BTLA와 PD-1 신호 전달 경로에 의한 인간 말초 혈액 γδ T 세포의 증식과 세포 독성의 개별적인 조절

Abstract

Background: B- and T-lymphocyte attenuator (BTLA) and programmed cell death-1 (PD-1) inhibit gamma-delta (γδ) T cell homeostasis and activation. This study was set up to examine whether BTLA and PD-1 signaling pathways were convergent or independent in human peripheral blood γδ T cells, where Vγ9Vδ2 T cells are majority. Materials & methods: Peripheral blood mononuclear cells (PBMCs) were isolated from adult healthy volunteers (HV) and patients with pediatric B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). CRISPR-Cas9 system was used to generate the herpesvirus entry mediator (HVEM)-deleted Jurkat or HL-60 cell lines. PBMCs and inactivated WT or HVEMlow tumor cells were co-cultured in the presence of interleukin (IL)-2 and zoledronate to induce BTLA/HVEM and PD-1/ PD-ligand 1 (PD-L1) signaling during expansion. The cell surface markers on those cells were analyzed by flow cytometry. The intracellular IFN-γ, and induction of phosphoproteins, such as protein kinase B (AKT), extracellular signal-regulated kinases (ERK) 1/2, and src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2), were also assessed by flow cytometry. Additionally, flow cytometry was used to evaluate the cell proliferation, degranulation, and cytotoxicity of. Results: PD-1 was extensively expressed in γδ T cells from patients with B-ALL, but not AML. However, the BTLA was similarly expressed in γδ T cells of three groups, suggesting an essential role of BTLA on γδ T cells. Proliferation of γδ T cells was increased when PBMCs were co-cultured with inactivated HVEMlow tumor cells compared with WT tumor cells. The cytotoxicity of the expanded γδ T cells was not affected by co-culture with inactivated HVEMlow Jurkat cells and was further increased in the presence of anti-PD-L1 monoclonal antibody (mAb). The inhibition of BTLA or PD-1 signaling increased the phosphorylation of AKT, whereas repressed phosphorylation of SHP-2 in γδ T cells. However, there were no synergistic or additive effects by a combined inhibition. Conclusion: Herein, I demonstrate that the interactions between BTLA/HVEM and between PD-1/PD-L1 regulated proliferation and cytotoxicity of human peripheral γδ T cells, respectively. The results suggest that inactivation of BTLA/HVEM signaling pathway during expansion could help produce more γδ T cells without compromising cytotoxicity.