Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells

Abstract

Background: Cervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.

Results: In this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711-20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-gamma ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-gamma-secreting CD8+ T cells in 5/5 healthy donors.

Conclusion: We show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Figure 1

Immunophenotypic analysis of dendritic cells. Plastic adherent monocytes were cultured in the presence of GM-CSF and IL-4 for 6 days, followed by activation with a cytokine cocktail consisting of IL-1, IL-6, TNF-α and PGE₂ for 24 hours (filled histogram). Analysis of typical DC membrane markers (CD80, 83, 86 and HLA-DR) and the monocyte marker CD14 was determined on R1 as shown in the forward vs side scatter dot plot, CD3 and CD19 on the ungated population. An isotype control is represented by the bold line.

Figure 2

IFN-γ production by an HPV type 16 E7 peptide-specific HLA-A2⁺ T cell clone shows that peptide-pulsed mature DC efficiently bind and present the E7 peptide to T cells. HPV-16 E7-peptide-pulsed or non-pulsed mature DC were used as stimulator cells. Mature DC were co-incubated with a HPV-16 E7-specific CD8⁺ CTL clone. After 6 h, IFN-γ released in the co-culture was determined by IFN-γ ELISA. The T cell clone produced 2364 ± 28 pg/mL IFN-γ upon E7 peptide-pulsed mature DC stimulation compared to 83 ± 21 pg/mL IFN-γ upon control stimulation (p-value = 0.001).

Figure 3

Representative example of the phenotypic analysis of the effector T cell population. CD8⁺ T cells were isolated by magnetic separation using CD8 microbeads. Dot plots were gated on the lymphocyte population based on forward and side scatter properties. The indicated percentages are within the lymphocyte population.

Figure 4

Representative presentation for each donor of a positive well (B) compared to a control well (A) in the IFN-γ ELISPOT plate. CD8⁺ T cells stimulated multiple times with HPV-16 E7 peptide-pulsed mature DC were analyzed for antigen-specific IFN-γ secretion by means of IFN-γ ELISPOT. For this analysis cultured CD8⁺ T cells were stimulated with HPV-16 E7 peptide-pulsed K562-HLA-A*0201 cells (B), while non-pulsed K562-A*0201 cells were used as control cells (A).