Article Figures & Data
Figures
- Fig. 1.
TAPP associated with U-251 cells by immunofluorescence microscopy and by intracellular flow cytometry. A, immunofluorescence of U-251 cells shows that the TAPP are present within their predicted intracellular locations. U-251 gliomas were allowed to attach to coverslips overnight. The cells were fixed, permeabilized, and then stained with the specific antibodies. Top, left to right, Her2/neu, GnT-V, and survivin. Top middle, tyrosinase Mage-1 and Trp-1 (which is as bright as the negative control; not shown). Bottom middle, EphA2, GP100, and IL13Rα2. Bottom, B-cyclin, Mart-1, and hTert. B, intracellular flow cytometry of TAPP within U-251 glioma cells. U-251 glioma cells (106) were fixed and permeabilized. The permeabilized cells were incubated with the primary antibodies for 1 h, washed, and subsequently incubated with a FITC-labeled secondary antibody directed against the primary antibody. Flow cytometric analysis of 104 glioma cells is shown. The isotypic control (dark line) and the anti-TAPP staining (either a fine line or dashed line) are shown. a, Her2/neu; b, GnT-V and survivin; b, tyrosinase and Mage-1; d, Trp-1 and EphA2; e, GP100 and IL13Rα2; f, Mart-1; g, B-cyclin and hTert.
- Fig. 2.
Cytotoxicity assays done with HLA-A2–restricted CTL or TIL effectors and peptide-loaded T2 cells or glioma target cells. CTL (top and middle rows) were directed against Mart-1, GP100, EphA2, and Her/neu peptides and incubated with peptide-loaded T2 target cells (top row) or glioma target cells (middle row). TIL (bottom row) were incubated with either peptide-loaded T2 cells or the glioma target cells. The effector TIL 1374 cells are specific for tyrosinase (366-377; left) and TIL 771 cells are specific for GP100 (154-162; right) and incubated with the target glioma cells. The phenotypes of the glioma cells used are, for SNB19: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+; U-251: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro+; U-373: HLA-A2+, Mart-1+, GP100+, EphA2+, Her2/neu+, tyro−; A172: HLA-A2−, Mart-1+, GP100+, EphA2+; T98G: HLA-A2+, tyro+; LN229: HLA-A2−, tyro+.
Tables
- Table 1.
Human glioma cell line neuroglial profile and HLA class I and II phenotypes
Glioma Neuroglial* Molecular phenotype Class I Class II HLA-A HLA-B HLA-C HL-DR HLA-DQ 04-11-MG V, F 1 8, 57 w6, w7 13, 17, 52 2, 6 12-11-MG N, GF 24, 32 27, 64 w2, w8 4, 7 2, 3 13-06-MG N, GF, V 1, 2 44, 57 Not tested NT NT A172 N, V 1, 3 8, 42 w7 NT NT D-54MG N, V 1, 3 7, 8 w7 NT NT D-645MG V 2, 23 35, 49 w4, w7 1, 4, 53 5, 8 DBTRG05-MG V, F 2, 68 35, 38 w12, w15 4, 14, 52, 53 5, 8 NR203 V, F 2 7, 60 w7, w10 4, 15, 51, 53 6, 7 NR206 GF, V 2 62 w10 4, 53 8 NR213 GF, V 2 44, 55 w5, w9 4, 15, 51, 53 6, 7 LN18 NT 2, 9 5, w35 Not tested w3 NT LN229 NT 3 35 w4, w12? NT NT LNZ308 N, V, F 24 35, 51 w4, 14 NT NT SF767 N 1, 32 8, 44 w5, w7 NT NT SNB19 N, V 2 18 w5 NT NT T98G N, V, F 2 35, 39 w4, w7 8, 12, 52 4, 7 U-87MG N, V, F 2 44 w5 NT NT U-118MG N, V, F 24, 29 39, 44 w7, 16 NT NT U-251MG N, V 2 18 w4 NT NT U-373MG GF, V 2 18 w5 NT NT NOTE: The gliomas were typed by a PCR-based molecular approach. NT, not tested; w4, w12? indicates that we cannot rule out in the presence of w4 in the presence of w12. Molecular specificities done by high-resolution HLA PCR: 04-11-MG—A0101; B08(01,19N)*, B5701; Cw0602, Cw07(01,06,18); DPB1 0101, DPB0301; DRB1 0301, DRB1302; DRB3 0101, DRB0301; DQB1 0201, DQB0604. D-645MG—A02(05,14), A23(01,02); B35(01,42), B4901; Cw04(01,09N), Cw07(01,06,18); DRB10101, DRB0405, DRB40103; DQB10302, DQB0501. DBTRG05-MG—A02(01,04,09), A68(01,11N,23); B35(01,42), B3801; Cw12(03,04), Cw15(02,07); DRB10402, DRB14(01,39); DRB3, DRB4; DQB10302, DQB0503. NR203—A0201, B07(02,05,06), B40(01,33); Cw0304, Cw0702; DPB10401, DRB10408, DRB1501; DRB40103, DRB0101; DQB10301, DQB0602. NR206—A0201; B1501; Cw0304; DPB11301; DRB10405; DRB40105; DQB10303. NR213—A0201; B44(02,10N,27), B5501; Cw0303, Cw05(01,03); DPB1 0401, DPB1101; DRB1 0401, DRB1501; DRB4 0103, DRB5(0101); DQB1 0301, DQB0602. T98G—A0201; B3503, B3906; Cw04(01,09N), Cw0702; DPB1 0301, DPB0401; DRB1 0801, DRB1201; DRB3 0202; DQB1 0302, DQB1 0402.
↵* The gliomas were profiled for neuroglial markers: neurofilament 160 (N), glial fibrillary acidic protein (GF), Galactocerebroside (GC), vimentin (V), and fibronectin (F).
- Table 2.
Summary of TAPP expression profiles by glioma cell lines
(A) Intracellular flow cytometric analysis of 12 TAPP expressed within the glioma cells* Antigen Glioma A172 D-54 LN18 LN229 LNZ308 SF767 SNB19 T98G U-118 U-251 U-373 U-87 B-cyclin 1 1 2 2 2 2 3 1 1 1 2 1 EphA2 1 1 1 2 2 1 1 0 1 2 3 1 Her2/neu 2 3 3 3 3 2 3 2 2 3 3 3 IL13Rα2 3 4 2 2 3 2 3 2 3 3 3 3 GnT-V 2 1 2 2 1 2 2 3 1 3 2 2 GP100 3 2 0 0 3 2 3 3 0 3 4 3 Mage-1 4 3 2 3 3 3 3 2 2 3 2 3 Mart-1 3 0 3 3 3 2 3 3 0 3 3 3 Survivin 3 2 2 2 3 2 2 3 1 3 2 3 hTert 4 4 3 3 4 4 2 4 4 4 3 4 Trp-1 0 2 1 1 0 0 1 1 0 0 1 0 Tyrosinase 2 0 2 3 1 2 2 3 0 2 0 0 Σ score 28 23 23 26 28 24 28 27 15 30 28 26 (B) Real-time PCR expression of Aim-2, Gage-1, Sart-1, and Trp-2 mRNA† Antigen Glioma A172 D-54 LN18 LN229 LNZ308 SF767 SNB19 T98G U-118 U-251 U-373 U-87 Aim-2 2 3 3 3 3 2 3 3 3 3 3 2 Gage-1 nd nd nd nd nd nd nd nd 2 nd nd nd Sart-1 2 2 3 2 nd 1 2 2 2 1 2 nd Trp-2 nd nd nd 2 nd 2 nd 2 2 nd 1 nd Σ score 4 5 6 7 3 5 5 7 9 4 6 2 Total‡ 32 28 29 33 31 29 33 34 24 34 34 28 ↵* The cells were fixed, permeabilized, and stained with the antibodies directed toward the various tumor antigens. Cells were scored for the intensity of immunofluorescence staining obtained by flow cytometry. A score of 0 was given when the staining was not above that of the negative control cells. A score of 1+ to 4+ reflects the mean peak channel intensity of the cells. In the last row, we assign a cumulative score for each of the 12 cell lines (Σ score to summarize the overall strength of the 12 TAPP analyzed).
↵† RNA was isolated from gliomas in exponential growth. The RNA was analyzed by real-time PCR after the RNA was converted into cDNA. The ΔCT value was calculated by taking the experimental values of the tumor antigen and subtracting the 18S value. nd indicates mRNA was not detected; the CT score was >35 cycles. A ΔCT value of 10 to 14 was a high degree of expression and assigned a 4; a value of 14 to 18 was assigned a 3; a value of 18 to 22 was assigned a 2; and a value of 22 to 26 was assigned a 1. In the next to last row, we assign a score for each of the 12 cell lines (Σ score) to summarize the overall strengths of the 4 TAPP analyzed.
↵‡ The sum total of the Σ score from A + Σ score from B = total Σ score‡. The Σ score provides overall evaluation of the strength of expression of the 16 TAPP associated with each of the glioma cell lines.
- Table 3.
Frequency comparisons of TAPP protein and mRNA in glioma cell lines and in surgically resected glioblastoma multiforme tissues as found in the studies presented here and elsewhere
TAPP gene Glioma cell lines/protein detection GBM specimens/protein detection GBM specimens/qPCR detection Glioma cell lines/qPCR detection GBM specimens/molecular detection GBM specimens/microarray detection Positive/total analyzed (% frequency) Positive/total analyzed (% frequency) reference source Positive/total analyzed (% frequency) Positive/total analyzed (% frequency) Positive/total analyzed (% frequency) reference source Positive/total analyzed (% frequency) B-cyclin 17/17 (100%) 10/10 (100%) 6/14 (43%) EphA2 16/17 (94%) 13/14 (93%), ref. 22 10/11 (91%) 10/14 (71%) 9/9 (100%), ref. 21 Her2/neu 17/17 (100%) (76%) ref. 16 11/11 (100%) N = 43 (81%), ref. 16 2/14 (14%) IL13Rα2 17/17 (100%) 11/11 (100%), ref. 44 10/11 (91%) 11/11 (100%), ref. 44 4/14 (29%) GnT-V 17/17 (100%) 11/11 (100%) 6/13 (46%), ref. 27 0/14 (0%) GP100 10/17 (59%) (45%) ref 16 9/11 (82%) 8/21 (38%), ref. 12; 43 (46%), ref 16 0/14 (0%) Mage-1 17/17 (100%) 12/14 (86%), ref. 45; (38%) ref. 16 11/11 (100%) 8/21 (38%), ref. 1243 = n (40%), ref. 16 3/14 (21%) Mart-1 13/17 (76%) 4/11 (36%) 0/14 (0%) Survivin 17/17 (100%) 45/56 (80%), ref. 25 10/11 (91%) 1/14 (7%) (83%) ref. 26 hTert 17/17 (100%) 10/10 (100%) 30/43 (70%), ref. 23 0/14 (0%) Trp-1 6/17 (35%) 10/11 (91%) 11/21 (52%), ref. 12 0/14 (0%) Tyrosinase 11/17 (65%) 10/11 (91%) 8/21 (38%), ref. 12 14/14 (100%) Aim-2 10/11 (91%) 17/17 (100%) N = 43 (93%), ref. 18 0/14 (0%) Gage-1 0/11 (0%) 2/17 (12%) 13/20 (65%), ref. 13 14/14 (100%) Sart-1 5/10 (50%), ref. 24 10/11 (91%) 17/17 (100%) 14/14 (100%) Trp-2 9/11 (82%) 7/17 (100%) 13/21 (62%), ref. 12 0/14 (0%) NOTE: Microarray based on http://www.broad.mit.edu/mpr/publications/projects/Cancer_Susceptibility/glioma_nutt_combo.gct.
Abbreviations: GBM, glioblastoma multiforme; qPCR, quantitative PCR.
Volume 13, Issue 2
