Differential Expression of CD3ζ Message and Protein in Tumor Infiltrating Lymphocytes from Solid Tumor Specimens and Malignant Ascites from Patients with Ovarian Carcinoma

The expression of the CD3ζ 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 CD3ζ immunoprecipitates using anti-CD3ζ rabbit serum revealed that in 6 out of 6 patients with EOC, the CD3ζ 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 CD3ζ. CD3ζ 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-CD3ζ rabbit serum revealed that CD3ζ protein was expressed in all 5. In addition, CD3ζ protein was expressed in 3 additional ASC-TIL specimens for which ST-TILs were not available. Therefore, the CD3ζ protein was expressed in ASC-TIL isolated from 8 out of 8 patients with EOC. CD3ζ 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 CD3ζ-specific primers, revealed that CD3ζ 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, CD3δ transcripts were present at comparable levels in all specimens. Treatment with recombinant interleukin-2 (rIL-2) (600 IU/ml) restored the expression of CD3ζ protein and transcripts in cultured ST-TILs, whereas fresh ST-TILs did not express CD3ζ, in contrast to fresh ASC-TILs. These results demonstrate differential expression of CD3ζ in ST- TILs versus ASC-TILs in patients with EOC. CD3ζ 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.