The human natural killer cell immune synapse

Abstract

N atural killer (NK) cells express a variety of inhibitory killer Ig-like receptors (KIR) that inhibit cytotoxicity upon recognition of class I MHC proteins (1). In this manner NK cells detect diseased cells through their loss of expression of self-MHC protein rather than by directly detecting foreign antigen, a conjecture known as the missing self-hypothesis (2). Inhibitory NK receptors containing two Ig domains, denoted KIR1 (or KIR2DL1) and KIR2 (or KIR2DL2), recognize the class I MHC proteins, HLA-Cw4 or -Cw6 and HLA-Cw3 or -Cw7, respectively (3, 4). Crystal structures of both class I MHC and KIR extracellular domains have been determined (5-9), and the appropriate binding sites have been mapped by site-directed mutagenesis (10-15). Although the binding kinetics between soluble KIR͞MHC proteins, determined by surface plasmon resonance, are extremely fast (16-18), video microscopy of NK cell immunosurveillance shows that intercellular contacts last for minutes (not shown). Thus, we set out to delineate the molecular mechanisms of NK cell recognition that occur over this time frame.An enhanced variant of green fluorescent protein (EGFP) (19), originally discovered and cloned from Aequorea victoria jellyfish (20, 21), was used to mark the location of HLA-C. Plasmids encoding EGFP attached to the intracellular C terminus of class I MHC protein were transfected into 721.221, a B cell line derived by mutagenesis that does not express class I MHC protein (22,23). These transfectants then were incubated with various NK cell lines for 20 min at 37°C, after which time many NK cell͞target cell conjugates were formed. Conjugates of living NK and target cells were imaged by laser-scanning confocal fluorescence microscopy. This methodology advances previous imaging of mouse T cell͞target cell intercellular contacts that used paraformaldehyde-fixed cells (24) or live T cells interacting with MHC protein-rich lipid bilayers (25). Here, immune synapses are shown to exist at the contact between two living human cells. Materials and MethodsCell Lines and Transfectants. Plasmids encoding EGFP attached to the C terminus of HLA-Cw3 or -Cw4 were prepared by PCR of the appropriate HLA-C allele to remove the stop codon and add an EcoRI restriction site at the 5Ј end and a NotI site at the 3Ј end. EGFP was prepared by PCR from the plasmid pEGFP (CLONTECH) to contain a NotI site at the 5Ј end and a BamHI site at the 3Ј end. These PCR products then were joined and amplified together by PCR by using primers at the 5Ј end of HLA-C and the 3Ј end of EGFP. The product was cloned first into pBABE and then into pcDNA3 (Invitrogen). Plasmids encoding other GFP-linked proteins were prepared by PCR of the appropriate HLA-C allele to remove the stop codon and cloned as KpnI͞NotI fragments into the vector initially encoding HLA-Cw3-GFP. All primers were purchased from Life Technologies (Gaithersburg, MD) and all plasmid inserts were sequenced by the Core Facilities, Dana-Farber Cancer Institute (Boston, MA). 721.221 cells were transfected with 10...

The human CD94 gene encodes multiple, expressible transcripts including a new partner of NKG2A/B

“…This model would explain the spontaneous ATP-independent segregation of proteins observed at the iNKIS. 29,33 The CD94-T4 protein is 21 amino acids shorter than the full-length CD94. Its preferred partner is NKG2B, which is 18 amino acids shorter than NKG2A.…”

Expression and role of phosphatidylcholine‐specific phospholipase C in human NK and T lymphocyte subsets

“…After specific ligand engagement, cytolytic cells bind to sensitive target cells and rapidly reorganize cell membrane proteins, accumulating actin, talin and lytic granules at the cell-cell contact site (immunological synapse) [2,3]. Serine proteases (granzymes) and membrane-perturbing proteins, perforins (PFN) and granulysins are then released at the immunological synapse to induce target cell death [4,5].…”

The CD200 and CD200 receptor cell surface proteins interact through their N‐terminal immunoglobulin‐like domains

“…In T cells these proteins often interact with other proteins with two IgSF domains in an end-to-end topology. For these interactions to occur, the opposing cell membranes need to come into close proximity and the size of the interacting proteins has been shown to be important in the formation of the immunological synapse and triggering of T cells [10][11][12]. Mutagenesis analysis of CD200 indicated that the N-terminal (membrane-distal) domain of CD200 contains the CD200R binding site [13].…”