Very Few Cytolytic T Cells are Detectable by a Non-Radioactive Long Term Fluorolysis Assay in Tumor Infiltrating Lymphocytes (TIL) from Cervical Cancer

Analysis of T cell cytolytic activity to specific antigens has become a routine procedure to investigate immunity to infection and vaccination. Peripheral blood and biopsy material from local tissues of patients is mostly available only in very limited amounts. Therefore, efficient procedures for analysis of small numbers of rare T cells from tissue samples is mandatory. We have adapted a highly sensitive cytolysis test, the fluorolysis assay (Kienzle et al., 2002, JIM), for the human system. The fluorolysis assay benefits from the use of small numbers of effector and target cells, a long incubation time without loss of specific signal, and is non-radioactive. Target cells (T2 cells) were stained with CFSE at two concentrations to discriminate between unstained effector cells, FL-1^low control, and FL-1^high specific target cells by flow cytometry. Target cells were incubated with recall antigen-specific T cells for 48 hours. Peptides for HLA-A2 restricted viral antigens used were of influenza virus A matrix protein-1 IMP_58–66 (GILGFVFTL), Epstein-Barr virus BMLF1_280–288 (GLCTLVAML) and Cytomegalovirus CMV_495–503 (NLVPMVATV). T cells were enriched using MHC-tetramer technology on a FACS-Vantage sorter or were unsorted peptide-restimulated T cell lines. As cervical cancer-specific antigens HPV16E7 _11–20 (YMLDLQPETT), E7_82–90 (LLMGTTLGIV), E7_86–93 (TLGIVCPI), and p16^INK4A _51–59 (VMMMGSARV), p16_53–62 (MMGSARVAEL) and as control HIV_94–102 (ILKRPVHGV) peptides were used. Recall antigen-specific lysis could be shown at a ratio of effector: target cells of 1: 100. The assay detected <100 antigen-specific effector cells by their lytic function. More importantly, specific lytic activity was detected in TIL lines to both antigens. The long term fluorolysis assay may therefore be a highly sensitive assay for lytic function of antigen-specific T cells.