A Novel Cytotoxic Mechanism by Leukemia-Specific Antibody in Immunotherapy with Leukemia Cell-Derived Heat Shock Protein 70.

Introduction: Tumor-derived heat shock proteins (HSPs), which bind the tumor-specific antigenic peptides, are good application for cancer vaccine. We previously reported that immunotherapy using leukemia cell-derived HSPs against leukemia cell in mice prolonged survival by leukemia-specific cellular immune responses through CD8 + cytotoxic T-cell (Sato et al. Blood, 2001; Iuchi et al. Int J Hematol, 2006). We also indicated that CD4+ as well as CD8+ T-cell is indispensable for the survival prolongation (Sato et al. Blood, 2001), suggesting that humoral immune response by CD4+ T-cell also contributes to eradicate leukemia cells. Contributions of humoral immune responses, including tumor-specific antibodies or cytotoxic activities, in anti-tumor immunity induced by tumor-derived HSP-based immunotherapy remain unclear. We therefore investigated humoral immune responses against leukemia cells in the leukemia cell-derived HSP70-immunized mouse 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 healthy mice liver tissue. After subcutaneous administration of A20-derived HSP70 (A20-HSP), liver-derived HSP70 (liver-HSP; control) 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 A20 secreted IgG (A20-Ig) as an A20-specific antigen. To investigate a contribution of A20-HSP70 specific CD4+ T-cell, expression of intracellular Th2-cytokine IL4 in the A20-HSP70 stimulated CD4+ T-cell in the HSP70-immunizaed mice was measured by flowcytometry. 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 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 dissociation of the antigenic peptide from A20-HSP after ATP-treatment. Additionally, IgG level against A20-Ig in the A20-HSP mice was significantly higher than that in the liver-HSP mice, and this reactivity was blocked by preincubation of the sera with A20-idiotype derived peptide (A20-IP), which is the A20-specific peptide. A20-HSP70-reactive IL4-producing CD4 + T-cells in the A20-HSP mice are extremely more than those in the liver-HSP mice. 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 binding to leukemia cell-derived HSP70, including B-cell leukemia idiotypic peptide, via activation of the leukemia-specific CD4+ T-cell. In addition, leukemia-specific antibody-mediated CDC contributes to the eradication of leukemia cells. To utilize the leukemia-specific CDC activities induced by HSP-based immunotherapy would be a novel therapeutic strategy to eradicate leukemia cells in the patients with leukemia.