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

Journal: bioRxiv

Article Title: Hypoimmunogenic human motor neurons induced from iPSCs in vivo substantially ameliorate ALS disease in large animal models

doi: 10.1101/2025.09.03.673895

Figure Lengend Snippet: (A) Schematic illustration of multiplex genetic-editing strategies for engineered HIP-NILB-iPSCs. (B) Indel analysis by Sanger sequencing for B2M , CIITA , IL6ST and TNFRSF1A in HIP-NILB-iPSCs. (C) Real-time PCR analysis of CD47 , CD24 , IgG1-Fc , CTLA4 and PD-L1 in HIP-NILB-iPSCs. (D) Western blot analysis and relative density quantification of B2M, CIITA, IL6ST, TNFR1, CTLA4-Ig, PD-L1, CD24 and CD47 in HIP-NILB-iPSCs of three independent samples. CIITA was detected in NILB-iPSCs and HIP-NILB-iPSCs upon IFN-γ and TNF-α treatment for 48 h. (E) Karyotyping of HIP-NILB-iPSCs. (F) Scatter plot comparison of transcriptomes for HIP-NILB-iPSCs and NILB-iPSCs. The raw counts for each gene were transformed to log10 value, and genes with more than 1 count in each sample were shown. The pluripotent-related genes were labeled in the diagram. Dashed lines depict the 10-fold changes. The R 2 was determined by Pearson’s correlation. The NILB-iPSCs were used as a control. (G) Heatmap showing expression level of stem cells-related and neuron development-related genes together with the enrichment analysis in WT iPSCs and HIP-NILB-iPSCs upon Dox induced for 0.5-, 4- and 7-days. Data are mean ± SEM; p values were determined using a two-tailed, unpaired Student’s t-test ; * p < 0.05; ** p < 0.01.

Article Snippet: The antibodies used were listed as follows: PE anti-human HLA-A,B,C (BioLegend, 311405, 1:20), PE anti-human HLA-DR,DP,DQ (BioLegend, 361716, 1:20), PE Mouse IgG2a, κ Isotype Ctrl (FC) antibody (BioLegend, 400213, 1:20), PE anti-human CD3 (BD Biosciences, 347347,1:50), FITC anti-human CD107a (BD Biosciences,555800,1:100), FITC Mouse IgG1, κ Isotype Control RUO (BD Biosciences, 555748,1:100), APC anti-human CD56 (BioLegend, 362504, 1:20), PE-Cy5 human CD56 (BD Biosciences, 555517, 1:10), APC Mouse IgG1, κ Isotype Ctrl (FC) antibody (BioLegend, 400122, 1:20), PE anti-mouse CD68 (BioLegend, 137014, 1:100), PE Rat IgG2a, κ Isotype Ctrl (BioLegend, 400508, 1:100), CoraLite® Plus 647-conjugated IBA1 monoclonal antibody (Proteintech, CL647-66827,1:200), CoraLite® Plus 647 Mouse IgG2a Isotype Control (Proteintech, CL647-65244,1:200).

Techniques: Multiplex Assay, Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Comparison, Transformation Assay, Labeling, Control, Expressing, Two Tailed Test