Phenotypic and Functional Characteristics of Human Natural Killer Cells Interacted with Cancer Cell Lines

Abstract

Natural killer (NK) cells play a critical role in innate immune responses against viruses and tumors. Matured human NK cells express multiple receptors to recognize target cells and perform immune response. Recently, NK cells have emerged as a tool in cancer immunotherapy. Thus, the present study investigated whether anti-tumor functions of NK cells differ according to the types of cancer. In addition, expression patterns of receptors on NK cells were determined when interacted with various tumor cells. This involved in examination of the changes on expression of surface marker and cytotoxicity on isolated NK cells from healthy-donor peripheral blood mononuclear cells (PBMCs) after being co-cultured with cancer cell lines. In some types of tumor cells, (Caco-2, HeLa, MCF-7 and OVCAR-3), showed morphological changes and inhibition of proliferation and/or colony forming by adding stimulated NK cells was observed, but it has not been affected in the other types of tumor cells. Furthermore, NK cells showed differential cytotoxicity in cancer cell lines. No cancer cells express interleukin(IL)-10, transforming growth factor(TGF)-βor tumor necrosis factor(TNF)-α cytokines, which influence NK cells activity, but mRNA level of IL-8varied among tumor cell types indicate if IL-8 level correlate with differential cytotoxicity. Moreover, human leukocyte antigen(HLA)-ABC, which binds to killer cell immunoglobulin-like receptor(KIR)on NK cells, was expressed on all cancer cells tested except Caco-2, and expression of ligands,(major histocompatibility complex class I-related chain (MIC)-A/B, UL16 binding protein (ULBP)-1 and ULBP-3),which interacted with natural killer group 2 member D(NKG2D)on NK cells, related to NK cells’ anti-tumor reactivity. Interestingly, NK cells lost CD56 expression by eliminating target cells. The proportion of CD16+CD56dimcells decreased after anti-tumor functions, followed by faint appearance of CD16 and/or CD56. NK cell had different immune activity depend on cancer cell type. In addition, malignant cells changed NK cell surface marker to immune-surveillance; thus, surface marker expression of NK cells should be a good candidate to develop cancer therapies based on anti-tumor immune responses. ;자연살해세포(NK cell)는 선천면역에서 항바이러스 및 항암반응에 중요한 역할을 하는 세포로 최근 암에 대한 면역치료법에서 치료제로서 활용되고 있다. 이 연구는 암세포 주와 NK cell을 공동배양 했을 때 표면단백질 발현과 기능변화를 확인하고자 하였다. 건강한 사람의 말초혈액 단핵세포로부터 NK cell을 분리하여 여덟 개의 암세포 주와 공동배양 한 뒤, 유세포 분석기로 NK cell의 단백질 발현을 분석하였다. 사이토카인을 처리했을 때, NK cell과 암세포 주를 공동배양 시 몇 세포 주들에서 LDH 방출 법을 통해 측정된 특정 세포독성이 증가하였으며, 세포부유물과 암세포 주의 성장이 억제되는 모습을 관찰하였다. 암세포 표면에 발현된 NK cell의 활성 및 억제 리간드의 균형 때문에 NK cell이 암세포 주마다 다른 세포독성을 보이는 것을 확인하였다. 흥미롭게도 NK cell이 암세포를 제거하였을 때, NK cell의 표면 단백질인 CD16과 CD56 발현이 감소되었고, 표적세포를 용해시키는 CD16+ CD56dim NK cell의 비율이 감소된 것을 확인하였다. 따라서 암세포 주는 NK cell과 상호작용을 통해 NK cell의 표면 단백질 발현을 변화시켜 표적세포에 대한 세포독성기능을 약화시킴으로써 면역으로부터 회피하는 것으로 생각된다. 따라서 NK cell의 표면단백질 발현변화 연구는 암세포에 대한 선천면역반응 및 면역치료를 기반으로 한 항암치료의 발달에 기여를 할 것으로 기대된다.