Modeling of immune elimination of highly antigenic tumor cells
Ellen Moore1, Megan Morisada1, Jay Friedman1, Clint Allen1,2*
1Head and neck Surgery Branch, NIDCD, NIH; 2Department of Otolaryngology, Johns Hopkins School of Medicine
Selective immune elimination of more antigenic cells during tumor development and possibly progression likely plays a major role in allowing tumors to reach the equilibrium and escape phases of immunoediting. We aimed to engineer a carcinoma model with a fluorescent marker linked to expression of a strong model antigen to study these processes. MOC1 oral carcinoma cells were stably transduced with a construct encoding mKate2, a monomeric far-red fluorescent protein, and the H2-Kb-rstricted ovalbumin class I epitope SIINFEKL in the same open reading frame. Surface presentation of SIINFEKL via H2-Kb in mKate2 positive but not negative cells was verified via flow cytometry with the 25-D1.16 mAb. Imperfect transduction yielded a cell line (MOC1mKate2SIIN) with a stable mixture of mKate2 high and low cells. Peptide-pulsed antigen presenting cell stimulation of TIL extracted out of MOC1mKate2SIIN-derived tumors grown in WT B6 mice revealed greater IFNg responses to SIINFEKL than to KSPWFTTL (p15E604-611), a known MOC1 tumor-associated antigen. Using a real-time impedance-based cytotoxicity assay with OT-1 CTLs as effectors and MOC1mKate2SIIN as targets at a 10:1 E:T ratio, outgrowth of mKate2 low/negative cells after approximately 48 hours suggested that mKate2 high/positive cells were selectively eliminated. Flow cytometric analysis of MOC1mKate2SIIN-derived tumors grown in WT B6 mice revealed near-complete loss of all mKate2 positive cells. Conversely, MOC1mKate2SIIN-derived tumors grown in Rag-/- B6 mice retained a mixture of mKate2 positive and negative cells, similar to the parental cell line. These experiments demonstrate in an experimental in vitro system and in a WT in vivo system the selective immune elimination of highly antigenic tumor cells, reinforcing the concepts of immunoediting. This model is a powerful tool for the study of therapeutics designed to induce polyclonal T-lymphocyte responses against both strong and weak tumor antigens.
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