KIR3DS1-Specific D0 Domain Polymorphisms Disrupt KIR3DL1 Surface Expression and HLA Binding

Abstract

KIR3DL1 is a polymorphic inhibitory receptor that modulates natural killer cell activity through interacting with HLA-A and HLA-B alleles that carry the Bw4 epitope. Amino acid polymorphisms throughout KIR3DL1 impact receptor surface expression and affinity for HLA. KIR3DL1/S1 encodes inhibitory and activating alleles, but despite high homology with KIR3DL1, the activating receptor KIR3DS1 does not bind the same ligand. Allele KIR3DL1*009 resulted from a gene recombination event between the inhibitory receptor allele KIR3DL1*001 and the activating receptor allele KIR3DS1*013. This study analyzed the functional impact of KIR3DS1-specific polymorphisms on KIR3DL1*009 surface expression, binding to HLA, and functional capacity. Flow cytometric analysis of primary human NK cells as well as transfected HEK293T cells show that KIR3DL1*009 is expressed at a significantly lower surface density compared to KIR3DL1*001. Using recombinant proteins of KIR3DL1*001, KIR3DL1*009, and KIR3DS1*013 to analyze binding to HLA, we found that while KIR3DL1*009 displayed some evidence of binding to HLA compared to KIR3DS1*013, the binding was minimal compared to KIR3DL1*001 and KIR3DL1*005. Mutagenesis of polymorphic sites revealed that the surface phenotype and reduced binding of KIR3DL1*009 are caused by the combined amino acid polymorphisms at positions 58 and 92 within the D0 extracellular domain. Resulting from these effects, KIR3DL1*009-positive NK cells exhibited less inhibition by HLA-Bw4 positive target cells compared to KIR3DL1*001-positive NK cells. The data from this study contribute novel insight into how KIR3DS1-specific polymorphisms in the extracellular region impact KIR3DL1 surface expression, ligand binding, and inhibitory function.

KIR3DL1 and HLA-B Density and Binding Calibrate NK Education and Response to HIV

“…In addition to the 80I versus 80T distinction in the Bw4 epitope, several polymorphic sites on KIR3DL1 titrate its affinity for HLA-Bw4 ligands (21, 31, 47). Therefore, binary categorization of KIR3DL1 and HLA-B molecules as “high” versus “low”, or Bw4-80I versus -80T, respectively, likely underestimates the variation in binding of KIR3DL1 to HLA-Bw4 generated by these highly polymorphic receptor and ligand families.…”

“…In instances where HLA expression is diminished, including HIV and HSV (25, 26, 73, 74), highly educated NK cells would be expected to mount the most efficient cytotoxic response. In pathologies where HLA expression persists, uneducated NK cells, lacking receptors to self-HLA and therefore refractory to inhibition, mediate superior clinical outcomes (8, 9, 31, 46, 58). In both pathologic circumstances, benefit may occur with an intracellularly retained receptor, which in the presence of its ligand, permits effector capacity while remaining immune to inhibition (Submitted for publication).…”

“…HLA -A, - B and -C epitopes were assigned to HLA-Bw4, -Bw6, -C1, and - C2 subtypes using the HLA Immunopolymorphism database version 3.14.0. KIR genotyping and KIR3DL1 subtyping were performed as previously described (19, 31, 32). Individuals with KIR3DL1*002 -group high alleles were examined using a Luminex-based KIR SSO platform according to the manufacturer’s instructions.…”

Deciphering the killer‐cell immunoglobulin‐like receptor system at super‐resolution for natural killer and T‐cell biology

“…Early work showed that HLA‐Bw4 allotypes with I80 formed more potent ligands for KIR3DL1 than those with T80,119 a functional difference reinforced by associations with disease outcome 27, 58, 120, 121, 122. However, recent high‐resolution studies have identified several I80 Bw4 allotypes that are poorly recognized by KIR3DL1, providing weaker KIR3DL1 ligands than selected T80 Bw4 allotypes 39, 41, 123. Compounding the difficulty of understanding the interactions between KIRD3DL1 and Bw4 is the extensive functional polymorphism of KIR3DL1 , which changes its cell‐surface expression25, 38 and capacity to recognize the Bw4 epitope 41, 65, 123…”

Killer cell immunoglobulin-like receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition

“…Namely, there are two diverse inhibitory lineages comprising KIR3DL1*005 -like and *015 -like alleles, whereas the third lineage is much more constrained at a population level and consists largely of the activating KIR3DS1*013 allele ( Norman et al, 2007 ; Parham et al, 2012 ). KIR3DL1 polymorphism was initially associated with differences in cell surface expression, with allotypes such as KIR3DL1*001, *002, and *008 being expressed at high levels, *005 and *009 at lower levels, and *004 being largely retained intracellularly ( Gardiner et al, 2001 ; Pando et al, 2003 ; Taner et al, 2011 ; Mulrooney et al, 2015 ). KIR3DL1 polymorphism can also affect recognition of HLA-I with polymorphisms at positions including 238 and 283 impacting recognition of various HLA-Bw4 ligands ( Carr et al, 2005 ; Yawata et al, 2006 ; Thananchai et al, 2007 ; O’Connor et al, 2014 ).…”