Fig 1.
Hypoxia downregulates MHC class I expression on tumor cells in vivo.
Whole body exposure of mice with 11 day established MCA205 pulmonary tumors (A, C; n = 6 per group per experiment) or 15 day established subcutaneous tumors (B, D; n = 5 per group per experiment) to 10% oxygen for 48h significantly downregulated MHC class I expression on tumor cells as compared with mice breathing 21% oxygen. MHC class I levels were determined by flow cytometry. Representative histograms (A and B) and associated quantification and statistics (C and D) of 2 independent experiments are shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.002 (C), p = 0.005 (D). Grey filled: Unstained control; Red: Hypoxia; Blue: Normoxia. MFI: mean fluorescence Intensity. Error bars indicate SD.
Fig 2.
Hypoxia downregulates MHC class I expression in vitro in 3D but not in 2D culture systems and requires the deeper hypoxia achieved in the 3D system.
(A-C): MCA205 tumor cells were cultured as 2D monolayers (A) or as 3D spheroids (B,C) and cultured under 21% O2 or 1% O2 for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms (B) and associated quantification and statistics (C) of 4 independent experiments are shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.013 (C). Grey filled: Unstained control; Red: Hypoxia; Blue: Normoxia. MFI: mean fluorescence intensity. Inset: 40X magnification of MCA205 grown at 1% O2 in 2D culture (A) or in 3D culture (B). Error bars indicate SD. (D) Representative flow cytometry histograms of Hypoxyprobe-1 (HP) indicating significantly increased levels of hypoxia in MCA205 cells grown under 1% oxygen for 48h as 3D spheroids (Red histogram) as compared with 2D monolayers (Blue histogram). n = 4. (E) Contour plots representing intensity of hypoxia within MCA205 cultures grown as 2D monolayers show 98% of the population was intermediately hypoxic. (F) MCA205 cells grown as 3D spheroids show two distinct populations of intermediately hypoxic (56%; HP MFI = 309) and severely hypoxic (39.5%; HP MFI: 1412) regions. (G, H) Gating on the 2 distinct hypoxic populations in the spheroid revealed inverse correlation between MHC class I and hypoxia levels. Less hypoxic cells had significantly higher percentage of MHC class I positive cells (G) and more hypoxic regions had lower percentage of MHC class I positive cells (H). (D-H) Representative data of 4 independent experiments.
Fig 3.
Hypoxia-mediated downregulation of MHC class I expression impairs recognition and killing of tumor cells by CTLs.
(A) Hypoxic-grown MCA205-OVA cells with downregulated MHC class I expression were poorly recognized and killed by effector OT.1 T cells compared to normoxic controls. OVA expressing MCA205 tumor cells were grown in 3D cultures for 48h in 21% O2 conditions. A subset of these cells were then moved to hypoxic (1% O2) conditions for an additional 24h. The hypoxic and normoxic spheroids were subsequently co-cultured with activated OT-I T cells. Tumor cells were identified by CellTracker staining (stained prior to co-culture) and cytotoxicity was assessed based on percent propidium idodide positive tumor cells. Each data point represents a replicate. Data is representative of 3 independent experiments. (B) Flow cytometry assessment of surface expression of MHC-SIINFEKL on OVA transfected MCA-205 cells. The tumor cells were grown as 3D spheroids for 48h in either 1% or 21% O2 conditions. Each data point represents an independent experiment. n = 4. Error bars indicate SD. The significance of differences was analyzed by the Student’s t-test (two-sided); *p = 0.02, ** p = 0.003 (A), p = 0.005 (B).
Fig 4.
Hyperoxia upregulates MHC class I expression equally in 2D and 3D cultures.
(A-D): MCA205 tumors were grown as 2D monolayers (A, C) or as 3D spheroids (B, D) at 21% O2 or 60% O2 for 48h. MHC class I levels were determined by flow cytometry. The magnitude of MHC class I upregulation was similar in 2D and 3D cultures. Representative histograms (A, B) and associated quantification and statistics (C, D) of 4 independent experiments shown. The significance of differences was analyzed by the Student’s t-test (two-sided); p = 0.002 (C), p = 0.001 (D). Grey filled: Unstained control; Blue: Normoxia (21% O2); Green: Hyperoxia (60% O2). Error bars indicate SD.
Fig 5.
Molecular oxygen regulates MHC class I expression transcriptionally.
(A) Mice bearing MCA205 pulmonary tumors were exposed to either respiratory hypoxia (10% O2), normoxia (21% O2), or respiratory hyperoxia (60% O2) for 48h. (B) MCA205 tumors grown in vitro in 3D spheroids under hypoxia (1% O2), normoxia (21% O2), or hyperoxia (60% O2) for 48h. Hypoxia significantly downregulated whereas hyperoxia significantly upregulated MHC class I transcripts as compared normoxic controls both in vivo and in vitro. RT-qPCR was used to analyze MHC class I (H-2Kb) transcript levels. Ribosomal protein L32 was used as internal control. Y- axis represents transcript levels relative to normoxic controls. n = 4. The significance of differences was analyzed by the Student’s t-test (two-sided); p values are as indicated in the figure. Error bars indicate SD.
Fig 6.
Hypoxia downregulates MHC class I expression via HIF transcription factors.
(A-C): siRNA mediated knockdown of HIF-1α reversed hypoxic downregulation of MHC class I expression as compared with the scrambled, non-targeting (NT) siRNA control. MCA205 tumor cells were reverse transfected with scrambled siRNA (NT; red histogram) or with HIF-1α specific siRNA (blue histogram) and cultured as 3D spheroids under 1% (A) or 21% (B) oxygen for 48h. Levels of MHC class I surface expression was determined using flow cytometry. Efficacy of gene knockdown was assessed using Western blot (C). β-Actin was used as the loading control. Representative data of 3 independent experiments shown. (D-F): Flow cytometry assessment of surface expression of HLA-ABC on paired isogenic renal cell carcinoma cell lines RCC4 (D), UMRC2 (E) and CAKI2 (F). Each pair had the parental cell line that lacked endogenous wild-type VHL (VHL null, transfected with empty vector) and one with vector stably expressing functional VHL (VHL restored). Restoring VHL function and thereby reducing HIF expression, significantly increased HLA-ABC expression on the cells. Representative histograms of 4 independent experiments are shown. Grey filled: unstained control; red: VHL null genotype; blue: VHL restored genotype. (D1-F1): Inactivation of HIF-1α by restoring VHL expression was verified by Western blotting for RCC4 (D1), UMRC2 (E1) and CAKI2 (F1) cells. β-Actin was used as the loading control.
Fig 7.
Hypoxia downregulates and hyperoxia upregulates expression levels of TAPs and LMPs.
(A, C) MCA205 tumor cells were cultured in vitro as 3D spheroids for 48h under 1%, 21% or 60% oxygen. (C) Relative band intensity, normalized to the loading control and 21% oxygen samples is shown. (B, D) For in vivo experiments, tumor nodules (MCA205 pulmonary tumors) were harvested from mice exposed to respiratory hypoxia (10% oxygen), normoxia (21% oxygen) or hyperoxia (60% oxygen) for 48h. (D) Relative band intensity, normalized to the loading control and 21% oxygen samples is shown. Protein levels were determined by Western blot. β-actin was used as loading control. Representative blots with samples from 2 independent experiments are shown.