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

Zeh, Herbert J.; Leder, Gerhard; Lotze, Michael T.; Salter, Russell D.; Tector, Matt; Stuber, György; Modrow, Susanne; Storkus, Walter J.

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

Cited by 69 publications

(60 citation statements)

References 31 publications

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“…[11][12][13] Briefly, the naturally HLApresented peptides were eluted from viable B-LCLs using a pH 3.3 acid elution technique. After washing the cells in Hank's balanced salt solution (HBSS Sigma, Deisenhofen, Germany), cells were incubated with 5 ml of pH 3.3 citrate-phosphate buffer (0.131 M citric acid, 0.066 M Na2HPO4) at room temperature for 2 min.…”

Section: Flow Cytometric Verification Of Peptide Bindingmentioning

confidence: 99%

“…To verify the presentation of naturally isolated peptides from recombinant HLA-A*3001/3014L molecules we used a flow cytometric peptide-binding assay [11][12][13] 4,6 Acid-treatment of the cell lines resulted in the dissociation of the naturally bound peptides and the release of b2-microglobulin from the HLA class I heavy chain. By adding synthetic FITC-labeled HLA peptide ligands and excess of recombinant b2-microglobulin the HLA class I molecules were reconstituted.…”

Section: Verification Of Peptide Binding To Hla-a*3001 and Hla-a*3014mentioning

confidence: 99%

“…Using the flow cytometric peptide-binding assay [11][12][13] we were able to confirm the binding of the 11-mer peptide (EITALAPSTMK) to HLA-A*3001 as well as to the HLA-A*3014L. We used B-LCLs naturally expressing the HLA molecules (A*02 and/or A*3001/14L) and showed that stable complexes of HLA-A*3014L and EITALAPSTMK are assembled after acid-treatment and reconstitution.…”

Section: -35mentioning

confidence: 99%

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The nature of peptides presented by an HLA class I low expression allele

Hinrichs

Föll²,

Bade‐Doeding³

et al. 2010

Haematologica

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Section: Flow Cytometric Verification Of Peptide Bindingmentioning

confidence: 99%

Section: Verification Of Peptide Binding To Hla-a*3001 and Hla-a*3014mentioning

confidence: 99%

Section: -35mentioning

confidence: 99%

See 1 more Smart Citation

The nature of peptides presented by an HLA class I low expression allele

Hinrichs

Föll²,

Bade‐Doeding³

et al. 2010

Haematologica

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“…The sensitivity of the stabilization assay was enhanced at the reduced (26%) temperature, a phenomenon similarly noted in the binding of peptides to RMA-S cells [15]. A minimum of 1-10nM peptide was required for stabilization; lower doses were insufficient, as also seen with the known CTL epitope influenza matrix 58-66 (Flu M1 58-66) [16]. …”

Section: Reconstitution Assaymentioning

confidence: 84%

“…We have identified a series of p53 peptides, conforming to the previously reported HLA-A2.1 binding motif [ 121, that bind to the HLA-A2.1 class I molecule using two independent flow cytometry-based assays. Recently, each of these assay systems has been demonstrated in titration experiments to have a detection limit of approximately 1-10 nM peptide for the binding of the CTL epitope influenza matrix 58-66 to HLA-A2.1 [16]. The effective concentration of peptide and catalytic mechanisms at the site of peptide loading are not known, and it is therefore difficult to speculate about the physiological implications of the peptide concentrations used in the assays.…”

Section: Discussionmentioning

confidence: 99%

Identification of wild‐type and mutant p53 peptides binding to HLA‐A2 assessed by a peptide loading‐deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay

et al. 1994

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Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay* Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2. l-restricted cytolytic T lymphocytes in vitro and in vivo.

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Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

et al. 1998

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By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4+ alphabeta T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4+ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

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

Cited by 69 publications

References 31 publications

The nature of peptides presented by an HLA class I low expression allele

The nature of peptides presented by an HLA class I low expression allele

Identification of wild‐type and mutant p53 peptides binding to HLA‐A2 assessed by a peptide loading‐deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay

Evidence that HLA class II-restricted human CD4+ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

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