Abstract
Glioblastoma multiforme (GBM) is the most common type of brain tumor and is a leading cause of mortality with mean patient survival 6-12 months following diagnosis. We have recently shownthat combined treatment of glioma bearing rats with Ad-Flt3L and Ad-TK+GCV dramatically improved survival in an intracranial syngeneic rat model. Ad-Flt3L encodes human soluble fms-like tyrosine kinase 3 ligand (Flt3L) and stimulates dendritic cell proliferation and improves antigen presentation. Ad-TK expresses herpes simplex type I-thymidine kinase and selectively kills rapidly dividing cells when used in combination with the non-toxic prodrug ganciclovir (GCV). We now show that combined treatment with Ad-TK and Ad-Flt3L improves long-term survival in C57BL/6 mice bearing syngeneic intracranial GL26 tumors when all individual therapies completely fail (p<0.001). Survival is not improved in RAG1 deficient mice (RAG1-/-), deficient for mature B and T cells, or mice deficient for either CD4+ and CD8+ cells, implicating that both CD4 and CD8 T cells play a critical role in tumor regression. Tumor antigen Trp2 is expressed by GL26 cells and clonal expansion of Trp2(180-188) reactive T cells was determined using ELISPOT, MHC I - Trp2(180-188) tetramers and T cell proliferation assays following treatment with Ad-Flt3L and Ad-TK+GCV. Furthermore, Ad-Flt3L and Ad-TK did not improve survival in tumor bearing Myd88- or TLR2-deficient mice, indicating that TLR2 and Myd88 signaling was involved in the development of effective anti-tumor immune responses. In summary, combined treatment with Ad-Flt3L and Ad-TK+GCV improves survival in a large syngeneic intracranial tumor model in mice. This was dependent on the establishment of tumor antigen specific T cell anti tumor immune responses that was dependent, in turn, on TLR2 signaling via the adapter protein Myd88.