Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross-presenting function

Abstract

Ability to cross-present exogenous antigens in the human leukocyte antigen class I pathway is key to the antigen presenting function of mature tumor cell-loaded dendritic cells (DC). Conditions of DC maturation have been shown to be important for DCs ability to produce proinflammatory cytokines and induce T cell effector functions. However, it remains unknown if the different pathways of maturation are associated with modulation of the ability of mature DCs to cross-present tumor antigens (TA). Here, we compare DC matured with 3 clinically relevant cytokine combinations including interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6 (termed DC-0), DC-0 cells incubated with prostaglandin-2 (termed DC-0+prostaglandin-2), or DC treated with interferon-gamma, interferon-alpha, tumor necrosis factor-alpha, Poly I:C, and IL1-beta (termed DC-1). We found that these DC vary in their ability to cross-present TA to cytotoxic T lymphocytes (CTL), with the DC-1 cytokine combination being significantly more effective than the other 2. TA cross presentation and CTL priming were strongly correlated with level of expression of the antigen processing machinery components, TAP1 and TAP2, indicating that these components could be used as biomarkers to standardize DC preparations for optimal function. However, the up-regulation of TAP1/TAP2 was not sufficient to explain the enhanced cross-presentation ability of DC-1 cells, as the use of IFN-gamma alone to up-regulate TAP1/TAP2 did not generate DC as effective at cross-presentation as the full DC-1 maturation cytokine combination. These data indicate for the first time that the pathways of DC maturation modulate antigen processing machinery component expression to different extents and that differently matured DC vary in the ability to cross-present TA to human leukocyte antigen class I-restricted CTL.

FIGURE 1

Expression of costimulatory molecules by DC after incubation with different cytokine combinations. After six days of culture in interleukin-4 and granulocyte macrophage colony stimulating factor supplemented media, human monocyte derived immature DC was incubated for 48 hr at 37°C in medium alone or in medium supplemented with 1 of the maturation cytokine combinations (Table 1). Cells were then harvested and stained with FITC labeled control IgG (gray histograms), anti-CD80, anti-CD83, anti-CD86, anti-CCR7 or HLA-DR-specific monoclonal antibodies (solid lines). Samples were analyzed by flow cytometry, adjusting the mean fluorescence intensity for control IgG-stained cells to 5 to permit comparison between DC culture conditions. DC indicates dendritic cell; IgG, immunoglobulin G.

FIGURE 2

Differential APM component up-regulation in DC matured with different cytokine combinations. A, representative flow cytometry histograms for APM component expression are shown, as performed for 12-healthy donor DC preparations. Separate experiments were repeated for 2 or 3 times per donor. After 6 days of culture in interleukin-4 and granulocyte macrophage colony stimulating factor supplemented media, human monocyte-derived immature DC were treated with medium alone or medium cultured with 1 of the cytokine combinations listed in Table 1 for 48 hour at 37°C. DC were then harvested and stained intracellularly with mAb specific for δ, LMP2, TAP1, TAP2 or tapasin (as described in Materials and Methods section). Stained cells were analyzed by flow cytometry, adjusting the MFI for control isotype matched mAb-stained cells to 5, to permit comparison between different DC culture conditions. B, MFI values summarized from 12 sets of healthy, HLA-A*0201⁺ donor DC that were matured with different cytokine combinations listed in Table 1. Mean MFI values (± SEM) obtained by flow cytometry are shown, where the isotype control staining was set to MFI = 5, to permit comparison between different DC culture conditions (*P<0.05). APM indicates antigen processing machinery; DC, dendritic cell; mAb, monoclonal antibody; MFI, mean fluorescence intensity.

FIGURE 3

Expression of antigen processing machinery components, δ, LMP2, TAP1, TAP2, and tapasin, after treatment with IFN-γ alone or with the DC-1 cytokine combination. Immature DC were incubated with IFN-γ alone (100 IU/mL for 48 h at 37°C) or with the DC-1 cytokine combination (tumor necrosis factor-α at 50 ng/mL, interleukin-1β at 25 ng/mL, IFN-α at 1000 U/mL, and IFN-γ at 1000 U/mL for 48 h at 37°C). DC were then harvested, stained intracellularly and analyzed by flow cytometry. Mean MFI values (+SEM) obtained from the flow cytometry analysis are shown. The isotype control monoclonal antibody staining was set to MFI = 5 to permit comparison between different DC culture conditions. DC indicates dendritic cell; IFN, interferon; MFI, mean fluorescence intensity.

FIGURE 4

A, Specificity of the HLA-A*0201-MAGE-3_271–279 specific CTL generated by in vitro stimulation. Naive CTL were stimulated as detailed in the Materials and Methods section and tested for their specificity using the TAP1/TAP2 deficient T2 cells pulsed with MAGE-3_271–279 or HIV POL_476–484 exogenous peptides. The HLA class I specific mAb W6/32 and the HLA-DR specific mAb L243 were used as controls for determination of class I restricted activity. B, Cross-presentation of MAGE-3_271–279 derived from HLA-A*0201⁻, JHU-029 cells to MAGE-3_271–279-specific effector cells by DC treated with different maturation conditions. On day 6 of the DC cultures, UV-B irradiated HLA-A*0201⁻, MAGE-3⁺ JHU-029 cells were added to the DC cultures, in the presence of each cytokine maturation combination, at 37°C for 48 hour. DC were used as targets in IFN-γ ELISPOT assays using HLA-A*0201-MAGE-3_217–279-specific CTL. The HLA class I antigen restricted CTL activity was monitored by incubating targets with HLA-A, HLA-B, HLA-C antigen-specific mAb W6/32 (gray bars). CTL were tested for specificity (as previously detailed) on every experiment where DC was used as targets (ns = non significant, P<0.05 1-tail permutation test). C, Apoptotic JHU-029 fed DC-1 cells were incubated with an interleukin-12 specific mAb or an Immunoglobulin G isotype control and used as stimulators in an ELISPOT assay. D, DC treated with the different maturation cytokine combinations were harvested and incubated with exogenous MAGE-3_271–279 peptide (10 μg/mL in AIM-V media for 1 hr at room temperature). IFN-γ ELISPOT assays were performed using the HLA-A*0201-MAGE-3_271–279 specific CTL. (ns indicates non significant, P<0.05 1 tail permutation test). CTL, cytotoxic T lymphocytes; DC, dendritic cells; mAb, monoclonal antibodies.

FIGURE 5

Cross-presentation of melanoma cell-derived MART-1 to CTL precursors by DC-1 cells leads to improved induction of melanoma-specific CTL response. DC-1, DC-0+PGE-2 or immature DC were cultured from a healthy HLA-A2⁺ donor and cultured with UV-treated apoptotic FEM-X melanoma cells for 48 hour at 37°C as a source of MART-1 antigen. These DC were then used to sensitize autologous CD8⁺ T cells and frequencies of IFN-γ producing melanoma antigen specific CTL were determined by ELISPOT. A, IFN-γ spots produced by DC-primed CTL recognizing the HLA-A2⁺ FEM-X melanoma cell targets. B, IFN-γ producing antigen specific CTL responding to HLA-A2⁺ DR4⁺ T2 target cells pulsed with the HLA class-I restricted melanoma peptide MART-1_27–35. CTL indicates cytotoxic T lymphocyte; DC, dendritic cell FEM-X; IFN, interferon; HLA, human leukocyte antigen.