Fig 2: Identification of TRIM21 as a ligand of CLEC-1.(A) Representative affinity chromatography elution products (SDS–polyacrylamide gel electrophoresis) after co-IP of UV-treated Raji or Hpb-all whole-cell extract (WCE) with protein G beads coupled to human Fc CLEC-1 or Fc alone (control) (three independent experiments). Arrowheads at 50 kDa indicate a band only present under the Fc CLEC-1 condition. MW, molecular weight. (B) LC-MS analysis (of the 50-kDa gel band) depicting the top hits of Fc CLEC-1 over the Fc alone control condition (n = 4, mean). (C) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji WCE (or without cells as control) with protein G beads coupled to human Fc CLEC-1 or irrelevant Fc CLEC7A (three independent experiments). Arrowheads correspond to TRIM21 detected in the co-IP in the retained and unretained fractions. (D) Representative Western blot of human CLEC-1 in elution product after co-IP of UV-treated Raji WCE and His–CLEC-1 with protein G beads coupled to antihuman TRIM21 mAb and revealed with either anti-His or anti–CLEC-1 mAb (three independent experiments). (E) Representative Western blot of human TRIM21 after co-IP of UV-treated Raji transduced with lentiviral TRIM21 (sh+) or control scrambled shRNA with protein G beads coupled to human Fc CLEC-1 (three independent experiments). (F) Flow cytometry analysis of human Fc CLEC-1 binding on UV-treated Raji transduced with lentiviral TRIM21 or control scrambled shRNA (Ctrl) (data expressed as fold change of binding of Fc CLEC-1 compared to binding of Fc alone) (n = 5, paired t test, *P < 0.05). (G) Sensograms generated by Biacore-based affinity measurement representing interaction of chip-immobilized recombinant human TRIM21 with five increasing concentrations of human Fc CLEC-1 (Fc alone as control) or His–CLEC-1 [irrelevant (irr) protein as control]. (H) Representative ELISA [density of optical (DO)] of recombinant human TRIM21 on coated human Fc CLEC-1 or Fc alone (n = 4, means ± SEM, unpaired t test, **P < 0.01 and ***P < 0.001).

Fig 3: Treatment with antihuman CLEC-1 antagonist mAb enhances antitumor immunity in CLEC-1 humanized mice.(A) CLEC1A or Clec1a mRNA expression in different subsets of myeloid cells from human or mouse lung tumors from Open-source data of scRNA-seq. AU, arbitrary units. (B) Two-dimensional Uniform Manifold Approximation and Projection and violin plot representations of CLEC1A expression (red scale projection) among clusters of myeloid cells and DCs from scRNA-seq dataset of pan-cancer (breast, colorectal, lung, and ovarian) TME. (C) Box plots representation of log2 [transcript count per million (TMP)+1] values for TRIM21 expression in different cancer types from TCGA analyzed by GEPIA tool. BRCA, breast-invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower-grade glioma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; READ, rectum adenocarcinoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors. Unpaired t test, *P < 0.05. (D and E) Tumor growth or survival curve of CLEC-1 humanized mice with subcutaneous MC38 adenocarcinoma in combination with CPA (150 mg/kg) (D) or with Hepa 1.6 LIHC (E) and treated with irrelevant isotype control or antihuman CLEC-1 antagonist mAbs #1 or #2 (100 µg per mouse) twice a week from days 4 to 21 (n = 14 to 17 of two independent experiments, unpaired t test, *P < 0.05 for tumor growth, and log-rank test, *P < 0.05 and **P < 0.01 for survival). (F) Percentage of PMNs/MDSCs (CD11b+Ly6G+MHC-II-), macrophages (CD11b+F4/80+Ly6C+), DCs (CD11c+MHC-II+), CD69+or Tmem (CD44+) CD8+, or CD4+ (CD3+CD4/8+) T cells evaluated by flow cytometry at day 13 after LIHC in the tumor-burdened livers of CLEC-1 humanized mice treated with isotype control or antihuman CLEC-1 antagonist mAbs #4 (100 µg per mouse) (data are expressed in the percentage of CD45+ or in MFI of MHC-II expression) (n = 8 to 13, means ± SEM of three independent experiments, unpaired t test *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig 4: CLEC-1 expression of in a mouse model of A. fumigatus keratitis. Typical corneal photos of A. fumigatus keratitis model in mice were prepared by intrastromal injection (1 × 105 conidia/cornea) at 0, 1 / 2, 1, 2, 3, 5, 7, 10, and 14 days after infection (A). Clinical scores were recorded (B). Corneas were excised from euthanized mice (6 in each group) at the corresponding time, and qRT-PCR (C) and Western blot (D) were performed to detect the expression of CLEC-1 and Dectin-1. The expression of CLEC-1 protein in the cornea of the control group and A. fumigatus keratitis mice models was observed by immunofluorescence staining (E, F) with 7 µm frozen sections. Red represents CLEC-1, and blue represents DAPI (A, epithelium; B, stroma; and C, endothelium). Magnification × 200.

Fig 5: CLEC-1 overexpression suppresses Dectin-1 and IL-1ß expression in response to A. fumigatus infection. The mice were randomly divided into four groups: control group, A. fumigatus infection group, CLEC-1 overexpression group, and CLEC-1 over-expression with A. fumigatus infection group. The qRT-PCR (A, B) and Western blot (C) were performed to detect the expression of CLEC-1 and IL-1ß. THP-1 macrophages were stimulated with A. fumigatus conidia (MOI = 1) for 16 hours after CLEC-1 overexpression. Cells were then subjected to RT-PCR (D, E) and Western blot (F) for CLEC-1 and IL-1ß.