BCR-ABL-specific CD4+ T-helper cells promote the priming of antigen-specific cytotoxic T cells via dendritic cells

Abstract

The advent of tyrosine kinase inhibitor (TKI) therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia (CML). However, the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges; therefore, an effective therapeutic has been sought. The BCR-ABL p210 fusion protein's junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy. BCR-ABL p210 fusion-region-specific CD4⁺ T-helper (Th) cells possess antileukemic potential, but their function remains unclear. In this study, we established a BCR-ABL p210 b3a2 fusion-region-specific CD4⁺ Th-cell clone (b3a2-specific Th clone) and examined its dendritic cell (DC)-mediated antileukemic potential. The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile. Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation, as indicated by upregulated production of CD86 and IL-12p70 by DCs, which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells. Moreover, in the presence of HLA-A*24:02-restricted Wilms tumor 1 (WT1)_235-243 peptide, DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes (CTLs). The expanded CTLs were cytotoxic toward WT1_235-243-peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo. However, the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α. Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

Figure 1

A b3a2-specific and HLA-DRB1*09:01-restricted CD4⁺ T-cell clone (SK). (a) Proliferative response of SK to graded concentrations of soluble b3a2 peptide. Autologous PBMCs were used as APCs. Proliferation was measured using the [³H]-thymidine incorporation assay. The presented values are the mean c.p.m. of duplicate cultures. (b) Inhibition of the proliferative response by anti-HLA antibodies. SK cells (5 × 10⁴) were cocultured with irradiated PBMCs (5 × 10⁵) in the presence of b3a2 peptide (0.1 μM) and indicated mAbs (10 μg/ml). (c) Proliferative response of SK cells to L-cells expressing the HLA-DR gene. SK cells (5 × 10⁴) were cocultured with irradiated L-cell transfectants (4 × 10⁴) prepulsed with the b3a2 peptide (10 μM). (d) IFN-γ secretion from SK cells (1 × 10⁵) cocultured for 24 h with THP-1 cells (5 × 10⁴) transduced with HLA-DR9 and/or BCR–ABL p210. (e) IFN-γ secretion from SK cells (5 × 10⁴) cocultured for 24 h with autologous DCs (2.5 × 10⁴) that were prepulsed with 150-Gy-irradiated THP-1 or BCR–ABL p210-transduced THP-1 cells (1 × 10⁴). (b–e) Data shown are means±s.d. of triplicate cultures and are representative of three independent triplicate experiments. (f) Expression of the genes TRAV and TRBV. (g) Representative flow cytometry profiles of TCR-Vβ22 and CD4 surface expression on SK cells. (h) TCR gene usage and V–(D)–J junction region sequences of SK. APCs, antigen-presenting cells; DCs, dendritic cells; IFN-γ, interferon-γ mAbs, monoclonal antibodies; PBMCs, peripheral blood mononuclear cells; TCR, T-cell receptor.

Figure 2

Th1 phenotype of the b3a2-specific CD4⁺ T-cell clone. (a) Representative flow cytometry profiles of surface αβTCR, γδTCR, CD4, CD8, CD45RO, CD45RA, CD62L and CCR7 on SK cells. (b) Surface expression of chemokine receptors (CCR4, CXCR3, CRTh2, CCR6 and CCR8). (c) Expression of transcription factors involved in Th1 and Th2 differentiation. After SK cells were stimulated for 24 h with plate-bound anti-CD3 mAbs (10 μg/ml), T-bet and GATA3 mRNA levels were quantified. To induce the Th1 and Th2 cells used as controls, PBMCs were stimulated with plate-bound anti-CD3 mAbs (10 μg/ml) under Th1 conditions (IL-12 plus anti-IL-4 Ab) and Th2 conditions (IL-4 plus anti-IL-12 Ab), respectively. The mRNA amounts (shown in a.u.) are normalized relative to the GAPDH mRNA amount. (d) Cytokine production by SK cells after 48-h stimulation with plate-bound control IgG or anti-CD3 mAbs (10 μg/ml). (c, d) Data shown are means±s.d. of triplicate cultures and are representative of three independent triplicate experiments. (e) PD-1 expression on SK. SK cells were stimulated with plate-bound control IgG or anti-CD3 mAbs (10 μg/ml) for 24 h. Staining histograms of PD-1 (solid line) and isotype-matched controls (dotted line) are shown. RFI is shown in the upper part of each panel. IL, interleukin; IgG, immunoglobulin G; IFN, interferon; mAbs, monoclonal antibodies; mRNA, messenger RNA; PBMCs, peripheral blood mononuclear cells; RFI, relative fluorescence intensity; Th, T helper; TCR, T-cell receptor.

Figure 3

Enhanced expansion of leukemia antigen-specific CTLs. (a) Representative flow cytometry profiles of surface CD40L on SK. SK cells were stimulated for 24 h with plate-bound control IgG or anti-CD3 mAbs (10 μg/ml). (b) Surface phenotype of DCs. Vehicle or b3a2 peptide DCs were cultured for 24 h with SK cells at a DC/SK ratio of 10:1. The expression of CD40, CD80, CD83, CD86, HLA-DR and CCR7 is shown. OK432 (10 μg/ml)-matured DCs and medium-control DCs served as controls. (a, b) Staining histograms of the indicated surface molecules (red) and isotype-matched controls (gray) are shown. RFI is shown in the upper part of each panel. (c) IL-12p70 production by DCs cultured with SK cells in the presence or absence of the soluble b3a2 peptide (1 μM). The controls for all other culture conditions were OK432-matured DCs and medium-control DCs. (d) HLA-DR- and CD40L-dependent production of IL-12p70 by DCs. b3a2 peptide DCs were cultured with SK cells for 24 h in the presence of the indicated blocking mAbs (10 μg/ml). (e) Schematic representation of the WT1-specific CTL priming assay. SK cells (5 × 10³) and DCs (1 × 10⁴)±b3a2 peptide (5 μM) were initially cocultured for 5 h to mature the DCs, after which the DCs and SK cells were irradiated and cultured with autologous CD8⁺ T cells (5 × 10⁴) in the presence of the WT1 peptide (5 μM). (f) Proliferation of CD8⁺ T cells at day 7 in the WT1-specific CTL priming assay was measured as [³H]-thymidine incorporation. (g) Frequency of WT1/HLA-A24-tetramer-positive CD8⁺ T cells at day 10 in the WT1-specific CTL priming assay. (h) Proliferation of CD8⁺ T cells at day 7 in the CMV-specific CTL priming assay was measured as [³H]-thymidine incorporation. (i) Frequency of CMV/HLA-A24-tetramer-positive CD8⁺ T cells at day 10 in the CMV-specific CTL priming assay. (c, d, f, h) Data are representative of three independent triplicate experiments. Error bars, s.d.; *P<0.05, one-way ANOVA. (g, i) Representative flow cytometry profiles of three independent experiments. HIV-env/HLA-A24 tetramer was used as a control. ANOVA, analysis of variance; b3a2, b3a2 peptide; CTL, cytotoxic T lymphocyte; CMV, cytomegalovirus; DC, dendritic cell; IL, interleukin; Ig, immunoglobulin G; IFN-γ, interferon-γ pWT1, WT1 peptide; RFI, relative fluorescence intensity; WT1, Wilms tumor 1.

Figure 4

Suppression of leukemia antigen-specific CTL responses by TKIs. (a) Effect of TKIs on IL-12p70 production by DCs. b3a2 peptide DCs were cultured in the presence of the indicated TKIs for 24 h. (b) Representative flow cytometry profiles of CD86 on DCs. (c) RFI values indicating CD86 expression. (a–c) OK432-matured DCs and medium-control DCs served as controls. (d) Proliferation of CD8⁺ T cells at day 7 in the WT1-specific CTL priming assay in the absence of SK. Irradiated DCs pulsed with the WT1 peptide were cultured with autologous CD8⁺ T cells, with the indicated TKIs added to the cultures. After 7 days, T-cell proliferation was measured using the [³H]-thymidine incorporation assay. (e) Effect of TKIs on IL-12p70 production by SK-conditioned DCs. b3a2 peptide DCs were cultured with SK cells in the presence of the indicated TKIs for 24 h. (f) Representative flow cytometry profiles of CD86 on SK-conditioned DCs. Vehicle or b3a2 peptide DCs were cultured for 24 h with SK cells at a DC/SK ratio of 10:1 in the presence of the indicated TKIs. (g) RFI values indicating CD86 expression on SK-conditioned DCs. (h) Proliferation of CD8⁺ T cells at day 7 in the WT1-specific CTL priming assay in the presence of the indicated TKIs was measured as [³H]-thymidine incorporation. (i) Frequency of WT1/HLA-A24-tetramer-positive CD8⁺ T cells (filled box) at day 10 in the WT1-specific CTL priming assay in the presence of the indicated TKIs. The HIV tetramer (open box) served as the control. (j) Representative optical microscopy images of SK/b3a2-matured DCs in the presence of the indicated TKIs. The arrows indicate DCs. Scale bar, 20 μm. DCs treated with dasatinib appear spherical (blue arrows), whereas DCs treated with the other TKIs present a spindle-shaped morphology (black arrows). (k) Proliferation of CD8⁺ T cells stimulated with plate-bound anti-CD3 mAbs in the presence of the indicated TKIs. (a, c, d, e, g–i, k) Data are representative of three independent triplicate experiments. Error bars, s.d.; *P<0.05, one-way ANOVA. ANOVA, analysis of variance; b3a2, b3a2 peptide; CTL, cytotoxic T lymphocyte; Dasa, dasatinib; DC, dendritic cell; Ima, imatinib; Nilo, nilotinib; pWT1, WT1 peptide; RFI, relative fluorescence intensity; TKI, tyrosine kinase inhibitor; WT1, Wilms tumor 1.

Figure 5

Suppression of leukemia antigen-specific CTL responses by IFN-α. (a) Effect of IFN-α on IL-12p70 production by DCs. (b) Representative flow cytometry profiles of CD86 on DCs. DCs were cultured for 24 h in the presence or absence of IFN-α (1 ng/ml). (c) RFI values indicating CD86 expression. (a–c) OK432-matured DCs and medium-control DCs served as controls. (d) Effect of IFN-α on IL-12p70 production by SK-conditioned DCs. b3a2-peptide-loaded DCs were cultured with SK cells in the presence of IFN-α. (e) Representative flow cytometry profiles of CD86 on SK-conditioned DCs. Vehicle or b3a2 peptide DCs were cultured for 24 h with or without SK cells at a DC/SK ratio of 10:1 in the presence or absence of IFN-α. (f) RFI values indicating CD86 expression. (g) Proliferation of CD8⁺ T cells at day 7 in the WT1-specific CTL priming assay in the presence or absence of IFN-α was measured as [³H]-thymidine incorporation. (h) Frequency of WT1/HLA-A24-tetramer-positive CD8⁺ T cells (filled box) at day 10 in the WT1-specific CTL priming assay in the presence or absence of IFN-α. The HIV tetramer (open box) served as the control. (i) Proliferation of CD8⁺ T cells stimulated with plate-bound anti-CD3 mAbs in the presence of IFN-α. (a, c, d, f–i) Data are representative of three independent triplicate experiments. Error bars, s.d.; *P<0.05, one-way ANOVA. ANOVA, analysis of variance; b3a2, b3a2 peptide; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; mAbs, monoclonal antibodies; WT1, WT1 peptide; RFI, relative fluorescence intensity; WT1, Wilms tumor 1.

Figure 6

Antileukemia activity of CTLs primed by SK-conditioned DCs. (a) Cytotoxic activities of expanded WT1-specific CD8⁺ T cells against an HLA-A24-positive cell line (PC9) loaded with vehicle or WT1 peptide. (b) Cytotoxic activities against K562-A24 cells that were mock transduced or transduced with a minigene encoding the HLA-A24-restricted WT1 epitope. (a, b) Cytotoxicity was measured at 4 h in ⁵¹Cr release assays at the indicated effector/target (E:T) ratios. Data are representative of three independent duplicate experiments. (c) Representative flow cytometry profiles of PD-1 on expanded WT1-specific CD8⁺ T cells. The CD8⁺ T cells were stimulated with soluble control IgG or anti-CD3 mAbs (1 μg/ml) plus anti-CD28 mAbs (1 μg/ml) for 24 h. Staining histograms of PD-1 (solid line) and isotype-matched controls (dotted line) are shown. RFI is shown in the upper part of each panel. (d) In vivo inhibition of leukemia. RJ mice were s.c. coinjected with mixtures of K562-A24-WT1 minigene cells and either saline or WT1-specific CTLs. Where indicated, mixtures of SK cells and DCs±b3a2 peptide were i.p. injected into mice at day −1. The average tumor size for each group from day 0 to day 35 is shown. Error bars, s.d.; *P<0.05, one-way ANOVA; NS (e) Kaplan–Meier survival curves plotted for treated and control mice. *P<0.05, log-rank (Mantel–Cox) test; NS. (WT1-CTL, n=8; WT1-CTL+SK+DC, n=8; WT1-CTL+SK+DC+b3a2, n=8; no treatment, n=7). ANOVA, analysis of variance; CTL, cytotoxic T lymphocyte; DC, dendritic cell; i.p., intraperitoneally; NS, not significant; s.c., subcutaneously; RFI, relative fluorescence intensity; WT1, Wilms tumor 1.