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Volume 68, Issue 4, Pages 1604-1612 (October 2005) Dendritic cells of IgA nephropathy patients have an impaired capacity to induce IgA production in naïve B cells Jan-Willem Eijgenraam, Andrea M. Woltman, Sylvia W.A. Kamerling, Francine Briere, Johan W. De Fijter, Mohamed R. Daha, Cees Van Kooten Kidney International Volume 68, Issue 4, Pages 1604-1612 (October 2005) DOI: 10.1111/j.1523-1755.2005.00572.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Immunoglobulin production is dependent on CD40L and interleukin (IL)-10. Highly purified IgD+ B cells (104) were cultured with 75 Gy irradiated nontransfected L cells (L-orient) or CD40L transfected L cells (L-CD40L) (0.25 × 104) in the presence of IL-2 (20 U/mL) or IL-10 (50 ng/mL) as indicated. After 14 days supernatants were harvested and tested for IgA, IgG, and IgM production using specific enzyme-linked immunosorbent assays (ELISAs). Data shown are the mean ± SD of quadruple cultures and represents one out of three independent experiments. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Dendritic cells (DCs) increase Ig production by naïve B cells. Highly purified IgD+ B cells (104) were cultured with 75 Gy irradiated CD40L transfected L cells (0.25 × 104) in the absence of dendritic cells or in the presence of dendritic cells (104), in the presence of interleukin (IL)-2 (20 U/mL) or IL-10 (50 ng/mL) as indicated. After 14 days, supernatants were harvested and tested for IgA, IgG, and IgM production using specific enzyme-linked immunosorbent assays (ELISAs). Data shown are the mean ± SD of fivefold cultures and are representative of three independent experiments. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Analysis of CD1a, CD14 and DC-SIGN expression on monocytes and immature dendritic cells (DCs) from controls and patients with IgA nephropathy (IgAN). (A) Surface expression of CD1a, CD14, and DC-SIGN on monocytes (upper panel) and monocyte-derived dendritic cells (lower panel) was determined using fluorescence-activated cell sorter (FACS) analysis. Specific staining is represented by the filled histograms and control staining by the open histograms. Shown is a representative example of dendritic cells from a control. (B) Surface expression of DC-SIGN on dendritic cells from control persons and dendritic cells from IgAN patients was determined using FACS analysis. Shown are the mean fluorescence intensity (MFI) ± SD of five control persons and five IgAN patients. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Irradiation of dendritic cells does not influence CD40L-induced interleukin (IL)-10 production and CD86 expression. (A) Dendritic cells (5 × 104) were either irradiated (33 Gy) or not irradiated and cultured for 72 hours in the presence of control L cells or CD40L-transfected L cells (104). IL-10 production was measured by specific enzyme-linked immunosorbent assay (ELISA). Shown are the mean ± SD of quadruple cultures. (B) Surface expression on denritic cells of CD86 was determined by fluorescence-activated cell sorter (FACS) analysis. Shown is the mean of three experiments. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Dendritic cells from IgA nephropathy (IgAN) patients induce less net IgA production compared with controls. (A) Highly purified IgD+ B cells (104) were cultured with 75 Gy irradiated CD40L-transfected L cells (0.25 × 104) in the presence of dendritic cells from controls (N = 12) or in the presence of dendritic cells (104) from patients with IgAN (N = 12), in the presence of interleukin (IL)-2 (20 U/mL) or IL-10 (50 ng/mL) as indicated. After 14 days, supernatants were harvested and tested for IgA. Data shown are the mean ± SEM of fivefold cultures of three independent experiments. (B) IgG and IgM production was measured using specific enzyme-linked immunosorbent assays (ELISAs). Data shown are the mean ± SD of fivefold cultures of three independent experiments. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Ratio of IgA production induced by dendritic cells (DCs) from patients relative to controls. Mean individual IgA production induced by dendritic cells from patients with IgA nephropathy (IgAN) is divided by the mean IgA production induced by dendritic cells from control persons and expressed as a ratio. Shown are the mean +SD of five measurements. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Dendritic cells from patients with IgA nephropathy (IgAN) induce less IgA1 and IgA2. Highly purified IgD+ B cells (104) were cultured with 75 Gy irradiated CD40L-transfected L cells (0.25 × 104) in the presence of dendritic cells from control persons (N = 12) or in the presence of dendritic cells (104) from patients with IgAN (N = 12), in the presence of interleukin (IL)-2 (20 U/mL) or IL-10 (50 ng/mL) as indicated. After 14 days supernatants were harvested and tested for IgA1 (A) and IgA2 (B). Data shown are the mean ± SD of fivefold cultures of three independent experiments. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 Supernatant of CD40L stimulated dendritic cells (DC) can induce IgA switch, but to a lesser extent than dendritic cells. (A) Dendritic cells (5 × 105) of control persons and IgA nephropathy (IgAN) patients were cultured in the presence of medium, control L cells (unstimulated supernatant) or CD40L-transfected L cells (stimulated supernatant) (105) in a total volume of 500 μL. After 72 hours, supernatants were harvested and 50 μL of each supernatant was added to naïve B cells (104) in the presence of interleukin (IL)-10 (50 ng/mL). After 14 days, supernatant was harvested and tested for IgA, using specific enzyme-linked immunosorbent assay (ELISA). (B) Dendritic cells (104) from the same control persons and IgAN patients were added to naïve B cells (104) in the presence of medium or IL-10 (50 ng/mL). After 14 days, supernatant was harvested and tested for IgA, using specific ELISA. Kidney International 2005 68, 1604-1612DOI: (10.1111/j.1523-1755.2005.00572.x) Copyright © 2005 International Society of Nephrology Terms and Conditions