1:Advanced Therapies Unit. Fundación para la Investigación Biomédica, Hospital Universitario Niño Jesús; 2:Pediatric Surgery Department, Hospital Niño Jesús; 3:Pathologic Department, Hospital Niño Jesús; 4:Pediatric Hematology-Oncology Department, Hospital Niño Jesús; 5:Tumor Immunology Laboratory, IdiPAZ, La Paz University Hospital

Tumor infiltrating lymphocytes (TILs) have shown efficacy in some adult cancers but it has not been yet explored in pediatric cancer.  Our group is assessing the feasibility of using TILs for the treatment of pediatric cancer. A total of 21 pediatric samples, treatment naïve or after chemotherapy, were received in the Advance Therapies Unit. After enzymatic digestion, tumor immune infiltrating profile were analyzed by spectral cytometry and also cultured to study their ability to effectively expand. TILs from resected tumors were cultured firstly using the standard method with IL2, but in order to optimize the TILs production time and to improve the successful expansion rate, we developed a new propagation method based on 3-signals-T-cell-activation. This new strategy differs from the traditional method in that cytokine exposure-signal was changed to IL7, IL15 and IL21, and the co-stimulation signals used were the agonistics of CD3 and CD28. We observed that adding costimulatory signal in phase 1 of culture lead to a faster increasing of absolute numbers and viability of TILs and a higher rate of TILs-successful expansion. This important issue will allow us to reduce the time of TILs manufacturing. Regarding TILs products, we observed an important decrease in CD4-phenotype-Treg cells with the new method but no differences were found in the expression of differentiation and activation markers or intracellular cytokines production. Our results shown that T cells from pediatric solid tumors could effectively be isolated and expanded and that the new expansion-method showed a more robust way to expand pediatric TILs.