Comparative Immunogenicity of a Cytotoxic T Cell Epitope Delivered by Penetratin and TAT Cell Penetrating Peptides

Abstract

Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.

Keywords: CPP; TAT; antigen delivery; antigen presentation; cytotoxic T cell epitope; immunogenicity; immunotherapy; membrane penetrating peptide; membrane translocating peptide; penetratin; vaccine.

Figure 1

In vitro stimulation of T cells by AntpSIIN or TATSIIN pulsed bone marrow derived dendritic cells (BMDC). DC were incubated with AntpSIIN, TATSIIN, OVACD8, Antp, TAT peptide or media for 8 h and added to B3Z T cells for 18 h. LacZ activity in B3Z T cells was assayed by total culture lysates with LacZ substrate CPRG. The absorbance (560 nm) of chlorophenol red released by β-galactosidase was read after 4 h incubation at 37 °C. Values are the mean ± SEM for 4 replicates.

Figure 2

Measurement of in vivo CD3⁺ T cell proliferation and CTL lysis. C57BL/6 mice were immunised i.d. with PBS, AntpSIIN or TATSIIN and purified CFSE labelled OT-I strain T cells were injected i.v. Splenocytes were subsequently assessed via flow cytometry. CD3⁺ OT-I T cells that have undergone 0–5 cell divisions are shown as representative dot plots (A) and as CFSE profile histograms (B); with viable CD3⁺ OT-I T cells (black line) and CFSE curve fitting generated by the Weasel curve fitting software (red line); (C) The percentage of SIINFEKL specific lysis was determined in immunised mice by the in vivo killing assay 8 days later, shown by representative histograms and mean percent killing ± SEM (n = 6).

Figure 3

In vivo IFN-γ response to AntpSIIN and TATSIIN immunisation. C57BL/6 mice were injected i.d. on days 0, 10 and 17 with PBS, 25 µg AntpSIIN or 25 µg TATSIIN. The number of IFN-γ secreting cells in response to stimulation by OVACD8 (SIINFEKL), OVACD4 (OVA323-339) or media was analysed by ELISpot assay. Results are shown as mean spot-forming units (SFU)/5 × 10⁵ cells ± SEM in triplicate wells.

Figure 4

(A) In vitro stimulation of SIINFEKL-specific T cells by AntpSIIN, TATSIIN and TATXSIIN pulsed DC2.4. Cells were incubated with the peptide antigens for 8 h and added to B3Z T cells for 18 h. Controls included cells alone, Antp, OVACD8 (SIINFEKL) and PEPCD8, a non-internalising peptide; (B) In vitro stimulation of SIINFEKL-specific cells by DC2.4 cells pulsed with tryptic digests (Tryp) of TATSIIN, TATXSIIN and SIINFEKL. Cells were incubated with the tryptic digests as in A. In both (A) and (B), LacZ activity in B3Z T cells was assayed by total culture lysates with LacZ substrate CPRG. The absorbance (560 nm) of chlorophenol red released by β-galactosidase was read after 4 h incubation at 37 °C. Values show the mean ± SEM for 4 replicates.

Figure 5

Uptake of AntpMUC1K^b and TATMUC1K^b peptides by DC in vitro. (A) AntpMUC1K^b and TATMUC1K^b staining by BC2 antibody after pulsing at 100 µM for 60 min with uptake assessed as the difference between surface (dotted line) and intracellular (bold line) staining by flow cytometry. Isotype controls are shown as filled grey areas. DC were pulsed with AntpMUC1K^b or TATMUC1K^b peptides at either varying concentrations (5 to 200 µM) for 60 min (B) or a constant dose of 100 µM for set times between 5 and 360 min (C). Uptake was determined by flow cytometry as the percent surface staining subtracted from the percent intracellular staining (mean ± SEM, for 3 replicates).

Figure 6

Uptake of AntpMUC1K^b and TATMUC1K^b by BMDC is via endocytosis. DC cultures were pre-treated for 45 min with cytochalasin D (10 µg/mL) or NaN₃/2-deoxyglucose (10 mM) before adding 100 µg/mL of AntpMUC1K^b or TATMUC1K^b for 60 min. Uptake was determined via flow cytometry and expressed as the percent surface staining subtracted from the percent intracellular staining (mean ± SEM, for 3 replicates).

Figure 7

Cellular immune responses in TATMUC1K^b and AntpMUC1K^b immunised mice, measured as in vivo CTL killing and ELISpot assays. (A) C57BL/6 mice were immunised i.d. with PBS, 25 µg AntpMUC1K^b or 25 µg TATMUC1K^b and the percent MUC1 SAPDTRPAP specific lysis was determined eight days after immunisation. Representative histograms from 2 mice are shown; (B) IFN-γ responses to AntpMUC1K^b and TATMUC1K^b peptides in C57BL/6 mice, injected as above, with the number of IFN-γ secreting cells analysed by ELISpot assay. Results are shown as mean spot-forming units (SFU)/5 × 10⁵ cells ± SEM with 3 replicates. Results are representative of two experiments.

Figure 8

Tumour growth is delayed by immunisation. C57BL/6 mice were immunised on days 0, 10 and 17 with PBS, 25 µg AntpMUC1K^b or 25 µg TATMUC1K^b then inoculated subcutaneously 7 days after final immunisation with 2 × 10⁵ B16-MUC1 melanoma cells into the abdomen. Tumour growth was recorded. Data showing the product of individual perpendicular measurements (mm²) and days post tumour inoculation. Number of tumour-free mice at day 28 is also shown (n = 8/group, ** p < 0.05).