In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3 delta gene expression: a phenotypic and functional analysis.
In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3 delta gene expression: a phenotypic and functional analysis.
复制标题
在选择性缺乏CD3 Delta基因表达的CD3/TCR-人胸腺细胞种群的体外扩张：一种表型和功能分析。
DOI：
10.1084/jem.172.5.1409
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发表时间：
1990-11-01
影响因子：
15.3
通讯作者：
Mingari, M C
中科院分区：
文献类型：
--
作者：
Poggi, A;Biassoni, R;Pella, N;Paolieri, F;Bellomo, R;Bertolini, A;Moretta, L;Mingari, M C
采用PAN技术结合抗体包被磁珠去细胞技术获得高纯度的CD1-3-4-8-人胸腺细胞。其中大多数细胞表达胞浆CD3抗原，通过已知与CD3 epsilon链反应的单抗进行鉴定。在低剂量PMA(0.5 ng/ml)和随后加入重组白介素2(rIL-2；100U/ml)培养24小时后，细胞发生广泛的增殖(2wk后是初始细胞输入的40-60倍)。增殖细胞以CD3-TCR-为主。其余细胞(5%~40%)为CD3+TCRγ/Delta+(BB3-A13+)细胞。进一步去除CD3+TCR-γ/Delta+细胞可获得高纯度的CD3-细胞群，在培养中进一步增殖，没有明显的表型变化。在相同实验条件下，CD3+胸腺细胞仅检测到CD3+TCR-α/β+细胞，说明PMA不影响CD3/TCR复合体的表面表达，而是诱导CD3-胸腺细胞优先生长。CD3-胸腺细胞的表面标记分析显示，CD7+细胞均一，而CD2和CD8抗原表达的细胞比例较低。在自然杀伤(NK)细胞标志物中，CD56在所有细胞中高表达，CD16、CD57、CD11b、NKH2和GL183缺失。重要的是，这些细胞不同于外周NK细胞，80-95%的细胞表达胞浆CD3抗原。功能分析显示对NK敏感的(K562)和NK耐药的(M14，Daudi)人靶细胞均有很强的杀伤活性。在对Fc-Gamma R+P815细胞的重定向杀伤实验中，CD3、CD2和CD16等分子的特异性单抗不能增强靶细胞的杀伤作用，而PHA能诱导强烈的细胞杀伤作用。此外，PHA单独或与PMA联合使用可诱导CD3-胸腺细胞产生肿瘤坏死因子-α(TNF-α)和干扰素-γ(干扰素-γ)(但不能产生IL-2)。在PMA和rIL-2存在下克隆新鲜的CD1-3-4-8-胸腺细胞，得到CD3-CD56+克隆，其细胞杀伤活性和淋巴因子产生模式与多克隆群体相似。对编码CD3/TCR分子的转录本的Northern杂交分析表明，CD3 Zeta、epsilon和Gamma转录本存在，而CD3 Delta未被检测到。可以检测到TCR链的成熟转录本，但没有发现TCR-αmRNA，只有截短形式(1.0kb)的TCR-βmRNA。
Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3 epsilon chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+ TCR gamma/delta+ (BB3- A13+) cells. Further removal of CD3+ TCR-gamma/delta+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR- alpha/beta+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80- 95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the Fc gamma R+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8- thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3 zeta, epsilon, and gamma transcripts, while CD3 delta was undetectable. Mature transcripts for both gamma and delta TCR chains could be detected, whereas no TCR-alpha mRNA and only a truncated (1.0 kb) form of TCR-beta mRNA were revealed.(ABSTRACT TRUNCATED AT 400 WORDS)