Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

<div><p>The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that <i>PEPP2</i> gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.</p></div

Recognition by PEPP2-specific CTLs.

<p>Antigen specificity of PEPP2-specific CTLs was analyzed. (A) IFN-γ secretion by CTLs responding to CIR-A24 cells pulsed with PEPP2^271-279 or HIV-derived peptide was evaluated by ELISpot assay. The CTLs used in the assays were induced by 3 stimulations and analyzed at the 5th to 7th days after last stimulation.Y-axis indicates the number of spots observed in each well, which contains 3x10⁴ CD8-positive cells. (B) Cytotoxicity of PEPP2-specific CTLs was examined by ⁵¹Cr release assay. (C) Cytotoxicity of PEPP2-specific CTLs against CIR-A24 cells pulsed with various concentrations of PEPP2^271-279 was examined at E/T ratio of 30. (D) Cytotoxicity of PEPP2-specific CTLs against cancer cell lines KMS11 (HLA-A*24:02-positive, PEPP2-negative), KMS21 (HLA-A*24:02-positive, PEPP2-positive), NB4 (HLA-A*24:02-negative, PEPP2-positive), and K562 (HLA-A*24:02- negative, PEPP2- positive), and PBMCs from HLA-A*24:02-positive HD was assessed by ⁵¹Cr release assay. (E) KMS21 cells were pre-treated with anti HLA class I antibody or mouse isotype IgG1 antibody and used for cytotoxicity assay. Experiments were performed in triplicate. *p < 0.05 (Students <i>t</i>-test).</p

Expression profile of PEPP2 gene in LSCs derived from leukemia cell.

<p>AML cell line KG1a (A) and CML cell line KU812 (D) were stained with monoclonal antibodies against CD34 and CD38. CD34⁺38^- cells and CD34^- cells were obtained by MoFlo cell sorter system. PEPP2 expression in unsorted samples, CD34⁺38^- fraction, and CD34^- fraction of KG1a were assessed by standard RT-PCR (B,E). PEPP2 protein expression of KG1a (C) and KU812 (F) was detected by immunohistochemistry using anti-PEPP2 monoclonal antibody. These figures show the representative data of 3 independent experiments.</p

Effects of pretreatment of target cells with 5-aza-2′-deoxycytidine.

<p>(A) PL21 cells or PBMNCs from HLA-A*24:02-positive healthy donor were pretreated with 5-aza-2′-deoxycytidine for 72 hours, then evaluated for their sensitivity to lysis by PEPP2-specific CTLs. (B) Expression of HLA class I, HLA-DR, and PD-L1 was assessed in PL21 cells before and after treatment with 5-aza-2′-deoxycytidine. Green line; isotype control. Blue line; pre-treatment. Red line; post-treatment. These figures show the representative data of 2 independent experiments.</p