19 research outputs found

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

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

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    <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 <i>in vitro</i> in 3D but not in 2D culture systems and requires the deeper hypoxia achieved in the 3D system.

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    <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% O<sub>2</sub> or 1% O<sub>2</sub> 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% O<sub>2</sub> 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

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

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    <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% O<sub>2</sub> 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% O<sub>2</sub> 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 MHC class I expression via HIF transcription factors.

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    <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.

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    <p><b>(A)</b> Mice bearing MCA205 pulmonary tumors were exposed to either respiratory hypoxia (10% O<sub>2</sub>), normoxia (21% O<sub>2</sub>), or respiratory hyperoxia (60% O<sub>2</sub>) for 48h. <b>(B)</b> MCA205 tumors grown <i>in vitro</i> in 3D spheroids under hypoxia (1% O<sub>2</sub>), normoxia (21% O<sub>2</sub>), or hyperoxia (60% O<sub>2</sub>) 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.

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    <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% O<sub>2</sub> or 60% O<sub>2</sub> 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% O<sub>2</sub>); Green: Hyperoxia (60% O<sub>2</sub>). Error bars indicate SD.</p

    IT Administration of A<sub>2A</sub>R Agonist Protects from Increased Death Rate upon Oxygenation of Mice with Acute Lung Injury

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    <p>Compensation for the oxygenation-associated loss of the hypoxia → adenosine → A<sub>2A</sub>R signaling pathway by IT injection of CGS21680 significantly decreased the oxygen-exacerbated death rate in mice with acute lung injury induced by IT injection of SEB and LPS. For further explanation, see legend for <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-g001" target="_blank">Figure 1</a>.</p

    Intratracheal Injection of A<sub>2A</sub>R Selective Agonist Mimics Protective Effects of Hypoxia

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    <div><p>(A) IT injection of the A<sub>2A</sub>R agonist CGS21680 into endotoxin-inflamed lungs provides protection similar to that observed in hypoxia-treated mice. Number of PMNs recovered after 48 h by BAL from endotoxin-injected animals that were kept at normal 21% oxygen atmosphere was significantly diminished by IT injections of CGS21680 compared to placebo-treated mice. Lung PMNs (left graph) from A<sub>2A</sub>R agonist-treated animals also produced lower levels of reactive oxygen metabolites (H<sub>2</sub>O<sub>2</sub>; right graph).</p> <p>(B) Significantly decreased lung vascular permeability (protein in BAL; left graph) and improved lung gas exchange (p<sub>a</sub>O<sub>2</sub>; right graph) in endotoxin-injected mice after treatment with the A<sub>2A</sub>R agonist CGS21680.</p> <p>(C) Histologic evidence for the lung tissue-protecting effects of A<sub>2A</sub>R agonist during endotoxin- and oxygenation-induced lung damage. Quantitative analysis of lung histopathology by a pathologist blinded to the experimental design revealed inhibition of PMN sequestration in IT LPS-injected mice after treatment with the A<sub>2A</sub>R-selective agonist CGS21680 for 48 h. The lung tissue damage was also significantly decreased as assessed by the LIS (<i>n</i> = 9, mean ± standard deviation). Representative H&E-stained slices in the right two photomicrographs show less intracapillary PMN sequestration and almost no intraalveolar accumulation of PMNs in CGS21680-treated mice. These CGS21680-induced changes are similar to those observed for the effects of hypoxia on endotoxin- injected animals (compare with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-g006" target="_blank">Figure 6</a>C).</p></div

    Evidence for the Up-Regulation of Immunosuppressive A<sub>2A</sub>R Expression on In Vivo-Activated Granulocytes Isolated from Inflamed Lungs

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    <div><p>(A) The selective A<sub>2A</sub>R agonist CGS21680 inhibited the fMLP-stimulated hydrogen peroxide production by granulocytes in blood of healthy mice to only a small degree, reflecting low levels of expression of A<sub>2A</sub>R on naïve blood granulocytes. In contrast, granulocytes recovered by BAL from inflamed lungs 48 h after IT LPS injection were much more inhibited by CGS21680, demonstrating functional up-regulation of A<sub>2A</sub>R on in vivo-activated cells.</p> <p>(B) CGS21680 induces cAMP accumulation in in vivo-activated granulocytes isolated from lungs 48 h after IT LPS injection. No effects of the A<sub>2A</sub>R agonist were observed in naïve granulocytes obtained from bone marrow of healthy mice (left graph) or in in vivo-activated granulocytes recovered from inflamed lungs of <i>A<sub>2A</sub>R</i> gene-deficient mice (right graph). The CGS21680-stimulated cAMP production observed in lung granulocytes obtained from wild-type mice could also be antagonized by the selective A<sub>2A</sub>R antagonist ZM241385. Naïve bone marrow granulocytes were used for cAMP measurements, since it was impossible to isolate naïve cells from blood of healthy mice in sufficient numbers.</p> <p>(C) Higher levels of A<sub>2A</sub>R-specific mRNA in in vivo-activated granulocytes. In parallel with the much stronger A<sub>2A</sub>R agonist-induced inhibition of hydrogen peroxide production and accumulation of cAMP in in vivo-activated granulocytes, the relative levels of A<sub>2A</sub>R-specific mRNA were much higher in in vivo-activated granulocytes obtained from inflamed lungs 48 h after IT LPS injection, as compared with naïve granulocytes isolated from the bone marrow of healthy mice (left graph). Up-regulation of A<sub>2A</sub>R mRNA in in vivo-activated granulocytes was confirmed in another set of experimental animals breathing 21% oxygen, but was increased to a much lesser extent in animals subjected to 100% oxygen (right graph). Levels of A<sub>1</sub>R mRNA did not change much in inflammatory lung granulocytes from animals breathing normal atmosphere, but were clearly increased in mice exposed to 100% O<sub>2</sub>. In the two sets of experiments (left and right graphs), granulocytes were pooled from five and six mice per treatment, respectively. Taken together, the results demonstrate that granulocytes recovered from alveolar spaces of inflamed lungs did, indeed, up-regulate their A<sub>2A</sub>R expression during these in vivo lung injury assays, thereby confirming and extending previous findings in other inflammation models [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-b11" target="_blank">11</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030174#pbio-0030174-b53" target="_blank">53</a>]</p></div
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