Article Figures & Data
Figures
- Fig. 1.
Patient's anti-p53 antibodies in immunoprecipitation assays react with both mutant (248) and wild-type (SN3) forms of the p53 protein. As a positive control, a murine monoclonal antibody to p53 was reacted with the wild-type p53 (lane a). Monoclonal antibody to I-CAM was reacted with mutant p53 as a negative control (lane b). Patient's sera without anti-p53 did not bind the labeled p53 (lanes c-f); however, sera from patients with p53 missense mutation bound both mutant and wild-type p53 (lanes g-h), respectively.
- Fig. 2.
Western blot assay using fragments of p53 as fusion proteins. No immunodominant regions were found. Examples of two patient's reactions. A, patient 296 reacted with the 35 and N5 fusion proteins. B, patient 309 had antibodies that recognized NC and N5 proteins. Oct 1 pou was an unrelated protein that served as a negative control.
- Fig. 3.
A, HLA-DQ7 (DQB1*0301) binding motif (1) and p53 mutant peptide sequences. The mutated amino acid residues are in italics and underlined. Amino acids that match predicted anchor residues are in bold letters. If the peptides were predicted to bind two or more anchor residues, antibody production was predicted. B, HLA-DR1 (DRB1*0101) binding motif and p53 mutant peptide sequences. The mutated amino acid residues are in italics and underlined. Amino acids that match predicted anchor residues are in bold letters. All patients had the DRB1*0101-DQB1*0501 haplotype. In patients wherein more than one anchor residue was predicted to bind, antibody production was seen (patients 66 and 296). Conversely, p53 serum antibodies were not seen in the patient where less than two anchor residues bound the motif (patient 245).
- Fig. 4.
IFN-γ and IL-5 ELISPOT assays to measure CD4+ T-cell responses in healthy HLA-DQ7+ donor PBMC after stimulation with wild-type p53210-223 or mutant p53220C peptides. A, after 1 wk of IVS using autologous, mature DC pulsed with the wild-type p53210-223 or p53220C peptide, respectively. HLA-DQ7+ healthy CD4+ T cells were assayed using IFN-γ ELISPOT assays. Stimulator cells in these assays were autologous, mature day 7 DC loaded with the same wild-type p53210-223 or p53220C peptides. Pretreatment with either HLA-A, HLA-B. HLA-C–specific mAb W6/32 (30), HLA-DR–specific mAb (L243), or HLA-DR, HLA-DP, HLA-DQ antigen–specific mAb (LGII-612.14) or without any mAb was done to block the recognition of CD4+ T cells as described in Materials and Methods. Columns, mean spots; bars, SD. A representative of experiments repeated at least twice with four separate HLA-DQ7+ healthy donor PBMC (P < 0.05). B, after 1 wk of IVS using autologous, mature DC pulsed with the wild-type p53210-223 or p53220C peptide, respectively. HLA-DQ7+ healthy CD4+ T cells were assayed using IL-5 ELISPOT assays. Stimulator cells in these assays were autologous, mature day 7 DC loaded with the same wild-type p53210-223 or p53220C peptides. Pretreatment with either HLA-A, HLA-B, HLA-C–specific mAb W6/32 (30), HLA-DR–specific mAb, (L243) or HLA-DR, HLA-DP, HLA-DQ antigen–specific mAb (LGII-612.14) or without any mAb was done to block the recognition of CD4+ T cells as described in Materials and Methods. Columns, mean spots; bars, SD. A representative of experiments repeated twice with four separate HLA-DQ7+ healthy donor PBMC (P < 0.05).
Tables
- Table 1.
Correlation of patient's anti-p53 antibody production with p53 mutations and immunodominant epitopes
Patient no. Mutation codon p53 antibody Western blot fragments 40 tyr→cys + 2C, 23 53 tyr→cys + NC, N5, 2C, 35, 23 168 tyr→cys + N5, NC, 35 66 arg→gln + 5, NC, 23 226 gly→glu + N5, NC 309 pro→ser + N5, NC 296 pro→leu + N5, 35 200 his→arg - — 192 arg→gly - — 69 ile→asn - — 245 cys→tyr - — 62 G insert - — 99 arg→leu - — 27 tyr→ser - — 71 glu→stop - — NOTE: Patients' sera reacted with evolutionarily conserved domains of the p53 protein to determine possible immunodominant epitopes on the p53 molecule. Tumors from the patients were sequenced to determine the exact p53 mutation. Sera was tested for the presence of anti-p53 antibodies using a nondenaturing immunoprecipitation assay. Sera containing anti-p53 antibodies were incubated with various p53 fragments that were created using glutathione S-transferase fusion proteins in E. coli. This Western blot assay determined if the patient's sera bound to particular p53 fragments more frequently than others, thereby establishing if possible immunodominant portions7 of the p53 protein existed.
- Table 2.
Correlation of patients' antibody production and p53 mutations with class II haplotypes
Patient no. Anti-p53 antibody p53 mutation codon/exon DR-DQ1 DR-DQ2 DPB1 DPB2 27 No Yes/220/6 1302-0609 0103-0501 0401 0201 40 Yes Yes/220/6 1201-0301 1201-0301 0101 3601 46 No No 1601-0502 0301-0201 0401 0402 49 No No 1501-0602 0101-0501 0401 0401 53 Yes Yes/220/6 1502-0601 0701-0303 1501 0301 55 No No 0701-0201 1305-0301 — — 56 No No 1501-0602 1101-0301 3301 4101 58 No No 1301-0603 0701-0303 - - 59 No No 0103-0301 0301-0201 0401 0201 62 No Yes/279/8 1501-0503 1401-0503 0401 1001 63 No No 0103-0501 0301-0201 0401 0401 66 Yes Yes/248/7 0101-0501 0701-0303 3701 4601 69 No Yes/251/7 1101-0301 1101-0301 3701 3701 71 No No 0701-0201 0301-0201 0201 1101 75 No No 1503-0602 0302-0402 0201 0101 77 No No 1302-0609 0701-0303 0402 0101 85 No No 0101-0501 0701-0303 0401 0401 93 No No 0701-0201 0701-0201 - - 98 No No 0301-0201 0402-0301 0402 0301 99 No Yes/306/8 1201-0301 0701-0201 — — 100 No No 0301-0201 0301-0201 0201 0402 102 No No 0301-0201 1602-0502 — — 107 No No 0801-0301 0701-0201 0402 0301 168 Yes Yes/220/6 1503-0602 1102-0301 0401 0201 180 No No 0701-0201 0402-0301 — — 191 No No 0401-0301 0101-0501 5101 1101 192 No Yes/249/7 0701-0201 0701-0201 1301 1401 200 No Yes/193/6 1401-0503 1503-0502 0301 1301 217 No No 0401-0302 0404-0303 0401 0201 226 Yes Yes/272/8 0403/06-0302 0407-0301 0401 0301 230 No No 1501-0602 0301-0201 0401 0301 233 No No 0701-0201 1302-0501 1801 0101 242 No No 0101-0501 1104-0301 0402 0402 244 No No 0101-0501 0101-0501 0401 0402 245 No Yes/275/8 0101-0501 0701-0201 0401 0401 253 No No 1602-0502 1303-0301 0201 1701 269 No No 1502-0601 1502-0601 2301 4801 270 No No 0102-0501 1103-0301 0401 0402 273 No No 0301-0201 1302-0501 1101 4001 278 No No 1302-0609 0901-0303 0101 1701 289 No No 0901-0303 0102-0501 0401 0201 296 Yes Yes/278/8 0701-0201 0101-0501 0401 0301 309 Yes Yes/278/8 1101-0602 0102-0501 0201 0101 NOTE: Tumor DNA was isolated PCR-amplified for the class II loci listed above and then probed with horseradish peroxidase–labeled oligonucleotides as described in Materials and Methods (—, not determined).
- Table 3.
Competitive binding assay of p53 mutant peptides to DQ7 (DQA1*0301, DQB1*0301, DQB 3.1)
Peptide IC50 (umol/L) AYK (positive control) <0.1 34P (negative control) >10 p53 mutant patient 168 0.58 p53 wild-type patient 168 4.5 p53 mutant patient 69 >10 p53 wild-type patient 69 >10 p53 mutant patient 226 >10 p53 wild-type patient 226 >10 NOTE: p53 peptides, both mutant and wild type, were sequenced (14-mers), and IC50 was determined.
Volume 13, Issue 23
