Differential presentation of a soluble exogenous tumor antigen, NY-ESO-1, by distinct human dendritic cell populations

Abstract

Dendritic cells (DCs) play a critical role in initiating antigen-specific immune responses, because they are able to capture exogenous antigens for presentation to naive T cells on both MHC class I and II molecules. As such, DCs represent important elements in the development of vaccine therapy for cancer. Although DCs are known to present antigens from phagocytosed tumor cells or preprocessed peptides, we explored whether they might also present soluble recombinant NY-ESO-1, a well characterized cancer antigen. We compared the abilities of human monocyte-derived DCs and DCs derived in vitro from CD34-positive stem cells to present NY-ESO-1 epitopes to MHC class I-restricted cytotoxic T cells. Although monocyte-derived DCs did not efficiently crosspresent free NY-ESO-1 protein, IgG-immune complexes containing NY-ESO-1 were avidly presented after uptake by Fcgamma receptors (FcgammaRII). In contrast, CD34-derived DCs were unable to process either soluble or immune complexed NY-ESO-1, although they efficiently presented preprocessed NY-ESO-1 peptides. This difference did not necessarily correlate with endocytic capacity. Although monocyte-derived DCs exhibited greater fluid-phase uptake than CD34-derived DCs, the two populations did not differ with respect to their surprisingly limited capacity for Fcgamma receptor-mediated endocytosis. These results indicate that monocyte-derived DCs will be easier to load by using protein antigen in vitro than CD34-derived DCs, and that the latter population exhibits a restricted ability to crosspresent soluble exogenous antigens.

Fig 1.

Phenotype of monocyte-derived DCs. Monocytes isolated by magnetic bead selection from PBMCs were harvested immediately (day 0) cultured with GM-CSF and IL-4 for 5 days. At this time, TNFα (10 ng/ml) or NY-ESO-1-containing immune complexes (1 μg/ml as protein) were added and the cells harvested 24 hr later. The cells were then analyzed by flow cytometry by using the indicated antibodies.

Fig 2.

Presentation of NY-ESO-1-derived epitopes to antigen-specific CD8+ T cells by monocyte-derived DCs exposed to NY-ESO-1-containing immune complexes. Monocyte-derived DCs were prepared from a HLA-A2 (A*0201)-positive healthy donor and used as antigen-presenting cells for different NY-ESO-1-specific CD8+ T cells. (A) ELISPOT assay using mo-DCs exposed in medium 12 hr (DC alone) or NY-ESO-1 synthetic peptide (p157–165 at 10 μg/ml) (Upper), free NY-ESO-1 protein (1 μg/ml), or NY-ESO-1 immune complexes prepared by using the ES121 monoclonal antibody (1 μg/ml of NY-ESO-1 protein, 1 μg/ml of IgG), NY-ESO-1 immune complexes prepared by using serum from a melanoma patient with antibody for ESO-1 (1 μg/ml of NY-ESO-1 protein, 1/1,000 dilution of sera) or with serum from antibody-negative patient, free recombinant SSX2 protein (1 μg/ml; negative control), and/or SSX2 immune complex generated by using the monoclonal antibody HM498 (1 μg/ml of SSX2 protein, 1 μg/ml of IgG). Shown are excised filters stained for γ-interferon secretion. Numbers at bottom refer to the number of CD8+ T cells added per well. (B) mo-DCs incubated with the HLA-A2-restricted NY-ESO-1 peptide p157–165, the HLA-Cw3-restricted peptide p92–100, free NY-ESO-1 protein (1 μg/ml) or NY-ESO-1 ICs (as in A). Data in Top represent number of positive ELISPOT spots using Cw3-restricted CTLs expanded from a melanoma patient; bottom using the A2-restricted CTL “clone 5.” (C) ELISPOT assay performed as above comparing presentation of peptide to clone 5 by mo-DCs alone, exposed to free NY-ESO-1 protein, NY-ESO-1 ICs, NY-ESO-1 ICs prepared by using a Fab fragment of the monoclonal antibody E978 (1 μg/ml) or exposed to mAb E978. For all experiments, mo-DCs were collected on day 5 and incubated with peptide, protein, or immune complexes overnight before ELISPOT assay. (D) ELISPOT assay performed as above except (where indicated) NY-ESO-1-IC-containing wells also contained 10 μg/ml or monoclonal IgGs against CD32 (FcγRII) or CD64 (FcγRI).

Fig 3.

Lactacystin inhibits crosspresentation of a NY-ESO-1 epitope. mo-DCs were harvested at day 5 and then cultured for an additional 12 hr in the presence or absence of lactacystin (10 μM) in normal growth medium or medium containing the NY-ESO-1 peptide p157–165 (10 μg/ml), free NY-ESO-1 (1 μg/ml), NY-ESO-1 ICs (using the monoclonal antibody E978 at 1 μg/ml), or the control protein SSX2. ELISPOT assays using clone 5 were performed as above. Open bars, no lactacystin; solid bars, lactacystin added.

Fig 4.

Macrophages do not crosspresent NY-ESO-1 immune complexes. mo-DCs and CD14+ macrophages were compared for their abilities to present exogenous NY-ESO-1 either as free protein or as an immune complex (as above) to the HLA-A2-restricted CD8+ T cell clone 5. ELISPOT data are shown. A contains data from mo-DCs; B contains data from macrophages. The NY-ESO-1 peptide was used for this experiment (p157–165, 1 μg/ml). Solid bars represent HLA-A*0201-positive mo-DCs or macrophages; open bars represent HLA-mismatched controls.

Fig 5.

CD34-derived DCs fail to crosspresent NY-ESO-1. Immature cd34-DCs were prepared and cultured overnight in the presence of the indicated antigens or peptides with or without the addition of heat-killed E. coli to induce maturation (17). A illustrates the surface expression by flow cytometry of HLA-DR and CD83, showing the ability of the E. coli to induce maturation (i.e., enhanced expression of both of these markers). B shows the results of a series of ELISPOT assays using clone 5 as the NY-ESO-1-specific T cell clone. Immature (−) or stimulated (+) cd34-DCs were cultured overnight in medium containing no addition, the HLA-A2-restricted peptide p157–165, free NY-ESO-1 protein (30 or 1 μg/ml), or NY-ESO-1 ICs (1 μg/ml of NY-ESO-1 protein). As positive controls, the ability of T2 cells (A*0201) to present p157–165 was measured, as was the ability of the melanoma cell line SK-MEL-37 to present endogenous NY-ESO-1.

Fig 6.

Endocytosis of fluid-phase FITC–dextran by monocyte- and CD34-derived immature DCs. Immature or mature DCs were incubated in medium containing FITC–dextran (40,000 molecular weight; 1.0 mg/ml) for 1 hr at 37°C. After washing, samples were analyzed by flow cytometry. Immature mo-DCs accumulated >10-fold more FITC–dextran than did cd34-DCs. Maturation reduced uptake by both cell populations to nearly control values. Dark line, cells exposed to FITC–dextran; thin line, no dextran control.

Fig 7.

Binding and endocytosis of NY-ESO-1-containing immune complexes by monocyte- and CD34-derived DCs. (A) Surface FcγRs were determined by flow cytometry on mo-DCs, cd34-DCs, and CD14+ monocytes by using antibodies to FcγRII (CD32) and FcγRIII (CD16). (B) ICs formed by combining recombinant NY-ESO-1 and affinity-purified rabbit polyclonal anti-NY-ESO-1 antibody (30 μg IgG/ml) were incubated with immature mo-DCs, cd34-DCs, or CD14+ monocytes for 1 hr at 37°C. The cells were washed with PBS four times, fixed, and permeabilized by using 0.05% saponin to detect both surface and intracellular ICs. The cell-associated ICs were then visualized by polyclonal anti-rabbit-Alexa488 antibody prior to analysis by flow cytometry. Thick lines, ICs added; thin lines, no ICs added. (C) Immature mo-DCs, cd34-DCs, or CD14+ monocytes were exposed to FITC–dextran (green) or biotinylated NY-ESO-1 ICs for 1 hr at 37°C. The cells were then fixed, permeabilized, and stained for the ICs (green) or the lysosomal membrane marker lgp/lamp (red). Both DC types internalized FITC-dextran, delivering it to lamp-positive lysosomes (which thus appeared yellow); the DCs internalized little or no IC (cd34-DCs were barely over background). In contrast, the monocytes internalized little dextran but significant amounts of the NY-ESO-1 ICs.