_:b207555022 . . . _:b207555017 . _:b207555016 . . _:b36885 . _:b207555019 . _:b207555009 . _:b207555018 . . _:b207555029 . . _:b207555028 . . _:b207554961 . _:b207555025 . _:b36925 "In several recent papers, the immune response of BALB/c mice to a Mu p53 peptide was contrasted with that same peptide containing a point mutation (Mu p53.234-240 and 236 M to I) (>>15<<\u201317). Although immunization with peptide in adjuvant resulted in responsiveness only to the mutated sequence (15, 16), a response to the WT peptide was obtained when these same mice were injected with dendritic cells pulsed with peptide" . _:b207555024 . _:b36875 . _:b207555027 . _:b207554988 . _:b207555026 . . _:b36926 "Although immunization with peptide in adjuvant resulted in responsiveness only to the mutated sequence (>>15<<, 16), a response to the WT peptide was obtained when these same mice were injected with dendritic cells pulsed with peptide (17)." . _:b36900 . . _:b207554953 . _:b36910 . _:b207554965 . . _:b36921 . . _:b36922 . . . . _:b36917 . _:b36918 . _:b207554977 . _:b36911 "Recent studies demonstrate that low avidity T cells can escape the negative selection process and emerge into the periphery (>>54<<\u201357)." . _:b207554974 . _:b36919 . _:b207554987 . . _:b207554939 . _:b207554933 . . . _:b207555011 . _:b36889 "at the base of the tail with 100 \u03BCg of the indicated WT p53 peptide and 120 \u03BCg of the I-Ab-binding synthetic T helper peptide representing residues 128-140 of the hepatitis B virus core protein (>>36<<) emulsified in 100 \u03BCl of IFA (23)." . . _:b207554926 . . . _:b36919 "In contrast to our results with Mu WT p53.261-269 and to studies with Hu PBL (>>19<<\u201321), we could not detect A2.1restricted CTL with even low avidity for the WT p53.187197 T cell epitope in p53+/+ A2.1/Kb-Tg animals." . . _:b36887 . _:b36886 . _:b36899 "Monolayers were disrupted by trypsinization, and cells labeled with 51Cr as previously described (>>27<<)." . _:b36884 . _:b207554938 . _:b36903 "2, C and D), a method that is known to facilitate TCR-mediated Ag recognition and target cell lysis by increasing the numbers both of MHC-peptide complexes and of adhesion molecules expressed on the cell surface (51, >>52<<). The failure of the particular p53\u2212/\u2212 A2Kb 187 CTL clone 4 to lyse untreated Saos-2/143 cells was consistent with the previous finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as" . . _:b36881 . _:b207554963 . _:b36894 "CTL clones specific for Hu WT p53.187-197 (p53\u2212/\u2212 A2Kb 187 clone 4) and 149-157 (A2 149 clone 5) were derived by limiting dilution (>>46<<) of effector cell populations originally obtained from peptide primed p53\u2212/\u2212 A2.1/Kb and A2.1Tg mice, respectively." . . . _:b36876 "The p53\u2212/\u2212 mice (>>26<<) were obtained from Tyler Jacks (Massachusetts Institute of Technology) and mated with line 6 A2.1/Kb-Tg (23, 25, 28)." . _:b36895 . _:b36905 "the previous finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu \u03B1-3 domain of the A2.1 molecule (23, 25, >>27<<, 28, 31). However, due to their in vivo selection and stimulation in the absence of a sufficient participation by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant" . . . _:b36893 . _:b207555012 . _:b36915 . _:b36892 . . _:b207554989 . _:b36902 "2, C and D), a method that is known to facilitate TCR-mediated Ag recognition and target cell lysis by increasing the numbers both of MHC-peptide complexes and of adhesion molecules expressed on the cell surface (>>51<<, 52). The failure of the particular p53\u2212/\u2212 A2Kb 187 CTL clone 4 to lyse untreated Saos-2/143 cells was consistent with the previous finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as" . . . . . _:b36904 "with the previous finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu \u03B1-3 domain of the A2.1 molecule (23, >>25<<, 27, 28, 31)." . . . _:b36918 "Whereas CTL from Tg mice were able to lyse Hu p53 transfectants and/or Hu tumor cells (23), this has not been reported for CTL from Hu PBL (>>19<<\u201321), thereby raising the possibility that the latter represent low avidity T cells that have survived p53-specific tolerance induction." . _:b36881 "(T2A2Kb), Jurkat-A2.1 (JA2), the H-2b/b thymoma line EL4, EL4-A2.1/Kb (EA2Kb), the naturally A2.1-expressing, p53-deficient osteosarcoma line Saos-2, and this same line transfected with a Hu mutant p53 gene, Saos2/143 (23, 25, >>27<<, 42, 43). To obtain EA2Kb." . _:b207554937 . . _:b207554986 . _:b36876 . _:b36889 . . . _:b36907 . _:b36924 "A2.1-restricted T cells specific for peptides with high binding affinity to A2.1 could differ considerably from each other in their selection of low avidity T cells specific for self-peptides with intermediate A2.1-binding affinity (>>38<<)." . _:b36929 . _:b207555015 . _:b207555008 . _:b36901 . _:b36928 . _:b207554993 . _:b36914 . . _:b36916 . _:b207554982 . _:b207554983 . . . . _:b207555000 . _:b207554973 . _:b36897 "The vaccinia recombinant, vv-A2.1, which expresses the HLA-A2.1 molecule (>>48<<) was kindly provided by Dr. M. Nishimura (Surgery Branch, National Cancer Institute)." . _:b36880 "T2-A2.1/Kb (T2A2Kb), Jurkat-A2.1 (JA2), the H-2b/b thymoma line EL4, EL4-A2.1/Kb (EA2Kb), the naturally A2.1-expressing, p53-deficient osteosarcoma line Saos-2, and this same line transfected with a Hu mutant p53 gene, Saos2/143 (23, >>25<<, 27, 42, 43). To obtain EA2Kb." . . _:b207554976 . _:b36900 "results" . _:b36924 . _:b36910 _:b36928 . _:b36910 _:b36929 . _:b36910 _:b36930 . _:b36914 . . . . _:b36890 "the priming WT p53 peptide at 5 \u03BCg/ml and Hu \u03B22-microglobulin at 10 \u03BCg/ml in complete RPMI media (RPMI1640 containing 10% vol/vol fetal calf serum, 25 mM Hepes, 2 mM glutamine, 5 \u00D7 10\u22125 M \u03B2-mercaptoethenol and 50 \u03BCg/ml gentamycin) (23, >>25<<, 28). After 6 d, the resultant effector cells were assayed in a 4-h 51Cr-release assay (27) at various E/T ratios for lytic activity against T2A2Kb and EL4 targets that had been pulsed with either the priming WT p53 peptide, an unrelated" . _:b36927 "Although immunization with peptide in adjuvant resulted in responsiveness only to the mutated sequence (15, >>16<<), a response to the WT peptide was obtained when these same mice were injected with dendritic cells pulsed with peptide (17)." . _:b36896 "Anti-A2.1 inhibition was performed by exposure of 51Cr-labeled target cells to the anti-A2.1 mAb PA2.1 at saturating, nontoxic concentrations (23, >>47<<)." . _:b207554970 . _:b36899 . _:b36913 "Such gaps may reflect lack of expression or positive selection of TCRs of the relevant specificity, or deletion attributable to the presence of crossreactive epitopes contributed by other proteins (10, >>11<<, 58\u201360)." . _:b207554992 . . _:b207554994 . . _:b207554975 . _:b36900 _:b36901 . _:b36900 _:b36902 . _:b36900 _:b36903 . . _:b36911 . _:b36893 "was established by weekly restimulation of effector CTL with irradiated JA2 cells that had been pulsed with 5 \u03BCg of the indicated p53 peptide, irradiated C57BL/6 spleen filler cells, and 2% (vol/vol) rat Con A supernatant (23, >>27<<). The polyclonal, A2.1restricted CTL line (A2 149) specific for Hu WT p53.149-157 has been established from peptide-primed A2.1-Tg mice as previously reported (23)." . _:b36878 "The p53\u2212/\u2212 mice (26) were obtained from Tyler Jacks (Massachusetts Institute of Technology) and mated with line 6 A2.1/Kb-Tg (23, 25, >>28<<). Progeny were interbred, and offspring screened for mice that were p53\u2212/\u2212 and expressed A2.1/Kb." . . _:b207555023 . _:b36900 _:b36908 . . _:b36900 _:b36909 . _:b36900 _:b36904 . _:b36900 _:b36905 . _:b36900 _:b36906 . _:b36900 _:b36907 . _:b36910 _:b36924 . _:b207554950 . _:b36910 _:b36925 . _:b36910 _:b36926 . _:b36910 _:b36927 . _:b36910 _:b36920 . _:b36910 _:b36921 . _:b36910 _:b36922 . . _:b36910 _:b36923 . . _:b36910 _:b36916 . _:b36902 . _:b36910 _:b36917 . _:b36910 _:b36918 . _:b207554948 . . _:b36910 _:b36919 . _:b36910 _:b36912 . _:b36905 . _:b36910 _:b36913 . _:b36910 _:b36914 . _:b36910 _:b36915 . _:b36910 _:b36911 . _:b36923 "Such conditions may promote expansion of low avidity CTL specific for peptides with intermediate A2.1-binding affinity, such as p53.187-197 (19\u201321, >>63<<). However, the in vivo priming of A2.1/Kb-Tg mice expressing low levels of A2.1/Kb, followed by one cycle of in vitro restimulation of responder spleen cells with peptide-pulsed A2.1/Kb stimulators (in the absence of exogenous cytokines)" . _:b207555020 . . _:b207555001 . . . _:b207555016 . _:b36876 . . _:b36877 . _:b207554964 . _:b36878 . _:b207554955 . _:b36908 . _:b36879 . _:b207554946 . _:b36904 . _:b207554984 . _:b207554971 . _:b36875 . _:b36884 . _:b207555017 . . _:b36879 "Line 22 A2.1-Tg mice were H-2b/b and heterozygous for the transgene, which contains the \u03B1-1, -2, and -3 domain of A2.1 (23, >>25<<). C57BL/6 mice were purchased from the breeding colony of The Scripps Research Institute (TSRI). Mice were propagated and maintained under specific pathogen free conditions in our vivarium at TSRI. All experimental procedures were" . _:b36885 . . . _:b36886 . . _:b36887 . _:b36886 "1p53 transfectants, EL4 cells were cotransfected with 10 \u03BCg of plasmid containing a genomic clone of A2.1/Kb (27) and 2 \u03BCg of pC53-4.2N3 containing a Hu mutant p53 cDNA linked to the neomycin resistance gene (44), as described (>>27<<). The corresponding parental and p53-transfected lines expressed similar levels of A2.1 (Saos-2, Saos2/143) or A2.1/Kb (EA2Kb, EA2Kb.1p53) as detected by flow cytometry (27). The 10(3) Balb/c fibroblast cell line, which lacks endogenous" . _:b36875 "materials and methods" . _:b36880 . _:b36875 _:b36876 . _:b36875 _:b36877 . _:b36875 _:b36878 . _:b36915 "Previous reports have demonstrated that the amount of Ag required for negative selection is less than that required for recognition by effector T cells (>>61<<, 62)." . _:b36881 . _:b36875 _:b36879 . _:b36885 "1p53 transfectants, EL4 cells were cotransfected with 10 \u03BCg of plasmid containing a genomic clone of A2.1/Kb (27) and 2 \u03BCg of pC53-4.2N3 containing a Hu mutant p53 cDNA linked to the neomycin resistance gene (>>44<<), as described (27)." . _:b207554945 . _:b36892 "After 6 d, the resultant effector cells were assayed in a 4-h 51Cr-release assay (>>27<<) at various E/T ratios for lytic activity against T2A2Kb and EL4 targets that had been pulsed with either the priming WT p53 peptide, an unrelated A2.1-binding peptide, or no peptide." . _:b36882 . _:b36880 . _:b36883 . _:b207554947 . _:b36875 _:b36888 . _:b36875 _:b36889 . _:b36875 _:b36890 . _:b36892 . . _:b36875 _:b36891 . _:b36875 _:b36892 . _:b207555019 . _:b36875 _:b36893 . _:b36875 _:b36894 . _:b36893 . _:b36896 . _:b36875 _:b36895 . _:b207554944 . _:b36875 _:b36880 . _:b36875 _:b36881 . _:b36930 . _:b36875 _:b36882 . _:b36894 . _:b36875 _:b36883 . _:b36875 _:b36884 . _:b36875 _:b36885 . _:b207555010 . _:b36875 _:b36886 . _:b36895 . _:b36875 _:b36887 . . . _:b36888 . _:b207554967 . _:b36890 . _:b36928 "Although immunization with peptide in adjuvant resulted in responsiveness only to the mutated sequence (15, 16), a response to the WT peptide was obtained when these same mice were injected with dendritic cells pulsed with peptide (>>17<<). These results indicated that it was more difficult to obtain a response to the WT versus the mutant p53 sequence, consistent with an effect on the repertoire due to self tolerance." . . _:b36889 . _:b36914 "Such gaps may reflect lack of expression or positive selection of TCRs of the relevant specificity, or deletion attributable to the presence of crossreactive epitopes contributed by other proteins (10, 11, >>58<<\u201360)." . . _:b36875 _:b36896 . _:b36875 _:b36897 . _:b36875 _:b36898 . _:b36890 . _:b36875 _:b36899 . _:b207554960 . _:b207555006 . _:b36891 . _:b36900 . _:b36901 . _:b36902 . _:b207554995 . _:b36903 . _:b36896 . _:b36913 . _:b36897 . _:b36911 . _:b36898 . _:b36899 . . _:b36908 . _:b36912 . _:b36909 . _:b36879 . _:b36910 . _:b36911 . _:b207555025 . _:b36877 . _:b36904 . _:b207554972 . _:b36905 . _:b36906 . . _:b36907 . _:b36916 . . _:b207554941 . _:b207554934 . _:b36917 . _:b207555004 . _:b36918 . . _:b36919 . _:b36907 "finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu \u03B1-3 domain of the A2.1 molecule (23, 25, 27, 28, >>31<<). However, due to their in vivo selection and stimulation in the absence of a sufficient participation by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant A2.1-peptide" . _:b36912 . _:b36913 . _:b207554935 . . _:b36914 . . _:b36915 . _:b36921 . _:b36924 . . _:b36922 . _:b36925 . . _:b207554958 . _:b36926 . _:b36882 "(T2A2Kb), Jurkat-A2.1 (JA2), the H-2b/b thymoma line EL4, EL4-A2.1/Kb (EA2Kb), the naturally A2.1-expressing, p53-deficient osteosarcoma line Saos-2, and this same line transfected with a Hu mutant p53 gene, Saos2/143 (23, 25, 27, >>42<<, 43). To obtain EA2Kb." . _:b207554929 . _:b36927 . _:b36917 . _:b36920 . . _:b36921 . _:b36918 . _:b36919 . _:b36922 . _:b36895 "1p53 as well as cytokine- and noncytokine-pretreated Saos-2 and Saos-2/143 targets by Hu WT p53-specific CTL lines and clones was determined in a 5-h 51Cr-release assay (>>27<<). Cytokine-pretreated targets had been exposed for 20 h to both rIFN-\u03B3 (R&D Systems, Minneapolis, MN) at 20 ng/ml and rTNF-\u03B1 (R&D Systems) at 3 ng/ml (23). Anti-A2.1 inhibition was performed by exposure of 51Cr-labeled target cells to the" . . _:b36923 . _:b36925 . . _:b207555007 . _:b207554998 . _:b207554964 . _:b207554965 . . _:b207554966 . . . _:b207554967 . . _:b207554960 . _:b207554961 . _:b207554962 . _:b36909 "by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant A2.1-peptide complex and thus require less peptide Ag for their CD8-independent target cell recognition (23, 25, >>53<<). A2 149 CTL clone 5 as opposed to p53\u2212/\u2212 A2Kb 187 CTL clone 4 was therefore able to lyse untreated Saos-2/143 targets." . _:b207554963 . _:b207554972 . _:b36923 . _:b207554973 . _:b207554974 . _:b207554975 . _:b207554968 . _:b207554969 . _:b36928 . _:b207554927 "4"^^ . _:b207554970 . . _:b207554971 . _:b207554948 . _:b207554949 . _:b36929 . _:b207554930 . _:b207554950 . _:b207554926 "5"^^ . _:b207554951 . _:b207554944 . _:b207554945 . _:b36930 . _:b207555003 . _:b207554946 . _:b207554947 . _:b207554956 . _:b207554957 . _:b207554958 . _:b207554959 . . _:b207554952 . _:b207554953 . _:b207554954 . _:b207554955 . _:b207554996 . _:b36906 "previous finding that CTL from A2.1/Kb-Tg mice are at a disadvantage in recognition of cells expressing A2.1 as compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu \u03B1-3 domain of the A2.1 molecule (23, 25, 27, >>28<<, 31). However, due to their in vivo selection and stimulation in the absence of a sufficient participation by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant A2.1-peptide" . _:b207554997 . _:b207554998 . _:b207554999 . _:b207554992 . _:b207554933 "3"^^ . _:b207554993 . . _:b207554994 . _:b36888 . . _:b207554995 . _:b207555004 . _:b207554932 "3"^^ . _:b207555005 . _:b207554927 . _:b36920 "Alternatively, the lower A2.1 binding affinity of p53.187-197 relative to Mu p53.261-269 (23) in conjunction with the low level of expression of the A2.1/Kb transgene relative to the level of expression of conventional MHC molecules (>>28<<), could be directly or indirectly involved." . _:b207555006 . _:b207555007 . _:b207554966 . _:b207555000 . _:b207554935 "3"^^ . _:b207555001 . _:b207555002 . _:b207555003 . _:b36898 . _:b207554980 . _:b207554934 "3"^^ . _:b207554981 . _:b207554982 . _:b36910 "discussion" . _:b207554983 . _:b36903 . _:b207554976 . _:b207554977 . _:b207554929 "4"^^ . _:b207554978 . . _:b36884 "1p53 transfectants, EL4 cells were cotransfected with 10 \u03BCg of plasmid containing a genomic clone of A2.1/Kb (>>27<<) and 2 \u03BCg of pC53-4.2N3 containing a Hu mutant p53 cDNA linked to the neomycin resistance gene (44), as described (27)." . _:b207554979 . _:b207554988 . _:b207554989 . _:b207554928 "4"^^ . _:b207554990 . _:b207554991 . _:b207554984 . _:b207554985 . _:b207554931 "4"^^ . _:b207554986 . _:b207554962 . _:b207554987 . _:b207555028 . _:b207555029 . _:b207554930 "4"^^ . _:b36917 "a previous study (23) to be immunogenic in Tg mice and to represent naturally processed CTL epitopes, have also been demonstrated to stimulate Hu PBL primarily in vitro and to induce an A2.1-restricted, peptide-specific CTL response (>>19<<\u201321). Whereas CTL from Tg mice were able to lyse Hu p53 transfectants and/or Hu tumor cells (23), this has not been reported for CTL from Hu PBL (19\u201321), thereby raising the possibility that the latter represent low avidity T cells that" . . _:b207555024 . . _:b207554941 "3"^^ . _:b207555025 . _:b207555026 . . _:b207555027 . _:b207554940 "3"^^ . . . _:b207554943 "3"^^ . _:b207554985 . _:b36882 . _:b207555012 . _:b207554942 "3"^^ . _:b36908 "by Mu CD8, CTL from A2.1Tg mice, such as A2 149 clone 5, express TCRs with unusually high affinity for the relevant A2.1-peptide complex and thus require less peptide Ag for their CD8-independent target cell recognition (23, >>25<<, 53). A2 149 CTL clone 5 as opposed to p53\u2212/\u2212 A2Kb 187 CTL clone 4 was therefore able to lyse untreated Saos-2/143 targets." . _:b207555013 . _:b207555014 . _:b207555015 . _:b36897 . _:b207555008 . _:b207554937 "3"^^ . _:b207555009 . _:b207555010 . _:b207555011 . _:b207554991 . _:b207555020 . _:b207554936 "3"^^ . _:b207555021 . _:b207555022 . _:b207555023 . _:b207555016 . _:b207554939 "3"^^ . _:b207555017 . _:b207555018 . _:b207554996 . _:b207555018 . _:b207555019 . . _:b207554938 "3"^^ . _:b207554949 "2"^^ . _:b207554978 . . . _:b207554948 "2"^^ . _:b207554951 "2"^^ . _:b207554951 . _:b207554950 "2"^^ . . . _:b207554945 "3"^^ . _:b207554944 "3"^^ . _:b207554968 . . _:b207554947 "2"^^ . _:b207554940 . _:b36927 . _:b207555028 . _:b36901 "A prerequisite for tolerance induction by any self peptide is its ability to be endogenously processed and transported into the endoplasmic reticulum for association with class I MHC molecules (>>50<<). To determine whether the homologous WT p53.187-197 peptide is actually presented as naturally processed T cell epitope by A2.1 on the surface of Mu and Hu cells, both a peptide-specific, polyclonal CTL line (p53\u2212/\u2212 A2Kb 187) and a CTL" . _:b207554942 . _:b207554946 "2"^^ . . _:b36911 . _:b207554952 . . _:b207554957 "2"^^ . _:b36925 . _:b207554956 "2"^^ . . _:b207554959 "2"^^ . _:b207554958 "2"^^ . . . . _:b207554953 "2"^^ . _:b207554927 . _:b207554932 . _:b207554952 "2"^^ . _:b207554926 . _:b207554955 "2"^^ . _:b207554954 "2"^^ . _:b207554965 "2"^^ . _:b207555026 . _:b36887 "1p53) as detected by flow cytometry (>>27<<). The 10(3) Balb/c fibroblast cell line, which lacks endogenous Mu p53 and a transfectant of the line that expresses a Mu p53 gene containing a mutation at residue 215, were kindly provided by Drs." . _:b36912 "Such gaps may reflect lack of expression or positive selection of TCRs of the relevant specificity, or deletion attributable to the presence of crossreactive epitopes contributed by other proteins (>>10<<, 11, 58\u201360)." . _:b207554964 "2"^^ . _:b36922 "Such conditions may promote expansion of low avidity CTL specific for peptides with intermediate A2.1-binding affinity, such as p53.187-197 (>>19<<\u201321, 63). However, the in vivo priming of A2.1/Kb-Tg mice expressing low levels of A2.1/Kb, followed by one cycle of in vitro restimulation of responder spleen cells with peptide-pulsed A2.1/Kb stimulators (in the absence of exogenous" . . _:b207554967 "2"^^ . _:b207554933 . _:b207554966 "2"^^ . _:b207554932 . . _:b207554956 . _:b207554981 . _:b207554961 "2"^^ . _:b207554935 . _:b207554960 "2"^^ . _:b36898 "The vPE16 (>>49<<) vaccinia recombinant, which expresses the gp160 of the human immunodeficiency virus type 1, was kindly provided by Drs." . _:b207554934 . _:b207554963 "2"^^ . . _:b207554929 . _:b207554962 "2"^^ . _:b207554999 . _:b207554928 . _:b207554973 "2"^^ . _:b207554931 . . _:b207554972 "2"^^ . _:b207554930 . _:b207554975 "2"^^ . _:b36916 "Previous reports have demonstrated that the amount of Ag required for negative selection is less than that required for recognition by effector T cells (61, >>62<<)." . _:b207554941 . _:b207555027 . _:b207554974 "2"^^ . _:b207554940 . _:b207554980 . _:b207554969 "2"^^ . _:b207554943 . . _:b207554968 "2"^^ . _:b207554942 . _:b207554971 "2"^^ . _:b207554937 . _:b207554970 "2"^^ . _:b207554932 . _:b207554936 . _:b207554933 . _:b207554934 . _:b36878 . _:b207554935 . _:b207554981 "2"^^ . _:b207554928 . _:b207554939 . _:b207554929 . _:b207554930 . _:b207554931 . . _:b207554940 . _:b207554957 . _:b207554980 "2"^^ . _:b207554938 . _:b207554941 . "PMC0" . _:b207554942 . _:b207554943 . _:b207554983 "2"^^ . _:b207554936 . _:b207554937 . _:b207554949 . _:b207554938 . _:b207554939 . . _:b207554982 "2"^^ . . _:b207554948 . . _:b207554977 "2"^^ . _:b207554951 . _:b207554976 "2"^^ . _:b207554950 . _:b207554926 . _:b207554927 . _:b207554979 "2"^^ . _:b207554945 . . _:b36909 . _:b207554978 "2"^^ . _:b207554944 . _:b36930 "These residual low avidity CTL, however, could provide an opportunity for immunotherapy of tumors that express high levels of p53 (17, >>18<<). Given the variability of the effect observed on the repertoire by selftolerance to different peptides, as well as variability of responsiveness due to different modes of immunization, it is likely that the success of the immunotherapy" . _:b207554989 "2"^^ . _:b36929 "These residual low avidity CTL, however, could provide an opportunity for immunotherapy of tumors that express high levels of p53 (>>17<<, 18). Given the variability of the effect observed on the repertoire by selftolerance to different peptides, as well as variability of responsiveness due to different modes of immunization, it is likely that the success of the" . _:b207554947 . _:b207554988 "2"^^ . _:b207554946 . . _:b207554991 "2"^^ . _:b207554957 . . _:b36891 . _:b207554990 "2"^^ . _:b207554956 . _:b207554985 "2"^^ . _:b207554959 . _:b207554984 "2"^^ . _:b207554958 . . _:b207554987 "2"^^ . _:b36877 "The p53\u2212/\u2212 mice (26) were obtained from Tyler Jacks (Massachusetts Institute of Technology) and mated with line 6 A2.1/Kb-Tg (23, >>25<<, 28). Progeny were interbred, and offspring screened for mice that were p53\u2212/\u2212 and expressed A2.1/Kb." . _:b207554953 . _:b207554986 "2"^^ . _:b207554952 . _:b207554997 "2"^^ . _:b207555014 . _:b207554955 . _:b207554931 . _:b207554996 "2"^^ . _:b207554954 . _:b207554954 . _:b207554999 "2"^^ . _:b207554965 . _:b207554969 . _:b207554998 "2"^^ . _:b207555021 . . _:b207554964 . _:b207554936 . _:b207554993 "2"^^ . _:b207554967 . _:b207554992 "2"^^ . _:b36921 "peptide-specific CTL involved repeated cycles of restimulation with Hu stimulators expressing high levels of A2.1 and pulsed with high concentrations of synthetic peptide in the presence of exogenous T cell growth promoting cytokines (>>19<<\u201321). Such conditions may promote expansion of low avidity CTL specific for peptides with intermediate A2.1-binding affinity, such as p53.187-197 (19\u201321, 63)." . _:b207554966 . . _:b207554995 "2"^^ . _:b207554961 . _:b207554994 "2"^^ . _:b207554960 . . _:b207555005 "2"^^ . _:b207554963 . _:b207555004 "2"^^ . _:b207554962 . _:b207554949 . . . _:b207555007 "2"^^ . _:b207554973 . _:b207555006 "2"^^ . _:b207554972 . _:b36906 . _:b207555001 "2"^^ . _:b207554975 . _:b207555005 . . . . _:b207555000 "2"^^ . _:b207554974 . _:b207555003 "2"^^ . _:b207554969 . . _:b207555002 "2"^^ . _:b207554968 . _:b207555013 "2"^^ . _:b207554971 . _:b207555012 "2"^^ . _:b207554970 . _:b36888 "Dirk Dittmer and Arnold Levine (Princeton University) (>>42<<, 45)." . _:b207555015 "2"^^ . _:b207554981 . . _:b207555014 "2"^^ . _:b207554980 . _:b207555009 "2"^^ . _:b207554983 . _:b207555008 "2"^^ . _:b207554982 . _:b207555011 "2"^^ . _:b207554977 . _:b36891 "priming WT p53 peptide at 5 \u03BCg/ml and Hu \u03B22-microglobulin at 10 \u03BCg/ml in complete RPMI media (RPMI1640 containing 10% vol/vol fetal calf serum, 25 mM Hepes, 2 mM glutamine, 5 \u00D7 10\u22125 M \u03B2-mercaptoethenol and 50 \u03BCg/ml gentamycin) (23, 25, >>28<<). After 6 d, the resultant effector cells were assayed in a 4-h 51Cr-release assay (27) at various E/T ratios for lytic activity against T2A2Kb and EL4 targets that had been pulsed with either the priming WT p53 peptide, an unrelated" . _:b36911 . . _:b207554997 . _:b207555010 "2"^^ . _:b207554976 . . _:b207555021 "2"^^ . _:b207554979 . . _:b36914 . _:b207555020 "2"^^ . . _:b207554978 . _:b207555023 "2"^^ . _:b207554989 . _:b207555022 "2"^^ . _:b207554988 . _:b207555017 "2"^^ . _:b207554991 . _:b207555016 "2"^^ . . _:b207554990 . _:b207554928 . _:b36920 . . _:b207555019 "2"^^ . _:b207554985 . _:b207555018 "2"^^ . _:b207554984 . _:b207554959 . _:b207555029 "2"^^ . _:b207554987 . _:b207555028 "2"^^ . _:b36883 "(T2A2Kb), Jurkat-A2.1 (JA2), the H-2b/b thymoma line EL4, EL4-A2.1/Kb (EA2Kb), the naturally A2.1-expressing, p53-deficient osteosarcoma line Saos-2, and this same line transfected with a Hu mutant p53 gene, Saos2/143 (23, 25, 27, 42, >>43<<). To obtain EA2Kb." . _:b207554986 . _:b207555002 . _:b207554997 . _:b207554996 . _:b207555025 "2"^^ . . _:b207554999 . _:b207555024 "2"^^ . _:b207554998 . . _:b207555027 "2"^^ . _:b207554993 . _:b207555026 "2"^^ . _:b207554992 . . _:b207554995 . _:b207554994 . . _:b207555005 . . _:b207555004 . _:b207555007 . _:b207555013 . _:b207555006 . _:b207555001 . _:b207555022 . _:b36926 . . _:b207555000 . _:b207554943 . _:b207555003 . _:b207554990 . _:b36883 . _:b207555002 . _:b36925 . . _:b207554979 . _:b207555013 . . . _:b207555012 . _:b207555015 . . _:b207555014 . _:b207555029 . _:b207555009 . _:b207555024 . . _:b207555008 . _:b207555011 . . _:b207555010 . . _:b207555021 . _:b36894 . _:b207555020 . _:b207555023 . . . .