Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) LAIR1 expression in total B cells (left) and the compositions of subsets in LAIR+ and LAIR1-B cells (right) (N=10). (B-D) LAIR1 expression in major B cell subsets (B; N=10), the different class of surface Ig (C; N=5) and major B cell subsets (D; N=9). (E) heatmap and volcano plot generated from RNA-seq data between LAIR1+ and LAIR1- SWM B cells (N=4). Differential gene expression was identified with Log 2 FC >0.5 and adjusted p value <0.05. Upregulated genes in LAIR1- are 278 genes and down-regulated genes in LAIR1- are 218 genes. (F) the potential of PC differentiation induced by TLR7 or TLR9 signaling. (G) the percentages of ANA+ autoreactive B cells in LAIR1+ and LAIR1- memory fraction (N=7). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (F and G) and further adjusted for multiple comparisons using the Benjamini-Hochberg method (B, C and D). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) Phosphorylated LAIR1 induced by its crosslinking with biotinylated anti-LAIR1 antibody and streptavidin (N=5). (B-D) The effects of co-crosslinking with BCR and LAIR1 in naïve B cells (B; N=5), IgM+ USWM B cells (C; N=5) and IgG+ SWM B cells (D; N=5). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests. Asterisks indicate significant differences (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques:

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A-B) Representative examples of LAIR1 expression in 7 subsets of B cells (A) and activated naïve B cells and DN2 B cells (B) from HD and SLE patients (each N=9). (C) The positivity of LAIR1 in ANA+ and ANA- B cells. (D) The expression level of LAIR1 in LAIR1 expressing ANA+ autoreactive B cells between HD (N=9) and SLE patients (N=8). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Mann-Whitney U test (A, B and D) or the Wilcoxon signed rank tests (C). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) LAIR1 expression on B cells after 5 days co-culture with cTfh cells activated with anti-CD3/28 Beads in the presence of IL-21R-Fc protein (20 μg/mL) or isotype control (N=5). (B) The effects of IL-21 on LAIR1 expression in naïve B cells co-stimulated with CD40 or CD40/BCR in 3 days culture (N=12). (C) The effects of IL-21 on LAIR1 expression in SWM B cells co-stimulated with CD40 in 5 days culture (N=5). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (C) and further adjusted for multiple comparisons using the Benjamini-Hochberg method (A and B). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Co-Culture Assay, Control

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) The effects of IL-21 on LAIR1 expression in naïve B cells co-stimulated with TLR9 or TLR9/BCR in 3 days culture (N=7) (B) The effects of IL-21 on LAIR1 expression in ABCs differentiation from naïve B cells in 3 days culture (N=7). (C) The effects of IL-21 on LAIR1 expression in SWM B cells co-stimulated with TLR9 in 5 days culture (N=7). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (B) and further adjusted for multiple comparisons using the Benjamini–Hochberg method (A and C). Asterisks indicate significant differences (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) The effects of IL-21 on LAIR1 mRNA expression in naïve B cells co-stimulated with TLR9 or CD40/BCR in 2 days culture (N=6) (B) The effects of IL-21 on LAIR1 mRNA expression in naïve B cells in 2-24 hours culture (N=7). (C) IL-21-induced phosphorylation (left) and nuclear translocation (right) of STAT1/3/5 (N=3). (D) Amplification of IL-21-induced pSTAT3 level by TLR9 or CD40/BCR in the naïve B cell culture (N=4-5). (E-G) the effect of SD36 (1 μM) against STAT family proteins for 3.5 hours (E; N=3), LAIR1 mRNA expression change induced by IL-21 (F; N=6), pSTAT3 and the protein levels of STAT3 (G; N=6) and LAIR1 (H; N=11). Data are shown as mean ± SEM with each symbol representing an individual subjects. P values were calculated with the Wilcoxon signed rank tests (A, B, D, F and G). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Expressing, Phospho-proteomics, Translocation Assay, Amplification, Cell Culture

Journal: bioRxiv

Article Title: IL-21-STAT3 axis negatively regulates LAIR1 expression in B cells

doi: 10.1101/2025.01.14.632971

Figure Lengend Snippet: (A) STAT3 putative binding site in LAIR1 promoter region and its sequence (B) the binding of STAT3 of nuclear extract from IL-21 treated naïve B cells to LAIR1 promoter region (sequence #3) (N=3). P values were calculated with the Paired T test (C). Asterisk indicates significant difference (* P < 0.05).

Article Snippet: B cells (2.0×10 6 cells) isolated with EasySep human B cell isolation kit (Stem cell technologies) were rested in X-vivo medium (Lonza) at 37°C for 30 min and subsequently stimulated with biotinylated anti-LAIR1 polyclonal antibody (1.0 μg/mL, R&D Systems) or biotinylated isotype antibody (1.0 μg/mL, R&D Systems) in the presence of streptavidin (10 μg/mL, R&D Systems) for 20 min.

Techniques: Binding Assay, Sequencing