Oxygen is a master regulator of the immunogenicity of primary human glioma cells

Abstract

With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (∼20% O₂) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (∼5% O₂) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O₂ more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O₂ took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O₂ or physiologic 5% O₂. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.

Figure 1. Tissue culture gene expression compared with tumor in situ

Heat maps showing log base 2-transformed data for each experiment with a p-value < 0.001 and a fold change >3 for the comparison of 5% versus 20% O₂.

Figure 2. Primary glioma cell lines cultured in 5% oxygen express higher levels of glioma-associated antigens and makers of stemness

A, cells from four gliomas (patients 3, 4, 6, and 7) were cultured in 5% or 20% O₂ and analyzed for mRNA expression by real time PCR (n=4/group) and B, protein by flow cytometry where mean fluorescent intensity (MFI) is shown (n=3–4/group) or C, by western blot. * P < 0.05; ** P < 0.01

Figure 3. Tumor Lysates from 5% oxygen prime CTLs with superior tumoricidal function

A, PBMCs were stimulated with DCs pulsed with TL from 5% or 20% O₂ and incubated with HLA-A2 matched target glioblastoma cells (Patients 3 and 4) or an ependymoma (Patient 5). B, to determine if the increased tumoricidal response was MHC-I-dependent, pan-MHC I blocking antibody was added to target cells prior combining with PBMC. C, DCs pulsed with TL from 5% or 20% O₂ from patient 3 and incubated with patient 3 target cells cultured in 20% or 5% O₂. All experiments were repeated at least twice with similar results. Error bars, ± SEM. * P < 0.05; ** P < 0.01.

Figure 4. Tumor lysates from 5% oxygen cultures increase CD8 T cell priming

A, Dendritic cells were pulsed with CFSE-labeled TLs isolated from three patients derived in 5% or 20% O₂, stained with a-HLA-DR and analyzed by flow cytometry. PBMCs were co-cultured with DCs pulsed with 5% or 20% O₂ TLs +/− pp65 peptide for, B, 48 h and analyzed for secreted IFNγ or C, 72 h to determine IFNγ production on a per-cell basis in CD8⁺pp65 pentamer⁺ cells as indicated by gating strategy shown in sera-positive donor. All experiments were repeated at least twice with similar results. Error bars, ± SEM. * P < 0.05; ** P < 0.01.