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

<div><p>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 <i>in vivo</i> and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures <i>in vitro</i>; (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.</p></div

Hypoxia-mediated downregulation of MHC class I expression impairs recognition and killing of tumor cells by CTLs.

<p>(<b>A</b>) 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>B</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).</p

Hypoxia downregulates and hyperoxia upregulates expression levels of TAPs and LMPs.

<p><b>(A, C)</b> MCA205 tumor cells were cultured <i>in vitro</i> as 3D spheroids for 48h under 1%, 21% or 60% oxygen. <b>(C)</b> Relative band intensity, normalized to the loading control and 21% oxygen samples is shown. <b>(B, D)</b> For <i>in vivo</i> 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. <b>(D)</b> 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.</p

Hypoxia downregulates MHC class I expression via HIF transcription factors.

<p><b>(A-C):</b> 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% <b>(A)</b> or 21% <b>(B)</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 <b>(C)</b>. β-Actin was used as the loading control. Representative data of 3 independent experiments shown. <b>(D-F):</b> Flow cytometry assessment of surface expression of HLA-ABC on paired isogenic renal cell carcinoma cell lines RCC4 <b>(D)</b>, UMRC2 <b>(E)</b> and CAKI2 <b>(F)</b>. 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. <b>(D1-F1):</b> Inactivation of HIF-1α by restoring VHL expression was verified by Western blotting for RCC4 <b>(D1)</b>, UMRC2 <b>(E1)</b> and CAKI2 <b>(F1)</b> cells. β-Actin was used as the loading control.</p

Molecular oxygen regulates MHC class I expression transcriptionally.

<p><b>(A)</b> Mice bearing MCA205 pulmonary tumors were exposed to either respiratory hypoxia (10% O₂), normoxia (21% O₂), or respiratory hyperoxia (60% O₂) for 48h. <b>(B)</b> MCA205 tumors grown <i>in vitro</i> 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 <i>in vivo</i> and <i>in vitro</i>. 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.</p

Hyperoxia upregulates MHC class I expression equally in 2D and 3D cultures.

<p><b>(A-D):</b> MCA205 tumors were grown as 2D monolayers <b>(A, C)</b> or as 3D spheroids <b>(B, D)</b> 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 (<b>A, B</b>) and associated quantification and statistics (<b>C, D</b>) 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.</p

Hypoxia downregulates MHC class I expression <i>in vitro</i> in 3D but not in 2D culture systems and requires the deeper hypoxia achieved in the 3D system.

<p><b>(A-C):</b> MCA205 tumor cells were cultured as 2D monolayers <b>(A)</b> or as 3D spheroids <b>(B,C)</b> and cultured under 21% O₂ or 1% O₂ for 48h. Levels of MHC class I expression was determined using flow cytometry. Representative histograms <b>(B)</b> and associated quantification and statistics <b>(C)</b> 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 <b>(A)</b> or in 3D culture <b>(B)</b>. Error bars indicate SD. <b>(D)</b> 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. <b>(E)</b> Contour plots representing intensity of hypoxia within MCA205 cultures grown as 2D monolayers show 98% of the population was intermediately hypoxic. <b>(F)</b> 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. <b>(G, H)</b> 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 <b>(G)</b> and more hypoxic regions had lower percentage of MHC class I positive cells <b>(H)</b>. <b>(D-H)</b> Representative data of 4 independent experiments.</p