Reovirus strain R-92 in in vitro generation of tumor-specific T-lymphocytes.

person Anastasia O. Sitkovskaya Rostov Research Institute of Oncology, Rostov-on-Don, Russian Federation info_outline Anastasia O. Sitkovskaya, Elena Yu. Zlatnik, Sergey A. Kolpakov, Elena P. Kolpakova, Irina V. Mezhevova, Elena S. Bondarenko, Inna A. Novikova, Andrey Dashkov, Dmitry O. Kaymakchi, Gapiz M. Chupanov, Nairi B. Oganyan, Liubov Yu Vladimirova

Authors person Anastasia O. Sitkovskaya Rostov Research Institute of Oncology, Rostov-on-Don, Russian Federation info_outline Anastasia O. Sitkovskaya, Elena Yu. Zlatnik, Sergey A. Kolpakov, Elena P. Kolpakova, Irina V. Mezhevova, Elena S. Bondarenko, Inna A. Novikova, Andrey Dashkov, Dmitry O. Kaymakchi, Gapiz M. Chupanov, Nairi B. Oganyan, Liubov Yu Vladimirova Organizations Rostov Research Institute of Oncology, Rostov-on-Don, Russian Federation, Rostov Research Institute of Microbiology and Parasitology, Rostov-on-Don, Russian Federation Abstract Disclosures Research Funding Other Background: The purpose of the study was to assess the potential of the reovirus strain R-92 for in vitro generation of tumor-specific T-lymphocytes. Methods: The human reovirus strain R-92 used as a master seed strain was previously isolated from a patient with infectious hepatitis and adapted to the growth on the pig embryo kidney cell culture (PEKC); it lost its pathogenicity after multiple passages and was characterized by electron microscopic, serological methods and PCR. HeLa lysate was prepared by the incubation with the R-92 reovirus in medium 199+L-glutamine during 5 days (37 о C, 5% СО 2 ), cytopathogenic effect was observed. Immature dendritic cells (DCs) were derived from blood monocytes cultivated for 7 days in presence of IL-4 and GM-CSF. For the DC loading during 48 hours we used: 1. HeLa lysate obtained by repeated freezing and thawing of cells (control); 2. HeLa lysate obtained by co-culturing with reovirus; 3. reovirus cultured in a standard PEKC. To assess the DC ability to activate lymphocytes, they were co-cultured for 5 days in a 3:1 ratio, 1.5x10 5 of target cells (HeLa) were added (1:1) for 48 hours; number of dead HeLa cells was count. At each stage of the generation of DCs and activated lymphocytes, they were phenotyped by flow cytometry. Results: DC samples 2 and 3 showed increased percentage of activated cells compared to the loaded with the control tumor lysate, CD86 and HLA-DR coexpression was 62 and 69% vs. 25%, respectively; CD83 expression was maximal after DC loading with reovirus (72.9%). After co-culturing of DC with autologous lymphocytes, the highest number of activated CD4+CD38+ and CD8+CD38+, was observed in sample 2, T-regs (CD4+CD25+CD127 low ) - in the control. The death of target cells cultured with activated lymphocytes obtained by DC samples 2 and 3, was 100%, control sample showed minimal cytotoxicity against HeLa. The number of T-lymphocytes in samples 2 and 3 cultured with HeLa exceeded the control values by 3-10 times. Conclusions: Reovirus R-92 used for the immature DC loading increases their presenting activity. DCs loaded with reovirus HeLa lysate, when co-cultured with lymphocytes, activate CD4+ and CD8+ and suppress T-regs, apparently leading to the total death of target tumor cells.