Fig. 1. TLR4 expression is much greater on macrophages than on DCs Fig. 1. TLR4 expression is much greater on macrophages than on DCs. (A) DCs were fractionated from whole spleen, stained for surface expression of CD11c or CD11b, MHC class II, and TLR4 or Cont. mAb. CD11c or CD11b, MHC class II double positive populations were gated and analyzed for TLR4 (or control mAb) expression. Data are from one representative experiment of three performed. (B) Splenocytes from a density gradient were stained for MHC class II, TLR4, or control mAb. MHC class II⁺ (based on unstained cells as a negative control), side scatter^hi cells were gated and expression of TLR4 was analyzed (upper panels). Cells expressing high levels of TLR4 were sorted and histologically stained as described in the Methods, and compared to CD8⁺ CD11c⁺ DCs. These data are from one experiment similar to two others, but are based on many preliminary experiments. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 2. Surface TLR4 expression on splenic macrophages is rapidly downregulated after treatment with LPS or SEA. (A) Two groups of mice were treated in vivo with either LPS (left column) or with SEA (right column). At the times indicated after treatment, side scatter^hi MHC class II⁺ macrophages (as described in Fig. 1B) were gated and expression of TLR4 (black line) was monitored and compared to a control mAb stain (gray line). These data are from one experiment and each timepoint has been assessed between three and seven times. (B) Mice were treated with 250 μg of LPS or given nothing; 4 h later innate APCs were fractionated from spleen and stained for TLR4 expression. The top panel shows data from the MTS510 mAb and the bottom from the Sa15-21 mAb with the dotted line representing no treatment and the thick line LPS treatment. This is data is from one of three comparable experiments. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 7. Conditioning DCs with SEA enhances LPS-induced production of TNF and IL-12p70. Primary stimulation with SEA or LPS was followed 24 h later by a SEA or LPS challenge. One-hour post secondary challenge direct ex vivo intracellular cytokine staining on CD11c+ DCs as shown in Fig. 4A was performed (TNF, left panel: IL-12p70, right panel). Each bar represents the mean percentages ± SEM from a total of four separate experiments with one mouse per treatment. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 3. Titration of SEA-induced TLR4 alteration on macrophages Fig. 3. Titration of SEA-induced TLR4 alteration on macrophages. Density-gradient fractionated cells from 0 μg (Nothing), 0.012 μg, 0.3 μg or 7.5 μg SEA-injected mice were tested for TLR4 expression with mAb MTS510 6 h after injection. The cells were gated as in Fig. 2 and the unfilled histogram is data from the control mAb stain. Similar data were obtained from a 3 h time point (not shown). From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 4. SEB-induced DC activation is T cell dependent Fig. 4. SEB-induced DC activation is T cell dependent. (A) Density-gradient fractionated cells from uninjected (upper panel) or mice injected with SEB 12 h earlier (lower panel) were stained and analyzed by flow cytometry. CD11c+ cells were gated and analyzed for MHC class II and ICAM-1 (CD54) expression with the number indicating the percent of high expressing MHC class II, ICAM-1 double positive cells. These data are from one experiment similar to a repeat experiment. (B) Wild-type (upper panel) or Nude mice (lower panel) were injected with nothing (dotted line) or SEB 12 h earlier (solid line), and gradient fractionated CD11c+ cells were analyzed for MHC class II, CD54 and CD86. Data are from one representative experiment of four performed. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 5. Optimal SEA-induced DC activation is dependent on T cell costimulation. After overnight in vivo stimulation density-gradient fractionated cells from normal, SEA (3 μg) + Control Ig, or SEA + CTLA4-Ig treated mice were stained for CD11c, MHC II and CD86. Cells were gated on CD11c low side scatter (data not shown) and then analyzed for MHC II CD86 expression. The percentage indicates the fraction of double positive cells in the upper left panel. These data are from one of three comparable experiments. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology

Fig. 6. Peak production of TNF and IL-12p70 by CD11c DCs occurs 1 h after LPS injection. An example of our intracellular cytokine staining analysis 1 h after in vivo LPS stimulation is given in (A). Spleen cells taken from density gradients were determined to be viable by forward scatter (upper left) and CD11c+ cells (upper right) were analyzed for intracellular TNF production by staining with a negative control mAb (lower left) versus anti-TNF mAb (lower right). (B) Cumulative data for 1 h (left panel) and 4 h (right panel) time points are shown after mice were treated with nothing, SEA or LPS. All data are given as mean ± SEM from individual mice for TNF (upper panel) or IL-12p70 (lower panel). The 0 (normal) and 1 h data are combined from at least five separate experiments, and the 4 h data are from at least three. From: Staphylococcal enterotoxins condition cells of the innate immune system for Toll-like receptor 4 stimulation Int Immunol. 2004;16(12):1751-1760. doi:10.1093/intimm/dxh176 Int Immunol | © 2004 The Japanese Society for Immunology