Identification of CD8+ T-cell epitope from multiple myeloma-specific antigen AKAP4

Abstract

Multiple myeloma (MM) is a malignant plasma cell disorder affecting mainly the elderly population. Revolutionary progress in immunotherapy has been made recently, including monoclonal antibodies and chimeric antigen receptor T cell (CAR-T) therapies; however, the high relapse rate remains problematic. Therefore, combination therapies against different targets would be a reasonable strategy. In this study, we present a new X-chromosome encoded testis-cancer antigen (CTA) AKAP4 as a potential target for MM. AKAP4 is expressed in MM cell lines and MM primary malignant plasma cells. HLA-A*0201-restricted cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transduced with an adenovirus vector encoding the full-length AKAP4 gene were demonstrated to lyse AKAP4⁺ myeloma cells. Seven of the 12 candidate epitopes predicated by the BIMAS and SYFPEITH algorithms were able to bind HLA-A*0201 in the T2 binding assay, of which only two peptides were able to induce CTL cytotoxicity in the co-culture of peptide-loaded human mature dendritic cells and the autologous peripheral blood mononuclear cells (PBMCs) from the same HLA-A*0201 donor. The AKAP4 630-638 VLMLIQKLL was identified as the strongest CTL epitope by the human IFN-γ ELISPOT assay. Finally, the VLMLIQKLL-specific CTLs can lyse the HLA-A*0201⁺AKAP4⁺ myeloma cell line U266 in vitro, and inhibit tumor growth in the mice bearing U266 tumors in vivo. These results suggest that the VLMLIQKLL epitope could be used to develop cancer vaccine or T-cell receptor transgenic T cells (TCR-T) to kill myeloma cells.

Keywords: cancer-testis antigen; cytotoxic T lymphocytes; dendritic cell; multiple myeloma; peptide epitope.

Figure 1

AKAP4 is expressed on primary MM cells and MM cell lines and induces specific immune responses targeting myeloma cells. (A) AKAP4 was expressed both intracellularly and on the cell surface in primary cells of myeloma patients (gated on the CD38⁺CD138⁺population) and in myeloma cell line U266. (B) Intracellular expression of AKAP4 in different cell line. (C) Expression of HLA-A*0201 in different cell lines after IFN- γ stimulation. (D) AKAP4 expression in DC cells of healthy donors after adenovirus transfection. (E) The killing effect of CTL on U266 cells (HLA-A 0201⁺ AKAP4⁺) induced by AKAP4 adenovirus transfection DC was higher than that of RPMI-8226 (HLA-A 0201^- AKAP4⁺) (summarized the results of three independent experiments). Friedman test showed the p < 0.05 for U266 and RPMI-8266. Then, the p-value was analyzed by Wilcoxon rank sum test. *p < 0.05, **p < 0.01. NC: negative control.

Figure 2

The T2 binding assay showed 7 of the 12 peptides synthesized bind the HLA-A*0201 molecule with different affinity. (A, B) Representative multi-parameter flow cytometry scatter diagram for T2 cell affinity detection. (C) FI value of 12 peptides at the concentration of 50 μM (summarized the results of three independent experiments). (D) FI of the 12 peptides at different concentrations (one independent experiment). Fluorescence intensity (FI) = the mean fluorescence intensity of peptide-pulsed T2 cells/the FI of T2 cells not loaded with peptide − 1. CMV: peptide derived from CMV pp65 495-503 NLVPMVATV, served as positive control; Blank: result of T2 cells not loaded with any peptide.

Figure 3

Peptides No. 5 and No. 8 of AKAP4 protein can induce T cells to secrete IFN-γ in vitro.(A) Images of the ELISPOT assay. NC: Negative control, No. 3, 4, 5, and 8: Peptide No. 3, Peptide No. 4, Peptide No. 5, and Peptide No. 8; CMV: CMV peptide (positive control). (B) The score of Peptide No. 5 and Peptide No. 8 was higher than NC in ELISPOT assay (summarized the results of three independent experiments). Kruskal–Wallis rank sum was performed to assess the significance in (B) *p < 0.05, **p < 0.01.

Figure 4

Tumor cell line killing assay shows the anti-myeloma effect of peptide No. 8 specific CTLs in vitro. (A) The killing efficiency of CTL stimulated by No. 8 antigen peptide on U266 cell line under different effect target ratio (the effective target ratio is 25 in the left figure, 5 in the middle figure, and 1 in the right figure). (B) Compared to CTLs induced by peptide No. 5 and No. 3, peptide No. 8 specific CTLs lyse U266 cell more efficiently. (C) Peptide No. 8 specific CTLs lyse HLA-A*0201⁺ AKAP4⁺ myeloma cell line U266, while the other HLA-A*0201^- or AKAP4^- cell line is not killed efficiently (summarized the results of three independent experiments). Friedman test showed the p < 0.05 for U266 to other cell lines. Kruskal–Wallis rank sum test was performed to assess the significance in C *p < 0.05.

Figure 5

Tumor model showed the anti-myeloma effect of the peptide No. 8 specific CTLs in vivo. U266 cells (5 × 10⁵) were inoculated s.c. into NTG mice (day 0) followed by No. 8 peptide induced CTLs (5 × 10⁶) or PBS injected intravenously on day 1 and day 7. After the mice were killed in the end point of the experiment (Day 30), the tumors were peeled off, weighed, and photographed. Independent experiment was performed two times. The number of animals in the first experiment was 3 and the second was 4. The results of the two experiments were the same. The summarized results of the two experiments are shown in (A) (the tumor growth curve) and (B) (tumor weights). (C) The results of the second experiment (excised tumors). Two-way-ANOVA showed that the p-value is <0.001 for CTL and PBS in (A) Unpaired t-test was performed to assess the significance in A and B **p < 0.01.