Leukemia Specific Humoral Immune Responses in Immunotherapy with Leukemia Cell-Derived HSP70 in Mice.

Background: Tumor-derived heat shock proteins (HSPs), which bind the tumor specific antigenic peptides and carry them onto MHC molecules, are used for the vaccination against cancers. We previously reported that immunotherapy using leukemia cell-derived HSPs against minimal residual leukemia in mice prolonged survival by leukemia-specific CD8 + cytotoxic T lymphocyte induction. In addition, we showed that CD4+ as well as CD8+ T cell is indispensable for survival prolongation (Sato et al. Blood, 2001), which suggests that humoral immune response by CD4+ T cell is also important to eradicate leukemia cells. Contributions of humoral immune responses in anti-leukemia immunity induced by HSP-based immunotherapy remain unknown and are important information to induce effective anti-leukemia immunity. In this study, we investigated the humoral immune responses and cytotoxic activities against leukemia cells in the leukemia cell-derived HSP70 immunization mice model in vitro.

Methods: Balb/c mice and syngeneic A20 B-cell leukemia cell line were used in this study. HSP70 was purified from A20 cells or healthy mice liver tissue. After subcutaneous administration of A20-derived HSP70 (A20-HSP), liver-derived HSP70 (liver-HSP) 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 mice sera and FITC-conjugated anti-mouse-IgG was analyzed by flowcytometry. The sera were subjected to ELISA to detect the specific IgG against A20-HSP, or IgG secreted by A20 cells (A20 Ig) as putative A20-specific antigen. 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-HSP immunized mice (A20-HSP mice) was significantly higher than that from liver-HSP immunized mice (liver-HSP mice). IgG level against A20-HSP by ELISA was significantly increased in the A20-HSP mice compared with liver-HSP mice. The reactivities of A20-HSP mice sera against A20-HSP were completely lost by ATP treatment, which treatment dissociates the antigenic peptide from A20-HSP. In addition, IgG level against A20 Ig in the A20-HSP mice was significantly higher than that in the liver-HSP mice, and this reactivity against A20 Ig were inhibited by preincubation of sera with A20 Ig-idiotipe-derived peptide (A20 IP) DYWGQGTEL, which is known as the A20-specific peptide. The sera from A20-HSP mice showed no cytotoxic activity itself but showed significantly high CDC activity with complement against A20 but not to YAC-1 or T27A in vitro.

Conclusions: Immunization with leukemia cell-derived HSP70 induces the leukemia specific antibodies against peptides bound with leukemia cell-derived HSP70, including an idiotipic peptide of IgG secreted by leukemia cells. In addition, CDC by these leukemia specific antibodies is though to be one of the mechanisms of anti-leukemia immunity induced by leukemia cell-derived HSP70 immunization. These findings enable the effective monitoring of therapeutic effects on the HSP-based immunotherapy for patients with leukemia by using the leukemia specific antibodies, and might develop a new strategy to enhance the leukemia specific CDC activities induced by HSP70-immunization.