Flow-cytometric determination of peptide-class I complex formation identification of p53 peptides that bind to HLA-A2

doi:10.1016/0198-8859(94)90105-8

Human Immunology

Volume 39, Issue 2, February 1994, Pages 79-86

https://doi.org/10.1016/0198-8859(94)90105-8 Get rights and content

Abstract

A novel class I-peptide-binding assay was developed and used to identify a series of peptides derived from the human p53 tumor-suppressor gene product capable of binding the HLA-A2 class I allele. Brief pH 3.3 acid treatment of human cell lines rapidly denatures pre-existing class I complexes, as detected by loss of binding of conformation-dependent mAbs, leaving only free class I heavy chains associated with the viable cell surface. These heavy chains may be induced to refold and be recognized by antibodies (in 2–4 hours) when acid-treated cells are coincubated with exogenous ß₂-microglobulin and peptides capable of binding the relevant class I allele examined. This assay, with a detection limit of 1–10 nM peptide, was used to screen the capacity of a panel of nine peptides bearing HLA-A2-binding motifs and derived from the human p53 tumor-suppressor protein sequence. Eight of the nine peptides bound to, and reconstituted, HLA-A2 on acid-treated cells. This assay system will enable the rapid identification of peptides binding to any class I allele, which is the initial prerequisite for elucidating potential CD8 + T-cell epitopes.

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Therefore, this sorting signal was fused to the cDNA encoding a 27-mer peptide, consisting of the immunodominant 9-mer epitope under evaluation, which is flanked at the 5′ end and 3′ end by additional protein 9-mers. We evaluated presentation of the HLA-DP4-restricted epitope of the shared antigen MAGE-A3 (TQHFVQENYLEY)34,35 and the HLA-A2-restricted epitopes of the shared antigens gp100 (YLEPGPVTA),36–39 NY-ESO-1 (SLLMWITQC),40–42 and p53 (LLGRNSFEV).43,44 We electroporated HLA-A2+ moDCs with SDT-mRNA or PDT-mRNA and co-cultured them at a 1:1 ratio with CD8+ T cells that were electroporated with mRNA encoding the corresponding TCRα and TCRβ chain to test antigen presentation to CD8+ T cells (Table S3).
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Human C1R-A2 cells (stably transfected with episomal vector pHebo-A2 to express HLA-A*0201) were kindly provided by Dr. R. Kiessling (Cancer Center Karolinska, Karolinska Institute, Stockholm, Sweden), and were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 2 mM l-glutamine, 25 μg/ml gentamycin, 10% FCS and 250 μg/ml selective antibiotic Hygromycin B (Roche Diagnostics GmbH, Mannheim, Germany). The HLA-A*0201 reconstitution assay using C1R-A2 cells was performed as described previously [19] with a few modifications. Briefly, C1R-A2 cells were washed in ice cold PBS and the cell pellet was cooled on ice for 5 min.
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Peptides were dissolved in DMSO and frozen at -80°C until use. Peptide-induced stabilization of HLA-A2.1 molecules on T2 cells was performed as described previously.14 The positive control consisted of T2 cells loaded with the influenza virus matrix peptide, GILGFVFTL.
To identify potential immunogenic peptides derived from CA125.
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CD8+ cytotoxic T-lymphocyte populations generated against 4 CA125-derived peptides were able to induce lysis of autologous peptide-loaded target cells. CA125 YTLDrDSLYV peptide-specific cytotoxic T lymphocytes were found to effectively kill ovarian tumors expressing CA125. Cytotoxicity was inhibited by antihuman leukocyte antigen A2.1 (BB7-2) and antihuman leukocyte antigen class I (W6/32) antibodies, whereas natural killer-sensitive targets were not lysed. YTLDrDSLYV peptide-specific cytotoxic T lymphocyte precursor frequency was low in peripheral blood leukocytes of normal donors and patients with ovarian cancer as determined by interferon-gamma production in ELISPOT assays. Intracellular cytokine expression measured by flow cytometry showed a type 1 cytokine profile in YTLDrDSLYV peptide-specific cytotoxic T lymphocytes.
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La proteína p53 está sobreexpresada en la mitad de los tumores humanos. Se ha descrito que se produce una respuesta inmunológica específica mediada por linfocitos T citotóxicos dirigidos contra el epítopo 264-272 de p53 en pacientes con cáncer de cabeza y cuello. Demostrar que se produce ese tipo de respuesta en otros tumores podría ser determinante para el desarrollo de vacunas antitumorales específicas anti-p53. El objetivo de este estudio fue demostrar in vivo la existencia de linfocitos T citotóxicos específicos anti-p53 en sangre periférica de pacientes con cáncer de mama.
Se realizó la determinación mediante citometría de flujo del recuento de linfocitos T citotóxicos específicos dirigidos contra los epítopos HLA A2 restringidos 264-272 y 149-147 de la proteína p53 en sangre periférica de pacientes con cáncer de mama.
El percentil 99 de la concentración de células T anti-p53 en los pacientes con HLA A2.1 negativo fue 1/5.634 (punto de corte). En los pacientes con HLA A2 positivo, la mediana de linfocitos anti-p53_264-272 fue de 1/2.383 y la de linfocitos anti-p53_149-157, de 1/2.335. Todos los pacientes con HLA A2 positivo presentaron recuentos de linfocitos anti-p53 por encima del punto de corte para al menos uno de los 2 epítopos: 13/14 (93%) para p53_264-272 y 11/12 (92%) para p53_149-157.
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p53 264-272-specific CD8+ T cells were directly enumerated in the peripheral circulation of patients with breast cancer using tetrameric p53 264-272/HLA-A2.1 complexes by multicolor flow cytometry. The same procedure was used to enumerate T cells specific for another HLA-A2.1 restricted wild type p53 epitope, p53 (149-157).
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Flow-cytometric determination of peptide-class I complex formation identification of p53 peptides that bind to HLA-A2

Abstract

Curr Opin Immunol

Immunol Today

Cell

Cell

Adv Cancer Res

J Immunol Methods

Mol Immunol

Mol Immunol

Cell

Antigen recognition by class I restricted T lymphocytes

Annu Rev Immunol

Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules

Nature

Identification of self peptides bound to purified HLA-B27

Nature

Isolation and analysis of naturally processed viral peptides as recognized by cytotoxic T cells

Nature

Lessons from T cell responses to virus induced tumors for cancer eradication in general

Cancer Surv

Recognition of human melanoma cells by HLA-A2. 1-restricted cytotoxic T lymphocytes is mediated by at least six shared peptide epitopes

J Immunol

Clonal deletion versus clonal anergy: the role of the thymus in inducing self tolerance

Science

Limit of T cell tolerance to self peptides by peptide presentation

Science