + , CD4FoxP3 + and CD8 + FoxP3 + cells in renal biopsy.

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques:

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: 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).

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques: Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: (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.

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques: Labeling, Incubation, Purification, Flow Cytometry

Journal: PLoS ONE

Article Title: CD8 + Treg Cells Associated with Decreasing Disease Activity after Intravenous Methylprednisolone Pulse Therapy in Lupus Nephritis with Heavy Proteinuria

doi: 10.1371/journal.pone.0081344

Figure Lengend Snippet: (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.

Article Snippet: CD8 + CD25 + T cells were isolated by CD8 + T cell enrichment kit followed by separation with CD25 microbeads (BD Biosciences).

Techniques: Cell Culture, Labeling, Expressing, Transfection, Derivative Assay