Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide-pulsed dendritic cells

Abstract

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24-restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.

Fig. 1

Number of specific IFN-γ–secreting spots by peptide-stimulated cells before and after vaccinations. Effector cells were stimulated as described in “Materials and methods.” Results are shown as the number of specific spots per 10⁵ ex vivo stimulated cells. Open squares indicate prevaccination, solid squares, postvaccination

Fig. 2a,b

Cytotoxicity after patients were vaccinated by initial method of CTL induction. a The CEA652[9]-specific bulk CTL culture from vaccinated patient (TT). CTL culture after two rounds of restimulations in vitro was used as effector to test the lysis of the following targets: C1R/A24-pulsed CEA652[9] (open squares); C1R/A24 without peptide (solid squares). b CTL culture from vaccinated patient (UA). TISI-pulsed CEA652[9] (open circles); TISI without peptide (solid circles). Target cells were pulsed with 10 μg/ml peptide overnight

Fig. 3

Cytotoxic activity against tumor cell lines. Cytotoxicity was tested after vaccinations. CTL was established with four rounds of restimulations by primary in vitro immunization with peptide-pulsed DCs. CEA-specific CTL line was used as an effector to test the lysis of the targets cell lines: open squares indicate MKN45 (stomach, A24⁺, CEA⁺), open circles HT29 (colon, A24⁺, CEA⁺), open triangles WiDr (colon, A24⁺, CEA⁺), solid squares KM12LM (colon, A24⁻, CEA⁺), solid circles MKN1 (A24⁺, CEA⁻). Tumor cells were pretreated with IFN-γ for 48 h

Fig. 4

Inhibition of specific cytotoxicity of anti-CEA CTL lines by MAbs. ⁵¹Cr-labeled HT29 tumor cell (HLA-A24⁺, CEA⁺) were preincubated with anti–HLA class I MAbs and anti-HLA class II MAbs, or the CTL line was preincubated with anti-CD4 and CD8 MAbs. CTL line and target were mixed at an E/T ratio of 1:50. Results are indicated as percentage of specific lysis

Fig. 5a

DTH reaction at the vaccinated site. Photograph shows the vaccinated inguinal site of the patient (MS) after 2 days of vaccination. Erythema and induration 30 mm in diameter were observed at the vaccinated site. b Histological analysis of the skin biopsy specimen. Massive infiltration of the lymphocytes was observed at the vaccinated site

Fig. 6

Tetramer analysis of patient UY. Left figure is the analysis of prevaccinated sample and right figure is the postvaccinated sample. Samples were double-stained with PE-labeled tetramer and FITC-labeled CD8