(A): TGC-PECs from C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^Lps2/Lps2 mice were stimulated with PBS (shade histogram), rough LPS (open histogram). MFI is shown adjacent each histogram (gray-PBS, back-LPS).
(B): Time course of the change of MFI between PBS treated and LPS treated cells for each genotye during LPS activation of TGC-PECs.
(C): TGC-PECs from C57BL/6 WT, TLR4^−/−, Myd88^−/−, Trif^Lps2/Lps2 and IRF-3^−/− mice were stimulated with smooth or rough LPS (100ng/ml) for 24h and CD40 and CD86 expression was examined. The data is representative of three independent experiments. The change in MFI between PBS treated and LPS activated cells for each genotype is shown.

(A): C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^LPps2/Lps2 BMDCs were stimulated with LPS. Both Myd88^−/− and Trif^Lps2/Lps2 BMDCs manifested a reduced production of IL-6, TNF-a, IL-12p40 vs. WT BMDCs (Myd88^−/−, p = < 0.0001 vs. WT, Trif^Lps2/Lps2 p < 0.009 vs. WT, for each of these cytokines). However, the response in Myd88^−/− BMDCs was inferior to Trif^Lps2/Lps2 BMDCs (p < 0.02, for each cytokine). With IFN-β both Myd88^−/− and Trif^Lps2/Lps2 BMDCs manifested an inferior response vs. WT (p = 0.001, Myd88^−/− vs. WT; p = 0.0003, Trif^Lps2/Lps2 vs. WT) but Trif^Lps2/Lps2 BMDCs manifest an inferior response vs. MyD88^−/− BMDCs (p = 0.009, Myd88^−/− vs. Trif^Lps2/Lps2). For LPS-induced IL-10 production, Myd88^−/− BMDCs manifested an inferior response vs. WT (p < 0.0001). IL-10 responses of Trif^Lps2/Lps2 BMDCs were similar to WT BMDCs.
(B): Both Myd88^−/− and Trif^Lps2/Lps2 TGC-PECs manifested reduced production of IL-6 and TNF-α vs. WT TGC-PECs in response to LPS (Myd88^−/− and Trif^Lps2/Lps2 p<0.0001 vs. WT for each cytokine). No significant differences were noted between MyD88^−/− and Trif^Lps2/Lps2 cells. Both Myd88^−/− and Trif^Lps2/Lps2 TGC-PECs manifested a reduced production of IFN-β vs. WT TGC-PECs (Myd88^−/−, p = 0.006 vs. WT, Trif^Lps2/Lps2, p<0.0001 vs WT). However, Trif^Lps2/Lps2 response was inferior to the Myd88^−/− response (p = 0.01).
(C): C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^Lps2/Lps2 mice were injected with 100μg LPS (or PBS as indicated). Serum IL-6 production was reduced in both LPS treated Myd88^−/− (p = 0.0001) and Trif^Lps2/Lps2 mice (p = 0.009) vs. WT counterparts. Myd88^−/− mice manifested an inferior response vs. Trif^Lps2/Lps2 mice (p = 0.002). Serum IFN-β production was reduced in LPS treated Trif^Lps2/Lps2 mice (p = 0.005) but was not impaired in LPS treated Myd88^−/− mice (p = 0.3) vs. WT counterparts. Results are representative of 3 independent experiments with a total of 6 mice / group.

C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^Lps2/Lps2 BMDCs were stimulated with LPS. Experiments were performed either after either 3h of stimulation (A) or 24h of stimulation (B). After each time point the BMDCs were washed, irradiated and then cultured with purified allogeneic BALB/c T cells. The graphs display the change in T cell proliferation (thymidine incorporation) and IL-2 production between PBS and LPS activated DCs for each experimental group. Data are representative of 3 independent experiments with similar results.
(A): LPS activated Myd88^−/− BMDCs induced inferior IL-2 production (p = 0.02) and T cell proliferation (p = 0.01) vs. WT BMDCs. Trif^Lps2/Lps2 BMDCs manifested a similar phenotype to WT DCs. The augmentation of IL-2 induced by LPS-activated Myd88^−/− BMDCs was similar to LPS-activated TLR4^−/− BMDCs, although LPS-activated Myd88^−/− BMDCs induced a higher level of T cell proliferation vs. LPS-activated TLR4^−/− BMDCs (p = 0.03).
(B): LPS-activated Myd88^−/− BMDCs and Trif^Lps2/Lps2 BMDCs induced reduced T cell IL-2 responses compared to WT BMDCs at 24h. These responses were greater than those induced by TLR4^−/− LPS-activated BMDCs. (IL-2: Myd88^−/−, p = 0.04, Trif^Lps2/Lps2 p = 0.0002). LPS-stimulated Myd88^−/− BMDCs induced similar enhancement of T cell proliferation compared to those induced by LPS-activated TLR4^−/− BMDCs, whereas LPS-induced Trif^Lps2/Lps2 BMDCs did not enhance T cell proliferation (p = 0.009) vs. LPS-activated TLR4^−/− DCs.

C57BL/6 WT, TLR4^−/− and Myd88/Trif DKO BMDCs were stimulated with LPS. (A) Upregulation of CD86 and CD40 on CD11c⁺ cells was assessed by flow cytometry. (B): Production of IL-6, TNF-α and IL-12 p40 was measured by ELISA.
(C): C57BL/6 WT, TLR4^−/− and Myd88/Trif DKO TGC-PECS were stimulated with LPS and the upregulation of CD86 and the production of IL-6 and IFN-β measured.
(D): After 3h or (E) 24h LPS stimulation, BMDCs were irradiated and then cultured with purified allogeneic BALB/c T cells. T cell proliferation (thymidine incorporation) and IL-2 production was measured. The change between PBS and LPS activated DCs for each experimental group was plotted. Data are representative of 3 independent experiments with similar results.

(A): C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^Lps2/Lps2 and TRIF^−/− BMDCs were stimulated for 6h with PBS (shaded histogram) or 100 ng/ml of smooth LPS (open histogram) and expression of CD40 and CD86 on CD11c⁺ populations was assessed by flow cytometry. Mean fluorescent intensity (MFI, arbitrary units) is shown for each condition (grey-PBS, black-LPS) adjacent to each histogram. All groups significantly upregulated CD86 with LPS treatment compared to PBS controls (p < 0.03) except for TLR4^−/− BMDCs in which p = NS. All groups significantly (p < 0.02) upregulated CD40 upon LPS activation as compared to PBS controls except for TLR4^−/− group in which p = NS. Histogams are gated on CD11c⁺ population.
(B): C57BL/6 WT, TLR4^−/− and Trif^Lps2/Lps2 BMDCs were stimulated with PBS or titrated doses of smooth LPS and the UCM was measured by flow cytometry. The change in MFI (arbitrary units is potted on y axis) between PBS treated and LPS treated cells for each genotype is shown.
(C): C57BL/6 WT, TLR4^−/−, Trif^Lps2/Lps2, Myd88^−/−, and IRF-3^−/− BMDCs were stimulated with 100 ng/ml smooth or rough LPS, 100 ng/ml lipid A or PBS and the expression of CD40 and CD86 in CD11c⁺ cells assessed by flow cytometry. The graph shows the change in MFI between BMDCs stimulated with PBS and LPS or lipid A and PBS for each genotype. The data is representative of 3 independent experiments with a total of 3 mice / group.
(D): C57BL/6 WT, TLR4^−/−, Myd88^−/− and Trif^Lps2/Lps2 BMDCs were stimulated with PBS or LPS and expression of CD40, CD80 and CD86 / time on CD11c⁺ cells was assessed by flow cytometry. The graphs show the change in MFI between BMDCs treated with PBS and LPS for each genotype. The data are representative of 3 independent experiments with similar results.

(A): C57BL/6 WT, TLR4^−/−, Myd88^−/−, Trif^Lps2/Lps2 and TRIF^−/− mice were injected with 100μg LPS (open histogram) or PBS (shaded histogram). MFI in arbitrary units is shown (grey-PBS, black-LPS). Samples were obtained 3h later. For CD86 upregulation, WT: PBS vs. LPS, p = 0.01; TLR4^−/−: PBS vs. LPS, p = 0.9; Myd88^−/−: PBS vs. LPS, p = 0.005; Trif^Lps2/Lps2: PBS vs. LPS, p = 0.005; TRIF^−/−: PBS vs. LPS, p = 0.001. For CD40 upregulation, WT: PBS vs. LPS, p = 0.003; TLR4^−/− PBS vs. LPS, p = 0.6; Myd88^−/−: PBS vs. LPS, p < 0.0001; Trif^Lps2/Lps2 PBS vs. LPS, p = 0.0007; TRIF^−/− PBS vs. LPS, p = 0.002. Histograms are gated on CD11c⁺ cells.
(B): Time course response of UCM after in vivo LPS administration to the experimental groups. SPC = spleen cells.
(C): Assessment of UCM after 3h in vivo LPS adminstration in pDCs (defined as CD11c⁺PDCA-1⁺), lymphoid DCs (defined as CD11c⁺CD8α⁺CD11b⁻) or myeloid DCs (CD11c⁺CD8α⁻CD11b⁺) cells. * p < 0.007
In B and C, the graph shows the change in MFI of cells obtained from mice injected with PBS vs. those injected with LPS for each genotype. The data are representative of 3 independent experiments with a total of 6 mice/group.

(A-B): Conditioned media was harvested from each of the indicated cells [BMDCs (A) or TGC-PECs (B)] after 24h of LPS stimulation and then added to TLR4^−/− cells for 6h for DCs and 24h for TGC-PECS (in A the TLR4^−/− cells were BMDCs, in B they were TGC-PECs). These time points were chosen based on the time to peak UCM during LPS stimulation in DCs (Figure 1D) and TGC-PECs (Figure 2B). After this point, expression of CD40 and CD86 was assessed. * P<0.01. The graphs show the change in MFI in TLR4^−/− cells cultured in harvested media (for each genotype shown on x axis) vs.TLR4^−/− cells cultured in PBS.
(C-D): BMDCs and TGC-PECs were stimulated with LPS in the presence of isotype control or IFN-α/β neutralizing antibody as detailed in the methods section. Expression of CD40 and CD86 was subsequently measured. * p < 0.01 The data are representative of 3 independent experiments with similar results.

C57BL/6 WT, Myd88^−/−, TLR4^−/− and Trif^Lps2/Lps2 mice were injected with 100μg LPS or PBS. Splenocytes were harvested from these mice at 0, 4.5 and 9h, irradiated and cultured with allogeneic BALB/c T cells. (A) The graph shows the change in T cell proliferation (thymidine incorporation) induced between PBS and LPS activated APCs of each genotype. Proliferation induced by Myd88^−/− splenocytes was inferior to that induced by WT cells (p = 0.004) and Trif^Lps2/Lps2 cells (p = 0.0001). (B) The graphs show the change in IL-2 production between PBS and LPS activated APCs for each genotype. Both LPS stimulated WT and Trif^Lps2/Lps2 splenocytes induced the production of IL-2 by allogeneic T cells as compared to PBS stimulated cells. This effect was abrogated in Myd88^−/− and TLR4^−/− splenocytes. The results are representative of 2 independent experiments with a total of 6 mice/group.