Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Abstract

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201(+) donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28((173-181)), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-gamma and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28((173-181) )pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28((173-181) )is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28((173-181)), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.

Fig. 1

Expression of CML28 gene in cell lines detected by reverse transcription-PCR and agarose gel electrophoresis. PCR amplification was performed with specific oligonucleotides (sense 5′-TGGAGGAGGAGACGCATAC-3′ and anti-sense 5′-TGAGTGGGTGGAGGCAAT-3′) and PCR products were demonstrated through electrophoresis on 1% agarose gel with ethidium bromide staining. Lane 1 COS7 transfected with PCI-neo; Lane 2 COS7 transfected with PCI/CML28; Lane 3 MCF-7; Lane 4 Jurkat-A2/Kb; Lane 5 A549; Lane 6 LB373-MEL; Lane 7 PCR marker

Fig. 2

Specific lysis of various cell lines by the CTLs generated from PBMCs of healthy donors. a Special lysis of LB373-MEL by CTLs induced by CML28_(183–191) (ALFCGVACA), CML28_(173–181) (ALVDAGVPM), CML28_(191–199) (ALDSDGTLV) and CML28_(161–169) (SLLACCLNA), respectively. Effector cells were induced from the PBMCs of HLA-A*0201⁺ healthy donors through three sequential rounds of stimulation with every peptide at the concentration of 10 μg/ml once a week. On day 21, 4-h ⁵¹Cr-release assays were performed to test their cytotoxic activity against LB373-MEL cell line (HLA-A*0201⁺, CML28⁺). PBMCs stimulated with IL-2 were only used as controls. b Specific lysis of various tumor cell lines by CTLs induced by peptide CML28_(173–181) (ALVDAGVPM). The cytotoxic activity of the CTLs was assessed against LB373-MEL cells (melanoma cell line, HLA-A*0201⁺, CML28⁺), A549 (lung adenocarcinoma cell line, HLA-A*0201⁻, CML28⁺), and MCF-7cells (breast carcinoma cell line, HLA-A*0201⁺, CML28⁻) at various E:T ratio

Fig. 3

ELISPOT assay to measure IFN-γ secretion by CTLs induced from PBMCs of healthy donors. PBMCs from healthy donors were separated routinely and stimulated with synthetic peptides and IL-2 in RPMI 1640 supplemented with 10% FCS for 21 days. Then, these PBMCs were collected and ELISPOT assay was performed to determine the IFN-γ production by these cells. PBMCs without peptide stimulation (control) were analyzed to estimate background spot formation. Results represent mean values of triplicate wells and SDs were always <5%

Fig. 4

Specific lysis of Jurkat-A2/Kb cells pulsed or not with tested peptides. HLA-A*0201 transgenic mice were immunized with 100 μg of PCI/CML28 or PCI-neo plasmid in tibialis anterior. a Immune spleen cells were pooled and stimulated in vitro with the four tested peptides respectively and on day 7, CTL activity was measured against Jurkat-A2/Kb cells pulsed with four tested peptides, respectively. Spleen cells from mice immunized with PCI-neo were used as control. b The immune spleen cells restimulated with CML28_(173–181) effectively lysed Jurkat-A2/Kb cells pulsed with the peptide, but not Jurkat-A2/Kb cells without peptide pulsed

Fig. 5

The inhibition of recognition of the CML28_(173–181)-specific CTLs generated from PBMCs of healthy donors by anti-HLA-A*0201 and anti-CD8 antibody. a LB373-Mel cells (1×10⁴ cells/well) were pretreated with anti-HLA-A*0201 and anti-HLA-A24 antibody, respectively, for 1 h. ⁵¹Cr-releasing assays were performed to determine the cytotoxicity of CML28_(173–181)-specific CTLs against these LB373-MEL cells at various E:T ratio. b The effector cells were preincubated with anti-CD8 and anti-CD4 antibody, respectively, and then added to ⁵¹Cr-labeled cells to perform 4-h ⁵¹Cr-release assays

Fig. 6

The inhibition of recognition of the CML28_(173–181)-specific CTLs generated in HLA-A*0201 transgenic mice by anti-HLA-A*0201 and anti-CD8 antibody. a Jurkat-A2/Kb cells pulsed with CML28_(173–181) were treated with anti-HLA-A*0201 and anti-HLA-A24 antibody , respectively, for 1 h. ⁵¹Cr-release assay was performed to demonstrate the cytotoxic activity of the effector cells generated from the HLA-A*0201 transgenic mice against these Jurkat-A2/Kb cells. b The effector cells were preincubated with anti-CD8 and anti-CD4 antibody, respectively, and then added to ⁵¹Cr-labeled cells to perform 4-h ⁵¹Cr-release assays