PD-1 AND TIM-3 BLOCKING CANNOT ENHANCE APOPTOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA CELLS INDUCED BY PERIPHERAL BLOOD CD8+ T CELLS.

Abstract

Aim: Given the invaluable success of immune checkpoint inhibitors for tumor immunotherapy, in this study, the effect of programmed cell death 1 (PD-1) and T cell immunoglobulin-3 (TIM-3) blocking was investigated to induce apoptosis of leukemic cells by exhausted CD8⁺ T cells in patients with chronic lymphocytic leukemia (CLL).

Materials and methods: Peripheral blood CD8⁺ T cells were positively isolated from 16 CLL patients using magnetic beads separation method. Isolated CD8⁺ T cells were treated with either blocking anti-PD-1, anti-TIM-3 and isotype-matched control antibodies and then co-cultured with CLL leukemic cells as target. The percentage of apoptotic leukemic cells and the expression of apoptosis-related genes were evaluated by flow cytometry and real-time polymerase chain reaction methods, respectively. Interferon gamma and tumor necrosis factor alpha concentration was also measured by ELISA.

Results: Flow cytometric analysis of apoptotic leukemic cells indicated that the blockade of PD-1 and TIM-3 did not significantly enhance the apoptosis of CLL cells by CD8+ T cells, which then were confirmed by analysis of BAX, BCL2 and CASP3 gene expression, which was similar in blocked and control groups. No significant difference was found between blocked and control groups in terms of interferon gamma and tumor necrosis factor alpha production by CD8+ T cells.

Conclusion: We concluded that the blockade of PD-1 and TIM-3 is not an effective strategy to restore the function of CD8+ T cells in CLL patients at the early clinical stages of the disease. Further in vitro and in vivo studies are needed to more address the application of immune checkpoint blockade in CLL patients.