Journal: bioRxiv

Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

doi: 10.64898/2025.12.11.693708

Figure Lengend Snippet: (A) Schematic showing the structure of PD-L2 (left) containing the extracellular IgV and IgC domains, the TM (transmembrane) domain and a short cytoplasmic domain. Sequences of the last 7 amino acids of the TM domain and the whole cytoplasmic domain are shown for the mouse C57BL/6J (B6) and CAST/EiJ ( Mus musculus castaneus ) strains, and several other mammals, indicating amino acid numbering and amino acids conserved between multiple mammalian species in green. Mutation of the stop codon in B6 mice to tryptophan (W248) in the corrected mouse B6 strain (X248W) results in a cytoplasmic domain very similar to that in the CAST/EiJ mouse. (B) Graphs of percentage of B-1a, B-1b and B-2 cells among all peritoneal cavity CD19 + B cells (gated as in ) in PD-L2ko, WT and X248W mouse strains (n=10). (C-H) Flow cytometric analysis of peritoneal cavity B-1a cells from the indicated strains, gated as in , showing representative plots for expression of PC1 (C), CD73 (E) and IgG3 (G) and graphs showing percentage of B-1a cells positive for the same three proteins (D, F, H). Gates set using B-2 cells which are largely negative for both proteins . Sample numbers: PC1, PD-L2ko (n=6), WT (n=9) and X248W (n=9); CD73, PD-L2ko (n=5), WT (n=7) and X248W (n=9); IgG3, PD-L2ko (n=8), WT (n=5) and X248W (n=6). (I, J) Flow cytometric analysis of splenocytes from indicated strains showing IgG1 expression on B-1a cells (I). Graph shows percentage of IgG1 + B-1a cells (J). Sample numbers: PD-L2ko (n=7), WT (n6), X248W (n=7). Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 3 (B, D, F, H) or 2 (J) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001.

Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

Techniques: Mutagenesis, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

doi: 10.64898/2025.12.11.693708

Figure Lengend Snippet: (A) Comparison of sequences of the last 7 amino acids of the transmembrane (TM) domain and the whole cytoplasmic domain of PD-L2 for the indicated mouse strains, including C57BL/6J (B6), the corrected C57BL/6J (B6) strain with the X248W mutation and CAST/EiJ ( Mus musculus castaneus ), indicating amino acid numbering and amino acids identical between strains in green. (B) Diagram showing the amino acid sequence of the last 7 amino acids of the TM domain and the reconstituted cytoplasmic domain of PD-L2 X248W with residues conserved between different mammals in green (from ) and numbers showing amino acid positions. Upper panel: predicted phosphorylation sites Y252 and S257 are indicated in red with kinases that may phosphorylate these listed below. Lower panel: predicted binding partners to these two residues and P259 indicated below. Confidence scores of predicted kinases (from NetPhos 3.1) and binding partners (from Scansite 4.0): INSR (0.521), PKA (0.770); ITK (0.580), NEK10 (0.655), SHC1 (0.691), PKA (0.693), NEK2 (0.910), 14-3-3 (0.733), AURKA (0.703), SRC (0.619). (C) Histograms showing PD-L2 expression on B-1a (CD11b + CD5 + ) and B-1b (CD11b + CD5 - ) cells from peritoneal cavity of WT and PD-L2 X248W mice, pre-gated on live CD19 + cells. (D) Graphs of PD-L2 expression on B-1a and B-1b cells (n=10). (E, F) Flow cytometric analysis of PC1 (E) and CD73 (F) expression on peritoneal B-2 cells (CD19 + CD11b - ) showing example flow plots and graphs of frequencies of PC1 + and CD73 + B-2 cells from mice of the indicated genotypes. These gates were used to set gates for PC1 + and CD73 + B-1 cells in . Sample numbers: n=4 for PC1 in PD-L2ko and all CD73 analyses, 5 for PC1 in X248W and 6 for PC1 in WT. (G) Graphs showing frequency of IgG3-secreting B-1 cells (spot forming units, SFU) and amount of IgG3 production (spot size) determined using ELISpot assays on peritoneal B-1 cells from mice of the indicated genotypes incubated for 48 h (n=4 for PD-L2ko and WT, 5 for X248W). (H) Flow cytometric analysis of intracellular Blimp-1 detected with an antibody in B-2, B-1a and B-1b peritoneal cavity cells from a mouse expressing Blimp-1-GFP. Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 4 (D) or 2 (E-G) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01.

Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

Techniques: Comparison, Mutagenesis, Sequencing, Phospho-proteomics, Binding Assay, Expressing, Enzyme-linked Immunospot, Incubation, MANN-WHITNEY