IgG regulates the CD1 expression profile and lipid antigen-presenting function in human dendritic cells via FcγRIIa by Anna Smed-Sörensen, Markus Moll, Tan-Yun Cheng, Karin Loré, Anna-Carin Norlin, Leif Perbeck, D. Branch Moody, Anna-Lena Spetz, and Johan K. Sandberg Blood Volume 111(10):5037-5046 May 15, 2008 ©2008 by American Society of Hematology

The CD1 profile of monocyte-derived dendritic cells is influenced by the type of serum they are cultured in. The CD1 profile of monocyte-derived dendritic cells is influenced by the type of serum they are cultured in. The expression of CD1a and CD1d was determined on blood myeloid DCs (MDCs) and Langerhans cells (LCs) in the epidermis of skin. MDCs were identified in PBMCs by gating on size, followed by a gate on cells negative for lineage markers (CD3, CD8, CD14, CD16, CD19, and CD56). CD11c+ HLA-DR+ MDCs were subsequently identified among the lineage-negative cells, and the CD1a and CD1d profile of MDCs was displayed. To assess the CD1 profile of LCs, cells were allowed to migrate out from epidermis sheets of skin biopsies for 2 to 3 days. The cells were subsequently analyzed by flow cytometry, and LCs were identified by size and high expression of HLA-DR and their expression of CD1a and CD1d was determined. The histograms show 1 representative donor of 10 for MDCs (black) and 1 of 4 for LCs (dotted) (A). The expression of CD1 molecules was determined on monocyte-derived DCs cultured in human adult serum (AS) or fetal calf serum (FCS) and IL-4 and GM-CSF for 6 days to allow differentiation of DCs from monocytes. The mean fluorescence intensity (MFI) of CD1a, CD1b, CD1c, and CD1d on DCs cultured in AS () or FCS () from 18 donors was measured (B). Average CD1 expression is depicted by a line in the graphs. The statistical difference between the CD1 expression on DCs grown in AS and FCS was determined by paired t test. DCs cultured in AS and FCS obtained a similar overall phenotype with respect to expression of MHC class II (HLA-DR), MHC class I (HLA-A2), and DC-SIGN, as well as their lack of CD14 expression as determined by flow cytometry (C). The numbers on the plots are the percentages of dendritic cells that fall within each rectangle. The graphs show data from 1 representative donor of 6. Immature DCs grown in either AS or FCS were irradiated (30 Gy) and cocultured with allogeneic CD3+ T cells at a ratio 1:10 for 2, 4, and 6 days, and T-cell proliferation was determined by incorporation of 3H-thymidine (1 μCi [0.037 MBq]/well for 6 hours) and presented as counts per minute (cpm) (D). The graph shows the average proliferation plus or minus standard deviation of triplicates from 1 of 2 donors. To assess the ability of the DCs to mature, both DCs cultured in AS and FCS were exposed to 100 ng/mL LPS for 16 hours and the expression of CD83 was determined by flow cytometry (E). Histograms show unstained DCs (filled), unstimulated DCs (dashed), and LPS-stimulated DCs (solid) on DCs grown in either AS or FCS. One representative experiment of 6 is shown. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

Reduction of IgG levels in human adult serum results in increased expression of CD1a, CD1b, and CD1c, and decreased expression of CD1d on dendritic cells. Reduction of IgG levels in human adult serum results in increased expression of CD1a, CD1b, and CD1c, and decreased expression of CD1d on dendritic cells. The expression of CD1 molecules was determined on monocyte-derived DCs after 6 days of culture in IL-4 and GM-CSF and FCS, AS, or AS with reduced IgG content. To reduce the levels of IgG in AS, the serum was diluted 10 times in RPMI and passed through a protein G column twice to allow binding of IgG. As a control, immunoglobulins were added back to the AS after protein G binding. After 6 days of culture, the cell surface expression of CD1a (A), CD1b (B), CD1c (C), and CD1d (D) on DCs was determined by flow cytometry. The graphs show the MFI of CD1 expression from 1 representative donor of 4. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

Addition of immunoglobulins to FCS-based cultures results in a similar CD1 profile of dendritic cells as observed after culture in adult serum. Addition of immunoglobulins to FCS-based cultures results in a similar CD1 profile of dendritic cells as observed after culture in adult serum. The expression of CD1a and CD1d was analyzed on DCs after 6 days of culture in IL-4 and GM-CSF and AS, FCS, or FCS with a high dose of IVIg (20 mg/mL). The IVIg was dissolved in 0.2 M glycine, which was included as control. The graphs show the average MFI of CD1a and CD1d cell surface expression (± standard deviation) from 6 individual donors (A). The expression of CD1 molecules on DCs cultured for 6 days in the presence of IL-4, GM-CSF, and FCS with increasing doses of IVIg (0.004 to 10 mg/mL IVIg) was measured by flow cytometry (B). Three representative donors of 6 are plotted as individual lines (■, ○, and ◆). As references, vertical lines depict the average levels of IgG added to the AS DC cultures (∣) and found in serum of healthy individuals (¦). Furthermore, the effect of adding Fc fragments instead of intact immunoglobulins, or equimolar amounts of bovine serum albumin (BSA) instead of IVIg, to the FCS cultures was assessed by determining the expression levels of CD1a and CD1d on DCs after 6 days of culture. The graphs show the average MFI of CD1a and CD1d cell surface expression (± standard deviation) from 4 individual donors (C). Statistical differences were assessed by paired t test and considered significant when *P < .05; **P < .01 and ***P < .001. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

Low concentrations of immobilized but not soluble IgG alter the CD1 profile of dendritic cells. Low concentrations of immobilized but not soluble IgG alter the CD1 profile of dendritic cells. The surface expression of CD1a (A) and CD1d (B) on DCs cultured for 6 days in the presence of IL-4, GM-CSF, and FCS with low doses of immobilized IgG (○) or soluble IgG (•) was measured by flow cytometry. The graphs show the average MFI of CD1a and CD1d cell surface expression (± standard error of the mean) from 9 (immobilized IgG) or 2 (soluble IgG) individual donors. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

The relative CD1 gene expression corresponds to the CD1 protein expression pattern of dendritic cells cultured in the absence or presence of immunoglobulins. The relative CD1 gene expression corresponds to the CD1 protein expression pattern of dendritic cells cultured in the absence or presence of immunoglobulins. The CD1 gene expression was analyzed in DCs after 6 days of culture in IL-4 and GM-CSF and AS, FCS, or FCS supplemented with 0.5 mg/mL IVIg in a 2-step-process. First, total mRNA was transcribed into cDNA using random hexamers and pT-primers. Second, the CD1 genes and GAPDH (as endogenous control) were amplified with gene-specific primers using a real-time PCR kit based on SYBRgreen technology. The real-time data were analyzed using the comparative Ct method and the data presented as fold-change in gene expression. The graph shows relative CD1 gene expression (± standard deviation) of DCs cultured in FCS (■), AS (), or FCS with IVIg () from 1 representative experiment of 2. The real-time PCR was done in triplicates and a no-template control was included. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

Blocking of the activating Fcγ receptor CD32a abrogates the IVIg-mediated regulation of CD1 expression on dendritic cells. Blocking of the activating Fcγ receptor CD32a abrogates the IVIg-mediated regulation of CD1 expression on dendritic cells. The effect of blocking the Fcγ receptors CD16, CD32a, CD32b, and CD64 using 2 μg/mL of each antibody (in the presence of 0.5 mg/mL IVIg) on CD1 expression on DCs was determined. The graphs show the relative MFI expression of CD1a, CD1b, CD1c, and CD1d cell surface expression (± standard deviation) from 9 individual donors after 6 days of culture (A). The reference line indicates the relative CD1 expression on DCs cultured in FCS and IVIg. The statistical difference between the CD1 expression on DCs grown in FCS and IVIg and DCs cultured in FCS, IVIg, and blocking FcγR antibodies was determined by paired t test or signed rank test if normality test failed. The surface expression of the activating Fcγ receptor CD32a (B) and the inhibitory Fcγ receptor CD32b (C) on CD14+ monocytes and on DCs cultured in IL-4, GM-CSF, and FCS for the indicated times was determined by flow cytometry. The histograms show the CD32 expression in black and the isotype control in gray. Statistical differences were considered significant when P < .05; *P < .05 and **P < .01. Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology

The ability of dendritic cells to stimulate CD1-restricted T cells is determined by their CD1 expression profile. The ability of dendritic cells to stimulate CD1-restricted T cells is determined by their CD1 expression profile. To determine the ability of DCs to present CD1-restricted antigens to T cells, human monocytes were cultured in IL-4, GM-CSF, and FCS (•) or AS (□) for 6 days and plated in 96-well plates and pulsed with decreasing concentrations of antigen presented by CD1a (dideoxymycobactin), CD1b (glucose monomycolate), CD1c (mannosyl-b-1-phosphomycoketide), or CD1d (α-galactosylceramide). CD1-restricted T cells were then added at a 1:1 ratio to T-cell lines J.RT-3/CD8–2 (CD1a), LDN5 (CD1b), CD8–1 (CD1c), or sorted NKT cells (CD1d). Activation of CD1a-, CD1b-, and CD1c-restricted T cells was determined by their production of IL-2 using the HT-2 cells bioassay (A-C). Activation of CD1d-restricted NKT cells was measured after 6 hours of coculture by determining the frequency of IFNγ-producing cells by intracellular cytokine staining and flow cytometry (D). The graphs A-C show the average IL-2 release (± standard deviation) of triplicates in 1 representative donor of 3. The average frequency of IFNγ+ NKT cells (± standard deviation) in 2 individual donors (1 representative experiment of 2; 4 donors tested in total) is presented (D). Anna Smed-Sörensen et al. Blood 2008;111:5037-5046 ©2008 by American Society of Hematology