Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA, USA.
Anticancer Res. 2009 Nov;29(11):4673-82.
The expression of the CD3zeta subunit was investigated in fresh (uncultured) tumor-infiltrating lymphocytes (TILs) isolated from either solid tumor (ST) specimens or ascites (ASC) from patients with epithelial ovarian carcinoma (EOC). Western blot analysis of CD3zeta immunoprecipitates using anti-CD3zeta rabbit serum revealed that in 6 out of 6 patients with EOC, the CD3zeta protein was absent from ST-TILs. Immunoprecipitation with anti-phosphotyrosine monoclonal antibody (anti-PY20) from ST-TILs from one patient revealed bands co-migrating with the phosphorylated CD3zeta. CD3zeta protein was found to be expressed in only 1 out of 7 ST-TILs from patients with EOC. ASC-TILs were available in 5 of these patients and immunoprecipitation/Western blotting experiments using anti-CD3zeta rabbit serum revealed that CD3zeta protein was expressed in all 5. In addition, CD3zeta protein was expressed in 3 additional ASC-TIL specimens for which ST-TILs were not available. Therefore, the CD3zeta protein was expressed in ASC-TIL isolated from 8 out of 8 patients with EOC. CD3zeta protein was also expressed on peripheral blood mononuclear cells (PBMCs) from patients with EOC and from normal donors. RT-PCR studies of fresh ST-TIL specimens, using CD3zeta-specific primers, revealed that CD3zeta transcripts were absent from 13 out of 21 patients with EOC, down-regulated in 4 patients and present at levels comparable to those found in PBMCs in 4 other patients. In contrast, CD3delta transcripts were present at comparable levels in all specimens. Treatment with recombinant interleukin-2 (rIL-2) (600 IU/ml) restored the expression of CD3zeta protein and transcripts in cultured ST-TILs, whereas fresh ST-TILs did not express CD3zeta, in contrast to fresh ASC-TILs. These results demonstrate differential expression of CD3zeta in ST-TILs versus ASC-TILs in patients with EOC. CD3zeta transcripts and protein were found to be absent from most ST-TILs from patients with EOC, whereas they were expressed in ASC-TILs and PBMCs from such patients.
我们研究了新鲜（未培养）肿瘤浸润淋巴细胞（TIL）中 CD3zeta 亚基的表达情况，这些 TIL 是从上皮性卵巢癌（EOC）患者的实体瘤（ST）标本或腹水（ASC）中分离得到的。使用抗 CD3zeta 兔血清对 CD3zeta 免疫沉淀物进行 Western blot 分析显示，在 6 名 EOC 患者中，6 名患者的 ST-TIL 中均不存在 CD3zeta 蛋白。从一名患者的 ST-TIL 中用抗磷酸酪氨酸单克隆抗体（抗-PY20）进行免疫沉淀显示，与磷酸化的 CD3zeta 共迁移的条带。仅在 7 名 EOC 患者的 1 名 ST-TIL 中发现 CD3zeta 蛋白表达。在这些患者中的 5 名患者中可以获得 ASC-TIL，使用抗 CD3zeta 兔血清进行免疫沉淀/Western blot 实验显示，所有 5 名患者的 CD3zeta 蛋白均表达。此外，对于无法获得 ST-TIL 的另外 3 例 ASC-TIL 标本中，也检测到 CD3zeta 蛋白表达。因此，CD3zeta 蛋白在 8 名 EOC 患者的 ASC-TIL 中表达。CD3zeta 蛋白也在上皮性卵巢癌患者和正常供者的外周血单个核细胞（PBMC）中表达。使用 CD3zeta 特异性引物对新鲜 ST-TIL 标本进行 RT-PCR 研究显示，在 21 名 EOC 患者中，有 13 名患者的 CD3zeta 转录本缺失，4 名患者的 CD3zeta 转录本下调，4 名患者的 CD3zeta 转录本水平与 PBMC 相似。相比之下，所有标本中的 CD3delta 转录本水平相当。用重组白细胞介素-2（rIL-2）（600 IU/ml）处理可恢复培养的 ST-TIL 中 CD3zeta 蛋白和转录本的表达，而新鲜的 ST-TIL 则不表达 CD3zeta，这与新鲜的 ASC-TIL 形成对比。这些结果表明，EOC 患者的 ST-TIL 与 ASC-TIL 中 CD3zeta 的表达存在差异。在大多数上皮性卵巢癌患者的 ST-TIL 中发现 CD3zeta 转录本和蛋白缺失，而在这些患者的 ASC-TIL 和 PBMC 中则有表达。
