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 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

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

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

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

Decreased bacterial burden in mice with T-cell targeted deletion of HIF-1 α.

<p><u>A: </u><i>T cell lineage specific HIF-1</i> α <i>deficient mice have much less bacterial burden in liver and spleen 72 hrs after CLP</i> *:p<0.05 vs. WT, means±SEM, N = 3 per group. <u>B: </u><i>Growth of gas-forming bacteria and tissue destruction during CLP-induced sepsis in mice with HIF-1</i> α<i>-expressing T cells, but not in mice with HIF-1</i> α <i>gene–deleted T cells.</i> Masses of bacteria form rings around gas bubbles in spleens of mice with HIF-1 α expressing T cells. Much less bacteria could be seen in the spleen taken 72 h after CLP from mice with HIF-1 α deficiency in T cells.<u> C: </u><i>T-cell specific deficiency in HIF-1 α enhances effector functions of bactericidal granulocytes. </i><u>Left Panel:</u> Much stronger upregulation of activation marker CD11b on tissue granulocytes (CD11b⁺/Gr-1^bright cells) isolated from HIF-1 α-deleted mice compared to HIF-1 α -expressing control mice. *:p<0.05 vs. WT, N = 3. <u>Right Panel:</u> Higher spontaneous production of hydrogen peroxide by tissue granulocytes (CD11b⁺/Gr-1^bright cells) in HIF-1 α-deleted mice than in HIF-1 α-expressing control mice. *:p<0.05 vs. WT, N = 3.</p

Increased NF-κB m-RNA expression and activity of <i>ex vivo</i> activated T cells of mice with T-cell targeted deletion of HIF-1 α.

<p>For all three panels, T-cells from spleens were isolated from age and sex matched lck Cre (−) and lck Cre (+) HIF-1α loxP mice and stimulated as described (WT-S, κΟ−S). Unstimulated cells served as controls (WT, κΟ).<u> A: </u><i>T cell specific disruption of HIF-1 α gene substantially increases NF-κB binding activity in ex vivo TCR-activated T cells.</i> Nuclear extracts were prepared from harvested cells and EMSA was conducted. The experiment was repeated and representative data of two experiments are shown. All lanes contain hot binding probe for NF-κB. Specificity of EMSA was tested in the presence of 50 fold excess of either unlabeled probe (Con 1) or CRE specific probe (Con 2), respectively.<u> B: </u><i>T cell specific disruption of HIF-1 α gene increases NF-κB p50 and p65 binding activity in ex vivo TCR-activated T cells.</i> NF-κB-ELISA was conducted with nuclear extracts. *:p<0.05 vs. WT, N = 4.<u> C: </u><i>T cell specific disruption of HIF-1 α gene increases NF-κB p50 mRNA expression in ex vivo TCR-activated T cells.</i> RNA was prepared and subsided to quantitative RT-PCR. *:p<0.01 vs. WT. N = 4.</p

HIF-1α is a negative regulator of TCR-triggered pro-inflammatory cytokine secretion <i>in vitro</i> and <i>in vivo</i>.

<p><u>A: </u><i>T lymphocytes deficient in HIF-1α undergo more cell divisions as compared to wild type T cells.</i> Splenic T cells were purified, stained using CFSE, activated for 72 hours and analyzed by flow cytometry as described. A representative dot plot of wild type CD4 T cells (A) and HIF-1α deficient T cells (B) showing both activation by CD25 expression and cell divisions. (C) Comparison of number of divisions by CD4 T cells.*: p<0.05 vs. WT, N = 3 per group.<u> B: </u><i>T cell specific disruption of HIF-1 α gene substantially increases pro-inflammatory cytokine secretion by ex vivo TCR-activated T cells.</i> Spleen T cells were derived from T cell lineage specific HIF-1 α deficient mice. Cells were activated for 24 h under hypoxic conditions (1% O₂). Extracellularly secreted cytokines were determined by ELISA. *:p<0.05 vs. WT, N = 5.<u> C: </u><i>Higher intracellular levels of IFN-γ production by inflamed peritoneum-located hypoxic HIF-1 α deficient CD8+ T cells as compared with similarly located in vivo hypoxic HIF-1 α expressing T cells after CLP</i>. Peritoneal lavage was performed 72 hrs after CLP and 1,5×10⁶ T cells were restimulated and stained with anti-IFN-<i>γ</i> mAb. <u>D: </u><i>Levels of proinflammatory cytokines TNF-α and IL-6 are significantly higher as compared to mice with selective disruption of HIF-1α gene in T-cells.</i> Peritoneal fluid (TNF-α, left panel) and serum (IL-6, right panel) were withdrawn at the indicated times after CLP and cytokines were determined by ELISA. Closed circles: HIF-1α KO, open circles: WT. *:p<0.05 vs. WT, N = 4</p