A large quantity of CD3^-/CD19^-/CD16^- lymphocytes in malignant pleural effusion from a patient with recurrent cholangio cell carcinoma

Tumor infiltrating lymphocytes (TILs) are candidates for adoptive cellular immunotherapy. Here we report on a patient whose TILs presented unusual lymphocyte antigens. Pleural effusions were collected from a 47-year-old man with recurrent cholangio cell carcinoma and malignant effusion. Effusion- associated lymphocytes (EALs) were separated by Ficoll-Hypaque gradient, and the EAL phenotype was determined by flow cytometry. The percentage of positive cells was determined for each lymphocyte-related differentiation antigen. The percentages of CD3⁺, CD19⁺, and CD16⁺ lymphocyte subpopulations among EALs were 20%, 7%, and 3%, respectively. Nearly 70% of EALs were CD3^-/CD19^-/CD56^-/CD16^- cells. The phenotypes of peripheral blood lymphocytes (PBLs) collected simultaneously from the patient's peripheral blood were CD3⁺ (52%), CD19⁺ (20%), and CD16⁺ (20%). When EALs were cultured in medium without pleural effusion, T cell-related antigens, but not B cell- or natural killer (NK) cell-related antigens, were newly expressed on EALs, and this expression reached a plateau after 48h in culture. The proportions of CD3⁺, CD19⁺, and CD16⁺ cells were 69%, 7%, and 3%, respectively. However, when EALs were cultured in medium with pleural effusion, increased expression of T cell-related antigens was not observed; the proportions of CD3⁺, CD19⁺, and CD16⁺ cells were 16%, 6%, and 1%, respectively. Neither total cell numbers nor cellular viability of EALs changed significantly after in-vitro culture, suggesting that significant proliferation or death of EALs did not occur during the culture period. Co-culture of the patient's PBLs with autologous pleural effusion for 96 h did not alter the expression of lymphocyte-related antigens on the PBLs. These results indicate that expression of T cell-related antigens, but not B cell- or NK cell-related antigens, on EALs was blocked temporarily by the malignant pleural effusion. This is the first report concerning the existence of a large quantity of unclassified lymphocytes in which the T cell-related antigens were reversibly masked in the malignant pleural effusion.