Induction of leukemia-specific antibodies by immunotherapy with leukemia-cell-derived heat shock protein 70

Abstract

Cancer immunotherapy using heat shock protein (HSP) derived from autologous tumor requires cluster of differentiation (CD)4(+) as well as CD8(+) T-cells for the prolongation of patient survival, suggesting that a humoral immune response through CD4(+) T-cells is important in addition to cellular immunity. However, the role of humoral responses in HSP-based autologous tumor immunotherapy remains unclear. In the present study, we investigated whether leukemia-specific antibodies and antibody-mediated cytotoxicity against autologous leukemia cells have a crucial role in a mouse A20 leukemia model by immunizing A20-derived HSP70. Immunization with A20-derived HSP70 induced the production of anti-A20-antibodies and the antibodies recognized HSP70-binding peptides derived from A20. One of those was a major histocompatibility complex (MHC) class-I binding peptide, which has been clarified as the target peptide of CD8+ cytotoxic T-cells (CTL) against A20. The anti-A20-antibodies produced by immunization with A20-derived HSP70 induced complement-dependent cytotoxicity (CDC) against A20 in vitro. In addition, immunization with A20-derived HSP70 increased intracellular interleukin-4 (IL4)-production of CD4(+) T-cells, confirming the activation of type-2 helper T-cells. Taken together, immunization with leukemia-cell-derived HSP70 induces antibodies against leukemia-cell-specific peptides and might play a crucial role in the eradication of leukemia cells by CDC in mice. These findings will enable future establishment of a novel therapeutic strategy using antileukemia antibodies in HSP-based autologous tumor immunotherapy.

Figure 1

Detection of anti‐A20‐antibodies in immunized mice sera. Five healthy BALB/c mice were subcutaneously injected with A20‐derived heat shock protein 70 (A20‐HSP70), liver‐derived HSP70 (liver‐HSP70) or phosphate‐buffered saline (PBS) on days 0, 5, 10 and 15. On the first, second or third week from the last administration (on day 22, 29 or 36), the sera were harvested and pooled. Fluorescence intensity (FI) of A20 with immunized mice sera and fluorescein isothiocyanate (FITC)‐conjugated antimouse‐immunoglobulin G (IgG) was analyzed by flow cytometry. MFI, mean fluorescence intensity. Data are representative of three separate experiments.

Figure 2

Detection of anti‐A20‐heat shock protein 70 (HSP70) immunoglobulin G (IgG) in immunized mice sera by to enzyme‐linked immunosorbent assay (ELISA). Five healthy BALB/c mice were subcutaneously injected with A20‐HSP70, liver‐HSP70 or PBS on days 0, 5, 10 and 15. Three weeks after the last injection (on day 36), the sera were harvested and pooled. The sera, diluted at 1:20 (×20) or 1:100 (×100), were added to A20‐HSP70‐immobilized ELISA plates and then reacted with horseradish peroxidase (HRP)‐labeled antimouse‐IgG. Anti‐HSP70 monoclonal antibodies (MoAb) diluted at 1:1000 (×1000) were used for the positive control. OD, optical density. Each value represents the mean of triplicate samples. *P‐value < 0.001 versus phosphate‐buffered saline (PBS) or liver‐HSP70; t‐test.

Figure 3

Loss of the reactivities of A20‐heat shock protein 70 (HSP70)‐immunized mice sera against A20‐HSP70 by dissociation with binding peptides. Peptides were dissociated from A20‐HSP70 by adenosine 5′‐triphosphate (ATP) agarose column, and the reactivity of immunized mice sera against peptide‐dissociated A20‐HSP70 was analyzed by enzyme‐linked immunosorbent assay (ELISA). Mice sera were added to A20‐HSP70‐immobilized ELISA plates and then reacted with horseradish peroxidase (HRP)‐labeled antimouse‐immunoglobulin G (IgG). OD, optical density. Each value represents the mean of triplicate samples. *P‐value < 0.05 versus phosphate‐buffered saline (PBS), liver‐HSP70 for peptide‐bound A20‐HSP70, or sera from A20‐HSP70 for peptide‐dissociated A20‐HSP70; t‐test.

Figure 4

The specific reactivity of A20‐heat shock protein 70 (HSP70)‐immunized mice sera against A20‐cell surface immunoglobulin G (A20‐Ig) and A20‐iditypic epitope peptide (A20‐IP). (a) To confirm whether A20‐HSP mice sera recognize A20‐Ig, which is the A20‐cell surface immunoglobulin and is the representative immunogen to syngeneic mice, A20‐Ig was concentrated from tissue culture supernatant of A20 by precipitation with 50% saturated ammonium sulfate and purified on a protein‐A column. Immunized mice sera were added to A20‐Ig‐immobilized enzyme‐linked immunosorbent assay (ELISA) plates and then reacted with horseradish protein (HRP)‐labeled antimouse‐IgG. Each value represents the mean of triplicate samples. *P‐value < 0.05 versus or phosphate‐buffered saline (PBS), or liver‐HSP70; t‐test. (b) To confirm whether anti‐A20‐Ig antibodies induced by A20‐HSP70 immunization recognize A20‐idiotypic peptide (IP; DYWGQGTEL), which exists on the variable region of the heavy chain of the A20‐Ig and is known to be the peptide that can induce cluster of differentiation (CD)8+ cytotoxic T‐cells (CTL) against A20 and the anti‐A20‐Ig‐antibodies, A20‐HSP70 immunized mice sera were preincubated without (preincubation [–]) or with 100 µg/mL of H‐2^d‐binding peptide (A20‐IP, or control peptide derived from mouse influenza hemagglutinin protein [Flu‐HA] peptide; IYSTVASSL), and then subjected to ELISA in the same method described in Figure 4(a). OD, optical density. Each value represents the mean of triplicate samples. *P‐value < 0.05 versus Flu‐HA, **P‐value < 0.01 versus preincubation (–); t‐test. NS indicates statistically not significant.

Figure 5

Intracellular interleukin 4 (IL4) production of cluster of differentiation (CD)4⁺ T‐cells in A20‐heat shock protein 70 (HSP70)‐immunized mice. Splenocytes of each immunized group of mice were harvested and pooled 1, 2 or 3 weeks after the last immunization. The splenocytes were stimulated by each reagent (A20‐HSP70, liver‐HSP70, phosphate‐buffered saline [PBS] or concanavalin A [ConA; positive control]), and then labeled by antimouse CD4‐fluorescein isothiocyanate (FITC) and anti‐IL4‐phycoerythrin (PE) as described in Materials and Methods. Intracellular IL4 production by CD4⁺ T‐cells was analyzed by flow cytometer. (a) Percentage of IL4‐producing CD4⁺ T‐cells stimulated by each reagent in A20‐HSP mice. Each value represents the mean of duplicate samples. *P‐value < 0.05 versus PBS or liver‐HSP70; t‐test. NS indicates statistically not significant. (b) Representative histograms of IL4⁺CD4⁺ cells stimulated by A20‐HSP70 in the immunized mice on the first week. The percentages indicated in the plot refer to IL4⁺CD4⁺ cells.

Figure 6

A20‐specific complement‐dependent cytotoxicity (CDC) activity by the sera of A20‐heat shock protein 70 (HSP70)‐immunized mice. Three weeks after administrations of A20‐HSP70, liver‐HSP70 or phosphate‐buffered saline (PBS) to the healthy mice, the sera were harvested. CDC activity was determined by trypan blue uptake of mouse target cells (A20, YAC‐1 or T27A) after incubation with mice sera and with or without rabbit complement. Anti‐B220 antibodies were used for the positive control of CDC activity against A20 cells. Each value represents the mean of triplicate samples.*P‐value < 0.05 versus PBS, or liver‐HSP70; t‐test.