Leukemia Specific Complement Dependent Cytotoxicity Contributes to Anti-Leukemia Immunity Induced by Leukemia Cell-Derived HSP70 Immunization in Mice.

Background: Tumor-derived heat shock proteins (HSPs) can be used for the vaccination against cancer because tumor-derived HSPs bind the tumor specific antigenic peptides and these peptides are carried onto MHC molecules by HSPs. We previously reported that immunotherapy using leukemia cell-derived HSPs against minimal residual leukemia after syngeneic bone marrow transplantation in mice prolonged survival by inducing leukemia-specific CD8 + cytotoxic T lymphocyte (Sato et al. Blood, 2001). It is known that induction of cell-mediated immune response plays a main role of tumor rejection in the HSP-based immunotherapy. However we also showed that CD4+ as well as CD8+ T cell is necessary for survival prolongation of HSP-immunized mice, suggesting that humoral immune response though CD4+ T cell contributes to the eradication of leukemia cells in our mouse model. To investigate the contribution of humoral immune responses in anti-leukemia immunity induced by HSP-based immunotherapy, we tried to detect the anti-leukemia cell antibody and analyzed cytotoxicities by the antibody in the leukemia cell-derived HSP70 immunization mice model.

Methods: Balb/c mice and syngeneic A20 B-cell leukemia cell line were used in this study. HSP70 was purified from A20 cells or liver tissue from healthy mice. Three weeks after subcutaneous administration of A20-derived HSP70 (A20-HSP70), liver-derived HSP70 (liver-HSP70) or PBS to the healthy mice, the sera were harvested to perform following experiments. To detect anti-A20 antibodies, mean fluorescence intensity (MFI) of A20 cells with immunized mice sera and FITC-conjugated anti-mouse-IgG was analyzed by flowcytometry. Additionally, the sera were subjected to ELISA using HRP labeled anti-mouse-IgG to detect specific antibody against A20-HSP70. Complement dependent cytotoxicity (CDC) activities were determined by trypan blue uptake of mouse target cells (A20, YAC1: lymphoma, or T27A: myeloid leukemia) after incubation with mice sera and rabbit complement.

Results: MIF of A20 with the sera from A20-HSP70 immunized mice was significantly higher than that from PBS or liver-HSP70 immunized mice. In addition, anti-A20-HSP70 IgG level by ELISA was significantly increased in the A20-HSP70 immunized mice. However, anti-A20-HSP70 IgG level in liver-HSP70 mice was not elevated, suggesting that A20-HSP70 immunization induced the specific IgG against not HSP70 itself but peptides bound for A20-HSP70. The sera of mice immunized with A20-HSP70 showed no cytotoxic activity without complement but showed significantly high CDC activity against A20 in vitro. This CDC activity was not observed against YAC-1 or T27A.

Conclusions: Leukemia specific CDC by the antibodies against the peptides bound for leukemia cell-derived HSP70 is suggested to be one of the mechanisms of anti-leukemia immunity induced by immunization with leukemia cell-derived HSP70 in mice. This would be an important finding to eradicate leukemia cells effectively in HSP-based immunotherapy for leukemia patients.