Identification of a new hTERT-derived HLA-A*0201 restricted, naturally processed CTL epitope

Abstract

By the use of a neural network capable of performing quantitative predictions of peptides binding to HLA-A*0201 molecules, we identified a number of nonamer peptides derived from the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). Five nonimmunogenic peptides with measured binding affinities for HLA-A*0201 ranging from 155 to 1,298 nM were modified at the P1, P2 and P9 positions, respectively, to achieve stronger HLA-A*0201 binding. One peptide, mp30-38 (mp30), with an L to V substitution at position 9 was subsequently found to be immunogenic in mp30 immunized HLA-A*0201/H2K(b) or HHD transgenic mice. The T cell reactivity obtained was directed against both the mp30 and against the unmodified p30. Anti-mp30 specific T cells generated in HLA-A*0201 transgenic mice were dependent on TCR-CD8/MHC-I alpha3 binding and therefore not capable of recognizing mp30-pulsed human HLA-A*0201(+) cells or murine HLA-A*0201 transfectants. In order to show reactivity against naturally processed peptide in human tumor cells, an hTERT positive HLA-A*0201 negative colon carcinoma cell line (CCL220) was transfected with an HLA-A*0201/H2K(b) cDNA construct and used as target in ELISPOT and cytotoxicity assays. The data show that T cells from mp30 immunized HHD transgenic mice react specifically against the CCL220 transfectant indicating that p30 is naturally processed. In conclusion, we have identified a new CTL HLA-A*0201 restricted hTERT epitope, which is now, included in an ongoing phase 2 vaccine trial of patients with disseminated cancer.

Fig. 1

Immunogenicity and cross reactivity in the efferent arm of the immune response. Groups of C57-A2K^b transgenic mice (n = 3) were vaccinated with 3 × 10⁶ syngenic DCs pulsed with p30, mp30 or mp540 (10 μg/ml), and PADRE (5 μg/ml). Ten days after immunization, the mice were sacrificed and the pooled splenocytes (from 3 mice) were pulsed with the peptide used for immunization (100 μg/ml) and expanded in vitro for 5 days, on day 5, the splenocytes were tested in ELISPOT wells for IFN-γ production after exposure to EL4-A2K^b cells pulsed with wildtype peptide, modified peptide and Mock (control), respectively, *P < 0.001 (t test)

Fig. 2

CD8-dependence of the generated CTLs. C57-A2K^b transgenic mice (3 mice) were immunized with 3 × 10⁶ syngenic DCs pulsed with mp30 (10 μg/ml) and PADRE (5 μg/ml). Ten days after immunization, the mice were sacrificed and the pooled splenocytes (from 3 mice) were pulsed with mp30 (100 μg/ml) and expanded in vitro for 5 days. Day 5, splenocytes (300,000/well) were tested in ELISPOT wells for IFN-γ production when stimulated with either EL4-A2 cells (100,000) pulsed with mp30 or an HLA-A*0201-binding irrelevant peptide (Mock) or EL4-A2K^b cells (100,000) pulsed with mp30 or an HLA-A*0201-binding irrelevant peptide (a). In some cases, splenocytes were tested in the 4 h cytotoxicity assay (b). Target cells were mp30-pulsed EL4-A2K^b and incubated with effector cells at ratio of 1:50 in the presence of anti-CD8 antibody or isotype control. *P < 0.001 (t test)

Fig. 3

Target cells are more efficiently sensitized for lysis by modified than by non-modified peptide. Spleen cells derived from the HHD transgenic mice immunized with mp30 pulsed DCs were studied for their capacity to lyse EL4-A2K^b cells pulsed with a dose titration of mp30–38 versus p30-38 peptide. The effector cells and target cells pulsed with different concentration of peptides were cultured at ratio of 50:1 for 4 h. Filled squares are EL4-A2K^b pulsed with mp30, open squares are EL4-A2K^b pulsed with p30. Error bars represent standard deviations

Fig. 4

T cells specific for mp30 epitope release IFN-γ after exposure to CCL220-A2K^b transfectants and kill CCL220-A2K^b transfectants. HHD transgenic mice (n = 4) were vaccinated with 3 × 10⁶ syngenic DCs pulsed with mp30–38 (10 μg/ml) and PADRE (5 μg/ml). Eleven days after immunization, the mice were sacrificed and the pooled splenocytes (from 4 mice) were pulsed with the vaccination peptide (100 μg/ml) and expanded in vitro for 5 days. On day 5, the splenocytes were harvested and left under resting conditions (IL-2 was added) for another 4 days. After resting, the splenocytes were tested in ELISPOT wells for IFN-γ production in response to CCL220-A2K^b and CCL220, respectively (a). For the cytotoxicity assay (b), the splenocytes were tested on day 5. Target cells were CCL220-A2K^b, p30 pulsed CCL220-A2K^b and CCL-220, respectively. *P < 0.001 (t test)

Fig. 5

Changes in the frequency of hTERTmp30 specific T cells in 7 days cultures of PBMCs from two renal cell carcinoma patients vaccinated for 6 (Patient 2) or 10 (Patient 1) times (week 1–16) with mp30 loaded autologous dendritic cells. Vaccination-induced T cell responses against mp30 were measured by ELISPOT analysis and data show the mean number of peptide specific IFN-γ spots in response to mp30 by 3 × 10⁴ in vitro stimulated PBMCs. Non-specific IFN-γ spots are subtracted. Bars, range of duplicates