Morphologic, flow cytometric, functional, and molecular analyses of S100B positive lymphocytes, unique cytotoxic lymphocytes containing S100B protein.

Little is known about the S100B⁺ lymphocytes, which are unique human peripheral blood lymphocytes (PBL) containing the S100B protein. It has recently been shown that S100B is released from various types of S100B⁺ cells and exhibits varied cytokine-like activities. In this study, we precisely characterized the S100B⁺ lymphocytes of healthy adults with respect to the proportion in the whole PBL, immunophenotypes, function, and their S100B mRNA expression and also evaluated their S100B-releasing activity upon stimulation. S100B⁺ lymphocytes were detected in all individuals examined, and the proportion of S100B⁺ lymphocytes in the whole PBL ranged from 0.42% to 16.15% (mean, 4.21%). In addition, two subtypes of S100B ⁺ lymphocytes, a CTL subtype (CD3⁺ CD8⁺ CD16⁻) and a NK subtype (CD3⁻ CD3⁻ CD16⁺), were detected. The majority of the CTL subtype of S100B⁺ lymphocytes expressed the αß-T-cell receptor. Surprisingly, S100B mRNA was detected not only in S100B⁺ lymphocytes, but also in every S100B⁺ lymphocytes, although the expression levels of S100B mRNA in S100B⁻ lymphocytes were much lower than those of S100B⁺ lymphocytes. The CTL subtype of S100B⁺ lymphocytes exhibited blastic morphological changes, proliferated and released S100B upon stimulation with phytohemagglutinin. The NK subtype of S100B⁺ lymphocytes exhibited morphological NK activity when cocultivated with NK-sensitive target, K-562 cells. Thus, the CTL subtype of S100B⁺ lymphocytes exhibit the biological characteristics of T cells, while the NK subtype of S100B⁺ lymphocytes exhibit the characteristics of NK cells. These results suggest that S100B⁺ lymphocytes are a particular subtype of cytotoxic lymphocytes that play a unique role in antitumor immunity.

Potent anti-tumor killing activity of the multifunctional Treg cell line HOZOT against human tumors with diverse origins.

The T cell line HOZOT has a unique FOXP3+CD4+ CD8+CD25+ phenotype, exhibits suppressive activity in allogeneic mixed lymphocyte reactions (MLR), and produces IL-10, defining HOZOT as regulatory T cells (Tregs). Interestingly, in addition to possessing a suppressive Treg ability, HOZOT was also found to show cytotoxicity against certain representative human cancer cell types. In order to disclose the range of anti-tumor activity by HOZOT, we screened it by using a panel of twenty human tumor cell lines with different origins. Consequently, HOZOT showed potent cytocidal effects against a wide spectrum of neoplastic cells including carcinomas, sarcomas, mesotheliomas and glioblastomas except for hematopoietic malignancies. Its anti-tumor activity was strong enough with an E:T ratio of 4:1, which is considered to be more effective than that by conventional CTLs. Furthermore, an in vivo representative mouse tumor model by implanting human colon adenocarcinoma cells revealed that adoptive transfer of HOZOT almost completely eradicated disseminated lesions on peritoneum, markedly reduced metastases in lung and liver, and dramatically decreased bloody ascites caused by peritoneal carcinomatosis. Treatment of the tumor model mice by HOZOT with an E:T ratio of 2:1 even indicated the prolongation of their survival, although not reaching obvious statistical significance. In vitro blocking experiments using antibodies and inhibitors suggested that the cytotoxic mechanism of HOZOT against tumors is different from conventional cytotoxic cells such as CTL, NK or NKT cells. Altogether, our studies demonstrated the potent killing activity of HOZOT against a broad range of human malignancies, which indicates that HOZOT is a powerful tool in immunotherapy for advanced stage tumors.