Induced Major Histocompatibility Class II (MHCII) Expression Does Not Alter Chemosensitivity, Radiosensitivity, Redox Potential, or Proliferation Rate in a CIITA-Transfected Diffuse Large B Cell Lymphoma (DLBCL) Cell Line.

As previously demonstrated by gene expression profiling, MHCII is an independent gene expression signature in DLBCL, which when expressed at low levels, is associated with poor patient survival. The mechanism of decreased survival in low- expressing DLBCL patients is presumably loss of tumor immunosurveillance. However, the possibility remains that MHCII expression is merely a marker of another aspect of tumor biology such as chemosensitvity, radiation sensitivity, or proliferation rate. MHCII status could also reflect a change in the cellular redox environment since we have previously found an association between decreased antioxidant defense enzyme expression, increased thioredoxin system function and poor prognosis in DLBCL. We therefore investigated whether there are alterations in these other aspects of tumor biology by using a MHCII (−) DLBCL cell line model in which we restored MHCII expression by transfection with CIITA, the master transactivator of MHCII. To do this, we stably transfected the MHCII (−) DLBCL cell line, DB, (American Tissue Culture Corp), with a CIITA expression vector pcDNA 3.1 (InVitrogen) and isolated MHCII (+) clones. The phenotype was confirmed by RT-PCR and flow cytometry using HLA-DR as the representative molecule of MHCII expression (reported as MnX or mean channel of fluorescence). The parental line DB, clones 1–5, 4–3, 6-1, vector only control (pcDNA 3.1), and a positive cell line (Raji), were then subjected to in vitro chemosensitivity assays with the individual components of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and dexamethasone, the latter was used in the place of prednisone). Chemosensitivity was measured with MTS cell survival assay by calculating the EC 50 (molar concentration of drug required to kill 50% of cells). Radiation sensitivity was measured as percent cell survival 48 hours after exposure to 5 Gy. Proliferation rate was assessed by cell doubling time (in hours) using manual cell counts. The potential of the cells to reduce oxidants was assessed using the AOP-490 kit, which measured anti-oxidant biomarkers as uM uric acid EQ/ug protein (Oxis Research Inc, Portland, OR). No significant differences were detected in sensitivity to any of the tested treatment modalities, including the 4 different chemotherapy agents or radiation, between the parental or transfected clones. Nor were reducing potential or doubling times significantly altered with induction of MHCII expression. Representative data for the DB cell line, vector only control, and a representative clone are listed in Table1. Results for the other clones were similar. Our results help to exclude altered chemosensitivity, radiosensitivity, anti-oxidant potential, or proliferation rates as factors contributing to the unfavorable prognosis in patients with MHCII(−) DLBCL. Therefore, diminished tumor immunosurveillance remains the most likely explanation for the poor outcome in these patients.