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. 1994 Aug 1;54(15):4155-61.

Insertion signal sequence fused to minimal peptides elicits specific CD8+ T-cell responses and prolongs survival of thymoma-bearing mice

B R Minev  1 , B J McFarland, P J Spiess, S A Rosenberg, N P Restifo
Affiliations

Insertion signal sequence fused to minimal peptides elicits specific CD8+ T-cell responses and prolongs survival of thymoma-bearing mice

B R Minev et al. Cancer Res. .

Abstract

CD8+ T-lymphocytes (TCD8+) recognize minimal peptides of 8-10 residues which are the products of intracellularly processed proteins and are presented at the cell surface by major histocompatibility complex class I molecules. An important step in this process is the translocation of processed proteins from the cytosol across the endoplasmic reticulum membrane, mediated by transporter associated with antigen-processing proteins or alternatively by endoplasmic reticulum-insertion signal sequences located at the NH2-terminus of the precursor molecules. We report here that the addition of an endoplasmic reticulum-insertion signal sequence at the NH2-terminus of TCD8+ epitopes from chicken ovalbumin (amino acids 257-264) or a naturally occurring tumor antigen expressed by the murine mastocytoma P815 (P1A amino acids 35-43) significantly enhanced the priming of specific TCD8+ in vivo. The signal sequence did not enhance peptide immunogenicity by merely increasing the hydrophobicity of the peptide, since ovalbumin amino acids 257-264 peptide with the signal sequence at its COOH-terminus did not demonstrate enhanced efficacy. The signal sequence did not act as a helper epitope, since TCD8+ responses were not diminished in class II-deficient transgenic mice or in mice depleted of CD4+ T-cells in vivo. Importantly, a single immunization with the fusion peptide significantly prolonged survival of mice challenged with E.G7OVA, a thymoma transfected with the complementary DNA of chicken ovalbumin.

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Figures

Fig. 1
Fig. 1
ESOVA but not OVAES or OVA enhances priming in vivo. Specific anti-OVA immune response was elicited by immunization with synthetic peptides in IFA (A) or PBS (B). C57BL/6N mice were immunized with 200 μg/mouse ESOVA (●), OVAES (▽), OVA (▼), or PBS (□). After 10 days, recipient spleen cells were stimulated against OVA minimal peptide in vitro for 6 days and their cytotoxic activity was determined with EL-4 cells (left), EL-4 cells pulsed with OVA257–264 for 90 min (middle), or E.G7OVA cells (right).
Fig. 2
Fig. 2
ESOVA enhances priming in vivo in mice depleted of TCD4+. A, FACS analysis of splenocytes isolated from normal nondepleted (left column), TCD4+-depleted (middle column), or TCD8+-depleted (right column) mice. Mice were given i.v. injections, three times at intervals of 6 days, of antibody GK1.5 (anti-CD4) or 2.43 (anti-CD8). FACS analysis was performed on the day of secondary stimulation of splenocytes, using fluorescein isothiocyanate-labeled anti-CD4 and anti-CD8 antibodies. B, cytotoxic activity in nondepleted (left), TCD4+-depleted (middle), or TCD8+-depleted (right) mice. C57BL/6N mice were depleted of TCD4+ or TCD8+ as described in the text. The mice were immunized with ESOVA (●), OVAES (▽), OVA (▼), or PBS (□). After 10 days, recipient spleen cells were stimulated against OVA minimal peptide in vitro for 6 days and their cytotoxic activity was determined with EL-4 cells pulsed with OVA257–264 for 90 min.
Fig. 3
Fig. 3
ESOVA enhances priming in vivo in the absence of MHC class II. MHC class II-deficient transgenic mice were immunized with ESOVA (●), OVA (▼), or PBS (□). After 10 days, recipient spleen cells were stimulated in vitro for 6 days against OVA minimal peptide and their cytotoxic activity was determined with EL-4 cells (left) or EL-4 cells pulsed with OVA257–264 for 90 min (right).
Fig. 4
Fig. 4
Immunoprotection of C57BL/6N mice challenged with E.G7OVA cells by a single immunization with ESOVA in IFA (A) or PBS (B). Mice were immunized with ESOVA, OVAES, OVA, or PBS 10 days before the challenge with 104 E.G7OVA cells.
Fig. 5
Fig. 5
ESP1A but not P1A enhances priming in vivo. Specific anti-P1A immune response was elicited by immunization with synthetic peptides in IFA (A) or PBS (B). DBA/2 mice were immunized with 200 μg/mouse ESP1A (●), P1A (▼), or PBS (▽). After 10 days, recipient spleen cells were stimulated against P1A minimal peptide in vitro for 6 days and their cytotoxic activity was determined with P815 cells (left), CT-26 cells (middle), or CT-26 cells pulsed with P1A35–43 for 90 min (right).
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