a. Typical example of Bodipy staining of DCs from cancer patient. b. Cumulative results of lipid levels in DCs from patients with head and neck cancer. P values between the levels of lipids in blood DCs from control donors are shown; n = the number of samples analyzed. c. Lipid levels in peripheral blood DCs from patients with non-small lung cancer (NSCLC) and renal cell cancer (RCC). P values between the levels of lipids in blood DCs from control donors are shown n = the number of samples analyzed. d. Lipid levels in DCs from lymph nodes obtained from patients with head and neck cancer and benign tumors. P values between the levels of lipids in DCs from cancer patients and patients with benign tumors.

a. Staining of CD11c⁺ from the spleens of naïve and EL-4 TB mice with Bodipy 493/503. 10μm scale bar shown. b. A typical example of the analysis of lipid level in CD11c⁺ DCs from spleens of control (red) or EL-4 TB mice (blue). c. Cumulative results of lipid staining of splenocytes from different tumor models. Geomean fluorescence intensity (MFI) of Bodipy 493/503 in CD11c⁺ DCs was measured. Each group includes 4-8 mice. Control and TB mice were evaluated in parallel in each experiment. d. Proportion of DCs with high lipid content in spleens during progression of CT-26 tumors. Each time point included 3 mice. Mean ± SD are shown. e. Lipid levels in different populations of DCs from the spleens of control or EL-4 TB mice. pDC –plasmacytoid DCs; cDC-conventional DCs. Each group had 3 mice. f. Lipid levels in CD11c⁺ DCs from different organs of CT-26 TB mice collected on week 3 after tumor inoculation (tumor size - 1.5 cm in diameter). Mean ±SD from 4 mice are shown. g. Lipid levels in CD11c⁺ DCs from naïve or CT-26 TB mice collected on week 3 after tumor inoculation. Mean ±SD from 3 mice per group are shown. h. Lipid level in CD11c⁺ DCs from draining (inguinal) and distant (contra-lateral axillary) LNs in EL-4 tumor-bearing mice. Three experiments with the same results were performed. i. The amount of triglycerides (TAG) per 10⁶ CD11c⁺ DCs isolated from spleens of naïve BALB/c mice and CT-26 TB mice and in sera of TB mice. Each group included 3 mice. * - statistically significant differences (p<0.05) between values in DCs from control and TB mice.

a. One-hour uptake of OVA-APC by DCs generated from HPC in the presence of TES. The level of APC fluorescence was evaluated within the DC-NL and DC-HL populations. Mean±SD of three performed experiments are shown. * - statistically significant differences (p<0.05) from control DCs. b. Uptake of OVA-APC by DCs from EL-4 TB mice 2 h after i.p. injection of 100 μg of OVA-APC. Mean±SD of three mice per group is shown. c. DCs were either generated in vitro and then cultured for 48 h in control medium or TES, or isolated directly from spleens of naïve or EL-4 tumor-bearing (TB) mice. Cells were loaded with either 1 mg/ml DQ-BSA or 10 mg/ml FITC-OVA. DQ-BSA/FITC-OVA ratio was calculated within CD11c⁺ DCs after 1 h incubation. DQ-BSA/FITC-OVA ratio in control samples was expressed as 100%. Two experiments with the same results were performed. d. DCs generated in vitro as described above were loaded with DQ-BSA or FITC-OVA and incubated for the indicated time. Fluorescence in CD11c⁺ DCs was evaluated as described above. e. CD11c⁺ DCs were isolated from spleens of tumor-free and EL-4 tumor-bearing mice and loaded overnight with 1 mg/ml OVA. Cells were then stained with APC conjugated 25-d1.16 antibody. A typical result of one experiment out of 4 performed is shown. f. DCs were generated from progenitors in the presence of EL-4 TES and then loaded overnight with 1 mg/ml OVA. DCs were stained with Bodipy 493/503 and labeled with 25-d1.16 antibody. DCs with high and normal lipid content were analyzed. The mean fluorescence of cells stained with isotype control Ig was less than 20. * - statistically significant differences (p<0.05) between DC-NL and DC-HL. Cumulative results of 3 independent experiments are shown. g. Binding of 25-d1.16 antibody to DC-NL and DC-HL DCs from LN cells of EG-7 TB mice. The fluorescence of cells stained with isotype control Ig was less than 20. h.i. DCs with high and normal lipid content were sorted from LN of EG-7 TB mice and incubated with T cells isolated from OT-II transgenic mice in the absence of (h) or in the presence of 10 μg/ml of OT-II specific peptide (i). T cell proliferation was measured in triplicate by ³[H]-thymidine uptake. Two experiments with the same results were performed.

a,b. Stimulation of allogeneic T cell proliferation by DCs with different lipid content isolated from CT-26 TB mice. a. Examples of gates set for the sort of CD11c⁺ cells. Blue- naïve mouse, red–TB mouse. b. Stimulation of proliferation of allogeneic T cells by sorted DCs with high and low lipid content. Typical results of 4 performed experiments are shown. T cells alone had ³[H]-thymidine uptake of less than 500 CPM. c. Stimulation of allogeneic T cell proliferation by DCs generated from enriched BM HPC from naïve BALB/c mice, exposed for 2 days to CT-26 TES and sorted for DC-HL (red) or DC-NL (green) and compared to DCs cultured in control medium (blue). Three experiments with similar results were performed. T cells alone had ³[H]-thymidine uptake of less than 200 CPM. d. DCs were generated from HPC of C57BL/6 mice and cultured for 3 days with EL-4 TES in the presence of control IgG or anti-MSR1 antibody. After that time cells were cultured in triplicate with allogeneic T cells at indicated ratios and cell proliferation was evaluated by ³[H]-thymidine uptake. T cells alone had ³[H]-thymidine uptake of less than 500 CPM. e. DCs generated from BM HPC of wild-type and MSR1 KO mice in the presence of TES as described in Fig. 3j were used as stimulators in an allogeneic mixed leukocyte reaction. T-cell proliferation was measured by ³[H]-thymidine uptake. Two independent experiments were performed in triplicate. The values of T-cell proliferation induced by wild-type or MSR1-deifient DCs generated without the presence of TES were set as 100%. Values of T-cell proliferation induced by DCs generated in the presence of TES are presented as percentage from control level. f,g. DCs with high and normal lipid content were sorted from spleens of naïve and EL-4 TB mice and incubated at indicated ratios with T cells from OT-II (f) or OT-I (g) transgenic mice in the presence of 10 μg/ml of specific peptides. T cell proliferation was measured in triplicate by ³[H]-thymidine uptake. In the absence of peptides ³[H]-thymidine uptake in all samples was less than 5×10³ CPM. Three experiments with the same results were performed. h. CD11c⁺ DCs isolated from spleens of naïve and EL-4 TB mice were cultured overnight with 1 mg/ml OVA or in medium alone and then used to stimulate T cells from OT-II transgenic mice. T cell proliferation was measured in triplicate by ³[H]-thymidine uptake. i.j. DCs with high and normal lipid content were sorted from the spleens of EL-4 TB mice and incubated overnight with 1 mg/ml OVA. Cells were used to stimulate T cells from OT-II (i) and OT-I (j) transgenic mice. T-cell proliferation was measured in triplicate using ³[H]-thymidine uptake. Two experiments with similar results were performed.

a. Lipid levels in DCs generated in vitro from HPC and cultured for 3 days with EL-4 TES in the presence of 5 μg/mL TOFA. b. Proliferation of allogeneic (BALB/c) T cells cultured with DCs generated from HPC in the presence of TES and TOFA. Proliferation was measured in triplicate by ³[H]-thymidine uptake. T cells alone had ³[H]-thymidine uptake of less than 10³ CPM. c. Tumor-free (control) and EL-4 TB (TB) mice were treated for 2 weeks with TOFA using osmotic pumps (rate of release 1 μg/hr). Pumps filled with water were used as controls. The proportion of DCs with high lipid content was calculated. For each experiment 2 mice per group were used. Experiments were performed twice and the Mean±SD of the cumulative results is shown. * - statistically significant differences between TB and TB+TOFA groups. d. CD11c⁺ DCs isolated from the spleen of mice as described in Fig. 6c were incubated with allogeneic T cells and cell proliferation measured in triplicate by ³[H]-thymidine uptake. T cells alone had ³[H]-thymidine uptake of less than 10³ CPM. e. EL-4 tumor was established in C57BL/6 mice.. Mice (5 mice per group) were immunized with 5×10⁵ DCs transduced Ad-surv 4 days after tumor inoculation.. Vaccination was repeated twice at weekly intervals. Fourteen day osmotic pumps (Alzet, Cupertino, CA) (1μg/hr rate release) containing water or TOFA were implanted on day 14 after tumor cell injection. Tumor size was monitored and is presented as Mean ± SD per group. f. B16F10 tumor was established in C57BL/6 mice. Five days after tumor inoculation mice (8 mice per group) were immunized with a mixture of specific peptide and two Toll-like receptor ligands (TLR-L) [50 μg poly-IC, 100 μg CpG-1826]. Fourteen day osmotic pumps (1μg/hr rate release) filled with PBS were used in combination with lynch coil polyethylene tubing containing water or TOFA. Pumps were implanted on day 14 after tumor cell injection. Tumor size was monitored and results are presented as the mean tumor size (area in mm²) ± SD for every treatment group at various time points until the termination of the experiment.

a. Lipid level in DCs generated from enriched BM HPC and cultured in the presence of different concentrations of TES for indicated period of time. b. The total level of TAG using ESI-MS in CD11c⁺ DCs generated from BM HPC in the presence of 20% TES. Measurements were performed in triplicate. c. Uptake of fluorescently labeled C16 fatty acid by DCs generated from BM HPC and cultured for 24 hr with control medium or TES. The left panel is a typical example of staining after 30 min incubation with C16 fatty acid. The right panel shows cumulative results of 3 performed experiments (Mean±SD). Ice – cells incubated with fatty acid at 4°C; 37°C – cells incubate with fatty acid at 37°C. d. Gene expression of indicated SR in DCs cultured for 48 h with control medium or TES. Samples were evaluated in triplicate by qRT-PCR and the results were normalized to the level of expression of β-actin. The fold changes between DCs generated with TES and control were calculated. The results of from 3 performed experiments are shown. e. Expression of Msr1 on the surface of DCs cultured with control medium (CM) or TES. IC – isotype control. The typical examples of three performed experiments are shown. f. Gene expression of indicated scavenger receptors in DCs from naïve and EL-4 TB mice by qRT-PCR. The results were normalized to the level of expression of β-actin. Fold changes between EL-4 TB DCs and control DCs were calculated. The results of 3 performed experiments are shown. g. Expression of MSR1 within the population of conventional (cDC) or plasmacytoid (pDC) spleen DCs from naïve and EL-4 TB mice. Shaded histogram – isotype control; grey line – naïve mice, bold black line – TB mice. h. Expression of MSR1 in spleen DCs with normal lipid content (TB-NL) and high lipid content (TB-HL) from EL-4 TB mice. Control – DCs from control mice. Mean ± SD of 4 performed experiments are shown. * - statistically significant differences (p<0.05) from values in control DCs. Fluorescence of cells labeled with isotype control IgG was less than 20 in all samples. i. Lipid level in DCs generated in vitro and cultured for 3 days with TES in the presence of control IgG, or indicated antibodies against scavenger receptors. Mean ± SD from three experiments is shown. * - statistically significant differences (p<0.05) from values in control DCs. j. Lipid levels in DCs generated from enriched BM HPC of wild-type (WT) or MSR1 knockout (KO) C57BL/6 mice and transferred i.v. into sub-lethally irradiated congenic tumor-free (control) or EL-4 TB (TB) recipients. Lipids were measured in donor’s cells isolated from spleens of recipients two days after the transfer. Each group includes 4–8 mice. Geometric mean of fluorescence is shown. P values between control and TB mice were calculated using two-tailed Mann-Whitney test.