Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: WT and CD5L − mice were subjected to cecal ligation and puncture (CLP) surgery to induce moderate disease severity. a Kaplan–Meier survival curves were generated to compare survival between the two groups and significance was determined by log-rank (Mantel-Cox) test. Graphical representation of pooled individuals ( n = 15 in each group) from 5 independent experiments. b Quantification of body weight loss in the experimental setting described in a . Data are presented as mean values ± SEM. c – e WT and CD5L − mice were subjected to mid-grade CLP; control mice (sham) underwent the same surgical procedure but without ligation and puncture of the cecum. Mice were sacrificed 6, 24, or 72 h after surgery. Pooled data from at least 2 independent experiments. c CFU counts of the indicated tissues from WT and CD5L − mice after mid-grade CLP. Statistical differences between groups were analyzed by two-tailed Mann-Whitney test. DL, detection limit = 20 CFU. Mice per group at 6, 24, and 72 h: Peritoneum WT: 7, 6, and 7; peritoneum CD5L − : 6, 6, and 8. Blood WT: 7, 6, and 7; blood CD5L − : 8, 6, and 8. Lung, liver, and kidney WT: 7, 7, and 7; lung, liver and kidney CD5L − : 7, 6, and 8. d Absolute number of total CD45 + leukocytes, CD45 + CD11b + Ly6G + neutrophils, CD45 + CD11b + CD11c - F4/80 + macrophages and Ly6C + and CD206 + subsets within macrophage population, assessed by flow cytometry of cell populations in the peritoneal cavity. Statistical comparisons were drawn after performing two-tailed unpaired t-tests with Welch’s correction. Mice per group at 6, 24, and 72 h: Sham, WT, and CD5L − : 4, 4, and 4. CLP WT: 7, 7, and 7; CLP CD5L − : 7, 6, and 8. Floating bars show the minimum, average (line), and maximum values within each group ( c-d ). e Fold change in the indicated cytokines between CD5L − and WT mice, quantified by bead-based multiplex immunoassay in samples from the peritoneal cavity (left panel) and blood serum (right panel).

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Ligation, Generated, Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Multiplex Assay

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Principal component analysis (PCA) of RNA-seq expression data for the 4 groups of peritoneal cells [WT and CD5L − mice, either naive (0 h) or 6 h after CLP; n = 3]. b Volcano plot depicting differentially expressed genes in CD5L − vs. WT mice, 6 h after CLP. Red dots represent genes expressed at higher levels in CD5L − mice, while black dots represent genes with higher expression levels in WT controls. Grey dots represent genes bellow the cutoff of significant (|log 2 fold change| ≥ 0.5 and adjusted P values ≤ 0.05). Differential expression was evaluated using the Wald’s test, followed by adjustment for multiple testing with the Benjamini-Hochberg correction. Log 2 -fold change values were shrunk with the apeglm method to increase the signal-over-noise ratio of the effect size. c Dot plot of gene set enrichment analysis (mouse hallmark gene set collection) for CD5L − vs. WT mice, 6 h after CLP. The diameter of the dot indicates the degree of significance of the ontology term. Red dots represent terms enriched in CD5L − mice, while black dots represent terms enriched in WT mice. d Heatmap of the relative expression values (z-score of each gene across samples) of the top 7 upregulated pathways. Only differentially expressed genes were represented, excluding genes belonging to more than one pathway.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of cytokines, CFUs and neutrophil recruitment for a perplexity of 8. Each ellipse describes a phenotypic subtype and are derived from 36 different measurements (seven for WT, five for IP-treated, and six for the remaining groups). Measurements were log 2 transformed and normalized by the mean value of the “untreated” or the CD5L − subgroups for each setup [intravenous (IV); intraperitoneal (IP) or WT/CD5L − ]. b Linear discriminant analysis (LDA) biplot showing the overall profile of treatment of CLP with rCD5L administered IV or IP, and mid-grade CLP in WT or CD5L − mice. Ellipses show 95% confidence intervals for each treatment and vectors represent the contribution of each variable to the overall variance.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Derivative Assay, Transformation Assay

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Normalized counts of Cd5l transcripts obtained from RNA-seq analysis of peritoneal cells of WT mice at indicated times after CLP. Mice per group: 3. b RT-qPCR quantification of Cd5l expression, normalized with Hprt1 , in peritoneal cells and liver tissue at indicated times after CLP. Mice per group: 3. c ELISA quantification of CD5L in peritoneum and blood of WT mice at indicated times after mid-grade CLP, or in sham operated animals. Mice per group: at 0 h: 8 in peritoneum, 9 in blood; CLP at 6, 24, and 72 h (peritoneum and blood): 7, 9, and 7; Sham at 6, 24, and 72 h (peritoneum and blood): 6, 6, and 4. d CD5L − mice were subjected to mid-grade CLP and 3 h later injected IV with 2.5 mg/kg rCD5L, or PBS. Fluids from peritoneum and serum were collected 1 h later and total CD5L was quantified by ELISA. DL, detection limit: 6.25 pg/ml. Mice per group: 4 for PBS; 5 for rCD5L. Pooled data from at least 2 independent experiments ( c , d ). e Scatterplot illustrates the correlation between peritoneal and blood rCD5L levels following IV injection, accompanied by Spearman’s rank correlation coefficient and the two-tailed P value ( n = 5). Statistical differences between groups analyzed by one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), two-tailed unpaired t-tests with Welch’s correction ( c ), or two-tailed Mann-Whitney test ( d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Injection, IV Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Determination of IgM-bound and free endogenous CD5L in peritoneal fluid and serum of WT mice at specified time points after CLP. Values were obtained through densitometric analysis of western blots normalized to baseline IgM-bound CD5L levels (100) in naïve mice. Each group comprised six mice. b ELISA quantification of total IgM in the peritoneum and serum of WT mice at specified times after CLP. Mice per group at 0, 6, 24, and 72 h were as follows: Peritoneum: 7, 9, 7, and 7; Blood: 8, 9, 6, and 7, respectively. c ELISA quantification of total CD5L in the peritoneal cavity of mice subjected to high-grade CLP, and IP- or IV-treated with rCD5L. At 6 h, mice had received one dose of rCD5L (2.5 mg/kg), or PBS, 3 h after CLP. At 24 h, mice had received 2 doses of rCD5L, or PBS, 3 and 6 h after CLP. Mice per group: IP treatment: 6 untreated and 6 treated, at 6 h; 7 untreated and 5 treated, at 24 h. IV treatment: 8 untreated and 6 treated, at 6 h; 8 untreated and 6 treated, at 24 h. d Determination of IgM-bound and free total (endogenous + recombinant) CD5L in the peritoneal cavity of WT mice treated with 2.5 mg/kg rCD5L, or PBS (untreated), via IP or IV routes, 3 h after high-grade CLP, and euthanized 3 h later. Values were obtained by densitometric analysis of western blot bands corresponding to IgM-bound and free CD5L. Mice per group: IP treatment: 6 untreated and 5 treated. IV treatment: 5 untreated and 6 treated. Pooled data from at least 2 independent experiments ( a – d ). Statistical differences between groups analyzed by two-tailed unpaired t-tests with Welch’s correction ( a ), one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), or two-tailed Mann-Whitney test ( c , d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a ELISA quantification of LPS in sera from WT and CD5L − mice 72 h after mid-grade CLP; and in sera from WT mice subjected to high-grade CLP followed by rCD5L treatment. In these, mice were IP- or IV-injected with PBS (untreated), or with two doses of 2.5 mg/kg rCD5L, 3 and 6 h after surgery. Sera was collected and analyzed at 24 h. DL, detection limit: 0.16 ng/ml. The experimental groups comprised 7 WT and 6 CD5L − mice for mid-grade CLP, and for high-grade CLP: 7 IP-untreated, 5 IP-treated, 8 IV-untreated, and 6 IV-treated mice. b , c HMGB1 protein expression in WT and CD5L − organs, determined by immunofluorescence. b Representative images of HMGB1 expression in paraffin-preserved sections of lung, liver, and kidney of WT and CD5L − mice. c Quantification of HMGB1-stained areas in 3 regions from 2 independent images, from 2 mice per CLP group (72 h), or from 1 naïve control mouse per group (0 h), normalized by the DAPI-stained area in the corresponding regions. d , e HMGB1 expression in organs of WT mice subjected to high-grade CLP, and IP- or IV-treated with two doses of rCD5L. d Representative images of HMGB1 expression in lung, liver, and kidney of treated and untreated mice. e Quantification of HMGB1-stained areas, as in ( c ). a , c , e Floating bars represent the minimum, average and maximum values within each group. Statistical differences between groups were analyzed using a two-tailed Mann-Whitney test. b , d Scale bar: 20 μm. f WT and CD5L − mice were IP-injected with a sublethal LPS dose (1.5 mg/kg). Survival was monitored for 6 days. g WT mice were injected with a lethal LPS dose (10 mg/kg), and 3 h later with 2.5 or 5 mg/kg rCD5L, or left untreated (0). Survival was monitored for 4 days. f , g Kaplan–Meier curves were generated to compare survival between groups. Significance was determined by log-rank (Mantel-Cox) test. The graphical representation includes pooled data from 3 independent experiments for ( f ) and 2 independent experiments for g .

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Expressing, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Generated

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Quantification of inflammatory chemokines in WT and CD5L − mice at indicated time points after mid-grade CLP, using bead-based multiplex immunoassays. b WT mice subjected to high-grade CLP were IV-injected with 2.5 mg/kg of rCD5L, or PBS (untreated), 3 h later. Inflammatory chemokines were quantified 6 h post-CLP. c CXCL1 concentrations in WT and CD5L − mice after mid-grade CLP (extracted from data presented in a ). d CXCL1 concentrations in untreated and IV-treated WT mice after high-grade CLP (extracted from data presented in b ). a – d Pooled data from at least 2 independent experiments, analyzed by two-tailed Mann-Whitney test. a , c Mice per group: Peritoneum: 4 WT and 4 CD5L − at 0 h, 4 WT, and 3 CD5L − at 3 h, 6 WT, and 6 CD5L − at 6 h; Blood, 9 WT and 9 CD5L − at 0 h, 3 WT, and 3 CD5L − at 3 h, 8 WT and 5 CD5L − at 6 h. b , d Mice per group: 6 in untreated, 5 in rCD5L IV-treated. e CD5L − mice were injected IV with rCD5L (2.5 mg/kg) 3 h after mid-grade CLP and peritoneal cells were recovered 3 h later. Percentage of CD5L-bound cells within CD45 - cells; neutrophils (CD45 + CD11b + Ly6G + ), macrophages (CD45 + CD11b + F4/80 + ), other CD11b + (CD45 + CD11b + F4/80 - Ly6G - ), B cells (CD45 + B220 + ), and T cells (CD45 + CD3 + ). f MFI values of the CXCL1 channel within CXCL1 + cells in each subset defined in e . e , f Pooled data from 2 independent experiments, analyzed by two-way ANOVA. Mice per group: 5 untreated and 6 rCD5L-treated. Ly6G and CD11b MFI in neutrophils collected from the peritoneal cavity of rCD5L IP- (left panel) or IV-treated (right panel) WT mice, after high-grade CLP ( g ), or WT vs. CD5L − mice after mid-grade CLP ( h ). Mice per group: IP treatment: 3 at 6 h, 4 at 24 h; IV treatment: 6 at 6 h, 6 at 24 h ( g ); IP and IV treatment: 4 ( h ). Pooled data from 2 independent experiments, statistical comparisons between groups were established by two-tailed unpaired t-tests with Welch’s correction.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Multiplex Assay, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: Intravenous administration of rCD5L leads to elevated levels of free bioactive protein in the peritoneal cavity (1). Upon binding to target cells of non-hematopoietic origin (2), rCD5L induces significant production of the CXCL1 chemokine (3). The production of CXCL1 establishes a chemotactic gradient, resulting in heightened neutrophil activation (4) and recruitment from the bloodstream (5). The exact mechanism behind neutrophil activation, whether through increased CXCL1 levels or direct action of rCD5L, remains unclear. Increased neutrophil presence in the peritoneal cavity, coupled with the synergistic effect of rCD5L in bacterial binding and enhanced phagocytosis, facilitates efficient bacterial clearance (6). rCD5L aids in the effective removal of DAMPs, likely from both the bloodstream and the peritoneum (7). Endogenous local release of CD5L from resident macrophages contributes to the overall pool of free CD5L at the site of infection. This illustration is for explanatory purposes and is not drawn to scale. Created with Biorender.com.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Binding Assay, Activation Assay, Infection

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: Mice were immunized with chicken collagen on day 0 followed by a boost administration of the antigen on day 21. A , Incidence rates of WT and CD5L − animals were compared using the Chi 2 -statistic as detailed in table 3, * p < 0.05. B , Macroscopic assessment of arthritis was performed by scoring the swelling of each paw as follows: 0 = normal, 1 = mild swelling and/or erythema, 2 = pronounced swelling, 3 = deformity and 4 = ankylosis. RA severity score of each mouse was obtained by adding the score of the four paws. C , Weight variation after arthritis induction. Data represent weight variation only in mice that developed arthritis, shown as mean ± SEM (n = 10 WT; n = 20 CD5L⁻), pooled from five independent experiments. B, Pearson correlation between weight variation and disease score from WT (open circles) and CD5L⁻mice (filled circles).

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques:

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: Mice were immunized with chicken collagen on day 0 followed by a boost administration of the antigen on day 21. A , Quantification of CD5L by ELISA in the serum of mice after immunization in the indicated time points. Statistical comparisons were made using Kruskal-Wallis test with Dunn’s multiple comparisons. B, The frequency of the indicated populations was analyzed in different time points after immunization in the blood of WT and CD5L − mice by flow cytometry (frequencies within total live cells). Markers used for phenotyping: monocytes (Siglec-F − Ly6G − F4/80 + CD11b + ), inflammatory monocytes (Siglec-F − Ly6G − F4/80 + CD11b + Ly6C + ) and Ly6C − monocytes (Siglec-F − Ly6G − F4/80 + CD11b + Ly6C − ). Data shown are mean with SEM of n = 10 (WT) and n= 10 (CD5L − ) mice. Statistical comparisons were made using Šídák’s multiple comparisons test. C , The concentration of the indicated cytokines was analyzed in different time points after immunization in the sera of WT and CD5L − mice by ELISA. Statistical differences between groups were analyzed by Mann-Whitney test. The concentration of RANKL and cross-linked C-telopeptide of type I collagen (CTX-I) ( D ) was analyzed 56 days after immunization in the sera of WT and CD5L − mice by ELISA. E , RT-qPCR quantification of the indicated genes expression, normalized with Actb (β-actin), in total spleen cells from WT or CD5L − naïve mice. Mice per group: 5. Statistical differences between groups analyzed by two-tailed unpaired t-test with Welch’s correction. Data was pooled from 2 independent experiments except in c) where one representative experiment from two is depicted. Graphical representation of median and 25 th – 75 th quartiles in A ), C ), D ), E ). * p < 0.05, ** p < 0.01, ns: not significant.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay, MANN-WHITNEY, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: A , Representative histograms showing Pacific Blue-CD11b and APC-CD29 expression in human CD11b⁺CD14⁺CD4⁻ peripheral blood leukocytes stimulated with LPS and ConA for 48 h, with the addition of human recombinant CD5L (1 μg/ml) or recombinant CD5L plus human IgG (equimolar) during the final 24 h. Expression was assessed by flow cytometry. Box plots depict mean fluorescence intensity (MFI) of CD11b and CD29 in cultured cells (n = 8). P values were calculated using a paired Wilcoxon test. B , Column plots of CD16 expression on intermediate (IM, CD14 + CD16 + ) and non-classical (NCM, CD14 low CD16 + ) monocyte subsets. C , Protein levels of IL-10, PDL1, IFN-ψ and GAS6 in the supernatants of cell cultures. D , Heat maps showing Spearman correlation (π) values between IL-10 concentrations in culture supernatants and the expression intensity of CD11b and CD16, as measured by flow cytometry. Statistical significance is indicated as * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Expressing, Recombinant, Flow Cytometry, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: A , Box plots showing CD5L, IL-10, PD-L1, IFN-ψ, and GAS6 protein levels in supernatants from human peripheral blood leukocyte cultures (n = 14) stimulated with LPS and ConA (5 μg/mL and 0.625 μg/m, respectively) for 48 h, with the addition of a histone deacetylase inhibitor (HDACi, 50 μg/ml) during the final 24 h. B , Heat maps displaying Spearman correlation (π) values between CD5L and IL-10 levels in supernatants, CD16 expression intensity measured by flow cytometry, IFN-γ production, the relative size of the non-classical monocyte (NCM) subset, and the mean fluorescence intensity (MFI) of CD16 and CD11b. Statistical significance is indicated as * p < 0.05; ** p < 0.01; *** p < 0.001. C, Box plots illustrating changes in classical (CM; CD14⁺CD16⁻), intermediate (IM; CD14⁺CD16⁺), and non-classical (NCM; CD14 low CD16⁺) monocyte subsets in stimulated human leukocyte cultures (n = 14), treated as described in panel ( A ). D, Representative histograms of Pacific Blue-CD11b expression in human CD11b⁺CD14⁺CD4⁻ blood leukocytes stimulated as indicated and analyzed by flow cytometry. Box plots summarize CD11b mean fluorescence intensity (MFI) across conditions. P values were calculated using a paired Wilcoxon test.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Histone Deacetylase Assay, Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: CD14⁺ cells were isolated from peripheral blood leukocyte cultures of 35 RA patients, and CD5L levels in culture supernatants were measured by ELISA. Cultures with CD5L protein levels above 0.6 pg/mL (double detection limit) were classified as CD5L hi , and the remaining as CD5L low . A, Box plots showing protein levels of CD5L, IL-10, and PD-L1 in supernatants, as well as patient white blood cell (WBC) and platelet counts, and serum IFN-ψ levels, comparing CD5L hi (n = 10) and CD5L low (n = 25) CD14⁺ cells. B, Box plots of normalized gene expression levels in CD14⁺ cells measured by RNA-seq. P values were calculated using DESeq2; nominal p values are indicated.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Gene Expression, RNA Sequencing

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: CD14⁺ cells were isolated from peripheral blood leukocyte cultures of 35 RA patients, activated with LPS for 2 h, and subjected to RNA sequencing (RNA-seq, Illumina). A, Bubble plot showing mean normalized expression of gene markers for four monocyte clusters identified in human blood leukocytes. B, Bar plot representing the distribution of monocyte clusters based on the sum of cluster-specific gene expression. C, Heat map of gene expression changes (log₂ fold change) in non-classical and IFN-primed monocyte clusters in regression to serum CD5L levels and joint skeletal damage quantified by vdH-Sharp score. P values were calculated using DESeq2; nominal P values are indicated as * p < 0.05; ** p < 0.01; *** p < 0.001. D, Heat map of gene expression changes (log₂ fold change, FC) for efferocytosis markers (1), resolution mediators (2), and immunoregulatory macrophage program genes (3) in regression to serum CD5L levels and vdH-Sharp score. E, Schematic representation of a monocyte conditioned by CD5L.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Isolation, RNA Sequencing, Expressing, Gene Expression

Journal: bioRxiv

Article Title: CD5L insufficiency exacerbates skeletal joint damage in rheumatoid arthritis

doi: 10.64898/2025.12.16.694616

Figure Lengend Snippet: A , Box plots of CD5L protein levels in serum of RA patients and healthy controls (1), paired serum and synovial fluid samples from RA patients (2), and anti-CD5L antibodies in serum of RA patients and controls (3). B, Heat map of Spearman correlations (ρ) between serum CD5L levels or vdH-Sharp joint damage scores and serum cytokine and growth factor levels. Statistical significance is indicated as * p < 0.05; ** p < 0.01; *** p < 0.001. C, Dot plots showing correlations between serum CD5L levels and radiographic joint damage in 80 RA patients (1), and correlation of a CD5L-dependent gene signature in non-classical monocytes with vdH-Sharp score (2). Enrichment with osteoclast-related markers enhanced the correlation, whereas inclusion of IFN-sensitive genes abolished it. D, Bubble plot of the percentage frequency of CD5L⁺ cells within synovial myeloid clusters. E, UMAP of the scaled expression intensity sum of the CD5L-dependent signature in synovial myeloid cells from single-cell transcriptomics. Further enrichment distinguishes IFN-primed versus osteoclast-fostering macrophages. F, Heat map of a CD5L-dependent osteoclastogenic signature in blood CD14⁺ cells, showing a positive correlation with vdH-Sharp joint damage scores.

Article Snippet: CD5L serum levels were quantified using the Mouse CD5L ELISA Pair Set (Sino Biological, SEK50020), according to the manufacturers’ instructions.

Techniques: Expressing, Single-cell Transcriptomics

Journal: EBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy.

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: Fig. 1: 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

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L),27 recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 ◦C.

Techniques: Expressing, Control, Activation Assay

Journal: EBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy.

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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.

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L),27 recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 ◦C.

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy.

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: Fig. 3: 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).

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L),27 recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 ◦C.

Techniques: Direct ELISA, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy.

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L),27 recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 ◦C.

Techniques: In Vivo, Control, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: bioRxiv

Article Title: The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

doi: 10.1101/2025.10.02.680021

Figure Lengend Snippet: (A) Schematic of experimental approach for RNA sequencing of islets treated with vehicle or pro-inflammatory cytokines (PIC). (B) RNA sequencing data showing relative expression of Gpr31b in WT and KO islets treated with PIC (n=3 mice per condition). (C) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with vehicle (n=3 mice per condition). (D) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with vehicle. Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (E) Principal components analysis (PCA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). (F) Volcano plot of differentially expressed genes in WT and Gpr31b KO islets treated with PIC (n=3 mice per condition). Genes which meet the threshold of absolute fold change ≥1.5 and adjusted P-value <0.05 are in red; genes which only meet the adjusted P-value threshold are in blue. (G) Analysis of CD5L concentration by enzyme-linked immunosorbent assay (ELISA) in the supernatant of islets treated with vehicle or PIC. (H) Gene set enrichment analysis (GSEA) of WT and Gpr31b KO islets treated with PIC (n=3 mice per condition) using hallmark gene sets. Both suppressed ( top panel ) and activated gene sets ( bottom panel ) are shown. Normalized enrichment score (bars) and log-transformed P-value (blue dots). All pathways meeting significance criteria of adjusted P-value <0.05 are shown. (I) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for differentially expressed genes meeting more stringent criteria of absolute fold change ≥2 with adjusted P-value <0.05. (J) Heatmap from WT and Gpr31b KO islets treated with PIC showing Z-scores for genes in the leading edge for the ROS hallmark gene set. Data presented as mean with SEM. Statistical significance determined by Student’s t-test or paired one-way analysis of variance (Friedman ANOVA).

Article Snippet: CD5L protein levels in islet supernatants were quantified using a mouse CD5L enzyme-linked immunosorbent assay (ELISA) kit (orb565622; Biorbyt, Cambridge, UK) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transformation Assay

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Normalized counts of Cd5l transcripts obtained from RNA-seq analysis of peritoneal cells of WT mice at indicated times after CLP. Mice per group: 3. b RT-qPCR quantification of Cd5l expression, normalized with Hprt1 , in peritoneal cells and liver tissue at indicated times after CLP. Mice per group: 3. c ELISA quantification of CD5L in peritoneum and blood of WT mice at indicated times after mid-grade CLP, or in sham operated animals. Mice per group: at 0 h: 8 in peritoneum, 9 in blood; CLP at 6, 24, and 72 h (peritoneum and blood): 7, 9, and 7; Sham at 6, 24, and 72 h (peritoneum and blood): 6, 6, and 4. d CD5L − mice were subjected to mid-grade CLP and 3 h later injected IV with 2.5 mg/kg rCD5L, or PBS. Fluids from peritoneum and serum were collected 1 h later and total CD5L was quantified by ELISA. DL, detection limit: 6.25 pg/ml. Mice per group: 4 for PBS; 5 for rCD5L. Pooled data from at least 2 independent experiments ( c , d ). e Scatterplot illustrates the correlation between peritoneal and blood rCD5L levels following IV injection, accompanied by Spearman’s rank correlation coefficient and the two-tailed P value ( n = 5). Statistical differences between groups analyzed by one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), two-tailed unpaired t-tests with Welch’s correction ( c ), or two-tailed Mann-Whitney test ( d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Injection, IV Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Determination of IgM-bound and free endogenous CD5L in peritoneal fluid and serum of WT mice at specified time points after CLP. Values were obtained through densitometric analysis of western blots normalized to baseline IgM-bound CD5L levels (100) in naïve mice. Each group comprised six mice. b ELISA quantification of total IgM in the peritoneum and serum of WT mice at specified times after CLP. Mice per group at 0, 6, 24, and 72 h were as follows: Peritoneum: 7, 9, 7, and 7; Blood: 8, 9, 6, and 7, respectively. c ELISA quantification of total CD5L in the peritoneal cavity of mice subjected to high-grade CLP, and IP- or IV-treated with rCD5L. At 6 h, mice had received one dose of rCD5L (2.5 mg/kg), or PBS, 3 h after CLP. At 24 h, mice had received 2 doses of rCD5L, or PBS, 3 and 6 h after CLP. Mice per group: IP treatment: 6 untreated and 6 treated, at 6 h; 7 untreated and 5 treated, at 24 h. IV treatment: 8 untreated and 6 treated, at 6 h; 8 untreated and 6 treated, at 24 h. d Determination of IgM-bound and free total (endogenous + recombinant) CD5L in the peritoneal cavity of WT mice treated with 2.5 mg/kg rCD5L, or PBS (untreated), via IP or IV routes, 3 h after high-grade CLP, and euthanized 3 h later. Values were obtained by densitometric analysis of western blot bands corresponding to IgM-bound and free CD5L. Mice per group: IP treatment: 6 untreated and 5 treated. IV treatment: 5 untreated and 6 treated. Pooled data from at least 2 independent experiments ( a – d ). Statistical differences between groups analyzed by two-tailed unpaired t-tests with Welch’s correction ( a ), one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), or two-tailed Mann-Whitney test ( c , d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a ELISA quantification of LPS in sera from WT and CD5L − mice 72 h after mid-grade CLP; and in sera from WT mice subjected to high-grade CLP followed by rCD5L treatment. In these, mice were IP- or IV-injected with PBS (untreated), or with two doses of 2.5 mg/kg rCD5L, 3 and 6 h after surgery. Sera was collected and analyzed at 24 h. DL, detection limit: 0.16 ng/ml. The experimental groups comprised 7 WT and 6 CD5L − mice for mid-grade CLP, and for high-grade CLP: 7 IP-untreated, 5 IP-treated, 8 IV-untreated, and 6 IV-treated mice. b , c HMGB1 protein expression in WT and CD5L − organs, determined by immunofluorescence. b Representative images of HMGB1 expression in paraffin-preserved sections of lung, liver, and kidney of WT and CD5L − mice. c Quantification of HMGB1-stained areas in 3 regions from 2 independent images, from 2 mice per CLP group (72 h), or from 1 naïve control mouse per group (0 h), normalized by the DAPI-stained area in the corresponding regions. d , e HMGB1 expression in organs of WT mice subjected to high-grade CLP, and IP- or IV-treated with two doses of rCD5L. d Representative images of HMGB1 expression in lung, liver, and kidney of treated and untreated mice. e Quantification of HMGB1-stained areas, as in ( c ). a , c , e Floating bars represent the minimum, average and maximum values within each group. Statistical differences between groups were analyzed using a two-tailed Mann-Whitney test. b , d Scale bar: 20 μm. f WT and CD5L − mice were IP-injected with a sublethal LPS dose (1.5 mg/kg). Survival was monitored for 6 days. g WT mice were injected with a lethal LPS dose (10 mg/kg), and 3 h later with 2.5 or 5 mg/kg rCD5L, or left untreated (0). Survival was monitored for 4 days. f , g Kaplan–Meier curves were generated to compare survival between groups. Significance was determined by log-rank (Mantel-Cox) test. The graphical representation includes pooled data from 3 independent experiments for ( f ) and 2 independent experiments for g .

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Expressing, Immunofluorescence, Staining, Control, Two Tailed Test, MANN-WHITNEY, Generated

Journal: eBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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).

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L), recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 °C.

Techniques: Expressing, Activation Assay, Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Staining, Fluorescence, Software, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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.

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L), recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY, Immunofluorescence

Journal: eBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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).

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L), recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 °C.

Techniques: Direct ELISA, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Macrophage CD5L is a target for cancer immunotherapy

doi: 10.1016/j.ebiom.2023.104555

Figure Lengend Snippet: 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 mm 3 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.

Article Snippet: Direct binding enzyme-linked immunosorbent assay (ELISA) was performed by immobilizing 5 μg/ml of recombinant human CD5L (rhCD5L), recombinant mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, Minneapolis, MN, USA), human DMBT1 (kindly provided by Dr. Martina Tuttolomondo), and human recombinant CD5 (1636-CD-050, R&D systems) in 96-well microtiter plates, overnight at 4 °C.

Techniques: In Vivo, Immunofluorescence, Expressing, MANN-WHITNEY, Immunohistochemistry, Staining, Software