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Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes.

Theobald M, Biggs J, HernΓ‘ndez J, Lustgarten J, Labadie C, Sherman LA - J. Exp. Med. (1997)

Bottom Line: Elevated levels of the p53 protein occur in approximately 50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer.A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells.The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Elevated levels of the p53 protein occur in approximately 50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer. A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells. The combination of p53 deficient (p53-/-) and p53+/+ HLA-A2.1/Kb transgenic mice was used as a model to explore the possibility that A2.1-restricted cytotoxic T lymphocytes (CTL) are functionally tolerant of self peptides derived from the wild-type p53 tumor suppressor protein. A2.1-restricted CTL specific for a naturally processed p53 self-epitope spanning residues 187-197 were completely aborted in p53+/+ as opposed to p53-/- transgenic mice. In contrast, CTL specific for a second self-epitope spanning residues 261-269 of the murine p53 sequence were detected in both p53-/- and p53+/+ A2.1/Kb transgenic mice. However, the avidity of the CTL effectors obtained from p53+/+ mice was 10-fold lower than that obtained from p53-/- mice, again suggesting elimination of CTL with high avidity for the A2.1-peptide complex. The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.

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Recognition of naturally processed WT p53 epitopes by  A2.1-restricted CTL. The responder CTL line and clone specific for WT  p53.187-197 and derived from p53βˆ’/βˆ’ A2.1/Kb-Tg mice was (A) p53βˆ’/βˆ’  A2Kb 187 and (C) p53βˆ’/βˆ’ A2Kb 187 clone 4, respectively. The responder  CTL line and clone specific for Hu WT p53.149-157 and derived from  (p53+/+) A2.1-Tg mice was (B) A2 149 and (D) A2 149 clone 5, respectively. CTL lines and clones were assayed for cytotoxicity against the following targets: EA2Kb (open circles), EA2Kb.1p53 transfectants (closed circles), untreated p53-deficient Saos-2 (open squares), Saos-2 pretreated with  IFN-Ξ³ and TNF-Ξ± (closed squares), untreated Saos-2/143 p53-transfectants  (open triangle), and Saos-2/143 pretreated with IFN-Ξ³ and TNF-Ξ± (closed  triangles).
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Figure 2: Recognition of naturally processed WT p53 epitopes by A2.1-restricted CTL. The responder CTL line and clone specific for WT p53.187-197 and derived from p53βˆ’/βˆ’ A2.1/Kb-Tg mice was (A) p53βˆ’/βˆ’ A2Kb 187 and (C) p53βˆ’/βˆ’ A2Kb 187 clone 4, respectively. The responder CTL line and clone specific for Hu WT p53.149-157 and derived from (p53+/+) A2.1-Tg mice was (B) A2 149 and (D) A2 149 clone 5, respectively. CTL lines and clones were assayed for cytotoxicity against the following targets: EA2Kb (open circles), EA2Kb.1p53 transfectants (closed circles), untreated p53-deficient Saos-2 (open squares), Saos-2 pretreated with IFN-Ξ³ and TNF-Ξ± (closed squares), untreated Saos-2/143 p53-transfectants (open triangle), and Saos-2/143 pretreated with IFN-Ξ³ and TNF-Ξ± (closed triangles).

Mentions: 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 (p53βˆ’/βˆ’ A2Kb 187) and a CTL clone (p53βˆ’/βˆ’ A2Kb 187 clone 4) were established from p53βˆ’/βˆ’ A2.1/KbTg mice and tested for p53-specific recognition of Mu and Hu cell lines transfected with Hu p53, EA2Kb.1p53 and Saos-2/143, respectively (Fig. 2, A and C). As a positive control for p53-specific, A2.1-restricted lysis, the polyclonal CTL line A2 149 and the A2 149 CTL clone 5, both of which were derived from A2.1-Tg mice and specific for the natural CTL epitope Hu WT p53.149-157 (23), were included in these studies (Fig. 2, B and D). Comparison of the levels of lysis of the p53 transfectants relative to the parental target lines indicated that both WT p53.187-197 and 149-157 were endogenously processed and presented by Mu and Hu cells. Recognition was A2.1-restricted, as lysis was inhibited by an A2.1-specific antibody (data not shown). To obtain substantial Ag-specific lysis of A2.1-expressing Saos-2/143 cells by p53βˆ’/βˆ’ A2Kb 187 CTL clone 4 as opposed to A2 149 CTL clone 5, targets had to be pretreated by IFN-Ξ³ and TNF-Ξ± (Fig. 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 p53βˆ’/βˆ’ 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 compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu Ξ±-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 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 p53βˆ’/βˆ’ A2Kb 187 CTL clone 4 was therefore able to lyse untreated Saos-2/143 targets.


Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes.

Theobald M, Biggs J, HernΓ‘ndez J, Lustgarten J, Labadie C, Sherman LA - J. Exp. Med. (1997)

Recognition of naturally processed WT p53 epitopes by  A2.1-restricted CTL. The responder CTL line and clone specific for WT  p53.187-197 and derived from p53βˆ’/βˆ’ A2.1/Kb-Tg mice was (A) p53βˆ’/βˆ’  A2Kb 187 and (C) p53βˆ’/βˆ’ A2Kb 187 clone 4, respectively. The responder  CTL line and clone specific for Hu WT p53.149-157 and derived from  (p53+/+) A2.1-Tg mice was (B) A2 149 and (D) A2 149 clone 5, respectively. CTL lines and clones were assayed for cytotoxicity against the following targets: EA2Kb (open circles), EA2Kb.1p53 transfectants (closed circles), untreated p53-deficient Saos-2 (open squares), Saos-2 pretreated with  IFN-Ξ³ and TNF-Ξ± (closed squares), untreated Saos-2/143 p53-transfectants  (open triangle), and Saos-2/143 pretreated with IFN-Ξ³ and TNF-Ξ± (closed  triangles).
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Figure 2: Recognition of naturally processed WT p53 epitopes by A2.1-restricted CTL. The responder CTL line and clone specific for WT p53.187-197 and derived from p53βˆ’/βˆ’ A2.1/Kb-Tg mice was (A) p53βˆ’/βˆ’ A2Kb 187 and (C) p53βˆ’/βˆ’ A2Kb 187 clone 4, respectively. The responder CTL line and clone specific for Hu WT p53.149-157 and derived from (p53+/+) A2.1-Tg mice was (B) A2 149 and (D) A2 149 clone 5, respectively. CTL lines and clones were assayed for cytotoxicity against the following targets: EA2Kb (open circles), EA2Kb.1p53 transfectants (closed circles), untreated p53-deficient Saos-2 (open squares), Saos-2 pretreated with IFN-Ξ³ and TNF-Ξ± (closed squares), untreated Saos-2/143 p53-transfectants (open triangle), and Saos-2/143 pretreated with IFN-Ξ³ and TNF-Ξ± (closed triangles).
Mentions: 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 (p53βˆ’/βˆ’ A2Kb 187) and a CTL clone (p53βˆ’/βˆ’ A2Kb 187 clone 4) were established from p53βˆ’/βˆ’ A2.1/KbTg mice and tested for p53-specific recognition of Mu and Hu cell lines transfected with Hu p53, EA2Kb.1p53 and Saos-2/143, respectively (Fig. 2, A and C). As a positive control for p53-specific, A2.1-restricted lysis, the polyclonal CTL line A2 149 and the A2 149 CTL clone 5, both of which were derived from A2.1-Tg mice and specific for the natural CTL epitope Hu WT p53.149-157 (23), were included in these studies (Fig. 2, B and D). Comparison of the levels of lysis of the p53 transfectants relative to the parental target lines indicated that both WT p53.187-197 and 149-157 were endogenously processed and presented by Mu and Hu cells. Recognition was A2.1-restricted, as lysis was inhibited by an A2.1-specific antibody (data not shown). To obtain substantial Ag-specific lysis of A2.1-expressing Saos-2/143 cells by p53βˆ’/βˆ’ A2Kb 187 CTL clone 4 as opposed to A2 149 CTL clone 5, targets had to be pretreated by IFN-Ξ³ and TNF-Ξ± (Fig. 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 p53βˆ’/βˆ’ 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 compared with A2.1/Kb, due to the inability of Mu CD8 to interact with the Hu Ξ±-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 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 p53βˆ’/βˆ’ A2Kb 187 CTL clone 4 was therefore able to lyse untreated Saos-2/143 targets.

Bottom Line: Elevated levels of the p53 protein occur in approximately 50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer.A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells.The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
Elevated levels of the p53 protein occur in approximately 50% of human malignancies, which makes it an excellent target for a broad-spectrum T cell immunotherapy of cancer. A major barrier to the design of p53-specific immunotherapeutics and vaccines, however, is the possibility that T cells may be tolerant of antigens derived from wild-type p53 due to its low level of expression in normal thymus and lymphohemopoetic cells. The combination of p53 deficient (p53-/-) and p53+/+ HLA-A2.1/Kb transgenic mice was used as a model to explore the possibility that A2.1-restricted cytotoxic T lymphocytes (CTL) are functionally tolerant of self peptides derived from the wild-type p53 tumor suppressor protein. A2.1-restricted CTL specific for a naturally processed p53 self-epitope spanning residues 187-197 were completely aborted in p53+/+ as opposed to p53-/- transgenic mice. In contrast, CTL specific for a second self-epitope spanning residues 261-269 of the murine p53 sequence were detected in both p53-/- and p53+/+ A2.1/Kb transgenic mice. However, the avidity of the CTL effectors obtained from p53+/+ mice was 10-fold lower than that obtained from p53-/- mice, again suggesting elimination of CTL with high avidity for the A2.1-peptide complex. The circumvention of functional tolerance of high avidity CTL may therefore be a necessary prerequisite for optimizing immunotherapy against A2.1-restricted wild-type p53 epitopes in humans.

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Related in: MedlinePlus