Fig 1: RImAb specifically binds to human and mouse CD5L and reverts the polarization induced by IL10. a) Direct ELISA of RImAb to hDMBT1, rhCD5, rmCD5L, rhCD5L, or BSA. A representative experiment of three performed is shown. b) RT-qPCR quantification of mRNA expression of CD80, TNFA, CD163, VEGF, MERTK, and CD5L in PB monocytes treated, when indicated, with 5 µg/ml of RImAb for 45 min before the addition of IL10 (50 ng/ml) for 24 h. mRNA levels relative to GAPDH, and fold induction levels were calculated using the expression of each gene in IL10-stimulated macrophages for each donor as a reference. Data are represented as mean ± SEM (n = 5 to 9). Significance was calculated using the Mann–Whitney t-test (*p = 0.05).
Fig 2: CD5L expression by TAMs is associated with poor prognosis in papillary lung adenocarcinoma. a) Clinical characteristics of patients diagnosed with Papillary Adenocarcinoma (PAC) who participated in this study. b) Representative immunohistochemistry images showing CD5L expression in early (I) and advanced (II–III) stages of papillary lung adenocarcinoma. Scale bar represents 10 µm. c) Graph shows the number of CD5L+ macrophages per field in early (I, n = 35) and advanced (II–III, n = 20) stages. Data are presented as the mean ± SEM, *p < 0.01 determined by the Mann–Whitney t-test. d) Kaplan–Meier analysis of recurrence-free survival in cases with lower and higher TAM CD5L expression. The mean number of CD5L+ macrophages from stage I was taken as the limit value, *p < 0.01 determined by the Log-rank (Mantel–Cox) test. e) Representative immunofluorescence image depicting CD68+ (red) (i) and CD5L+ (green) macrophages, and their co-expression (orange), (ii). Scale bar represents 25 µm.
Fig 3: Lung and liver cancer cell-conditioned media (CM) induce an IL10-like phenotype and CD5L expression in macrophages.a) Principal Component Analysis (PCA) scatterplot of PB monocytes treated for 72 h with medium alone (control), reference activation stimuli (IFN/LPS, IL4, and IL10), or cancer cell-CM. Projection based on the expression profile of surface markers characterized by multicolor flow cytometry. PC1 and PC2 represented in each axis depict the first and second principal components, respectively. b) Expression of CD80, CD163, CD206, VEGF, MERTK and c)CD5L mRNA was assessed by RT-qPCR in PB monocytes treated for 24 h with the indicated stimuli. mRNA levels normalized to GAPDH, and fold induction levels were calculated using the average expression of each gene in control macrophages as a reference. Data are represented as mean ± SEM (n = 5 to 9). d) CD5L immunofluorescence staining (green) in macrophages treated with the indicated stimuli for 72 h. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar represents 20 µm. CD5L mean fluorescence intensity (MFI) was calculated with ZenLite software and is represented as MFI ± SEM of 50 macrophages scored in random fields (right) (n = 3). Significance was calculated using the Mann–Whitney t-test (*p = 0.05, **p = 0.01, ***p = 0.001).
Fig 4: Blockade of CD5L slows tumor growth in vivo and reprograms TAMs towards an antitumor profile. a) Study design and timeline for mouse model. b) LLC tumor growth in mm3 in mice treated with control (PBS) or the anti-CD5L RImAb. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the two-way repeated measures ANOVA test. c) Left: Immunofluorescence demonstrating CD5L expression (red) by F4/80+ TAMs (green) in control (PBS) and RImAb-treated mice. Nuclei were counterstained with Hoechst 33258 (blue). Scale bars represent 25 µm. Right: graph representing the number of F4/80+ TAMs and F4/80+ TAMs expressing CD5L per field in control and RImAb-treated animals. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test. d) Immunohistochemistry depicting expression of F4/80, iNOS, and Arg-1 in tumor samples from control (PBS) and RImAb-treated mice. Scale bar represents 10 µm. Average stained area obtained from five random areas was calculated using Image J (color deconvolution) software. Graphs illustrate the average stained area for F4/80, iNOS, Arg-1 and ratio of iNOS/Arg-1 in control (PBS) and RImAb-treated mice. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test.
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