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Cited by 121Immunotherapy and Immune ResponsesCancer, Hypoxia, and MetabolismCancer Immunotherapy and Biomarkers

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Hypoxia and hypoxia-inducible factor (HIF) downregulate antigen-presenting MHC class I molecules limiting tumor cell recognition by T cells

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Sethumadhavan, Shalini, Silva, Murillo, Philbrook, Phaethon, Nguyen, Thao, Hatfield, Stephen M., Ohta, Akio, Sitkovsky, Michail V.·2017·PLOS ONE
DOI 10.1371/journal.pone.0187314PMC PMC5695785
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Abstract

Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME in vivo and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures in vitro; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.

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Figures

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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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).CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Metadata

Authors
Sethumadhavan, S., Silva, M., Philbrook, P., Nguyen, T., Hatfield, S. M., Ohta, A., & Sitkovsky, M. V.
Affiliations
Northeastern University (US)
Corresponding author
Sitkovsky, Michail V.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2017-11-20
Volume
12
Issue
11
Pages
e0187314
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0187314
PMC
PMC5695785
OpenAlex
W2769873734
Cited by
119
References
48
Citations by year
2026: 6 · 2025: 20 · 2024: 12 · 2023: 19 · 2022: 11 · 2021: 15 · 2020: 19 · 2019: 13 · 2018: 4
FWCI
4.46
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • National Institutes of Health (R01GM)
  • National Cancer Institute (R01CA112561, R01CA111985)
  • National Institute of General Medical Sciences (R01GM097320)
  • National Center for Complementary and Integrative Health (R21AT002788)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Health Sciences › Medicine › Oncology › Cancer Immunotherapy and Biomarkers
Keywords
tumor hypoxia, hypoxia-inducible factor, MHC class I downregulation, antigen presentation, tumor microenvironment, immune evasion, T cell recognition, VHL, LMP7, hyperoxia, renal cell carcinoma, cancer vaccines, immune epitopes
MeSH terms
ATP Binding Cassette Transporter, Subfamily B, Member 2, Antigen-Presenting Cells, Genes, MHC Class I, Humans, Kidney Neoplasms, Oxygen, T-Lymphocytes, Transcriptional Activation, Cell Hypoxia, Gene Expression Regulation, Neoplastic, Proteasome Endopeptidase Complex, Hypoxia-Inducible Factor 1, Von Hippel-Lindau Tumor Suppressor Protein, Tumor Microenvironment

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