HLA class I-restricted T cell epitopes isolated and identified from myeloid leukemia cells

Abstract

Leukemia-associated antigens (LAAs) and HLA-I epitopes published previously have shown promise in inducing leukemia-specific T cell responses. However, the clinical responses are limited, and clinical effectiveness is yet to be achieved. Limitations, among others, being the LAAs themselves, the indirect approach to HLA-I epitope identification by reverse immunology, and the use of single or few LAAs and HLA-I epitopes, which limits the spectrum of inducible tumor-specific T cells. Use of a direct approach to identify naturally processed and presented HLA-I epitopes from LAAs, and higher numbers of antigens for T cell-mediated immunotherapy for leukemia may enhance clinical responses and broaden clinical effectiveness. In a prior study we used immunoaffinity purification of HLA-I peptide complexes from the differentiated myeloid tumor cell lines MUTZ3 and THP1 coupled to high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). From this we identified in the current study seven new HLA-I epitopes and the corresponding LAAs for myeloid leukemia. In comparison, the myeloid HLA-I epitopes reported here were generally stronger HLA-binders that induce stronger T cell responses than those previously published, and their source LAAs had higher immunogenicity, higher expression levels in myeloid tumors cells compared to normal hemopoietin and other major normal tissues, and more protein interaction partners, and they are targeted by CD8 T cells in CML patients. This study analyses and compares the LAAs and HLA-I epitopes based on various immunotherapeutic targets selection criteria, and highlights new targets for T cell-mediated immunotherapy for leukemia.

Figure 1

Expression of established leukemia-associated antigens (eLAAs) in MUTZ3 DCs and THP1MФ. RT-PCR showing expression of Proteinase 3, WT1, PRAME, Survivin, RHAMM, hTERT and CML66 in MUTZ3 DCs and THP1MФ. β actin was used as a control for the PCR reactions, a positive control (cDNA from cells positive for the respective antigen), and negative control (PCR minus cDNA) were included. All positive bands were of the predicted amplicon size. Proteinase 3 (363 bp), WT1 (380 bp), PRAME (154 bp), Survivin (129 bp), RHAMM (381 bp), hTERT (270 bp), CML66 (214 bp) and β actin (454 bp). The thick black lines on the gels delineate grouping of gels cropped from different parts of the same gel, or from different gels. Full length gels in Supplementary Fig. 1. Cropping: Proteinase, WT1 (Supplementary Fig. 1B), PRAME, Survivin (Supplementary Fig. 1C), RHAMM (Supplementary Fig. 1C,E), hTERT (Supplementary Fig. 1D,F), CML66 (Supplementary Fig. 1F) and β actin (Supplementary Fig. 1F). Expression of Proteinase and WT1 are also shown in Supplementary Fig. 1A, and CML-66 Supplementary Fig. 1D.

Figure 2

Peptide binding based on (A) experimental T2 cell line HLA A*02:01 stabilization assay (B) SYFPEITHI positional scoring matrix and (C,D) artificial neural networks-based affinity predictions in the immune epitope database IEDB^,. Dark and light grey filling represent eLAAs and pLAAs, respectively. (E) Correlation of peptide ranking based on HLA-A*02:01 stabilization assay with the T2 cell line, SYFPEITHI scoring and artificial neural networks-based affinity predictions in the immune epitope database IEDB.

Figure 3

IFNγ ELISpot assay of PBMCs from 4 HLA-A*02:01 healthy donors (white bars) and 8 HLA-A*02:01 positive CML patients (dark grey bars). The cells were tested against the eLAA HLA-I peptides P540-hTERT, P300-PRAME, P187-WT1, P165-RHAMM and P169-PROTEINASE 3 previously identified by reverse immunology (Table 2) and the pLAA HLA-I peptides P207-PININ, P130-LARP1, P378-TRRAP, P308-ROS1, P114-PSME3, P326-URP2, P141-MBOA7, P57-UHRF1 identified from the MUTZ3 DCs and THP1MФ HLA-I peptidomes by LC-MS/MS (Table 1). PBMCs without peptides served as a negative control. Data represented as Spots Forming Units (SFU) per 5 × 10⁴ CD8+ T cells. Symbols represent individual data points.

Figure 4

Immunogenicity of the LAAs for the most frequent HLA alleles. HLA-A*01:01, HLA-A*02:01, HLA-A*11:01, HLA-A*24:02, HLA-C*06:02, HLA-C*07:01 and HLA-C*07:02 that together represent more than 90% of the human population. The immunogenicity was determined with NetMHCpan 4.0 in IEDB using a binding affinity threshold of IC₅₀ (500 nM). The immunogenicity scores are presented as 1/IC₅₀ (500 nM) with values of 0 to 1 for low to high immunogenicity. Dotted and continuous line represent eLAAs and pLAAs, respectively.

Figure 5

Gene expression profiles of the established leukemia-associated antigens (eLAAs) and potential leukemia-associated antigens (pLAAs) in CML, AML and normal human hematopoietic cells determined using Bloodspot and the datasets GSE13159 and GSE42519 for human CML and AML, and normal human hematopoietic cells, respectively.

Figure 6

Gene expression profiles of the LAAs established leukemia-associated antigens (eLAAs) and potential leukemia-associated antigens (pLAAs) in normal human tissue analyzed and visualized using BioGPS (http://biogps.org/) and geneAtlas U133A gcrma datasets. The dotted line indicated low gene expression intensity cutoff of 10%.

Figure 7

Known protein interaction partners of the LAAs established leukemia-associated antigens (eLAAs) and potential leukemia-associated antigens (pLAAs) determined using STRING version 11.0 for Homo sapiens with a medium score of 0.400 and a cutoff of 10 interaction partners. (A) Protein interaction partners of the eLAAs. (B) Protein interaction partners of the pLAAs.