Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19 associates with CD5L to form a putative heterodimer of p19/CD5L. ( a ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC and cultured for 3 days, and total cell lysates or culture supernatants were immunoprecipitated with anti-FLAG or anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-c-MYC or biotin-conjugated anti-p19, respectively. Immunoprecipitated protein was confirmed by western blotting with the antibody used for immunoprecipitation. ( b ) HEK293T cells were transiently transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, or both and p3 × FLAG-CMV-14-Hyper-p19/CD5L, then cultured for 3 days. The culture supernatants were then subjected to p19/CD5L-specific ELISA in triplicate. Data are shown as the mean ± SD. ( c ) Naive CD4 + T cells were stimulated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) under pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 8 times, and subjected to immunoprecipitation with anti-CD5L followed by western blotting with biotin-conjugated anti-p19 and subsequently anti-CD5L. ( d ) Naive CD4 + T cells were also similarly stimulated with plate-coated anti-CD3 and anti-CD28 under Th, Th0 and pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 10 times, followed by p19/CD5L-specific ELISA in triplicate. ( e ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 WT or p3 × FLAG-CMV-14-p19 C55S and pCMV3-CD5L-c-MYC and cultured for 3 days. The culture supernatants were immunoprecipitated with anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-p19, then anti-c-MYC. Data are shown as the mean ± SD and representative of four ( a ) or two ( b – e ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05.

Article Snippet: The concentration was determined using CD5L as a standard in ELISA kit for CD5L (Sino Biological).

Techniques: Cell Culture, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD5L-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. ( a – d ) WT mice or CD5L-deficient mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 15, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). Mononuclear cells were also isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF, and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Blood was also taken over time and serum levels of p19/CD5L and CD5L were determined by ELISA ( e , f ). Data are shown as mean ± SD (n = 4–6) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test ( a , d ) or one-way ANOVA ( e , f ). * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: The concentration was determined using CD5L as a standard in ELISA kit for CD5L (Sino Biological).

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: Differentiation into GM-CSF-producing CD4 + T cells is impaired in CD5L-deficient CD4 + T cells in vitro. Naive CD4 + T cells from WT mice or CD5L-deficient mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 4 days under various Th-polarizing conditions; Th, Th0, Th1, ThGM, non-pathogenic Th17, and pathogenic Th17. These cells were then restimulated with PMA and ionomycin, and the intracellular cytokine staining was performed. Representative dot plots for GM-CSF, IL-17A, IFN-γ, and IL-10 in CD4 + T cells are shown ( a ), and average frequencies of respective CD4 + T cells were calculated and compared ( b ). Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The concentration was determined using CD5L as a standard in ELISA kit for CD5L (Sino Biological).

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone efficiently induces cell proliferation, STAT5 phosphorylation. ( a , b ) HEK293T cells were transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, p3 × FLAG-CMV-14-hyper-p19/CD5L, and p3 × FLAG-CMV-13-hyper-p40/CD5L. The cells were also cotransfected with expression vectors of p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC, together with those of p3 × FLAG-CMV-14-p19 and p3 × FLAG-CMV-14-p40, which produced IL-23 as the positive control. The total amount of DNA in each transfection sample was adjusted to be kept equal with the empty vector. Three days later, culture supernatants were collected, and 10% or 30% of them were used for stimulation of Ba/F3 cells expressing gp130/IL-12Rβ1/IL-12Rβ2/IL-23Rα. For p19 + p40 (IL-23), only 10% culture supernatant was added; therefore, “ – ” means that no data exist for the 30% lane ( a ). Ba/F3 cells were also stimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L (all 20 ng/ml), and hyper-p19/CD5L (0.1 – 20 ng/ml) ( b ). Proliferative activity of these cells was determined by measuring 3 H-thymidine incorporated into the DNA. ( c , d ) Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 3 days under Th conditions, washed, and rested in 10% FBS medium for 6 h. These cells were unrestimulated ( – ) or then restimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L, hyper-p19/CD5L, IL-27 (positive control for phosphorylation of STAT1 and STAT3, all 20 ng/ml), or IL-2 (positive control for phosphorylation of STAT5, 100 U/ml) for 5, 15 and 60 min, and subjected to western blotting with anti-pY-STATs and subsequently anti-total STATs ( c ). FACS analysis was also performed using anti-pY-STAT5 and its control antibody after stimulation for 20 min ( d ). Data are shown as the mean ± SD in triplicate and are representative of three ( a , c , d ) or two ( b ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The concentration was determined using CD5L as a standard in ELISA kit for CD5L (Sino Biological).

Techniques: Transfection, Plasmid Preparation, Expressing, Produced, Positive Control, Purification, Recombinant, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone induces augmentation of GM-CSF expression. Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence of recombinant p19, CD5L (all 20 ng/ml), and hyper-p19/CD5L (2–20 ng/ml) under Th conditions for 3 days, and subjected to intracellular staining of GM-CSF and IFN-γ ( a , b ). Representative dot plots were shown ( a ). Culture supernatants were analyzed for GM-CSF by ELISA ( c ). Similarly, naive CD4 + T cells from CD5L-deficient mice were stimulated and subjected to intracellular staining of GM-CSF and IFN-γ ( d , e ). Representative dot plots were shown ( d ). Culture supernatants were analyzed for GM-CSF by ELISA ( f ). Data are shown as mean ± SD in triplicates and are representative of more than two independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The concentration was determined using CD5L as a standard in ELISA kit for CD5L (Sino Biological).

Techniques: Expressing, Recombinant, Staining, Enzyme-linked Immunosorbent Assay