Involvement of suppressors of cytokine signaling in toll-like receptor–mediated block of dendritic cell differentiation by Holger Bartz, Nicole M. Avalos, Andrea Baetz, Klaus Heeg, and Alexander H. Dalpke Blood Volume 108(13):4102-4108 December 15, 2006 ©2006 by American Society of Hematology

TLR stimulation inhibits GM-CSF– and IL-4–induced development of DCs from human CD14+ monocytes. TLR stimulation inhibits GM-CSF– and IL-4–induced development of DCs from human CD14+ monocytes. Human CD14+ monocytes were cultivated for 6 days with GM-CSF and IL-4 (indicated as GI) in either the presence or absence of 10 ng/mL LPS (indicated as GI+LPS). CD14+ cells left unstimulated for 6 days are indicated as “none.” (A) Cell size and granula, and expression of CD1a and CD14, were measured by flow cytometry (representatives of at least 20 experiments). (B) Histograms of indicated surface molecules from either control DCs (open) or LPS-treated cells (filled). Data are from one representative experiment out of 4 experiments. (C) Expression of CD1a and CD14 was measured in cultures that were treated with graded amounts of LPS as indicated (1 out of 4 experiments). (D) Monocytes from 3 different donors (marked by circles, rectangles, and triangles) were cultivated either with GM-CSF+IL-4 alone (GI) or in the presence of an additional 10 μg/mL Pam3 CysSK4, 1 μg/mL FSL, 50 μg/mL poly[(I:C)], 50 μg/mL zymosan, or 10 ng/mL LPS. Cells were analyzed for expression of CD1a at day 6. (E) Cell numbers from LPS-treated cultures were determined as mean values of triplicate counts (n = 8). (F) Cells were analyzed for phagocytosis of FITC-labeled latex beads. Shown is ΔMFI (37°C-4°C) of 1 of 4 experiments with similar results. (G) Cells were restimulated at day 6 with 50 ng/mL LPS (▦) or left unstimulated (□) and analyzed for secretion of IL-6, TNF, IL-12p40, and IL-10 after overnight incubation (displayed as mean and SD from 1 of 3 donors with similar results). (H) DCs either treated with LPS or not during the differentiation period and completely untreated CD14+ cells were assayed for their capacity to induce proliferation of allogenic CD3-sorted T lymphocytes (triplicates of 1 of 3 similar experiments). (I) Similarly, cells were assayed in an autologous MLR with CD3-sorted T lymphocytes (triplicates of 1 of 3 similar experiments). To induce proliferation, 0.1 ng/mL superantigen SPEC was added to autologous cocultures. Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology

TLR stimulation inhibits murine dendritic cell development. TLR stimulation inhibits murine dendritic cell development. Murine bone marrow cells (Balb/c) were differentiated for 9 days with GM-CSF in the absence or presence of 100 ng/mL LPS or 100 nM CpG-ODN. (A) Cells were analyzed for expression of CD11b, CD11c, MHC class II (n = 5), and F4/80 (n = 2) expression by FACS (mean+SD). (B) Cells were restimulated at day 9 with 1 ng/mL LPS and analyzed for secretion of IL-12p40 (n = 3) and TNF-α (n = 2) after overnight incubation (mean+SD). (C) Cells were analyzed for phagocytosis of FITC-labeled latex beads. Shown is ΔMFI (37°C-4°C) (mean+SD, n = 4). (D) Day-10 Balb/c DCs treated with LPS or CpG-ODN during the differentiation period were assayed for their capacity to induce proliferation of CD90-sorted T lymphocytes (C57BL/6, triplicates of 1 of 3 experiments). Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology

TLR stimulation directly inhibits DC development. TLR stimulation directly inhibits DC development. (A) Bone marrow cells from either TLR9–/– or wt (C57BL/6) mice were differentiated with GM-CSF in the absence or presence of LPS or CpG-ODN. Cells from the different mice were either combined in a transwell format (left) or incubated alone (right). Cells were analyzed at day 9 for CD11c expression by FACS. (B) Experiments were done as above with C3H/HeJ (LPS-unresponsive) and C3H/HeN (control) mice (mean + SD; n = 4, *P < .05 against the respective control stimulation). Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology

TLR stimulation induces the expression of SOCS TLR stimulation induces the expression of SOCS. Freshly isolated human CD14+ monocytes were stimulated with 10 ng/mL or 30 ng/mL LPS and analyzed for expression of (A) SOCS-1, (B) SOCS-3, and (C) CIS by RT-PCR at the indicated time points (mean of duplicate... TLR stimulation induces the expression of SOCS. Freshly isolated human CD14+ monocytes were stimulated with 10 ng/mL or 30 ng/mL LPS and analyzed for expression of (A) SOCS-1, (B) SOCS-3, and (C) CIS by RT-PCR at the indicated time points (mean of duplicate determinations from 1 of 3 typical donors). (D) Murine bone marrow cells (Balb/c) were stimulated with 100 ng/mL LPS immediately after preparation and were analyzed for expression of SOCS-1, SOCS-3, and CIS by RT-PCR at the indicated time points (n = 4, mean+SD). Expression of SOCS1 (E) or SOCS-3 (F) was determined in monocytes stimulated with 10 μg/mL Pam3CysSK4,1 μg/mL FSL, 50 μg/mL poly[(I:C)], or 10 ng/mL LPS for 7 hours (1 representative of 3 experiments). Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology

SOCS proteins inhibit GM-CSF signaling. SOCS proteins inhibit GM-CSF signaling. (A) Human monocytes were preincubated with 100 ng/mL LPS for the indicated time. Subsequently, cells were stimulated with 500 U/mL GM-CSF for 30 minutes and were analyzed for phosphorylation of STAT-5 by FACS (mean fluorescence intensity of untreated cells was set as 100%). (B) Murine RAW264.7 macrophages stably overexpressing the indicated SOCS proteins were stimulated with 100 ng/mL GM-CSF for 30 minutes. Cells were analyzed for phosphorylation of STAT-5 by FACS. Data are mean + SD. Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology

SOCS-1 inhibits DC differentiation. SOCS-1 inhibits DC differentiation. (A) Murine bone marrow cells were forced to overexpress SOCS-1, SOCS-2, SOCS-3, or CIS by means of retroviral transfer of SOCS/GFP bicistronic constructs on days 1, 2, and 3 of the differentiation period. Cells were differentiated with GM-CSF for a total of 9 days and then analyzed for expression of CD11c by FACS either in the GFP-positive, transfected population or in the GFP-negative control population (mean+SD, n = 6, **P < .01). (B) FACS analysis of CD11c expression of one typical experiment from panel A either on mock (filled) or SOCS-1–transfected cells (open). Shown are overlay graphs for GFP-positive, transfected cells and GFP-negative control cells within one experiment. Holger Bartz et al. Blood 2006;108:4102-4108 ©2006 by American Society of Hematology