Fig 2: KGYY15binding is partially blocked by antibodies to CD11a and CD11b as well as by recombinant CD11a/CD18, CD11b/CD18, and CD40 proteins, but not recombinant CD28 protein. NAMALWA cells were stained with KGYY15-FITC in the absence/presence of competition. A, competition with anti-CD11a, anti-CD11b, or recombinant CD11a/CD18 or CD11b/CD18 proteins. B, competition with recombinant CD40 protein. C, competition with recombinant CD28 protein. Data are represented as mean ± SEM. p-Values were calculated by two-tailed t test and are shown in the figure.

Fig 3: Glycosylation pattern of CD11b expressed on T cells.a Western blot of CD11b on T cell lysates treated with PNGase F, O-glycosidases or Neuraminidase A for 4 h as indicated. b Lectin blot analysis of purified CD11b-flag from transduced human CD3+ T cells. Complete membrane blots are included in Supplementary Fig. 18. a, b One representative western blot from n = 2 biologically independent experiments. c Model of the interaction between Siglec-15 present on the surface of interacting cells and CD11b/CD18 integrin from the cell surface of T cells. The full extracellular domain of Siglec-15 (cyan) was manually built using the crystal structure of the V-set domain and the C2-type Ig-like domain of CD22 (PDB ID:5VKJ). The full extracellular domain of CD11b (blue)/CD18 (wheat) (PDB ID 7USM)73 heterodimer (from the cell surface of T cells) is represented. The I domain (taken from PDB ID 3K72) and the thigh region of CD11b are colored in gray and pink, respectively. The N-linked glycans present in CD11b are represented as blue spheres. In this model, Siglec-15 binding pocket at V domain is interacting with the sialylated N692-linked glycan of CD11b.

Fig 4: Glycosylation accounts for the different size bands of CD11a, CD11b, and CD18.A, NOD cell lysates were either assayed as is (NOD whole cell lysates) or subjected to immunoprecipitation by an OVA-peptide (OVA-pep IP; a control for KGYY15 peptide) or by CD3 antibody (CD3-ab IP; a control for CD40 antibody 4F11) then assayed for CD11a, CD11b, CD18, or CD40 in Western blots. CD3-ab immunoprecipitation eluates were also assayed for CD3. B, NOD PBMC lysates were subjected to deglycosylation by PNGase F (removal of N-linked deglycosylation) or by PNGase F and total O-linked deglycosylation (Total deglyc.), or not (Lysates), then Western blots for CD11a, CD11b, and CD18 were performed. Completely deglycosylated bands are indicated (compl. deglyc.). C, lysates were subjected to deglycosylation by PNGase F alone, PNGase F and total O-linked deglycosylation (Total deglyc.), deglycosylation with four different enzymes that remove O-linked glycosylations (O-linked deglyc.), or the four different enzymes that remove O-linked glycosylations separately (O-glycosidase, neuraminidase, β-galactosidase, or glucosaminidase). Western blots were then done for CD11a. Data are representative of three experiments.

Fig 5: Siglec-15 binds to CD11b in a sialic acid-dependent manner.a OD values corresponding to an ELISA performed with serially diluted IgG1-Fc (gray), Siglec-15-Fc WT (red) or Siglec-15-Fc R143A (blue) against plate-coated CD11b/CD18. Averages of triplicates are shown. b Co-immunoprecipitation showing the interaction of Siglec-15-Fc or Siglec-15-Fc R143A with CD11b. Assay repeated twice with similar results. c Bar graph representing the absolute STD-NMR intensities corresponding to proton signals of STn-Ser + Siglec-15 before the addition of CD11b/CD18 (gray), and after (orange). d Representative contour plots showing the expression of CD11b on activated CD4+ and CD8+ T cells by flow cytometry. e Quantitation of Siglec-15-Fc binding to CD4+ and CD8+ T cells in the presence or absence of anti-CD11b blocking mAb (clone M1/70), measured by flow cytometry (CD4+: p = 0.0052, CD8+: 0.0098, n = 4 donors). f Representative flow cytometric histograms (gMFI) and pooled data showing the fold change in the binding of Siglec-15–Fc to T cells transfected with indicated siRNAs (CD4+: p = 0.046, CD8+: p = 0.036, n = 5 donors). g Representative flow cytometric histograms (gMFI) and pooled data of the fold change in the binding of Siglec-15-Fc to T cells overexpressing CD11b/CD18 compared to untransduced cells (CD4+: 0.0007, CD8+: 0.036, n = 6 donors). Error bars denote SEM. *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001) as determined by two-tailed, unpaired Student’s t test. Source data are provided as a Source Data file.