Identification of a new HLA-A2-restricted T-cell epitope within HM1.24 as immunotherapy target for multiple myeloma

Abstract

Objective: The aim of this study was identification of human leukocyte antigen (HLA)-A2-restricted T-cell epitopes within the HM1.24 antigen as target for multiple myeloma (MM)-directed specific peptide-based immunotherapy.

Methods: The HM1.24 sequence was scanned for immunogenic peptides using the HLA-binding prediction software SYFPEITHI and BIMAS. Peripheral blood mononuclear cells from HLA-A2(+) healthy volunteers/blood donors (ND) were stimulated with autologous HM1.24-peptide-loaded dendritic cells, and expanded in vitro. Activation of T cells was assessed by ELISpot and cytotoxicity by (51)Chromium ((51)Cr)-release assays. T2-cells pulsed with irrelevant peptide, the HM1.24(-)/HLA-A2(+) breast carcinoma cell line MCF-7 and the HM1.24(+)/HLA-A2(-) myeloma cell line RPMI-8226 were used as controls. Expression of the HM1.24 gene (BST2) was assessed using purified plasma cells and Affymetrix-U133A+B microarrays. Frequency of peptide-specific CD8(+) T cells was detected using the flow-cytometric tetramer technique.

Results: Of eight nona-peptides with the highest probability of binding to HLA-A2, the HM1.24 aa22-30 peptide (LLLGIGILV) showed the most frequent activation of CD8(+) T cells in healthy volunteers (specific activation in 8 of 11 [73%] ND; compared with 5-19% for the 7 other HM1.24 peptides). Antigen recognition by the HM1.24 aa22-30-specific CD8(+) T cells was HLA-A2-restricted (ELISpot with HLA-A2-blocking antibodies: median, 15; range, 14-18 spots/well; isotype-control antibodies: median, 47; range, 44-48). HM1.24-aa22-30-specific CD8(+) T cells lysed HLA-A2(+) myeloma-derived cell lines ((51)Cr-release assay: XG-1 vs MCF-7, 91% vs 0%; U266 vs MCF-7, 38% vs 4.2%; IM-9 vs RPMI-8226, 22% vs 0%). Using the cross-reactive Neisseria meningitidis peptide LLSLGIGILV-specific CD8(+) T cells recognizing target cells loaded with the HM1.24 aa22-30 peptide (LLLGIGILV) as well as the myeloma-derived cell line U266 could be expanded from MM patients. The HM1.24 gene was expressed at comparable levels by plasma cells from 65 MM patients, 7 patients with monoclonal gammopathy of undetermined significance, and 7 ND.

Conclusions: HM1.24 aa22-30 is a newly identified HLA-A2-restricted T-cell epitope that is processed and presented by major histocompatibility complex class I. Specifically activated CD8(+) T cells are able to lyse MM cell lines. We conclude that HM1.24 aa22-30 represents a suitable candidate target for a specific peptide-based immunotherapy of MM.

Figure 1. Relative expression levels of the HM1.24-gene (BST2) according to the probe-set Affymetrix201241_at

The HM1.24-gene expression of CD138⁺ enriched plasma cells from normal donors (ND, n=7), donors with monoclonal gammopathy of undetermined significance (MGUS, n=7), patients with multiple myeloma (MM, n=65) and human myeloma cell lines (HMCL, n=20) was analysed by Microarray-analysis. The median of the relative expression level of each group is denoted as grey bar.

Figure 2. T cell activation with HM1.24 aa22-30

ELISpot-assays with HM1.24 aa22-30 pulsed T2 cells as target cells were performed with an effector:target ratio of 4:1. T2 cells pulsed with HM1.24 aa126-134, T2 cells alone pulsed with HM1.24 aa22-30, and T-cells alone were used as negative controls. Shown are representative results (mean of triplets) from 3 of 8 different donors.

Figure 3. HM1.24 aa22-30 specific CD8⁺ T-cells against peptide pulsed T2 cells with various effector:target ratios

HM1.24 aa22-30 peptide specific CD8⁺ T-cells were incubated with T2 target cells pulsed with HM1.24 aa22-30 (■) or with an irrelevant peptide (▲) using various effector:target ratios ranging from 1:1 to 0.125:1. The figure shows the results of the IFN-γ specific ELISpot assays (mean of triplets).

Figure 4. HLA-A2 restricted presentation of HM1.24 aa22-30

HM1.24 aa22-30 peptide specific CD8⁺ T-cells were incubated with HM1.24 aa22-30 pulsed T2 target cells together with HLA-A2 blocking antibodies or isotype-matched control antibodies. The figure shows the results of the IFN-γ ELISpot assays (mean of triplets).

Figure 5. Cytotoxicity assay with HM1.24 aa22-30 specific CD8⁺ T-cells

HM1.24 aa22-30 peptide specific CD8⁺ T-cells were incubated with HM1.24 aa22-30 (■) or HM1.24 aa171-179 (▲) pulsed T2 cells as target cells in varying effector:target ratios ranging from 7.5:1 (0.5 × 10⁴ HM1.24 aa22-30 pulsed T2 cells incubated with 3.75 × 10⁴ HM1.24 aa22-30 specific CD8⁺ T-cells) to 1.875:1. The figure shows results of the ⁵¹Chromium release-assay with the specific lysis in %.

Figure 6. Activation of HM1.24 aa22-30 specific CD8⁺ T-cells using the Myeloma Cell Lines IM-9 and U266 as targets

HM1.24 aa22-30 peptide specific CD8⁺ T-cells were incubated with the myeloma derived HLA-A2⁺ cell lines IM-9 (■) and U266 (●) in effector:target ratios ranging from 0.125:1 to 1:1. The HLA-A2⁺ mamma carcinoma cell line MCF7 (▲) was used as a negative control. The figure shows the results of the IFN-γ ELISpot assays.

Figure 7. Lysis of Myeloma Cell Lines mediated by HM1.24 aa22-30 specific CD8⁺ T-cells

HM1.24 aa22-30 peptide specific CD8⁺ T-cells from 3 different donors were incubated with the myeloma derived HLA-A2⁺ cell lines XG-1, U266 and IM-9 in varying effector:target ratios ranging from 1.25:1 to 5:1. The mamma carcinoma cell line MCF7 (HLA-A2⁺, HM1.24⁻) and the myeloma cell line RPMI8226 (HLA-A2⁻, HM1.24⁺) were used as negative controls. The figure shows results from ⁵¹Chromium release-assays with the specific lysis in %.