Table 1.
Summary of blood donors.
Fig 1.
Morphology of CD14-ML and CD14-ML-DC generated by the current procedure.
(A) Phase-contrast images of live cells (left) and cytospin samples stained with May–Grünwald Giemsa (right) of the human monocytes and monocyte-derived myeloid cell lines (CD14-ML) are shown. (B) Morphology of OK432-stimulated mo-DC and CD14-ML-DC are shown. mo-DC and CD14-ML-DC were stimulated with OK432 for 2 days and subjected to microscopic analysis. The data are representative of 2 experiments.
Fig 2.
Cell surface molecules of CD14-ML-DC and the production of IL-12p70 by CD14-ML-DC.
(A) CD14-ML-DC, before and after treatment with OK432, were analyzed for the expression of CD40, CD80, CD83, CD86, HLA class I and HLA class II. As a control, results of the analysis of OK432-stimulated mo-DC (monocyte-derived DC) are also shown. The staining profiles of the specific mAbs (black lines) and isotype-matched control mAbs (gray area) are shown. (B) CD14-ML-DC and mo-DC were cultured in 96-well flat-bottomed culture plates (1×105 cells/200 μl medium/well) in the presence OK432 (10 μg/ml). After 60 h, the concentration of IL-12p70 in the culture supernatant was measured by ELISA. The data are representative of 2 experiments.
Fig 3.
T-cell stimulation and antigen presentation by CD14-ML-DC.
(A) CD14-ML (rhombuses), CD14-ML-DC stimulated with IL-4 (squares), OK432 (triangles), or mo-DC (circles) were irradiated and co-cultured with allogeneic peripheral blood T cells (4×104 cells/well) or alternatively, only CD14-ML-DC (circles) were irradiated and cultured in a 96-well round-bottomed culture plates for 5 days. T cell proliferation was measured according to [3H]-methylthymidine-uptake in the last 16 h. The experiments were conducted on CD14-ML derived from 2 different donors. (B) The indicated numbers of CD-14-ML-DC were loaded with GAD65111-131 peptide (rhombuses) or those left unloaded (squares), X-ray-irradiated, and co-cultured with GAD65-specific HLA-DR53-restricted clonal human CD4+ T cells (3×104 cells/well) for 3 days. The proliferative response of the T cells was measured by incorporation of [3H]-methylthymidine in the last 16 h of culture. The experiments were conducted on CD14-ML derived from 2 different donors. (C) CD14-ML-DC were pulsed with recombinant GST-fused GAD65 protein or GST protein for 16 h, X-ray-irradiated, and subsequently added to GAD65-specific T cells. The proliferative response of the T cells was analyzed. The experiments were conducted on CD14-ML derived from 2 different donors.
Fig 4.
Induction of CD8+ T cell lines that are reactive to cancer antigens by CD14-ML-DC.
(A) Protocol for the induction of cancer antigen-specific CD8+ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with peptides for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD8+ T cells. Cells were cultured with rIL-7 (10 ng/ml) in AIM-V with 5% human decomplemented plasma. On days 7 and 14, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and on days 9 and 16, and were supplemented with rIL-2 (20 IU/ml). CD14-ML-DC were prepared each time, and we only added IL-4 (did not add OK432). IFN-γ ELISPOT assay and flow cytometry were performed after 6 or 7 days from the third round of peptide stimulation. (B, C) Peripheral blood CD8+ T cells were obtained from a HLA-A*24:02-positive healthy donor (healthy donor 1) and were co-cultured with 4 peptides (CDCA156-64, KIF20A66-75, LY6K177-186 and IMP-3508–516)-loaded autologous CD14-ML-DC. (B) On day 21, the number of IFN-γ producing CD8+ T cells were analyzed by ELISPOT assay (Day 21). The results of the T cells before stimulation culture are also shown (Day 0). The HIV-peptide was used as a control peptide. (C) On day 21, the T cells were recovered and stained with anti-CD8 mAb and the HLA-A*24:02/CDCA156-64 or HLA-A*24:02/LY6K177-186 tetramer. The numbers in the figure indicate the percentage of the CD8+ T cells that were positively stained with the tetramer of the HLA-peptide complex (Day 21). The results of the T cells before stimulation culture are also shown (day 0). (D, E) A similar experiment as in (B, C) was done with the cells obtained from a HLA-A*02:01-positive donor (healthy donor 2). We used 4 peptides (CDCA1351-359, KIF20A809-817, MART126-35 and IMP3515-523) for the stimulation of the T cells. (D) The number of IFN-γ producing CD8+ T cells was analyzed by ELISPOT assay. (E) The T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*02:01/MART126-35 dextramer, HLA-A*02:01/CDCA1351-359 tetramer or HLA-A*02:01/IMP3515-523 tetramer. The numbers in the figure indicate the percentage of the CD8+ T cells that were positively stained with the dextramer or tetramer of HLA-peptide complex.
Fig 5.
Induction of CD4+ T cell lines that are reactive to cancer antigens by CD14-ML-DC.
(A) Protocol for the induction of cancer antigen-specific CD4+ T cells by CD14-ML-DC. In order to generate CD14-ML-DC, we added IL-4 to CD14-ML. After 3 days, we added OK432. CD14-ML-DC were pulsed with a mixture of 6 peptides (CDCA139-64, CDCA155-78, KIF20A60-84, KIF20A809-833, LY6K119-142 and LY6K172-191) for 3 h, X-ray-irradiated (45 Gy), and subsequently mixed with autologous CD4+ T cells in AIM-V with 5% human decomplemented plasma. On day 7, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rIL-7 (5 ng/ml). After two days, these cultures were supplemented with rIL-2 (10 IU/ml). CD14-ML-DC were added with only IL-4 (did not add OK432). On day 14, the stimulated CD4+ T cells in each well were analyzed for specificity in IFN-γ ELISPOT assays. The T cells showing a specific response to the cognate peptide were transferred to 24-well plates and restimulated with the autologous peptide-pulsed CD14-ML-DC, and subsequently supplemented with rhIL-7 (5 ng/ml) and rhIL-2 (20 IU/ml). On day 21, the T cells were restimulated with the autologous peptide-pulsed CD14-ML-DC and supplemented with rhIL-7 and rhIL-2. IFN-γ ELISPOT assays were performed after 6 or 7 days from the fourth round of peptide stimulation. (B, C) After the stimulation (more than three times), the number of CD4+ T cells reacting to each peptide was analyzed with an IFN-γ ELISPOT assay (Day 28). The results of the T cells before stimulation culture are shown (Day 0). Dimethyl sulfoxide was used as a control. The results for the healthy donor 3 (B) and donor 2 (C) are shown.
Fig 6.
Induction of CD8+ T cell lines that are reactive to cancer antigens by CD14-ML-DC obtained from HNC patients.
Peripheral blood CD8+ T cells obtained from HLA-A*24:02-positive HNC patients were co-cultured with autologous CD14-ML-DC pre-loaded with a peptide mixture (CDCA156-64, KIF20A66-75, LY6K177-186 and IMP-3508–516) to induce T cell lines that were reactive to the peptides under a schedule similar to that shown in Fig 4A. On days 7 and 14, the CD8+ T cells were re-stimulated with peptide-loaded CD14-ML-DC. (A, C) On day 21, the number of CD8+ T cells responding to the peptides were analyzed by an IFN-γ ELISPOT assay. An HIV-peptide was used as a control peptide. (B, D) On day 21, the T cells were recovered and stained with an anti-CD8 mAb and a HLA-A*24:02/CDCA156-64, HLA-A*24:02/KIF20A66-75 or HLA-A*24:02/LY6K177-186 tetramer. The numbers in the figure indicate the percentage of the CD8+ T cells that were positively stained with the tetramer from the HLA-peptide complex. The results for the cancer patient 1 (A, B) and the cancer patient 2 (C, D) are shown.
Fig 7.
Induction of CD4+ T cell lines that are reactive to cancer antigens by CD14-ML-DC obtained from HNC patients.
CD4+ T cells isolated from PBMCs of HNC patients were stimulated with CD14-ML-DC that were pulsed with a mixture of 6 kinds of peptides (CDCA139-64, CDCA155-78, KIF20A60-84, KIF20A809-833, LY6K119-142 and LY6K172-191). After more than three rounds of stimulation, the number of CD4+ T cells that reacted to each peptide was analyzed by ELISPOT assay. CD14-ML-DC were used as stimulators in the assay, because only a few amount of blood samples could be obtained from cancer patients. The results for cancer patient 1 (A) and cancer patient 2 (B) are shown.
Fig 8.
Induction of CD8+ T cell lines that are reactive to antigens by CD14-ML-DC that express antigenic proteins.
(A) The lentivirus constructs for CMVpp65 (EF-CMV-IP) and MART1 (EF-MART1-IP) are shown. CD14-ML were transduced with the lentivirus vector and cultured in the presence of puromycin (5 μg/ml) to select and expand the cell population carrying the transgene, resulting in the generation of CD14-ML/CMV and CD14-ML/MART1. (B) Expression of CMVpp65 by CD14-ML/CMV was analyzed by flow cytometric analysis. The staining profiles of the specific mAb (black lines) and isotype-matched control mAb (gray area) are shown. (C) CD14-ML-DC/CMV derived from a HLA-A*24:02-positive healthy donor were cultured with autologous CD8+ T cells. On day 9, the number of T cells reactive to the CMVpp65341-349 peptide was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are shown (Day 0). (D) On day 9, the T cells were recovered and stained with an anti-CD8 mAb and a tetramer of HLA-A*24:02/CMVpp65341-349 complex. The numbers in the figure indicate the percentage of the CD8+ T cells positively stained with the tetramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (E) CD8+ T cells obtained from an HLA-A*24:02-negative healthy donor were co-cultured with autologous CD14-ML-DC/CMV. On day 9, the number of IFN-γ producing CD8+ T cells was analyzed by ELISPOT assay, using CD14-ML and CD14-ML/CMV as stimulators. The results of the T cells before stimulation culture are also shown (Day 0). (F) Expression of MART1 by CD14-ML/MART1 was analyzed by flow cytometric analysis. The staining profiles of the specific mAbs (black lines) and isotype-matched control mAbs (gray area) are shown. (G) CD8+ T cells obtained from an HLA-A*02:01-positive healthy donor were co-cultured with autologous CD14-ML-DC/MART1 cells. On day 21, the frequency of CD8+ T cells reactive to MART126-35 was analyzed by ELISPOT assay. The HIV-peptide was used as a control peptide. The results of the T cells before stimulation culture are also shown (Day 0). (H) On day 21, the T cells were recovered and stained with an anti-CD8 mAb and HLA-A*02:01/MART126-35 dextramer. The numbers in the figure indicate the percentage of the CD8+ T cells that were positively stained with the dextramer of the HLA-peptide complex. The results of the T cells before stimulation culture are also shown (Day 0). (I) CD8+ T cells obtained from an HLA-A*02:01-negative healthy donor were co-cultured with autologous CD14-ML-DC/MART1. On day 21, the frequency of CD8+ T cells reactive to MART1 was analyzed by ELISPOT assay, using CD14-ML-DC and CD14-ML-DC/MART1 as stimulators. The results of the T cells before stimulation culture are also shown (Day 0).