Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1(+) leukemias - PubMed (original) (raw)

Mapping of novel peptides of WT-1 and presenting HLA alleles that induce epitope-specific HLA-restricted T cells with cytotoxic activity against WT-1(+) leukemias

Ekaterina Doubrovina et al. Blood. 2012.

Abstract

The Wilms tumor protein (WT-1) is widely recognized as a tumor antigen that is expressed differentially by several malignancies. However, WT-1 peptides known to induce tumoricidal T cells are few. In the present study, we evaluated T-cell responses of 56 healthy donors to in vitro sensitization with autologous APCs loaded with a pool of overlapping 15-mer peptides spanning the sequence of WT-1. Thereafter, we mapped the WT-1 peptides eliciting responses in each individual, defined the immunogenic peptides, and identified their presenting HLA alleles. We report 41 previously unreported epitopes of WT-1: 5 presented by class II and 36 by class I alleles, including 10 that could be presented by more than 1 class I allele. IFNγ(+) T cells responding to 98% of the class I and 60% of the class II epitopes exhibited HLA-restricted cytotoxicity against peptide-loaded targets. T cells specific for 36 WT-1 peptides were evaluable for leukemocidal activity, of which 27 (75%) lysed WT-1(+) leukemic targets sharing their restricting HLA allele. Each epitope identified induced T-cell responses in most donors sharing the epitopes' presenting allele; these responses often exceeded responses to flanking peptides predicted to be more immunogenic. This series of immunogenic epitopes of WT-1 should prove useful for immunotherapies targeting WT-1(+) malignancies.

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Figures

Figure 1

Figure 1

WT-1–specific responses of CTLs generated from PBMCs of healthy donors (n = 56) by stimulation with autologous APCs loaded with total pool of WT-1–derived pentadecapeptides. (A) production of IFNγ in PBMCs alone (as a background), PBMCs coincubated overnight with the total pool of pentadecapeptides spanning the whole sequence of WT-1 protein (PBMC + WT-1 pool), and pregenerated WT-1–specific T cells coincubated overnight with WT-1 peptide–loaded PBMCs. (B) Cytotoxic activity of the WT-1–specific CTLs generated in vitro by stimulation with WT-1 total pool against WT-1− (autologous PHA-stimulated blasts) and WT-1+ (autologous PHA-stimulated blasts loaded with the total pool of WT-1 pentadecapeptides) targets at a 50:1 effector: stimulator ratio. (C) IFNγ response measured by FACS staining in different responder cell populations (PBMCs, pregenerated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pregenerated CTLs sensitized with the total pool of WT-1 15-mers) after secondary overnight stimulation with autologous PBMCs either unmodified or loaded with one of the following: RMF peptide, dominant epitopes of WT-1 identified by the epitope-mapping approach in the WT-1–total pool sensitized CTLs, or the WT-1 total pool of the 141 pentadecapeptides. (D) Cytotoxic activity of the WT-1–specific T cells generated in vitro by sensitization with autologous CAMs loaded with the RMF 9-mer or with the total pool of the WT-1 15-mers. The cytotoxicity of the T cells was assessed against autologous WT-1− targets (PHA-activated blasts) and the same targets loaded with RMF peptide, the total pool of WT-1 15-mers, or the dominant WT-1 epitope identified for the same T-cell line.

Figure 2

Figure 2

Strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT-1 protein and epitope mapping. (A) The sequence of the WT-1 protein consisting of 575 amino acids and the principle of 11 amino acid–overlapping pentadecapeptides are illustrated. A total of 141 pentadecapeptides are required to span the entire protein. The sequence of 575 amino acids published by Gessler et al was used. This sequence includes an additional 126 amino acids in the N-terminus. To match the sequential numbers of amino acids within the WT-1 sequence used with the longest, most frequently described WT-1 isoform D, we numbered the first 126 amino acids with negative values and used the positive values to number the subsequent 449 amino acids described in the longest isoform D. (B) Mapping grid consisting of 24 subpools each containing up to 12 WT-1–derived pentadecapeptides. Each peptide is uniquely contained within 2 intersecting subpools: for example, peptide 75 is uniquely shared by subpools 3 and 19. (C) IFNγ production by WT-1–sensitized CTLs in response to secondary overnight stimulation with the subpools of WT-1 pentadecapeptides loaded on autologous PBMCs. Dominant responses are observed for the subpools no. 3 and 19, both containing 1 common pentadecapeptide no. 75. (D) IFNγ production by the WT-1 CTLs in response to secondary overnight stimulation with the single pentadecapeptide contained within the subpools eliciting the highest responses as per the analysis determined in panel C confirms that the dominant immunogenic sequence is contained within pentadecapeptide no. 75. (E) HLA restriction of the WT-1–specific T cells responding to peptide no. 75 identified by 51Cr-release assay against a panel of allogeneic CAMs or PHA blasts matching single HLA alleles expressed by the WT-1 CTL donors. These are presented along the x-axis of the graph. The CAMs or PHA blasts used in the assay are unmodified (gray bars) or loaded with the WT-1–dominant epitope (black bars). The WT-1–specific cytotoxic activity of the WT-1 CTLs is restricted by the B3501 HLA allele.

Figure 3

Figure 3

HLA class I– and class II–restricted, WT-1–specific T cells respond to the same immunodominant peptide 15-mer derived from WT-1 protein in the WT-1 CTL sensitized with the WT-1 total pool of overlapping 15-mers loaded on autologous CAMs. (A) Production of IFNγ by the CD8+ and CD4+ WT-1–specific T cells in response to secondary overnight stimulation with the same dominant WT-1–derived 15-mer no. 41. (B) Identification of the immunogenic sequence of amino acids within pentadecapeptide no. 41 by IFNγ production after secondary overnight stimulation with autologous PBMCs loaded with a panel of 9-mers either unique for the peptide no. 41 (LDFAAPGAS [LDF]) or contained within the neighboring overlapping 15-mer no. 40 (PVLDFAPPG [PVL] or VLDFAPPGA [VLD]) and no. 42 (DFAPPGASA [DFA]). Only the 9-mer uniquely presented within the 15-mer no. 41, LDF, elicited an IFNγ response. (C) Peptide-specific cytotoxic activity of WT-1 CTLs against the panel of 9-mers and 11-mers contained within peptide no. 41 and loaded on autologous PHA-stimulated blasts is observed against both the 11-mer LDF and 9-mer LDF contained within the 11-mer LDF, as determined in a standard 51Cr-release assay at a 25:1 effector: stimulator ratio. (D) HLA restriction of the cytotoxic activity of the WT-1 CTLs. T cells restricted by HLA-A0201 lyse targets loaded with either the 11-mer or the 9-mer, whereas those restricted by HLA DRB10402 only lysed targets loaded with the 11-mer.

Figure 4

Figure 4

IFNγ+ T-cell responses to equimolar mixtures of 9-mer peptides identified by epitope mapping of in vitro responses and peptides within the same 15-mer or adjacent overlapping 15-mer peptides predicted to have higher binding affinity and immunogenicity. (A) Responses to a mixture of nonamers spanning amino acids +2 to +31 including the 6-15RDL and 22-31GGC peptides to which HLA A0201+ donors responded in epitope-mapping studies. (B) Responses to the in vitro–mapped (−75)-(−67)AILDFLLLQ epitope and a flanking peptide (−78)-(−70)LLAAILDFL with higher predicted binding affinity. (C) Responses to the in vitro–mapped 38-46LDFAPPGAS epitope and the overlapping 37-45VLDFAPPGA predicted to have higher binding affinity.

Figure 5

Figure 5

Schema of WT-1. (A) Schema of WT-1 DNA. (B) Schema of WT-1 mRNA (includes region encoding first 126 amino acids within the N-terminus reported by Gessler et al). (C) Localization of the previously described immunogenic epitopes presented through HLA class I (dark gray) and HLA class II (light gray) either predicted (p) by the computer algorithm or identified (i) by the comparative analysis of the immunogenicity of a panel of neighboring peptides. The 126 amino acids at the N-terminus are numbered with negative values from the first AUG codon in exon 1. (D) Localization of the immunogenic epitopes identified in this study by the epitope-mapping approach presented by HLA class I (yellow) and HLA class II (pink) alleles and marked within the schema of the WT-1 protein 575 amino acids in length, including 126 amino acids labeled with negative numbers at the N-terminus upstream from the first AUG codon initiating exon 1. (E) Number of healthy donors responding to the identified epitopes (yellow) or cluster of epitopes (gray). (F) Distribution of the functional activities among the corresponding regions of the WT-1 protein.

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