超抗原SEB活化的NKT细胞亚群形态和分化途径的研究

Morphology and differentiation pathway of superantigen SEB-activated NKT subsets

目的:以NKT细胞形态和分化途径为焦点,研究超抗原SEB活化的NKT细胞亚群的特征。方法:C57BL/J小鼠脾细胞分别经SEB或者ConA诱导,收集体外扩增10d和5d的淋巴细胞以及获取正常黏附性巨噬细胞。用荧光抗体染色,流式细胞仪测定淋巴细胞和大淋巴细胞群表面CD69的表达、NKT细胞亚群百分数和不同NKT细胞亚群的分化途径。倒置显微镜400倍下比较大淋巴细胞的形态。结果:SEB活化的淋巴细胞和大淋巴细胞表面CD69分子表达由正常值的0.11%分别提高到55.00%和68.95%。大细胞群中CD8+和TCRVB8+NKT细胞亚群的百分数由原始的0.36%和0.81%分别提高到30.29%和31.48%。这些细胞位于流式细胞图的上部,是大体积细胞群。显微镜下显示SEB活化的大淋巴细胞体积不仅大于ConA活化的T淋巴细胞,而且比正常巨噬细胞大5倍以上,是非黏附性细胞。胞内质粒松散、胞质与核之比>1。SEB活化的CD8+和TCRVβ8+NKT细胞亚群均由T细胞直接分化而来。结论:SEB活化10d的NKT细胞亚群主要是CD8+NK1.1+和TCRVβ8+NK1.1+的NKT细胞。这些细胞是大体积淋巴细胞,应属于T细胞的亚群。

AIM： This study is to explore the biological characteristics of NKT subsets through investigating their morphology and differentiation pathways. METHODS： Splenic lymphocytes from C57BL/J mice were treated with either Staphylococcal entertoxin B （SEB） or ConA and then cultured in vitro. On day .5 and day 10, the lymphocytes were harvested. Normal adherent macrophages and normal lymphocytes were harvested on day 0. Expression of CD69 molecule on the cell surfaces of the lymphocytes and large lymphocytes by SEB-activated was measured using staining of fluorescent antibody and flow cytometry （FACS）. And the percent （%） of NKT subsets was determined and analyzed the differentiation pathways of the NKT subsets. Appearance of subsets was observed and compared under a light microscope at ×400 magnification. RESULTS： Expression levels of CD69 molecule on the SEB-activated lymphocytes and the giant lymphocytes were significantly increased to 55% and 68.95% respectively as compared to the level （0.11%） detected on the normal lymphocytes （both P 〈 0.01; n = 3）. The percent （%） of CD8^＋ NK1.1^＋ and TcRVβ8^＋ NK1.1^＋ NKT subsets in the giant lymphocytes was significantly enhanced to 30.29% and 31.48% （84.0 or 38.9 folds） originally from 0.36% and 0.81%, respectively. Based on the cell distribution shown in the upper part of the FACS picture, they should be large-scale selection. The SEB-activated lymphocytes in size were larger than not only the ConA-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. They are no-adherent cells. There were a few granule seen in cytoplasm . The value of cytoplasm vs nuclei was less than 1.0. The SEB-activating CD8^＋ and TcRVβ8^＋ NKT subsets were not relative to a NK source. They were produced directly from T cells differentiation. CONCLUSION： The SEB-activating NKT cells are giant lymphocytes in size, consisting predominantly of CD8^＋ NK1.1^＋ NKT cells. They are considered as a subsets of T lymphocytes.