Figure 1. Identification of Siglec-15 as a T-cell suppressive molecule in the TCAA.

(a) Schematic representation of TCAA for rapid screening of cell surface molecules with co-stimulatory and co-inhibitory activity. cDNA plasmids coding human membrane proteins were individually transfected into an artificial antigen presenting cell line (aAPC) overnight together with a pre-expressing transmembrane form of anti-human CD3 antibody (OKT3) scFv. Jurkat-NFκb/ NFAT-reporter T-cells were added into the wells and the effect of each transmembrane protein on OKT3-stimulated reporter activity is indicated as intensity of GFP fluorescence. The function of the candidate genes is further validated on primary human T-cells. Siglec-15 is one of the molecules selected for further study.

(b) A representative result of TCAA. GFP signals of Jurkat-NFκb reporter cells were quantified based on the GFP positivity of the objects (y -axis) and the GFP density (x -axis) in each well of the array. The results of ~1,500 genes in the TCAA shown as different dots are displayed. The GFP signal in the well transfected with the mock plasmid is shown as a black dot. The activity of several genes with known T-cell stimulatory (red), apoptotic or inhibitory (light blue) activity, as well as Siglec-15 (dark blue) is indicated. Data are representative of two independent experiments.

(c) A representative reporter activity of Jurkat-NFAT cells after co-culture with aAPC transfected with Fas ligand (FASL), full length Siglec-15 (S15FL), Siglec-15 ectodomain fused with B7-H6 transmembrane motif (S15ATM), or mock plasmid is displayed. Data are mean ± s.e.m. (n=4 cell cultures). P values by two-tailed unpaired t-test (n.s., not significant; P = 0.9462).

(d) The homology of human Siglec-15 with B7 family members. Shown are the % identity or identity plus similarity of amino acid sequences in the extracellular domains.

See also Extended Data Fig. 1.