Natural killer (NK) cells are innate lymphocytes that aid in the protection of the host from infectious diseases and cancer. Regulation of cytotoxicity in NK cells is mediated by inhibitory receptors that bind major histocompatibility complex class I (MHC-I) molecules on target cells. Human myelogenous leukemia K562 cells are readily attacked by NK cells, because K562 cells expressed very low levels of MHC-I molecules for inhibitory NK cell receptors. In this study, we compared the ability of γ-irradiated- or mitomycin C (MMC)-treated K562 feeder cells to support expansion and activation of canine NK cells. We isolated CD5 negative cells from canine peripheral blood mononuclear cells by immunomagnetic separation and co-cultured with γ-irradiated (100 Gy)- or MMC (20 μg/mL)-treated K562 cells in the presence of interleukin (IL)-2, IL-15 and IL-21 for 21 days. As a result, number of CD5 negative cells, co-cultured with γ-irradiated- or MMC-treated K562 cells (56.72 ± 13.77 fold or 32.99 ± 10.83 fold), was increased than those of CD5 negetive cells (2.99 ± 1.42 fold). Also, we found that gene expression markers of activated NK cells such as NKp30, NKp44, NKp46, Ly49, NKG2D, CD244, perforin, and granzyme B and production of interferon gamma were similarly upregulated in NK cells co-cultured with γ-irradiated- or MMC-treated K562 cells, respectively. Next, we observed that cytotoxicity of NK cells co-cultured with γ-irradiated K562 cells was more sensitively reacted to canine mammary carcinoma cells than those of MMC-treated K562 cells. These results revealed that γ-irradiated K562 cells are more efficient feeder cells than MMC-treated K562 cells for enhancing NK cells expansion and activation.

Abstract
서 론
재료 및 방법
    영양세포(feeder cells) 준비
    CD5 음성 세포 분리 및 NK 세포 선택적 증식
    정량적 중합효소연쇄반응 (quantitative real-time reversetranscriptase-polymerase chain reaction, qPCR)을 이용한 유전자 분석
    Enzyme-linked immunosorbent assay (ELISA)를 이용한 IFN-γ 생성 분석
    표적세포에 대한 NK 세포독성 분석
    통계학적 분석
결 과
    CD5 음성세포 분리 및 영양세포와 공배양
    CD5 음성세포의 체외 증식 및 NK 세포 특성 분석
    활성화된 NK 세포의 IFN-γ 생성 분석
    활성화된 NK 세포의 표적세포에 대한 세포독성
고 찰
REFERENCES

• Se-A Lee(Viral Disease Research Division, Animal and Plant Quarantine Agency)
• Na-Yeon Gu(Viral Disease Research Division, Animal and Plant Quarantine Agency)
• Siu Lee(Viral Disease Research Division, Animal and Plant Quarantine Agency)
• Jienny Lee(Viral Disease Research Division, Animal and Plant Quarantine Agency) Corresponding Author.
• Yoon-Hee Lee(Viral Disease Research Division, Animal and Plant Quarantine Agency)
• Bang-Hun Hyun(Viral Disease Research Division, Animal and Plant Quarantine Agency)