LILRB1 transcription initiation sites in CD8 T cells, B cells, and monocytes. (A) Schematics of the LILRB1 locus on human chromosome 19 and selected mRNA sequences currently posted to NCBI. Lines are introns, and boxes are exons (roughly to scale). Exons 1 and 2 are separated by approximately 13 kb as indicated by a gap. The LILRB1 coding region is flanked by a start (ATG) and stop (TAG) codon. (B) PCR of 5′-RACE products generated from monocyte, B-cell, and CD8 T-cell RNA isolated from magnetic bead–separated cells. The enzyme TdT was excluded (—) or included (tag) when tagging the 5′ end of LILRB1 cDNA. PCR products were amplified with a tag-specific sense primer and an LILRB1-specific antisense primer (top) or LILRB1-specific sense and antisense primers (bottom). Templates for lanes indicated by clone M22 and clone B6 were the 5′-RACE clone from monocytes and B cells, respectively, used to obtain the sequences shown in panel C. (C) Sequencing of 5′-RACE products. Total PCR products were TOPO-cloned and sequenced. The sequence corresponding to the major tag-specific band for each cell type in panel B is compared with LILRB1 sequences BC015731 and AF283984. Boxed sequences indicate contiguous sequences upstream of exon 2 in the genome. (D) Luciferase reporter assay of sequences upstream of LILRB1 exon 1. The 500-bp and 2000-bp sequences found immediately upstream of LILRB1 exon 1 on chromosome 19 were amplified and placed upstream of the firefly luciferase ORF of pGL4.10. Freshly isolated primary T cells and monocytes were cotransfected with the reporter constructs and a control Renilla luciferase expression vector. Data represent firefly luciferase activity, normalized to Renilla luciferase activity, relative to that seen with the promoter-less basic pGL4.10 vector. Data from 3 donors are represented as mean + SD.