Oxidized lipids block antigen cross-presentation by dendritic cells in cancer

Abstract

Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.

Figure 1. Effect of tumor-derived factors on cross-presentation in DCs

A. Stimulation of OT-1 CD8⁺ T cell proliferation by CD11c⁺ DCs cultured with TES from EL-4 tumors for 48 h. Cells were cultured at indicated ratios in the presence of 0.1 μg/ml of control (RAHYNIVTF) or specific (SIINFEKL) peptides. Proliferation was measured in triplicate by ³H-thymidine uptake. Proliferation of T cells, in the presence of control peptide, was less than 1,000 CPM and is not shown. Three experiments, with the same results, were performed. B. Expression of pMHC in DCs loaded with SIINFEKL. CM – DCs incubated in complete medium. TES – DCs incubated with EL-4 TES. Three experiments, with the same results, were performed. C. Stimulation of OT-1 T cells by DCs, incubated for 2 days in complete medium (CM), or TES and loaded for 24 h with 100 μg/ml OVA. Cell proliferation was measured in triplicate by ³[H]-thymidine uptake. Typical results of 4 performed experiments are shown. D. Cumulative results of pMHC expression of DCs surface, after loading of cells with 100 μg/ml OVA. TES were obtained from EL-4 tumors. pMHC expression was measured within gated CD11c⁺ DCs. E. Typical example of TES effect on pMHC expression in DCs loaded with 5 μg/ml long OVA peptide. Three experiments, with the same results, were performed. F. pMHC staining of CD11c⁺ DCs, cultured for 24 h with EL-4 TES, and loaded with long peptide for an additional 24 h. Cells were stained with a 25-d1.16 primary and goat Alexa 488 conjugated anti-mouse secondary antibody. Scale bar = 100 μm. G. Effect of TES on the ability of DCs cross-present long gp100 derived peptide. Pmel-1 transgenic T cells were used as responders. Cell proliferation was measured in triplicate by ³[H]-tyhymidine uptake. Two experiments, with the same results, were performed. H. Effect of EL-4 TES (48 h incubation) on the presentation of endogenous antigen in OVA-Tg DCs or after loading of wild-type DCs with OVA. OT-1 T-cell proliferation was measured in triplicate. One DCs:OT-1 splenocytes ratio (1:20) is shown. Three experiments, with the same results, were performed. I. Effect of TES on presentation of pMHC by OVA-Tg DCs after 48 h incubation. For each TES, at least three experiments, with the same results, were performed. J. Effect of mild acid treatment (0.263 M citric acid and 0.123 M disodium phosphate, pH 3.0 for 2 min followed by extensive wash in PBS) on the removal of peptide from MHC class I on OVA-Tg DCs. Two experiments, with the same results, were performed. K. The effect of TES on pMHC expression in OVA-Tg DCs after mild acid treatment. For each TES, two experiments, with the same results, were performed. In all experiments, * designates statistically significant (p<0.05) differences from control.

Figure 2. Cross-presentation in DCs isolated from tumorbearing mice

A. Expression of pMHC on the surface of CD11c⁺ DCs isolated from spleens of naïve or EL-4 TB (tumor ≈ 1cm in diameter) mice and loaded with 5 μg/ml of OVA long peptide. On the left, typical example of staining. On the right, the cumulative results of four experiments in indicated populations of DCs. B. Expression of pMHC on the surface of DCs isolated from spleens of naïve or EL-4 TB (tumor ≈ 1cm in diameter) OVA-Tg mice. On the left, typical example of staining. On the right, the cumulative results of four experiments in indicated populations of DCs. In all experiments, * designates statistically significant (p<0.05) differences from control.

Figure 3. Effect of TES on antigen processing in DCs

A. Typical example of lipid level in gated CD11c⁺ DCs treated with EL-4 TES for 48 h after staining with BODIPY. Seven experiments with similar results were performed. B. Cross presentation of antigens by DCs with different levels of lipids. DCs treated with TES and loaded with OVA were stained with BODIPY. Left panel - example of gates set for discriminating DCs with normal lipid levels (NL-DCs) from DCs with high lipid levels (HL-DCs). DCs were considered NL-DCs when their fluorescence overlapped the fluorescence of the control DCs. Control DCs in red; DCs treated with TES in blue. Three experiments, with the same results, were performed. C. Cross presentation of long OVA peptide by DCs with different levels of lipids. Left panels show the gate of NL-DCs and HL-DCs and pMHC expression is shown in the right panel. D. TES does not affect cross-presentation in CD204 deficient DCs. DCs generated from Msr1^−/− mice were treated with TES, loaded with OVA, and used for stimulation of OT-1 T cells as described in Fig. 1C. Proliferation of OT-I T cells was measured in triplicate. Typical results of 4 performed experiments are shown. E. Typical examples of pMHC (left panel) and H2K^b (right panel) expression in CD204 deficient DCs. Grey shaded line - DCs without OVA treatment; blue line - DCs loaded with OVA in control medium (CM); red, green, and orange lines - DCs loaded with OVA and pre-treated with TES from EL-4, MC38, and CT26 tumors, respectively.

Figure 4. Lipid accumulation in mouse DCs

A-D. Co-localization of LB and cell compartments involved in cross-presentation after 48 h culture of DCs with TES. Lipids were stained with HCS LipidTOX (red fluorescence); lysosomes with LAMP2 antibody (A); trans-Golgi complex with giantin antibody (B); early endosomes with Rab5a antibody (C); ER with calnexin antibody (D). Alexa Fluor® 488 (green fluorescence) labeled secondary antibody, was used in all cases; except for D, where Alexa Fluor® 594 (red fluorescence) and BODIOY lipid dye (green fluorescence) were used. Scale bar = 100 μm. Four experiments with the same results were performed. E. The proportion of different classes of free FA (left panel) and individual unsaturated FA (right panel) in control DCs; F. The presence of different unsaturated FA in DCs. G. Oxidized C_18:2 LA and C_20:4 AA in spleen DCs and sera from EL-4 TB mice (three mice per group). H. The presence oxLA in DCs incubated with TES. I. Accumulation of mono-oxygenated TAGs in mouse DCs cultured in the presence of control medium (CM) or EL4, MC38 TES. Typical LC-ESI-MS profiles of TAG with m/z 916 (upper left panel), its MS² spectrum (lower left panel) and its suggested structure (middle panel). Right panel - amount of oxTAG 54:6, 54:5, 54:3 and 56:5 at m/z 912, 914, 918 and 942. J. Accumulation of truncated oxTAGs in mouse DCs, cultured in the presence of control medium (CM) or EL4, MC38 TES. Typical LC-ESI-MS profile of truncated TAG with m/z 764 (upper left panel), its MS² spectrum (lower left panel), and possible structure (middle panel). Fragmentation of the parent ion at m/z 764 [M+NH₄]⁺ reveals product ions at m/z 603 and 465. The product ion, at m/z 603, was formed by loss of the truncated acyl chain (that corresponded to 7-oxo-heptanoic acid). The product ion, at m/z 465, was formed by loss of oleic acid. Right panel – amount of truncated TAGs. K. Content of LA-CE (left panel) and oxLA-CE (C_18:2-2O) (right panel) in DCs treated with TES.

Figure 5. Accumulation of oxidized lipids in human DCs

A. Total lipids in donor’s DCs treated with SK-MEL TCM. Typical example of BODIPY staining. B. Major FFAs detected in DCs; C. oxLA in DCs. MS² spectrum of parent ions at m/z 295 (left panel), its possible structure (middle panel), the amount of oxLA (9-HODE) in DCs (right panel); D. Cholesteryl Esters (CE) levels in human DCs cultured with and without TCM; E. oxCE in DCs. Possible structure of CE 18:2-OOH (upper left panel); MS1 and MS2 spectra of CE 18:2-OOH (lower left panel); amount of CE 18:2-OOH in DCs (right panel). ND - not detected. F. Accumulation of individual molecular species of TAGs in DCs. G. OxTAGs in DCs. Typical LC-ESI-MS profile (left panel); MS² spectrum of TAG at m/z 932 (middle panel); amount of oxTAGs in DCs (right panel). Fold increase over values in DCs cultured in CM are shown. B-G Two experiments were performed.

Figure 6. Effect of oxidized LA on cross-presentation by DCs

A. Accumulation of large LB in DCs cultured with LA and LA+AMVN. DCs were cultured for 24 h in serum free medium with 10μg/ml LA and then treated with AMVN (0.2mM) for 2h, prior to washing and loading with OVA (100 μg/ml). Cells were analyzed 18 h later. Staining with BODIPY (scale bar = 100 μm); B. Amount of oxLA in the absence and in the presence of AMVN, inset - LC-MS profile and MS2 spectrum; C. Contents of major molecular species of oxTAGs containing LA; D. Amount of oxCE C_18:2-2O in DCs after loading with LA in the absence and in the presence of AMVN; E. Stimulation of OT-1 T cells, by DCs pre-treated with LA and AMVN and loaded with OVA. Experiments were performed in triplicate and repeated twice. F. Stimulation of OT-1 T cells, by DCs pre-treated with LA and AMVN and loaded with SIINFEKL peptide. Experiments were performed in triplicate and repeated three times. G. pMHC expression in CD11c⁺ DCs, pre-treated with LA and AMVN and loaded with long OVA-derived peptide. Experiments were performed twice. H. Stimulation of pmel-1 T cells by DCs, pre-treated with LA and AMVN and loaded with long gp100-derived peptide. Experiments were performed in triplicate and repeated twice.

Figure 7. The role of oxidized lipids in cross-presentation

A. The protection effect of vitamin E on cross-presentation. DCs were treated with vitamin E (50μM) for 24 h, followed by 2 h treatment with 0.5 μM AMVN. Cells then were loaded with 100 μg/ml OVA overnight and used for stimulation of OT-1 T cells. Typical results of T-cell proliferation of 4 performed experiments are shown. T cells alone had ³[H]-thymidine uptake of less than 500 CPM. B. Percentage of CD11c⁺ DCs expressing pMHC, after the treatment with vitamin E and AMVN, as described above. Left panel - DCs loaded with 100μg/ml OVA; right panel – DCs loaded with 5μg/ml OVA-derived long peptide. Cumulative result of 4 experiments is shown. C. pMHC expression on DCs, generated from bone marrow of OVA-Tg mice and treated with 0.5 mM AMVN. Three experiments with the same results were performed. D. Proliferation of OT-1 T cells, stimulated with DCs from wild-type mice loaded with OVA and from OVA-Tg mice. DCs were pre-treated with AMVN (0.5 mM) for 2 h. Typical results of 4 performed experiments are shown. In all experiments, * designates statistically significant (p<0.05) differences from control.

Figure 8. The mechanisms of oxidized lipids effect on cross-presentation by DCs

A. pMHC expression on DCs treated with 5μM of proteasome inhibitor lactacystin for 48 h. Wild-type DCs were directly loaded with 100 μg/ml OVA or 5μg/ml long OVA-derived peptide, during the last 24 h of culture, in the presence of lactacystin. Two experiments, with the same results were perfroemd. B. The effect of lactacystin on the ability of DCs, loaded with OVA (100μg/ml), to stimulate proliferation of OT-1 T cells. C, D. The effect of IFN-γ on cross-presentation of long OVA-derived peptide, by DCs treated with TES. DCs were cultured with IFN-γ (250 ng/ml) for 1 day, followed by TES treatment and loading with long OVA-derived peptide. C. Top panel – pMHC expression; bottom panel MHC class I (H2K^b) expression. CT26, LLC, and EL-4 designate type of TES used. D. The effect of IFN-γ on the ability of DCs to stimulate proliferation of OT-1 T cells after loading with OVA. E, F. The effect of IFN-γ on cross-presentation of DCs, loaded with LA and treated with AMVN. E. Top panel – MHC class I (H2K^b) expression; bottom panel - pMHC expression. F. The effect of IFN-γ on the ability of DCs to stimulate proliferation of OT-1 T cells after loading with OVA.