Efficient induction of a Her2-specific anti-tumor response by dendritic cells pulsed with a Hsp70L1-Her2(341-456) fusion protein

Abstract

Heat shock proteins (HSPs) have been shown to interact with antigen-presenting cells (APCs), especially dendritic cells (DCs). HSPs act as potent adjuvants, inducing a Th1 response, as well as antigen-specific CD8(+) cytotoxic T lymphocytes (CTL) via cross-presentation. Our previous work has demonstrated that Hsp70-like protein 1 (Hsp70L1), a new member of the Hsp70 subfamily, can act as a powerful Th1 adjuvant in a DC-based vaccine. Here we report the efficient induction of tumor antigen-specific T cell immune response by DCs pulsed with recombinant fusion protein of Hsp70L1 and Her2(341-456), the latter of which is a fragment of Her2/neu (Her2) containing E75 (a HLA-A2 restricted CTL epitope). The fusion protein Hsp70L1-Her2(341-456) promotes the maturation of DCs and activates them to produce cytokines, such as IL-12 and TNF-α, and chemokines, such as MIP-1α, MIP-1β and RANTES. Taken together, these results indicate that the adjuvant activity of Hsp70L1 is maintained in the fusion protein. Her2-specific HLA-A2.1-restricted CD8(+) CTLs can be generated efficiently either from the Peripheral blood lymphocytes (PBL) of healthy donors or from the splenocytes of immunized HLA-A2.1/K(b) transgenic mice by in vitro stimulation or immunization with DCs pulsed with the Hsp70L1-Her2(341-456) fusion protein. This results in more potent target cell killing in an antigen-specific and HLA-A2.1-restricted manner. Adoptive transfer of splenocytes from transgenic mice immunized with Hsp70L1-Her2(341-456)-pulsed DCs can markedly inhibit tumor growth and prolong the survival of nude mice with Her2(+)/HLA-A2.1(+) human carcinomas. These results suggest that Hsp70L1-Her2(341-456)-pulsed DCs could be a new therapeutic vaccine for patients with Her2(+) cancer.

Figure 1

Increased phenotypic maturation and activation of DCs by recombinant Hsp70L1–Her2_341–456 fusion protein stimulation. DCs were treated with 7.5 µg/ml Hsp70L1–Her2_341–456, Her2_341–456, Hsp70L1 or PBS for 48 h and then collected for FACS analysis of HLA-DR, CD80, CD86 and CD83 expression. The representative schematic depicts flow cytometric analysis of a sample from PBS-treated DCs (first line), Her2_341–456-stimulated DCs (second line), Hsp70L1-stimulated DCs (third line) and Hsp70L1–Her2_341–456-stimulated DCs (fourth line). HLA-DR⁺ positive cells were gated at the Q2-1 and Q4-1 areas, CD80⁺, CD86⁺ and CD83⁺ positive cells gated at the Q1-1 and Q2-1 areas, and the double positive cells gated at the Q2-1 area. All positive cell populations were labeled with the percentage of total DCs. DC, dendritic cell; HLA, human leukocyte antigen; HSP, heat shock protein; PBS, phosphate-buffered saline.

Figure 2

Increased cytokine and chemokine secretion from DCs stimulated with the recombinant Hsp70L1–Her2_341–456 fusion protein. DCs cultured for 5 days were stimulated with 7.5 µg/ml Hsp70L1–Her2_341–456, Her2_341–456, Hsp70L1 or PBS for 72 h. The levels of IL-12p70, TNF-α, MIP-1α, MIP-1β and RANTES present in the supernatants were measured by ELISA. Data are displayed as the mean cytokine or chemokine concentration (pg/ml)±s.e.m. For chemokine and cytokine production, * indicates a significant difference (P<0.05) between Hsp70L1–Her2_341–456 and Hsp70L1 stimulation, and ** indicates a significant difference (P<0.05) between Hsp70L1–Her2_341–456 and Her2_341–456 stimulation. DC, dendritic cell; HSP, heat shock protein; PBS, phosphate-buffered saline.

Figure 3

In vitro induction of human Her2-specific CTL responses by Hsp70L1–Her2_341–456-pulsed DCs. PBLs from healthy donors were stimulated with autologous DCs pre-pulsed with Hsp70L1–Her2_341–456 or Her2_341–456 to assess the Her2-specific CTL responses by a standard 4-h lactate dehydrogenase (LDH) release assay. CD8⁺ CTLs purified from the PBLs stimulated with three rounds of DCs pulsed with Hsp70L1–Her2_341–456 or Her2_341–456 were used as effector cells (E), and SW620 cells (Her2⁺, HLA-A2.1⁺) and T2 cells pulsed with Her2-E75 were used as target cells (T). Control targets included SK-BR-3 cells (Her2⁺, HLA-A2.1⁻), irrelevant CAP-1 peptide pulsed or unpulsed T2 cells. Various E/T ratios were tested as indicated. Data are presented as the mean±s.e.m. of three independent experiments. CTL, cytotoxic T lymphocyte; DC, dendritic cell; HLA, human leukocyte antigen; HSP, heat shock protein.

Figure 4

Induction of Her2-specific Th1 responses and CTLs in HLA-A2.1/K^b transgenic mice immunized with Hsp70L1–Her2_341–456-pulsed DCs. Splenocytes from immunized HLA-A2.1/K^b transgenic mice were used to assess Her2-specific immune responses by a CTL assay. The splenocytes were stimulated with Her2_341–456 and then used as effector cells (E), and SW620 cells (Her2⁺, HLA-A2.1⁺) cells and Her2-E75 peptide pulsed T2 cells were used as Her2-specific and HLA-A2.1-restricted targets (T), with SK-BR-3 and T2 cells as control targets. Various E/T ratios were tested as indicated. Data are presented as mean±s.e.m. of three independent experiments. CTL, cytotoxic T lymphocyte; DC, dendritic cell; HLA, human leukocyte antigen; HSP, heat shock protein.

Figure 5

Suppression of tumor growth by adoptively transferred splenocytes from HLA-A2.1/K^b transgenic mice immunized with Hsp70L1–Her2_341–456-pulsed DCs. Athymic nude mice were injected s.c. with 2×10⁶ SW620 cells. After 3 days, 1×10⁸ Hsp70L1–Her2_341–456 restimulated splenocytes derived from each group of immunized HLA-A2.1/K^b transgenic mice was transferred by i.v. injection in a volume of 0.2 ml. Additionally, mice received 2000 IU IL-2 i.p. in a volume of 0.5 ml every 2 days. The control groups received either IL-2 administration alone or no treatment. Following SW620 tumor inoculation, tumor growth was monitored by measuring the diameter of the tumor every 2 days and recorded as the average tumor diameter. HLA, human leukocyte antigen; HSP, heat shock protein; PBS, phosphate-buffered saline. s.c., subcutaneous injection; i.v., intravenous injection; i.p., intraperitoneal injection.

Figure 6

Prolonging survival of SW620 tumor-bearing mice receiving adoptively transferred splenocytes from HLA-A2.1/K^b transgenic mice immunized with Hsp70L1–Her2_341–456-pulsed DCs. Nude mice were injected s.c. with 2×10⁶ SW620 cells. After 3 days, 1×10⁸ Hsp70L1–Her2_341–456-restimulated splenocytes derived from each group of immunized HLA-A2.1/K^b transgenic mice was transferred by i.v. injection in a volume of 0.2 ml. Additionally, mice received 2000 IU IL-2 i.p. in a volume of 0.5 ml every 2 days. The control groups received either IL-2 administration alone or no treatment. The survival of mice receiving adoptive transfer of splenocytes after SW620 tumor inoculation was recorded. Each group contained eight mice. s.c., subcutaneous injection; i.v., intravenous injection; i.p., intraperitoneal injection. HLA, human leukocyte antigen; HSP, heat shock protein; PBS, phosphate-buffered saline.