CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity

Abstract

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169⁺ monocytes. Here, we have investigated the phenotype of human CD169⁺ monocytes in different diseases, their capacity to activate CD8⁺ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14⁺ CD16^- classical and CD14⁺ CD16⁺ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169⁺ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14⁺ monocytes and boosted their capacity to cross-present antigen to CD8⁺ T cells. Furthermore, we observed CD169⁺ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169⁺ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8⁺ T cells. In conclusion, our data indicate that CD169⁺ monocytes are activated monocytes with enhanced CD8⁺ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

Keywords: CD169; CD8+ T cell; COVID-19; Siglec-1; antigen-presentation; cancer; monocyte; nanovaccine.

Figure 1

CD14⁺ CD169⁺ monocytes display enhanced maturation status. (A) Unsupervised high dimensionality reduction analysis of circulating monocytes and dendritic cells (HLA-DR⁺ CD3/CD19/CD56^–) using CD14, CD16, CD88, CD163, HLA-DR, FcϵR1α, CD123, CD11c, CD1c, CD141, Axl, Siglec-6, BTLA, and CD169 using opt-SNE, and overlaid with conventional gating. (B) The expression of CD14, CD16, CD88, and CD169 on tSNE plots. (C) Gating strategy identifies classical (CD14⁺ CD16^-), intermediate (CD14⁺ CD16⁺), or non-classical (CD14^- CD16⁺) monocytes, and CD169⁺ cells. (D) Percentage of CD169⁺ cells within monocytes subsets (n = 13) in healthy donors. (E) Heatmaps comparing the relative expression of markers defining monocytes and DC subsets. (F, G) Expression of co-stimulatory and HLA molecules are compared between CD169⁺ and CD169^- classical monocytes shown as (F) representative histograms and (G) quantification (n = 4). Paired t-tests were used. **P < 0.01.

Figure 2

CD169 expression in monocytes is driven by IFN-I and CD14⁺ CD169⁺ monocytes are present in COVID-19 patients. (A) Wanderlust trajectory analysis of monocyte population using CD14⁺ CD169^- monocytes as starting population overlaid by conventional gating of monocyte subsets. (B, C) CD14⁺ monocytes were isolated and treated with 1,000 IU/ml IFNα for 24h unless indicated otherwise. Percentage of CD169⁺ cells or CD169 median fluorescence intensity (MFI) of CD169⁺ population are shown. (n = 4). (D–G) Analysis of public scRNA-seq dataset of PBMCs from patients with COVID-19 (n = 9), severe influenza (n = 5) and healthy controls (n = 4) using Seurat pipeline. (D) UMAP analysis showing the expression of CD14 and SIGLEC1. (E) SIGLEC1 expressing monocytes as shown as percentages or MFI in different groups. (F) Violin plot of SIGLEC1 expression in clusters of monocytes and DC subsets from each patient group. (G) Violin plots of IFN-I score, TLR activation score, maturation markers CD83 and CD86, in monocytes and DC clusters of all groups. (H, I) Spectral flow cytometry analysis of COVID-19 patients (ArtDECO cohort) of CD169-expressing monocytes/macrophages in circulation or bronchoalveolar space. (H) Percentage of CD169⁺ cells within monocytes subsets or alveolar macrophages (AM) (n = 16) in the (left panel) blood or (right panel) BALF of COVID-19 patients. (I) Expression of HLA-DR compared between CD169⁺ and CD169^- subsets of monocytes or alveolar macrophages (AM). Paired t-tests were used. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

CD14⁺ CD169⁺ monocytes are present in cancer patients. (A) Analysis of public scRNA-seq dataset of PBMCs from PDAC patients (n = 12) and healthy controls (n = 4) using Seurat algorithm and projected onto UMAP space where cell types are indicated. The expressions of CD14 and SIGLEC1 are visualized on UMAP. (B) Analysis of public scRNA-seq dataset of PBMCs from patients with lung cancer (n = 7) using Seurat and UMAP clustering. The expression of SIGLEC1 and CD14 are shown. (C) Percentage of CD169⁺ cells within classical (CD14⁺ CD16^-), intermediate (CD14⁺ CD16⁺), or non-classical (CD14^- CD16⁺) monocytes in patients with pancreatic ductal adenocarcinoma (PDAC, n = 4), hepatocellular carcinoma (HCC n = 7), colorectal liver metastasis (CRLM, n = 4), and melanoma (n = 4). Monocytes were gated on live, HLA-DR⁺ Lin(CD3/CD19/CD56)^– cells. (D) Expression of HLA-DR between CD169⁺ and CD169^- classical or intermediate monocytes in cancer patients. Paired t-tests were used. ****P < 0.0001.

Figure 4

IFN-α treated monocytes showed enhanced peptide presentation to CD8⁺ T cells. (A–C) After CD14⁺ isolation, monocytes were incubated with IFNα overnight, loaded with different concentrations of gp100, washed, and gp100-specific CD8⁺ T cells were added. After 24h, IFNγ secretion by CD8⁺ T cells after co-culture with monocyte loaded with (B) short peptide or (C) long peptide was measured by ELISA. (D, E) Targeted antigen delivery to CD14⁺ CD169⁺ monocytes using antibody conjugated with gp100 peptide. (D) IFNα-treated CD14⁺ CD169^high monocytes or (E) freshly-isolated total CD14⁺ monocytes were loaded with different doses of αCD169-gp100 or control IgG-gp100 antibody-conjugates, washed, and gp100-specific CD8⁺ T cells were added. After 24h IFNγ secretion was measured by ELISA. Data are mean from four donors. Paired t-tests were used. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Ganglioside-liposomes deliver antigen/adjuvant to CD14⁺ CD169⁺ monocytes for presentation to CD8⁺ T cells. (A) Gangliosides GM3 and GT1b were incorporated into DiD-labeled liposomes and uptake was determined by flow cytometry. GalNAc, N-acetyl galactosamine; Cer, ceramide; Ctrl, control. Additionally, toll-like receptor 7/8 agonist R848 was incorporated and tumor-associated peptide was encapsulated. (B, C) Ganglioside liposome uptake by human CD14⁺ CD169⁺ monocytes as (B) representative plots and (C) quantification (n = 4) are shown. (D) Ganglioside-liposome uptake by human splenic autofluorescence (AF)^- CD14⁺ Lin(CD3/CD19/CD56)^– monocytes, repartitioned as CD169⁺ or CD169^- cells. (E) Reanalysis of ganglioside-liposome uptake on circulating classical CD169⁺ monocytes (HLA-DR⁺ Lin^- CD14^high CD1c^-) and DC3 (HLA-DR⁺ Lin^- CD14^int CD1c⁺). (F, G) Ganglioside-liposome uptake by CD169⁺ classical monocytes of cancer patients as (F) representative plot and (G) quantification are shown. Friedman test using a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 0.05, was used. **adjusted P < 0.01, ****adjusted P < 0.0001. (H, I) PBMCs were incubated with R848-containing ganglioside-liposomes at 37°C for 45 min, washed, and cultured for five hours in complete medium, with the addition of brefeldin-A for the final three hours. TNFα production by classical monocytes was measured by intracellular flow cytometry, gated on live CD14⁺ CD16^- HLA-DR⁺ Lin(CD3/CD19/CD56)^– cells. (H) Representative plot from one donor and (I) quantification as fold change over control are shown. Data are mean ± SEM from 6-7 donors. (J, K) After CD14⁺ isolation, monocytes were incubated with different concentrations of Ganglioside/WT1/R848 liposome or control (Ctrl) liposome, washed, and WT1-specific CD8⁺ T cells were added. (J) IFNγ secretion after 24h was determined by ELISA. (K) Fold change of IFNγ secretion over Ctrl liposome is shown for GM3/WT1, GT1b/WT1, or GM3 devoid of peptide, at 1.0 µM dose. Paired t-tests were used. *P < 0.05, **P < 0.01, ****P < 0.001. (L) Two nanovaccine platforms, antibody- and liposome-based, deliver antigen to CD169+ monocytes for antigen-specific CD8⁺ T cell activation.