Figure 2. Tumor reactive T cells in tumor digest can be enriched by sorting and subsequent in vitro expansion of CD8⁺ PD-1⁺ population in tumor digests.

(A) CD8⁺PD-1⁺ and CD8⁺PD-1^- populations in tumor digests were enriched by FACS sorting. Sorted cells and un-sorted bulk tumor digests were immediately stimulated and expanded by placing in a T25 flask with irradiated 2.5×10⁷ PBMCs from 3 allogeneic donors in the presence of 30ng/ml OKT-3 Ab. IL-2 was added the next day (REP; rapid expansion). PD-1 expression level of each sorted population, immediately after sorting and after the expansion was determined by FACS analysis. Lymphocyte, PI^-, and CD3⁺ population are shown. At day 18 – day 24, reactivity of each population was examined by IFN- γ release assay or (B) intracellular IFN- γ staining assay by tumor stimulation. Lymphocyte, PI^-, and CD8⁺ population are shown. Representative data of 2 independent experiments for each case are shown.