Cell-surface C-type lectin-like receptor CLEC-1 dampens dendritic cell activation and downstream Th17 responses

Abstract

Dendritic cells (DCs) represent essential antigen-presenting cells that are critical for linking innate and adaptive immunity, and influencing T-cell responses. Among pattern recognition receptors, DCs express C-type lectin receptors triggered by both exogenous and endogenous ligands, therefore dictating pathogen response, and also shaping T-cell immunity. We previously described in rat, the expression of the orphan C-type lectin-like receptor-1 (CLEC-1) by DCs and demonstrated in vitro its inhibitory role in downstream T helper 17 (Th17) activation. In this study, we examined the expression and functionality of CLEC-1 in human DCs, and show a cell-surface expression on the CD16^- subpopulation of blood DCs and on monocyte-derived DCs (moDCs). CLEC-1 expression on moDCs is downregulated by inflammatory stimuli and enhanced by transforming growth factor β. Moreover, we demonstrate that CLEC-1 is a functional receptor on human moDCs and that although not modulating the spleen tyrosine kinase-dependent canonical nuclear factor-κB pathway, represses subsequent Th17 responses. Interestingly, a decreased expression of CLEC1A in human lung transplants is predictive of the development of chronic rejection and is associated with a higher level of interleukin 17A (IL17A). Importantly, using CLEC-1-deficient rats, we showed that disruption of CLEC-1 signaling led to an enhanced Il12p40 subunit expression in DCs, and to an exacerbation of downstream in vitro and in vivo CD4⁺ Th1 and Th17 responses. Collectively, our results establish a role for CLEC-1 as an inhibitory receptor in DCs able to dampen activation and downstream effector Th responses. As a cell-surface receptor, CLEC-1 may represent a useful therapeutic target for modulating T-cell immune responses in a clinical setting.

Graphical abstract

Figure 1.

Human DCs express cell-surface CLEC-1. (A) Western blot analysis of CLEC-1 expression in human moDCs, HAECs, HUVECs, and HEKs. Cell extracts were immunoprecipitated with anti-human CLEC-1 mAb (D6 clone) and then analyzed by western blot with a second in-house anti-human CLEC-1 mAb (IgG1). Arrows indicate human CLEC-1 and IgG HC and LC at the expected size of 32, 50, and 25 kDa, respectively. M line represents molecular-weight size markers. (B) Representative dot plots and histograms of IgG1 isotype or CLEC-1 (IgG1) staining in non-permeabilized (non-perm) and permeabilized (perm) conditions, evaluated by flow cytometry for human blood: (i) CD16⁺ and CD16⁻ subpopulation of CD45⁺CD14⁻CD11c⁺HLA-DR^high DCs; (ii) CD45⁺CD14⁺CD16⁺ monocytes; (iii) SSC^highCD16⁺ neutrophils; and (iv) HAECs. Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (C) Representative dot plots or histograms of IgG1 isotype or CLEC-1 (IgG1) staining vs DC-SIGN or CD16 staining for human moDCs in non-perm or perm conditions and evaluated by flow cytometry (i-ii). Histograms represent the overlay image of CLEC-1 staining (gray filled histogram) matching the isotype control IgG1 staining (open histogram). (iii) Cell-surface expression of CLEC-1 vs HLA-DR on unstimulated (US), TLR 4-L (LPS), TLR 3-L (Poly I:C), TLR 7-L (R848), and TGF-β–stimulated moDCs. (iv) Histogram represents MFI ± standard error of the mean (SEM) of CLEC-1 staining of 6 independent experiments. Statistical analysis of CLEC-1 MFI staining was performed between US and each stimuli. Panel Di shows representative confocal microscopy images, and (ii) quantitation of CLEC-1 protein in non-perm and perm conditions for human HUVECs, moDCs, CD16⁺ monocytes and neutrophils. Panels exhibiting DAPI (blue) and CLEC-1 (green) staining revealed by anti-human CLEC-1 mAb (D6 clone) followed by secondary anti-mouse Alexa-488 antibody. Original magnification ×600. Images are representative of 4 independent experiments. CLEC-1 protein quantitation was performed by velocity software and expressed as histogram of mean ± SEM of numbers of fluorescent spots per cell (n ≥7). *P < .05; **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; MFI, mean fluorescence intensity; mono, monocytes; neutro, neutrophils.

Figure 2.

CLEC-1 triggering on human moDCs prevents downstream Th17 activation. (A) Human moDCs were stimulated with pb anti–CLEC-1 or IgG1 isotype control mAb for 5 minutes. CLEC-1 and binding partners were immunoprecipitated in low-stringent conditions (D6 clone) and were revealed by western blot using anti-phosphotyrosine mAb (4G10). Representative image of western blot with arrows indicating bands with changes in phosphorylation intensity between isotype control and anti–hCLEC-1 mAb stimulation, and IgG HC and LC chains of immunoprecipitating antibody (at the expected size of 50 and 25 kDa, respectively). M line represents molecular-weight size markers. Data are representative of 3 independent experiments. (B) Human moDCs were incubated with or without (−) pb anti–hCLEC-1 or IgG1 isotype control mAbs, and were alternatively stimulated simultaneously with TLR 4-L (LPS) or zymosan for 24 hours, and CD80, CD86, CD83, and HLA-DR were evaluated by flow cytometry (overlays are representative of 8 independent experiments). (C) Tumor necrosis factor-α, IL-12p70, IL-6, IL-23, and IL-10 were assessed by ELISA in supernatants (histograms represent mean ± SEM of 8 independent experiments). (D) Human moDCs were stimulated with pb anti–hCLEC-1 or IgG1 Iso control mAbs for 20 minutes and with or without zymosan. Representative images of western blot revealing phosphorylation of IκBα (PSer32/36) or the degradation of total IκBα at the expected size of 40 and 39 kDa, respectively. Data are representative of 3 independent experiments. M line represents molecular-weight size markers. (E) Following 24 hours of CLEC-1 triggering, human moDCs were extensively washed and subjected to MLR with allogeneic T cells for 5 days. (i) T-cell proliferation was assessed (CFSE dilution) by flow cytometry in allogeneic T cells, and (ii) IL-17 and IFN-γ production was evaluated by ELISA in supernatants. Data were expressed in histograms as mean ± SEM of 8 independent experiments. **P < .01; ***P < .001. IgG HC, IgG heavy chain; IgG LC, IgG light chain; Iso, isotype; UT, untreated.

Figure 3.

Decreased CLEC1A expression in lung transplants is predictive of CR. Lung transplants from stable patients or from patients prior to the development of CR were subjected to qRT-PCR for HPRT, CLEC1A, IL17A, IFNG, and TGFB1. Results were expressed in histograms as mean ± SEM of 7 samples in each group and were expressed in AU of specific cytokine/HPRT ratio. *P < .05; **P < .01. mRNA, messenger RNA.

Figure 4.

Rat CLEC-1–deficient BMDCs enhance Th17-cell activation. (A) BMDCs from WT and CLEC-1–deficient rats were stimulated with TLR 4-L or zymosan for 24 hours, and CD80, CD86, and Class I and II major histocompatibility complex (MHC) were assessed by flow cytometry. Data were expressed in histograms as mean ± SEM of 6 independent experiments. (B) BMDCs were stimulated with TLR4-L or zymosan for 8 hours, and Il12p40, Il12p35, Il23p19, Il6, Il10, and Tgfb1 were assessed by qRT-PCR. Results were expressed in histograms as mean ± SEM of 6 independent experiments and were expressed in AU of specific cytokine/Hprt ratio. (C) BMDCs were incubated for 4 days in MLR with allogeneic purified CD4⁺ T cells. (i) Histogram and representative staining of proliferation (CFSE dilution) assessed in CD4⁺ T cells by flow cytometry, and (ii) histogram and representative dot plots of percentage of IL-17⁺ and IFN-γ⁺ cells among gated CD4⁺ T cells assessed by flow cytometry. Data were expressed in histograms as mean ± SEM of 6 independent experiments. *P < .05; **P < .01. KO, knockout; MFI, mean fluorescence intensity; mRNA, messenger RNA; NS, nonstimulated.

Figure 5.

Blocking CLEC-1 Fc fusion protein enhances rat BMDC-mediated Th17-cell activation. (A) BMDCs from naïve rats were incubated for 4 days in MLR with allogeneic purified CD4⁺ T cells, together with CLEC-1 Fc or irrelevant hSEAP-Fc fusion proteins (produced and purified under the same conditions) (10 μg/mL). (i) Histogram of proliferation (CFSE dilution) of CD4⁺ T cells assessed by flow cytometry, and (ii) IL-17 and IFN-γ cytokine production assessed in supernatants of MLR by ELISA. Data were expressed in histograms as mean ± SEM of 4 independent experiments. (B) Purified CD4⁺ T cells from naïve rats were stimulated with pb anti-CD3 (5 μg/mL) in combination with CLEC-1–Fc or irrelevant hSEAP-Fc fusion proteins (10 μg/mL) for 4 days. (i) Proliferation (CFSE dilution) was assessed by flow cytometry, and (ii) IL-17 cytokine production was assessed in supernatants by ELISA and in CD4⁺ T cells by flow cytometry as FSC vs isotype or IL-17 staining. Data were expressed as histograms as mean ± SEM of 4 independent experiments. **P < .01; ***P < .001. FSC, forward scatter.

Figure 6.

CLEC-1–deficient rats exhibit an exacerbation of in vivo DC-mediated CD4⁺Th1/Th17 responses. (A) WT, CLEC-1–deficient rats (i-ii) and chimeric rats reconstituted with BM from WT or CLEC-1–deficient rats (iii) were immunized subcutaneously in the footpad with CFA plus KLH protein (100 μg/mL). At day 10 after immunization, popliteal LNs were harvested and total LN cells or purified CD4⁺ T cells were re-stimulated in vitro with KLH or control ovalbumin (25 μg/mL) for 3 days. Histograms and representative plots of proliferation (CFSE dilution) and percentage of IL-17⁺, IL-17⁺ IFN-γ⁺, and IFN-γ⁺ cells in gated CD4⁺ T cells assessed by flow cytometry. Data were expressed as histograms as mean ± SEM of 4 independent experiments. Staining of isotypes was indicated as control. (B) WT and CLEC-1–deficient rats were transplanted with cardiac allografts. (i) At day 5 after transplantation, purified CD4⁺ T cells from spleen were re-stimulated in vitro with donor T-cell–depleted splenocytes (MLR) for 3 days. Histograms of proliferation (CFSE dilution) in gated CD4⁺ T cells assessed by flow cytometry and expressed as mean ± SEM of 4 independent experiments. (ii) Il17a and Ifng were assessed by qRT-PCR in cardiac allografts harvested at day 5 after transplantation. Results were expressed in histograms as mean ± SEM of 4 independent experiments and were expressed in AU of specific cytokine/Hprt ratio. *P < .05; **P < .01. mRNA, messenger RNA.