Detection of peptide-specific CTL-precursors in peripheral blood lymphocytes of cancer patients

Abstract

Development of therapeutic vaccines is one of the major areas of tumour immunotherapy today. However, clinical trials of peptide-based cancer vaccines have rarely resulted in tumour regression. This failure might be due to an insufficient induction of cytotoxic T lymphocytes in the current regimes, in which cytotoxic T lymphocytes-precursors in pre-vaccination peripheral blood mononuclear cells are not measured. Initiation of immune-boosting through vaccination could be better than that of immune-priming with regard to induction of prompt and strong immunity. If this is also the case for therapeutic vaccines, prevaccination measurement of peptide-specific cytotoxic T lymphocytes-precursors will be important. In the present study, we investigated whether cytotoxic T lymphocytes-precursors reacting to 28 kinds of peptides of vaccine candidates (13 and 15 peptides for HLA-A24 + and HLA-A2 + patients, respectively) were detectable in pre-vaccination peripheral blood mononuclear cells of 80 cancer patients. Peptide-specific cytotoxic T lymphocytes-precursors were found to be detectable in peripheral blood mononuclear cells of the majority of cancer patients (57 out of 80 cases, 71%). The mean numbers of positive peptides were 2.0 peptides per positive case. Peripheral blood mononuclear cells incubated with positive peptides, not with negative peptides, showed significant levels of HLA-class-I-restricted cytotoxicity to cancer cells. The profiles of positive peptides entirely varied among patients, and were not influenced by the cancer origin. These results may provide a scientific basis for the development of a new approach to cancer immunotherapy, e.g.) cytotoxic T lymphocytes-precursororiented peptide vaccine.

Humoral Responses to Peptides Correlate with Overall Survival in Advanced Cancer Patients Vaccinated with Peptides Based on Pre-existing, Peptide-Specific Cellular Responses

“…Stock solutions were diluted with saline just before use. For the peptide screening, prevaccination PBMCs were provided for assays of peptide-specific CTL precursors using methods reported previously (29). Peptide-specific IFN-␥ production was calculated by subtraction of IFN-␥ production of the peptide-stimulated PBMCs in response to a negative control (HIV peptide) from that in response to a corresponding peptide in quadricate assays, and a two-tailed Student's t test was used for the statistical analyses.…”

A phase I trial of cytotoxic T-lymphocyte precursor-oriented peptide vaccines for colorectal carcinoma patients

“…We have previously reported 14 vaccine candidate peptides that can induce HLA-A24-restriced and tumour-specific CTL in cancer patients (Gomi et al, 1999;Kikuchi et al, 1999;Yang et al, 1999;Harashima et al, 2000;Kawano et al, 2000;Nakao et al, 2000;Nishizaka et al, 2000). We have also shown that most cancer patients have peptide-specific CTL precursors for some of these peptides, and that peripheral blood mononuclear cells (PBMCs) stimulated with positive peptides show HLA-class-I-restricted and tumour-specific cytotoxicity Maeda et al, 2002;Suzuki et al, 2002). In the present study, patients with advanced stages of colorectal cancer were immunised with up to four peptides identified in pre-vaccination measurement of peptidespecific CTL precursors to evaluate the toxicities and responses to CTL precursor-oriented peptide vaccination.…”

Identification of Peptide Vaccine Candidates Sharing among HLA-A3+, -A11+, -A31+, and -A33+ Cancer Patients

“…Cytotoxicity Assay. The peptide-stimulated PBMCs were incubated for Ͼ21 days, and cytotoxicity was tested by a standard 6-h 51 Cr release assay (17). A two-tailed Student's t test was used for the statistical analysis.…”

“…The method used for the detection of peptide-specific CTLs has been reported elsewhere (17). In brief, PBMCs (1 ϫ 10 5 cells/well) were incubated with 10 M of each peptide in the wells of a U-bottomedtype 96-well microculture plate (Nunc, Roskilde, Denmark) in 200 l of culture medium.…”

“…The concentration of IFN-␥ was assessed by ELISA as reported previously (16). For the inhibition assay, the peptide-reactive CTLs were positively purified using a CD8 Isolation kit (DYNAL, Oslo, Norway), and their peptidespecific IFN-␥ production was measured in the presence of 20 g/ml of anti-HLA class I (W6/32, IgG2a), anti-CD8 (Nu-Ts/c, IgG2a), or anti-HLA class II (H-DR-1, IgG2a) mAbs, as reported previously (17). The background IFN-␥ production (Ͻ100 pg/ml) was subtracted from the data.…”