Detection of Human PD‑1 by Western Blot. Western blot shows lysates of HEK293 human embryonic kidney cell line either mock transfected or transfected with human PD-1. PVDF membrane was probed with 1 µg/mL of Mouse Anti-Human PD‑1 Monoclonal Antibody (Catalog # MAB10861) followed by HRP-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # HAF018). Specific bands were detected for PD‑1 at approximately |
40-80 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
|Detection of PD‑1 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) either (A) untreated or (B) treated with 5 ng/mL PHA for 2 days were stained with Mouse Anti-Human PD‑1 Monoclonal Antibody (Catalog # MAB10861) followed by Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B) and Mouse Anti-Human CD3 epsilon PE‑conjugated Monoclonal Antibody (Catalog # FAB100P). Quadrant markers were set based on control antibody staining (Catalog # MAB002).|
Programmed Death-1 (PD-1) is a type I transmembrane protein belonging to the CD28/CTLA-4 family of immunoreceptors that mediate signals for regulating immune responses (1). Members of the CD28/CTLA-4 family have been shown to either promote T cell activation (CD28 and ICOS) or downregulate T cell activation (CTLA-4 and PD-1) (2). PD-1 is expressed on activated T cells, B cells, myeloid cells, and on a subset of thymocytes. In vitro, ligation of PD-1 inhibits TCR-mediated T cell proliferation and production of IL-1, IL-4, IL-10, and IFN-gamma. In addition, PD-1 ligation also inhibits BCR mediated signaling. PD-1 deficient mice have a defect in peripheral tolerance and spontaneously develop autoimmune diseases (2, 3). Two B7 family proteins, PD-L1 (also called B7-H1) and PD-L2 (also known as B7-DC), have been identified as PD-1 ligands. Unlike other B7 family proteins, both PD‑L1 and PD‑L2 are expressed in a wide variety of normal tissues including heart, placenta, and activated spleens (4). The wide expression of PD-L1 and PD-L2 and the inhibitor effects on PD-1 ligation indicate that PD-1 might be involved in the regulation of peripheral tolerance and may help prevent autoimmune diseases (2). The human PD-1 gene encodes a 288 amino acid (aa) protein with a putative 20 aa signal peptide, a 148 aa extracellular region with one immunoglobulin-like V‑type domain, a 24 aa transmembrane domain, and a 95 aa cytoplasmic region. The cytoplasmic tail contains two tyrosine residues that form the Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) and Immunoreceptor Tyrosine-based Switch Motif (ITSM) that are important in mediating PD-1 signaling. Mouse and human PD-1 share approximately 60% aa sequence identity (4).