Poggi A, Biassoni R, Pella N, Paolieri F, Bellomo R, Bertolini A, Moretta L, Mingari M C
Instituto Nazionale per la Ricerca sul Cancro, Genova, Italy.
J Exp Med. 1990 Nov 1;172(5):1409-18. doi: 10.1084/jem.172.5.1409.
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)
通过淘选技术结合用抗体包被的磁珠去除细胞，获得了高度纯化的CD1 - 3 - 4 - 8 - 人胸腺细胞。如用已知与CD3ε链反应的单克隆抗体评估，这些细胞中的大多数表达细胞质CD3抗原。在用低剂量佛波酯（PMA，0.5 ng/ml）培养并在24小时后添加重组白细胞介素2（rIL - 2，100 U/ml）后，细胞经历了广泛增殖（2周后初始细胞输入量增加40 - 60倍）。大多数增殖细胞为CD3 - TCR - 。其余细胞（5 - 40%）为CD3 + TCRγ/δ + （BB3 - A13 + ）细胞。进一步去除CD3 + TCR - γ/δ + 细胞得到了高度纯化的CD3 - 群体，其在培养中进一步增殖且无明显表型变化。当CD3 + 胸腺细胞在相同实验条件下培养时，仅能检测到CD3 + TCR - α/β + 细胞，这表明PMA不影响CD3/TCR复合物的表面表达，而是诱导CD3 - 胸腺细胞优先生长。对培养的CD3 - 胸腺细胞的表面标志物分析表明，它们均一性地表达CD7 + ，而低比例细胞表达CD2和CD8抗原。在自然杀伤（NK）细胞标志物中，所有细胞均高表达CD56，而CD16、CD57、CD11b、NKH2和GL183均不存在。重要的是，这些细胞与外周NK细胞不同，因为其中80 - 95%表达细胞质CD3抗原。功能分析显示，它们对NK敏感（K562）和NK抗性（M14、Daudi）的人靶细胞均具有强大的细胞溶解活性。在针对FcγR + P815细胞的重定向杀伤试验中，针对包括CD3、CD2和CD16在内的触发分子的特异性单克隆抗体未能增强靶细胞裂解，而PHA诱导了强烈的细胞溶解作用。此外，单独的PHA或PHA与PMA联合诱导CD3 - 胸腺细胞产生肿瘤坏死因子α（TNF - α）和干扰素γ（IFN - γ）（但不产生IL -2）。在PMA和rIL - 2存在的情况下对新鲜的CD1 - 三 - 4 - 8 - 胸腺细胞进行克隆，得到了CD3 - CD56 + 克隆，其表现出与多克隆群体相似的细胞溶解活性和淋巴因子产生模式。对编码CD3/TCR分子的转录本进行Northern印迹分析，发现存在CD3ζ、ε和γ转录本，而未检测到CD3δ。可检测到γ和δTCR链的成熟转录本，而未发现TCR - αmRNA，仅发现截短的（1.0 kb）TCR - βmRNA形式。（摘要截断于400字）
