Fig 1: CNS-specific Mcl/Mincle silencing, as well as blockage of the endogenous ligand SAP130, protects from EAE.(A) MOG-immunized rats injected with Mcl (n = 8), Mincle (n = 8), Mcl/Mincle (n = 8), or scrambled control siRNA (n = 8) i.t. and i.c. at 7, 9, and 12 days p.i. were followed for clinical signs of EAE (representative of 3 experiments). (B) Characterization of proliferation and cytokine production of infiltrating cells isolated from spinal cord on day 12 p.i., stimulated in vitro with PMA/ionomycin/brefeldin A for 5 hours (proliferation and cytokine production) in rats injected with Mcl/Mincle (n = 5) or scrambled control siRNA (n = 6) and (C) quantification of IFN-γ– and IL-17–producing CD4+ T cells (representative of 2 experiments). (D) Clinical signs of EAE and disease parameters in DA rats treated with anti-SAP130 (n = 18) or rabbit IgG isotype control (n = 18) antibody i.t. and i.c. on days 2 and 7 p.i. (2 pooled experiments). Data are presented as the mean ± SEM. The following statistical tests were used: 1-way ANOVA with Dunnett’s multiple-comparisons test (A, for area under the curve [AUC] of clinical EAE and weight change), Mann-Whitney U test (A–D [for average, cumulative, and max EAE score], B, and C), unpaired 2-tailed t test (D, for AUC of clinical EAE and weight change), and χ2 test (A and D, for EAE incidence). *P < 0.05.
Fig 2: Attenuated response to Mcl/Mincle stimulation in CLRc BMMas resulting in altered CD4+ T cell extravasation and activation.(A) Bone marrow–derived macrophages (BMMas) from DA (n = 4) and CLRc (n = 4) rats were stimulated with receptor-specific ligands (TDM and TDB) for 18 hours (representative of 3 experiments). Genes downstream of the Mcl/Mincle pathway were analyzed by qPCR. (B) MOG-specific CD4+ effector cells were reactivated for 4 days with DA (n = 6) or CLRc (n = 6) BMMas at a ratio of 40:1 (T cell/BMMa) in the presence of MOG peptide. Flow cytometry analysis of CD4+ T cell assessing cytokine production and proliferation (representative of 2 experiments). (C) Transendothelial extravasation of MOG-specific CD4+ effector cells toward DA (n = 4) or CLRc (n = 4) BMMas. Flow cytometry analysis of CD4+ T cell assessing transmigration (representative of 2 experiments). (D) Adoptive transfer of GFP+ MOG-specific CD4+ effector cells injected i.v. into DA (n = 7) or CLRc (n = 7) recipients 6 days p.i. (representative of 2 experiments). Characterization of proliferation and cytokine production in both GFP– as well as GFP+ infiltrating cells isolated from spinal cord on day 13 p.i. stimulated in vitro with PMA/ionomycin/brefeldin A for 5 hours. Data are presented as the mean ± SEM. All comparisons were analyzed with the Mann-Whitney U test. *P < 0.05; **P < 0.01.
Fig 3: The MCL/ MINCLE signaling pathway is upregulated in MS patients and correlates with disease activity and progression.(A) Representative immunofluorescent staining of MS lesions: MCL or MINCLE (green), HLA-DR expression (LN3, red). Original magnification, ×40. (B and C) Gene expression analysis of PBMCs from MS patients and noninflammatory neurological disease controls (NINDCs) using RNA sequencing. (B) Expression of MCL, MINCLE, and CARD9 comparing MS patients in remission (n = 73), MS patients in relapse (n = 14), CIS patients (n = 28), and NINDCs (n = 36). (C) Expression of MCL, MINCLE, CARD9, and IL8 according to Expanded Disability Status Scale (EDSS) score. (D) PBMCs from RRMS patients in relapse (n = 9) and healthy controls (HC) (n = 11) were stimulated in vitro with TDB, LPS, or media. qPCR analysis of IL8 expression of adherent CD14+ fraction after 24-hour stimulation. (E) Flow cytometry analysis of IL-8 production gated on CD14+ monocytes after 48-hour stimulation. Data are presented as the mean ± SEM or box plots with whiskers representing 5th to 95th percentile. The following statistical tests were used: 1-way ANOVA with Dunnett’s multiple-comparisons test (B), Mann-Whitney U test (C and D), and unpaired 2-tailed t test (E). *P < 0.05; **P < 0.01.
Fig 4: CLRc regulates expression of Mcl and Mincle in monocytes and macrophages.(A) Expression of Mcl and Mincle assessed by microarray in spleens of (DA × PVG) × DA backcrossed rats subjected to EAE, affected (n = 95, score ≥1) and nonaffected (n = 51, score 0). (B) Gene expression in DA (n = 6) and CLRc (n = 6) spleens determined by qPCR (representative of 2 experiments). (C) Flow cytometry analysis of cells isolated from blood and spinal cord of naive DA (n = 5) and CLRc (n = 5), as well as DA (n = 7) and CLRc (n = 6) rats on day 13 p.i. assessing Mcl and Mincle protein expression by mean fluorescence intensity (MFI) (representative of 2 experiments). Ly, lymphocyte; Mi, microglia; Mo/Ma, monocyte/macrophage; Gr, granulocyte. (D) qPCR analysis of Mcl and Mincle expression in BMMas and MoDCs (BM-APCs) derived in vitro and meninges from naive and 7-day-p.i. DA (n = 4) and CLRc (n = 4) rats (representative of 2 experiments). (E) Immunofluorescent staining of rat spinal cord 11 days p.i. Representative images of staining for Mcl, Mincle, and nuclei (DAPI) (original magnification, ×40). Data are presented as the mean ± SEM or box plots with whiskers representing 5th to 95th percentile. All comparisons were analyzed with the Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 5: CLEC4D knockdown inhibits the proliferation in GC cells. (a) Analysis of DepMap Database (https://depmap.org/portal/download/) suggested that knockout of CLEC4D could attenuate the proliferation in a panel of GC cell lines. (b) We depleted the endogenous CLEC4D expression in HGC-27 and NCI-N87 cells using shRNA. Immunoblot assays were used to verify the knockdown efficiency. (c) Membrane expression of CLEC4D of GC cells was determined using flow cytometry. (d) CCK-8 assays showed that CLEC4D knockdown compared with control attenuated the propagation in HGC-27 and NCI-N87 cells. (e) HGC-27 and NCI-N87 cells with CLEC4D deficiency gave rise to far less colonies, compared with control cells did (P < 0.001). (f) Tumor xenografts model demonstrated that the growth of NCI-N87 cells was remarkably inhibited by CLEC4D knockdown in vivo. **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-CLEC4D antibody