Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies

Abstract

Background: Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8(+) and CD4(+) T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65(495-503)-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for ex vivo expansion of CMV-specific CTLs.

Methods: CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65(495-503) peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65(495-503) complex.

Results: About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65(495-503) peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8(+) T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC.

Conclusion: This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.

Figure 1

HLA-A*02:01/CMV-pentamer staining, number of A2/CMV-pentamer-positive CD8⁺ T cells, and cytolytic function of antigen-specific CTLs stimulated with HSP70/CMV-PC and CMVpp65_495-503 peptide. Frequency (A) and number (B) of HLA-A*02:01/CMV-pentamer-positive CD8⁺ CTLs from 16 healthy HLA-A*02:01/CMV-seropositive platelet donors on day 0 and 7, 14, and 21 days after stimulation with recombinant HSP70, CMVpp65_495-503 peptide, or HSP70/CMV-PC. Cells cultured in the presence of the HSP70-peptide-binding buffer served as negative controls (NC). The 16 donors were divided into three groups (weak: n = 5, medium: n = 5, strong: n = 6) according to the frequency of generated A2/CMV-pentamer-positive CD8⁺ T cells on day 7. Cytolytic activity (C) of expanded T cells against antigen-loaded PBMCs after one (day 14) and two (day 21) restimulation cycles in cells from 16 healthy HLA-A*02:01/CMV-seropositive donors. CFSE-labeled PBMCs unloaded or loaded with CMVpp65_495-503 peptide were used as target cells. T cells generated for 14 or 21 days in the presence of the CMVpp65_495-503 peptide or HSP70/CMV-PC (effector cells) were co-cultured with target cells for 5 h at a cell ratio of 10:1 or 1:1, respectively. The basal cytotoxic activity of effector T cells induced by CMVpp65_495-503 peptide or HSP70/CMV-PC against the unloaded target cells was subtracted from the cytotoxic values measured after incubation of effector T cells against CMVpp65_495-503 peptide-loaded PBMCs. The results of independent experiments are expressed as mean ± SD. Asterisks shown in the Figure indicate only statistically significant differences between levels in CMVpp65_495-503 peptide- and HSP70/CMV-PC-stimulated cells (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Effects of HSP70, CMVpp65_495-503 peptide, and HSP70/CMV-PC in the presence/absence of α2-macroglobulin on T-cell proliferation. PBMCs were labeled with CSFE and cultured with HSP70-peptide-binding buffer (NC), HSP70, CMVpp65_495-503 peptide, or HSP70/CMV-PC for 4 days. Cell proliferation within the CD8⁺ (A) and the A2/CMV-pentamer-positive CD8⁺ (B) T-cell populations was evaluated by CFSE dilution. In order to determine whether antiviral T-cell induction occurred due to cross-presentation of peptides by HSP70, the uptake of HSP70/CMV-PC was blocked by adding α2-macroglobulin (A2M). The results of 5 independent experiments using cells from medium responder are expressed as mean ± SD. Asterisks shown in the Figure indicate only statistically significant differences between levels in CMVpp65_495-503 peptide- and HSP70/CMV-PC-stimulated cells (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

mRNA expression levels of HSP70 and HO-1 determined by quantitative real-time RT-PCR. mRNA expression levels of HSP70 (A-C) and HO-1 (D-F) for antigen-specific T cells of donors divided in weak (A, D), medium (B, E), and strong (C, F) after stimulation with HSP70, CMVpp65_495-503 peptide, and HSP70/CMV-PC were determined by real-time RT-PCR. Constitutively expressed GAPDH gene was used as the reference standard for normalization of mRNA levels. Expression levels of mRNA were measured every second day after stimulation/restimulation and, finally, on day 21. The results of independent experiments are expressed as means (weak: n = 5, medium: n = 5, strong: n = 6). RQ values were calculated by the delta-delta CT method.

Figure 4

Detection of IFN-γ and granzyme B protein secretion by ELISA. The capacity of T cells to secrete cytokines and effector molecules was assessed by determining the secretion levels of (A) the Th type 1 (Th1) cytokine IFN-γ and (B) granzyme B by ELISA. After stimulation with HSP70, CMVpp65_495-503 peptide, and HSP70/CMV-PC the T-cell culture supernatants were harvested on days 7, 14, and 21 and used for analysis. The results of independent experiments are expressed as means (weak: n = 5, medium: n = 5, strong: n = 6) are expressed as mean ± SD. Asterisks shown in the Figure indicate only statistically significant differences between levels in CMVpp65_495-503 peptide- and HSP70/CMV-PC-stimulated cells (* p < 0.05, ** p < 0.01, *** p < 0.001).