To investigate the methods of expansion of CD8 positive tumor-infiltrating lymphocytes (CD8+TILs) obtained from human hepatocellular carcinoma (HCC) and to study their immunological characteristics.

Thirty-eight tumor tissue specimens were obtained from radical liver cancer surgery. Immunohistochemistry staining was performed on cancer nests and side tissues to study the distribution of TILs, and TIL precursor cells were extracted by mechanical shearing, mixed enzyme digestion and discontinuous density gradient centrifugal separation. Activated with high dose of recombinant human interleukin 2 at first, the isolated cells were co-stimulated of anti-CD3 and anti-CD28 antibody. The CD8+TILs were sorted by magnetic beads. The effect of CD8+TILs on the growth of Hep3B and HepG2 cells lines was observed in CCK8 cytotoxicity experiment in vitro.

TILs were enriched in paracancer tissues, 12 cases were successfully extracted, 6 cases were achieved a large amplification among them. The number of amplificated cells was (1.2-2.5)×10⁸ after 17-50 times expansion fold, and the proportion of CD3+ cells in the paracancer tissues was (77.53±16.37)%, CD3+CD4+ ratio was (27.08±21.56)%, CD3+CD8+ ratio was (44.55±12.73)%, and the cell viability was (90.5±3.0)%. Moreover, CD8+TILs showed strong growth inhibition effect on Hep3B and HepG2 cell lines.

This study successfully establishes a method for efficient extraction and cultivation of CD8 + TILs, and obtains CD8 +TILs with high antitumor activity, which provides a research basis for adoptive immunotherapy of solid tumors such as advanced liver cancer.