Fig. 4

ILT-4 is identified as a receptor for human SEMA4A. a Binding of SEMA4A-Fc to activated T cells. Freshly isolated naive (lin⁻CD45RA⁺), memory Th2 (CD45RO⁺CRTH2⁺) and CD45RO⁺CRTH2⁻CD4⁺ T cells were stained with SEMA4A-Fc (upper), or activated with anti-CD3 mAb⁺ anti-CD28 mAb for 1 day before staining with SEMA4A-Fc (down). SEMA4A-Fc binding was analyzed by flow cytometry. Open histograms represent the staining of indicated cell subsets with SEMA4A-Fc; filled histograms represent the isotype. Numbers in the histograms indicate the mean fluorescence intensity for the markers indicated below histograms. Data represent one of five independent experiments. b Expression cloning of SEMA4A receptor. H9 cells were infected with retroviral cDNA library of activated CD4⁺ T cells, and the SEMA4A-Fc-binding cells were enriched by flow cytometry. c Flow cytometry enriches a cDNA insert of about 2.5 kb. The genome for the enriched cells were isolated and the inserts were amplified by PCR with a pair of primers designed according to the sequence of the retroviral expression vector. d Identification of interaction between ILT-4 and SEMA4A by high-throughput screening of a receptor array. 293T cells were transfected with plasmids of different human transmembrane genes in different wells of 384-well plates. SEMA4A-Fc was added into each well to evaluate its binding to the transfects by Applied Biosystems 8200 Cellular Detection System. The Fc receptor transfectants are used as positive control. Scale bars represent 50 μm. e Interaction of SEMA4A and hILT-4 was tested by FACS. 293T cells were transfected with ILT-4 cDNA and stained with hIgG (filled histogram), SEMA4A-Fc (dash line), SEMA4A-Fc+ an excess of ILT-4-Fc (full line), or SEMA4A-Fc+ an excess of mIgG (dotted line) (left); H9 cells were transfected with SEMA4A cDNA and stained with hIgG (filled histogram), ILT-4-Fc (dash line), an excess of anti-SEMA4A mAb + ILT-4-Fc (thick line), or an excess of mIgG+ SEMA4A-Fc (dotted line) (right). Data represent one of five independent experiments