The inducible Hsp70 as a marker of tumor immunogenicity

Abstract

Growing evidence indicates that the stress response in general and heat shock proteins (Hsps) in particular have a profound impact on tumor immunogenicity. In this study, we show that tumor cells subjected to a nonlethal heat shock stress are unable to form tumors in syngenic mice, whereas they do so in athymic nude mice. Moreover, heat-shocked MethA immunity is tumor specific. Enhancement of T-cell-mediated immunogenicity correlates with the expression of the inducible Hsp70 but not the constitutive Hsc70. These observations have a bearing on the proposed functional role of Hsp-peptide association in antigen processing and presentation by major histocompatibility complex I molecules under normal and stressful conditions.

Fig. 1.

(A) Expression of inducible Hsp70 and constitutive Hsc70 after heat shock. MethA cells were incubated at 37°C or 42°C for 20–60 minutes followed by incubation at 37°C for 16 hours, washed twice in cold phosphate-buffered saline, homogenized by a cold detergent solution (0.5% NP40, 0.5% N-octyl-β-d-glucopyranoside, in 50 mM Tris, 150 mM NaCl pH 7.3), and centrifuged (14 000 × g) for 10 minutes at 4°C. Supernatants (30 μg of protein per lane) were analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Gels were fixed and Coomassie stained or immunoblotted as previously described (Ménoret and Bell 2000) using monoclonal antibodies specific for the inducible Hsp70 (StressGen, clone C92F3A–5), the constitutive Hsc70 (StressGen, clone 1B5), and actin (SIGMA, clone AC-40). (B) In vitro survival of heat-shocked MethA cells. Tumor cells were incubated at 37°C (no treatment) or 42°C for 20, 40, 60, and 90 minutes, washed, counted, and seeded at 20,000 cells per flask in RPMI supplemented with 5% fetal calf serum. An aliquot of the culture was counted daily for 5 days. (C) Immunogenicity of heat-shocked MethA. Tumor cells were incubated at 37°C (no treatment) or 42°C for 20, 40, and 60 minutes, washed, and diluted to 5 × 10⁵ cells/mL. Viability was determined before and after injection by trypan blue exclusion. Cells (1 × 10⁵ in 200 μL) in plain RPMI were injected intradermally in either immunocompetent BALB/cJ mice or immunocompromised athymic BALB/cJ nude/nude mice. The viability of the cells was assessed after injection by trypan blue exclusion. The growth of the tumors was recorded every 2–3 days by caliper measurement of the diameter of the tumors. Each line represents the growth of a tumor in a single animal

Fig. 2.

Injection of heat-shocked cells induces TNF-α release in splenocytes. BALB/cJ mice were injected with either culture medium (RPMI), 1 × 10⁵ MethA cells or 1 × 10⁵ MethA cells incubated for 60 minutes at 42°C (MethA 42°C) as described in the legend of Figure 1C. Spleen cells harvested 48 hours after tumor injection were plated on ELISPOT 96-well plates previously coated with anti–TNF-α antibody. The incubation was performed at 37°C for 24 hours without exogenous stimulation (white background) or with MethA cells (black background). After incubation, the plates were washed and incubated with anti–TNF-α antibody conjugated with alkaline phosphatase and developed with 3-amino-g-ethylcarbazole (AEC) substrate. Spots were counted by ELISPOT analyzer (Autoimmune Diagnostica)

Fig. 3.

Heat-shocked MethA immunity is tumor specific. BALB/cJ mice were injected intradermally at day −7 in the right flank, with either culture medium (RPMI) or 1 × 10⁵ MethA cells incubated for 60 minutes at 42°C as described in the legend of Figure 1C. Positive control of immunization was performed by injecting 2 × 10⁷ lethally γ-irradiated MethA cells subcutaneously at day −14 and −7. All mice were challenged intradermally in the left flank at day 0 with 1 × 10⁵ live MethA or with 1 × 10⁵ live CT26-E (a clone derived from the BALB/c colon adenocarcinoma CT26). The growth of the tumors was recorded every 2–3 days by caliper measurement of the diameter of the tumors. Each line represents the growth of a tumor in a single animal