Article Figures & Data
Figures
- Fig. 1.
Identification of HLA-A2-restricted CTL epitopes of GPC3 by using HLA-A2.1 (HHD) Tgm. A, protocol for identification of GPC3-derived and HLA-A2-restricted CTL epitopes. We primed the HLA-A2.1 (HHD) Tgm with BM-DCs (5 × 105) pulsed with the mixture of GPC3-derived peptides carrying HLA-A2 (A*0201) binding motif into the peritoneal cavity once a week for two weeks. Seven days after the last DC vaccination, spleens were collected and CD4− spleen cells (2 × 106/well) were stimulated with syngeneic BM-DCs (2 × 105/well) pulsed with each peptide in vitro for 6 days. We used these cultured CD4− spleen cells as responder cells in ELISPOT assay to evaluate GPC3-specific response of CTLs. B, bar graph, IFN-γ ELISPOT counts/2 × 104 CD4− spleen cells cocultured with peptide pulsed BM-DCs subtracted with those cocultured with BM-DCs without peptide loading (left). Bar graph, summation of IFN-γ ELISPOT diameters/2 × 104 CD4− spleen cells cocultured with peptide-pulsed BM-DCs subtracted with those cocultured with BM-DCs without peptide loading (right). Columns, mean of triplicate assays; bars, SE. All assays were done thrice with similar results.
- Fig. 2.
Immunohistochemical staining with anti-CD4 or anti-CD8 mAb in tissue specimens of HLA-A2.1 (HHD) Tgm immunized with the GPC3 144-152 peptides. These tissue specimens were removed and analyzed 7 days after the second DC vaccination (original magnification, ×200).
- Fig. 3.
CTL induction from PBMCs of HLA-A2- or HLA-A24-positive HCC patients. A and B, GPC3 peptide-reactive CTLs were generated from CD8+ T cells of HLA-A2+ and/or HLA-A24+ HCC patients. After three or four stimulations with autologous monocyte-derived DCs pulsed with the GPC3144-152 or GPC3298-306 peptide, the CTLs were subjected to a standard 51Cr release assay at the indicated effector/target ratio. Their cytotoxicity against the GPC3298-306 peptide pulsed C1R-A2402 cells or T2-A0201 cells, and each unpulsed cells (A), or GPC3− HLA-A2+, HLA-A24+ HCC cell line SK-Hep-1, GPC3− HLA-A2+, HLA-A24+colon cancer cell line SW620, and those cell lines transfected with the human GPC3 gene; SK-Hep-1/GPC3 or SW620/GPC3 (B) were examined by a 51Cr release assay. C and D, GPC3+ HLA-A2+, HLA-A24+ HCC cell line HepG2, GPC3+ HLA-A2−, HLA-A24− HCC cell line HuH-7, and GPC3− tumor cell lines SW620 and SK-Hep1 were used as target cells (left). Points, percentage of specific lysis calculated based on the mean values of a triplicate assay. D, inhibition of cytotoxicity by anti-HLA class I mAb (right). After the target HepG2 cells were incubated with anti-HLA class I mAb (W6/32, IgG2a) or anti-HLA DR mAb (H-DR-1, IgG2a), respectively, for 1 hour, the CTLs generated from PBMCs of patient A2-8 by stimulation with GPC3144-152 peptide (top) or CTLs generated from patient A24-6 using the GPC3298-306 peptide (bottom) were added. IFN-γ production (top; IFN-γ ELISPOT assay) and cytotoxicity (bottom; 51Cr release assay) were markedly inhibited by W6/32, but not by H-DR-1.
- Fig. 4.
Marked inhibition of growth of a GPC3-transfected human HCC cancer cell line, SK-Hep1/GPC3, engrafted into NOD/SCID mice after adoptive transfer of human CTLs induced by the GPC3 peptides. A, when tumor size reached 25 mm2 on day 9 after s.c. tumor implantation, human CTLs (3 × 106) reactive to HLA-A2-restricted (▪) GPC3 peptide and generated from one HLA-A2+ donor, or those reactive to HLA-A24-restricted (□) GPC3 peptide and pooled from two HLA-A24+ donors were i.v. inoculated. On day 14, the inoculation of CTLs generated from the donors distinct from those at the first injection was repeated. The control CD8+ T cells stimulated with irrelevant HLA-A2-restricted (▴) or HLA-A24-restricted (▵) HIV peptides were also injected into mice as a control. Tumor volumes in NOD/SCID mice given twice on days 9 and 14 with GPC3 epitope peptide–induced CTL lines (n = 4), control CD8+ T cells (n = 4), or saline alone (n = 4). Tumor size was expressed in square millimeters. B, points, mean tumor sizes in each group of mice; bars, ±SD (n = 4). Statistical significance was evaluated using t test.
Tables
- Table 1.
GPC3-derived peptides conserved between human and mouse GPC3 and predicted to be bound to HLA-A2 (A*0201)
A2-binding peptide Position Subsequence residue listing HLA-A2 binding score* GPC3A2-1 44-52 RLQPGLKWV 879 GPC3A2-2 102-110 FLIIQNAAV 319 GPC3A2-3 144-152 FVGEFFTDV 828 GPC3A2-4 155-163 YILGSDINV 162 GPC3A2-5 169-177 ELFDSLFPV 1055 GPC3A2-6 254-268 RMLTRMWYC 1259 GPC3A2-7 281-289 VMQGCMAGV 196 GPC3A2-8 326-334 TIHDSIQYV 496 GPC3A2-9 522-560 FLAELAYDL 402 ↵* Binding scores were estimated by using BIMAS software (http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken_parker_comboform).
- Table 2.
Expression of GPC3 in HCC tissue, quantification of serum-soluble GPC3, and GPC3-specific CTL induction in HCC patients
Age Gender State of tumor* GPC3 expression† Serum GPC3‡ HLA expression§ CTL induction∥ HLA-A2 (A*0201)–positive patients Pt-A2-1 80 F IIIa + + + + Pt-A2-2 72 M II + + + + Pt-A2-3 67 F II ND + ND + Pt-A2-4 54 M I + − + + Pt-A2-5 57 M I ND − ND − Pt-A2-6 66 M I − + − − Pt-A2-7 54 M IIIa + − + − Pt-A2-8 73 M II ND − ND + Pt-A2-9 68 F IIIa + − + − Pt-A2-10 54 M II + + + − HLA-A24 (A*2402)-positive patients Pt-A24-1 60 M IVa + + + + Pt-A24-2 57 M IVa + + + − Pt-A24-3 75 F IIIa + + + + Pt-A24-4 59 M IIIa ND − ND + Pt-A24-5 52 M IVb − − + − Pt-A24-6 65 M I ND + ND + Pt-A24-7 61 M I ND − ND + Pt-A24-8 74 M II ND − ND − Pt-A24-9 59 M IVb − − − − Pt-A24-10 69 M IVa + + + − Pt-A24-11 72 M II − − + − Pt-A24-12 61 M IIIa + + + + Abbreviations: F, female; M, male; ND, not determined.
↵* Tumor-node-metastasis classification.
↵† Positive (+) or negative (−) staining of tumor cells in contrast with peritumor normal tissue as background staining.
↵‡ Serum levels >106 ng/mL were evaluated as positive.
↵§ Immunohistochemical staining of the membrane of tumor cells was evaluated as positive.
∥ Specific lytic activity (≧20%) at E:T ratio = 20 against HepG2 target cells was evaluated as positive by 51Cr release assay.
Volume 12, Issue 9
