Interferon-a-mediated enhancement of TRAIL expression on CD49a/b-based human natural killer cell subsets

Abstract

선천 면역 반응에서 자연살해세포(NK cell)는 세포 독성 반응에 매우 중요한 역할을 한다. 자연살해세포는 암 세포들이나 외부 병원균들을 제거하는데 효과적인 역할을 할 수 있다. 활성화된 자연살해세포는 세포독성 과립들을 통해 표적 세포들을 죽일 수 있고 또는 Fas 리간드나 종양괴사인자(TNF)-관련 세포사멸 유도 리간드(TRAIL)를 통한 세포자연사(apoptosis)를 일으킬 수도 있다. TRAIL은 또한 자연살해세포의 항암, 항바이러스 기능을 조절하는데 중요한 분자라고 밝혀져 왔다. 이러한 관점들을 통해 자연살해세포의 중요성을 인식하였고, 비교적 활발히 연구가 이루어진 CD56bright, CD56dim으로 나뉜 자연살해세포보단 덜 알려진 CD49a와 CD49b로 나뉜 자연살해세포의 표현형에 대해 알아보고자 하였다. 바이러스 감염된 상태에서CD49a와 CD49b로 나뉜 자연살해세포 중 특히, CD49a+CD49b+ 세포는 높은 활성화 수용체들을 발현했고 사이토카인 생성 능력과 세포독성 능력이 강화 되어있는 것을 다른 논문을 통해서 확인하였다. 이를 통해, 본 연구에서는 CD49a+CD49b+ 세포가 말초혈액 상태에서도 우수한 세포독성 능력이 있을 것이라 가정하고 CD49a+CD49b+ 세포에서 다른 세포그룹들과 표현형 차이가 있는지 확인하고자 하였다. TRAIL 발현에 특히 집중하기 위해 IFN-α로 자연살해세포를 자극하여 자연살해세포에 TRAIL 발현을 극대화하였다. 본 연구에서는 자연살해세포에 발현하는 CD49a, CD49b, TRAIL, CXCR6, CD25 수용체들의 표현형을 유세포분석기를 통해 확인하였다. 분석해본 결과, CD49a, CD49b로 나뉘어진 자연살해세포에서는 바이러스 감염 상태가 아닌 말초혈액에 존재하는 상태에서도 CD49a+CD49b+ 세포가 적지만 존재하고 있고, CD49a+CD49b+ 세포는 IFN-α로 자극하기 전에도 높은 TRAIL 발현율을 갖고 있었다. 게다가, CD49a+CD49b+ 세포는 CXCR6와 CD25 발현율도 높았다. 그러나, CXCR6와 CD25에서는 IFN-α 자극에 의한 발현의 변화는 발견되지 않았다. 결과적으로, 잘 알려지지 않은 CD49a+CD49b+ 세포를 TRAIL과 접목시켜 이후의 응용 연구들을 통해 자연살해세포의 세포독성을 향상시키도록 할 수 있고 이를 활용하여 항바이러스 치료 또는 항암치료에 적용시킬 수 있을 것으로 기대된다.;Natural Killer (NK) cells are essential cytotoxic innate lymphocytes and are able to eliminate tumors and intracellular microbes such as viruses. Activated NK cells kill target cells by releasing cytotoxic substances and inducing apoptosis which is mediated by the surface expression of Fas ligand and TNF-related apoptosis inducing ligand (TRAIL). TRAIL+ murine NK cells in the liver are capable of suppressing tumor metastasis. In addition, TRAIL plays essential roles in enhancing NK cell cytotoxicity against viruses such as hepatitis viruses. Having the critical functions of NK cells in mind, I focused my research on the phenotype of the CD49a/b NK cell subsets, which are based on the surface expression of CD49a and CD49b. Although CD56-based human NK cell subsets were well- known, the knowledge of the CD49a/b NK cell subsets was insufficient. It was reported that the CD49a+CD49b+ subset expressed more NK cell activation markers and had ability to exert cytotoxicity and secrete cytokines in viral infections. Thus, I hypothesized that the CD49a+CD49b+ NK cell subset could show enhanced functions, especially in terms of TRAIL-mediated cytotoxicity. To measure TRAIL expression on CD49a/b NK cell subsets, I stimulated human NK cells with IFN-α, which induce TRAIL on the surface of NK cells. To determine the phenotype of the NK cell subsets, I measured the expression of CD49a, CD49b, TRAIL, CXCR6, CD25 on NK cells. In some experiments, NK cells were pre-treated with low concentrations of IL-12 and IL-15 to rejuvenate NK cells which had been frozen for storage and thawed for experiments. IFN-α was used to examine whether it affects the expressions of above molecules on NK cells. In addition, to verify the influence of IL-12 and IL-15 stimulation, I performed experiments employing fresh, unfrozen NK cells which were not pre-treated with IL-12 and IL-15. The phenotype of NK cells was determined and analyzed by flow cytometry. I found that human NK cells could be divided into four subsets on the basis of CD49a/b expression. The CD49a+CD49b+ NK cell subset existed in the peripheral blood mononuclear cells of healthy, uninfected human donors, although the frequency was low. The CD49a+CD49b+ NK cell subset expressed more TRAIL than other three subsets when NK cells were not treated with IFN-α. TRAIL expression was significantly increased after IFN-α treatment on all four CD49a/b-based NK cell subsets. The CD49a+CD49b+ subset also expressed more CXCR6, which is a chemokine receptor, and CD25, which is an NK cell activation marker, than other subsets. In conclusion, the CD49a+CD49b+ human NK cell subset showed high TRAIL expression in resting state and its TRAIL expression was increased by IFN-α. The IFN-α-mediated enhancement of TRAIL expression on the CD49a/b-based NK cell subsets may be applied to the development of immunotherapy for control virus infections and malignant tumors.