Human in vivo-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway

Abstract

Presentation of exogenous antigens on MHC-I molecules, termed cross-presentation, is essential for cytotoxic CD8⁺ T cell responses. In mice, dendritic cells (DCs) that arise from monocytes (mo-DCs) during inflammation have a key function in these responses by cross-presenting antigens locally in peripheral tissues. Whether human naturally-occurring mo-DCs can cross-present is unknown. Here, we use human mo-DCs and macrophages directly purified from ascites to address this question. Single-cell RNA-seq data show that ascites CD1c⁺ DCs contain exclusively monocyte-derived cells. Both ascites mo-DCs and monocyte-derived macrophages cross-present efficiently, but are inefficient for transferring exogenous proteins into their cytosol. Inhibition of cysteine proteases, but not of proteasome, abolishes cross-presentation in these cells. We conclude that human monocyte-derived cells cross-present exclusively using a vacuolar pathway. Finally, only ascites mo-DCs provide co-stimulatory signals to induce effector cytotoxic CD8⁺ T cells. Our findings thus provide important insights on how to harness cross-presentation for therapeutic purposes.

Fig. 1

Ascites DCs are distinct from classical DCs. Purified ascites DCs, ascites macrophages, tonsil DCs, and blood monocytes were analyzed by single-cell RNA-seq using a Drop-seq approach. Combined single-cell transcriptomes were analyzed. a, b t-SNE representation of cell clusters identified using unsupervised clustering. Each dot represents an individual cell. a Colors represent sample origin; don donor. b Colors represent identified clusters. Clusters are manually ordered and cell numbers for each cluster is indicated. c Heatmap of scaled expression (log values of UMI) for the top 20 differentially expressed genes of each cluster (based on log fold change)

Fig. 2

Ascites DCs are monocyte-derived cells. Purified ascites DCs, ascites macrophages, tonsil DCs, and blood monocytes were analyzed by single-cell RNA-seq using a Drop-seq approach. Combined single-cell transcriptomes were analyzed. a Signature scores in individual cells for indicated gene signatures. b Annotation of cell clusters

Fig. 3

Human mo-DCs and mo-Mac both cross-present efficiently. Purified DCs and macrophages from tumor ascites (a) or in vitro culture of monocytes (b, c), or DCs derived in vitro from CD34⁺ precursors (d) were incubated with serial concentrations of MelanA long or short peptide (a, b, d) or MelanA-coated beads (c). After washing, antigen-specific CD8⁺ T cells were added. After 24 h, IFN-γ secretion was assessed as a measure of T cell activation. Background level was subtracted. Mean ± SEM of three (a, d), six (b), or five (c) independent experiments

Fig. 4

Human mo-DCs and mo-Mac are inefficient for the transfer of exogenous proteins into their cytosol. a, b Purified DCs and macrophages from tumor ascites, derived in vitro from monocytes, or DCs derived in vitro from CD34⁺ precursors were loaded with a cell-permeable FRET-sensitive substrate of β-lactamase, and incubated with or without exogenous β-lactamase. After 3 h, cleavage was measured by flow cytometry. a Representative results of six (tumor ascites), ten (in vitro monocyte-derived), or eight (in vitro CD34⁺ cell-derived) independent experiments. b Quantification of β-lactamase transfer. Symbols represent individual donors. N = 6 for tumor ascites, N = 10 for in vitro monocyte-derived cells, and N = 8 for CD34⁺ cell-derived cells. **p < 0.01, Wilcoxon non-parametric test. c Purified DCs or macrophages were incubated with β-lactamase coupled to Atto dye 633 at 4 or 37 °C during 3 h. Representative results of three independent experiments

Fig. 5

Human mo-DCs and mo-Mac use the vacuolar pathway for cross-presentation. Purified in vitro-generated mo-DCs (a, d), mo-Mac (b, e), or CD34⁺ cell-derived CD1a⁺ DCs (c, f) were incubated with serial concentrations of MelanA long or short peptide, in the absence or presence of lactacystin (a–c) or pan-cathepsin inhibitor (d–f). After washing, antigen-specific CD8⁺ T cells were added. After 24 h, IFN-γ secretion was assessed as a measure of T cell activation. Background level was subtracted. Mean ± SEM of three (a), five (b, d, e), or three (c, f) independent experiments

Fig. 6

Human mo-DCs, but not mo-Mac, are efficient inducers of effector cytotoxic CD8⁺ T cells. Purified DCs and macrophage from tumor ascites were cultured with allogeneic CellTrace Violet-stained naïve CD8⁺ T cells for 7 days, in the absence (a, b) or presence (c, d) of naïve CD4⁺ T cells autologous to CD8⁺ T cells. Expression of Granzyme A, Perforin, and IFN-γ was assessed by intracellular flow cytometry. a, c Representative results of eight independent experiments. Gated on live CD8⁺ T cells. b Number of proliferating CD8⁺ T cells is shown. Symbols represent individual donors. N = 8. Median is shown. d Number of CD8⁺ T cells expressing effector molecules is shown. Symbols represent individual donors. N = 8. Median is shown. *p < 0.05, **p < 0.01, Wilcoxon non-parametric test

Fig. 7

Human mo-DCs, but not mo-Mac, provide co-stimulatory signals for the differentiation of cytotoxic CD8⁺ T cells. a Heatmap of scaled expression for selected co-stimulation genes. d donor. b Purified DCs and macrophage from tumor ascites were cultured with allogeneic naïve CD4⁺ T cells for 7 days, in the presence of naïve CD8⁺ T cells. Total number of live CD4⁺ T cells at the end of the culture is depicted. N = 8. c Purified DCs and macrophage from tumor ascites were cultured in the absence or presence of CD40-L, IFN-γ, and R848 for 24 h. Secretion of IL-12p70 was measured in the supernatant. N = 5. b, c Symbols represent individual donors. Median is shown. ** p < 0.01, Wilcoxon non-parametric test