Fig 2: Identification of a coding polymorphism in the first EGF-like domain of Cd93 in NOD and NZB/W F1 mice. a The Cd93 gene was PCR amplified from genomic DNA. Sequencing revealed a point mutation at cDNA nucleotide position 790. The AAC→CAC codon change in NOD and NZB/W F1 mice led to an amino acid substitution in the first EGF-like domain of Cd93 at position 264. The first EGF-like domain in which the defined polymorphism resides is stippled. b Bone marrow macrophage lysates (left) were separated on an 8% SDS-PAGE under reducing conditions, transferred to PVDF, and probed with 5 μg/ml polyclonal anti-CD93 cytoplasmic tail Ab 1150 (black arrow). Isolated bone marrow B-cell lysate immunoblots (right) were probed with 0.1 μg/ml polyclonal anti-CD93 (anti-C1qR1). The blots were stripped and re-probed for β-actin or β-tubulin to control for similar protein loading. The blots are from one experiment, representative of two, using different animals as a source of bone-marrow-derived macrophages or B cells. c sCD93 is detected in sera from NOD and NZB/W F1 mice. Sera were diluted (1:50) and tested for the presence of sCD93 by sandwich ELISA. Shown is the average of triplicate wells (± SD) from one experiment, representative of two, using different animals as a source of sera

Fig 3: sCD93 in peritoneal lavage fluid and urine, and its expression in the glomeruli of the db/db mice. (A,B) The level of serum sCD93 at age 8, 20, and 31 weeks of age (n = 4 per group) (A) and the sCD93 of the peritoneal lavage fluid (n = 5~7 per group) (B) were measured by ELISA. (C–G) The kidney and urine samples of 31-week-old db/db mice were analyzed for the CD93 expression and sCD93 level. PAS staining (C), the degree of albuminuria (n = 4~5 per group) (D), urinary sCD93 level (n = 4~5 per group) (E), immunofluorescence staining with CD31 and CD93 (F) and the quantification (n = 5 per group) (G) were presented. The glomeruli and tubule were isolated from the kidney of db/db and db/m mice and processed for CD93 immunoblot with β-actin as loading control (H) and the quantification was presented (n = 4 per group) (I). The difference was compared between the db/db and db/m+ group. Data are presented as the mean ± SE. * p < 0.05. Scale bars, 50 µm.

Fig 4: The NOD CD93 defect maps to Idd13. a Chromosomal map showing regions of either NOD chromosome 2 (white bar) or B6 chromosome 2 (black bar) introgressed onto the B6 or NOD background for each congenic strain obtained from Jackson Laboratories. b The left column shows AA4.1 staining on B-cell progenitors (B220lo) in the BM for each congenic strain. The middle column divides B cells in the bone marrow into mature recirculating ( B220hi/IgM+), immature (B220lo/IgM+), and pro-/pre-B cells (B220lo/IgM−) for each of the congenic strains. The histograms resolve AA4.1 expression by pro-/pre- and immature B cells (black line) in contrast with mature recirculating B cells (gray line). The ratio of the mean fluorescence indices (MFIs) of AA4.1 expression by pro-/pre- and immature B cells to the AA4.1 expression by mature recirculating B cells is shown as MFI fold Δ. The MFI fold Δ is an average of at least n = 4 mice per strain shown, and *p < 0.05 compared with B6 mice

Fig 5: iNKT cell analysis in B6, B6 CD93−/−, B6.NODIdd13 and NOD mice. a The left panel shows the frequency of iNKT cell staining (CD3lo, PBS57-loaded CD1d Tetramer+) in the liver of B6 (n = 9, mean 15.9 ± 5.79%) B6 CD93−/− (n = 7, mean 9.78 ± 4.20%), B6.NODIdd13, (n = 5, mean 7.71 ± 4.29%), and NOD (n = 5, mean 8.15 ± 3.11%) mice. The right panel further resolves CD4+ iNKT and CD4− iNKT cells. b The bar graph indicates the frequency of CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in the livers of B6 CD93−/−, B6.NODIdd13, and NOD mice. c The bar graph shows the absolute number of thymic DN and CD4SP iNKT cells (CD3lo, PBS57-loaded CD1d Tetramer+) in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. d The bar graph indicates the total number of splenic CD4+ and CD4− iNKT (CD3lo, PBS57-loaded CD1d Tetramer+) cells in B6, B6 CD93−/−, B6.NODIdd13, and NOD mice. Error bars indicate ± SD, *p < 0.05 compared with B6 mice