Regression of intestinal adenomas by vaccination with heat shock protein 105-pulsed bone marrow-derived dendritic cells in Apc(Min/+) mice

Abstract

Heat shock protein (HSP) 105 is overexpressed in various cancers, but is expressed at low levels in many normal tissues, except for the testis. A vaccination with HSP105-pulsed bone marrow-derived dendritic cells (BM-DC) induced antitumor immunity without causing an autoimmune reaction in a mouse model. Because Apc(Min/+) mice develop multiple adenomas throughout the intestinal tract by 4 months of age, the mice provide a clinically relevant model of human intestinal tumor. In the present study, we investigated the efficacy of the HSP105-pulsed BM-DC vaccine on tumor regression in the Apc(Min/+) mouse. Western blot and immunohistochemical analyses revealed that the tumors of the Apc(Min/+) mice endogenously overexpressed HSP105. Immunization of the Apc(Min/+) mice with a HSP105-pulsed BM-DC vaccine at 6, 8, and 10 weeks of age significantly reduced the number of small-intestinal polyps accompanied by infiltration of both CD4(+) and CD8(+) T cells in the tumors. Cell depletion experiments proved that both CD4(+) and CD8(+) T cells play a critical role in the activation of antitumor immunity induced by these vaccinations. These findings indicate that the HSP105-pulsed BM-DC vaccine can provide potent immunotherapy for tumors that appear spontaneously as a result of the inactivation of a tumor suppressor gene, such as in the Apc(Min/+) mouse model.

Figure 1

Overexpression of heat shock protein (HSP) 105 in adenomatous polyps of Apc^Min/+ mice. (a) Macroscopic polyps in the small intestine of 4‐month‐old Apc^Min/+ mice. (b) A microscopic analysis of polyps in the small intestine of 12‐week‐old Apc^Min/+ mice stained with hematoxylin–eosin (left) and anti‐HSP105 monoclonal antibody (middle). A normal small intestine was stained with anti‐HSP105 monoclonal antibody as a negative control (right). Objective magnification was ×100. (c) Western blot analysis of HSP105 in the small intestine of 4‐month‐old Apc^Min/+ mice. The samples were small intestines of Apc^Min/+ and C57BL/6J mice homogenized in lysis buffer. The small intestines of three mice per group were pooled.

Figure 2

Vaccination with heat shock protein (HSP) 105‐pulsed bone marrow‐derived dendritic cells (BM‐DC) decreased the number of polyps in the small intestine of the Apc^Min/+ mice. (a) The Apc^Min/+ mice were inoculated intraperitoneally with HSP105‐pulsed BM‐DC (5 × 10⁵), BM‐DC alone, or myelin basic protein‐pulsed BM‐DC or phosphate‐buffered saline (PBS) at 6, 8, and 10 weeks of age. At 12 weeks of age, the small intestines of the Apc^Min/+ mice were excised, stained with methylene blue, and the number of tumors was counted by the naked eye. Each group consisted of 10 Apc^Min/+ mice. The statistical significance of the differences in results was determined using an unpaired t‐test. (b) The survival rate of Apc^Min/+ mice immunized with HSP105‐pulsed BM‐DC, BM‐DC alone, or PBS as a control. The immunization protocol was the same as that of (a). The overall survival rate was calculated using the Kaplan–Meier method, and statistical significance was evaluated using Wilcoxon's test.

Figure 3

Both CD4⁺ and CD8⁺ T cells are involved in the antitumor immunity elicited by the heat shock protein (HSP) 105‐pulsed dendritic cell vaccine. (a) The protocol for the vaccination and the depletion of T cell subsets. (b) The number of polyps in the small intestine of Apc^Min/+ mice with various treatments. The number of tumors was counted as described in the legend for Fig. 2. Each group consisted of eight Apc^Min/+ mice. The statistical significance of the difference between the results was determined using the unpaired t‐test.

Figure 4

Induction of heat shock protein (HSP) 105‐specific T cells via immunization with HSP105‐pulsed bone marrow‐derived dendritic cells (BM‐DC). (a) The Apc^Min/+ mice were inoculated with HSP105‐pulsed BM‐DC or BM‐DC at 6 and 8 weeks of age. The spleen cells were harvested from 10‐week‐old Apc^Min/+ mice and depleted with either CD4⁺ or CD8⁺ cells using magnetic cell‐sorting system. CD4⁻ cells were used as a source of CD8⁺ T cells and antigen‐presenting cells, and CD8⁻ cells were used as a source of CD4⁺ T cells and antigen‐presenting cells. Thereafter interferon‐γ enzyme‐linked immunospot (ELISPOT) assays were carried out. Briefly, CD4⁻ or CD8⁻ T cells (5 × 10⁵) in each well were cultured together with 2 µg/mL HSP105, myelin basic protein, or medium alone for 24 h. The statistical significance of the difference in results was determined using the unpaired t‐test. The spleens of three mice from each group were pooled. This experiment was carried out three times, with similar results. (b) The Apc^Min/+ mice were inoculated with HSP105‐pulsed BM‐DC or BM‐DC at 6 and 8 weeks of age. The small intestines were excised from 10‐week‐old Apc^Min/+ mice and then were analyzed after immunohistochemical staining with anti‐CD4 monoclonal antibody or anti‐CD8 monoclonal antibody (magnification ×200).