The production of KIR–Fc fusion proteins and their use in a multiplex HLA class I binding assay

https://doi.org/10.1016/j.jim.2015.06.012Get rights and content

Highlights

  • KIR and HLA class I ligand interactions modulate NK cell reactivity.

  • Soluble recombinant KIR–Fc fusion proteins are made efficiently in insect cells.

  • Production method described is simple and scalable.

  • KIR–Fc used in multiplex HLA class I binding assay to characterize ligand specificity

  • Results inform functional and evolutionary studies of NK cell immunity.

Abstract

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR–Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR–Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I.

Introduction

Killer-cell immunoglobulin like receptors (KIR) are a family of germ-line encoded cell surface receptors that regulate the activity of natural killer (NK) cells and T cells in immunity and reproduction through interaction with HLA class I molecules (Parham and Moffett, 2013). Both KIR and HLA are encoded by polymorphic genes, which have numerous alleles encoding unique KIR and HLA class I proteins, which are known as allotypes. HLA class I proteins are expressed on the surface of most cell types and present a diverse repertoire of peptides that furnish ligands for KIR and other immune system receptors (Bjorkman et al., 1987, Colonna et al., 1992, Colonna and Samaridis, 1995, Moretta et al., 1993). The HLA class I locus contains three highly polymorphic genes, called HLA-A, B and C. HLA-C is the most recently evolved and the only one for which all the variant forms are KIR ligands (Guethlein et al., 2007, Older Aguilar et al., 2010, Older Aguilar et al., 2011). Dimorphism at position 80 in HLA-C defines two epitopes, C1 (asparagine 80) and C2 (lysine 80), which are ligands for two different forms of two-domain KIR (Mandelboim et al., 1996, Winter and Long, 1997). KIR2DL1 encodes methionine at position 44 and binds to C2 bearing HLA-C, KIR2DL2/3 encodes lysine at position 44 and binds to C1 bearing HLA-C allotypes.

Because the genes encoding KIR and HLA class I are on different chromosomes, their independent segregation during meiosis produces diversity in the number and type of KIRHLA gene combinations inherited by individuals (Norman et al., 2013, Wilson et al., 2000). Further, NK cells can express more than one KIR at a time (Lanier, 1997, Valiante et al., 1997). This inherent diversity has complicated the investigation of the specific KIR–HLA class I interactions that modulate immune response. Development of soluble KIR proteins for which the reactivity for single HLA class I molecules was determined by direct binding assay, facilitated understanding of how particular receptor–ligand combinations contributed to NK cell reactivity (Winter et al., 1998). These recombinant proteins were made in a mammalian cell expression system by fusing the extracellular domains of a two-domain KIR with two Fc domains of a human IgG1 to form a soluble homodimer (Winter and Long, 2000).

We have adapted this method for the production of soluble KIR–Fc fusion proteins by using baculovirus-infected insect cells. The advantage of this approach is that insect cells are simple to culture. They have short doubling times that facilitate scaling and they are capable of higher protein yields than mammalian cell systems of expression. Because of these advantages, the baculovirus–insect cell system is now one of the most widely used methods for the production of recombinant proteins (Hitchman et al., 2009). Although not equivalent to higher eukaryotic cells, most post-translational modifications are made correctly in insect cells, and proteins unable to be expressed in E. coli have been successfully expressed in the insect cell system (Victor et al., 2010). The baculovirus family are species-specific double-stranded DNA viruses that infect insects as their natural host (Kost and Condreay, 1999). Once inserted into the host nucleus, the baculovirus is packaged into flexible nucleocapsids, into which foreign DNA may readily be inserted. The target gene, in this case the KIR–Fc fusion construct, is inserted into a transfer vector and positioned between sequences that are homologous to the ones in the baculoviral genome. When the viral genome and transfer vector are transfected into insect cells, recombination occurs, and produces intact viral genomes harboring the target gene sequence. The target gene replaces the non-essential baculoviral polyhedrin gene. The strong promoter of the polyhedrin gene is co-opted for production of recombinant target protein.

We have also developed a multiplex assay that tests the binding of soluble KIR–Fc to 97 HLA class I allotypes. This assay uses the Luminex platform, in which the antigenic targets are microbeads, each coated with a defined HLA class I allotype. Such beads were developed originally for studying the specificity of human alloantibodies (Pei et al., 1998, Pei et al., 2003), but our group has successfully adapted this platform for use with recombinant two-domain KIR–Fc fusion proteins and monoclonal antibodies (Hilton and Parham, 2013, Moesta et al., 2008). By adjusting the relative concentration of two fluorescent dyes, a set of 100 individually identifiable beads is generated. Each bead is then coated with a different HLA class I allotype, allowing the results of the immunoassay to be correlated with HLA class I specificity.

The goal of the KIR–Fc HLA-bead binding assay is to determine the strength and specificity of the interactions between HLA class I and KIR using defined purified proteins. The results can be used to predict the reactivity of KIR expressing NK cells and T cells when their KIR recognize cognate HLA class I ligands. This assay represents a major advance from the cell-based direct binding assay in which the reactivity of only a few KIR and HLA class I combinations could be determined at any one time (Winter and Long, 2000). Moreover, the KIR–Fc HLA bead-binding assay is designed to inform cellular assays of lymphocyte function in which receptor deficient effector NK cells transfected with a specific KIR are incubated with ligand-deficient target cells transfected with a specific HLA class I molecule (Moesta et al., 2008).

The HLA class I specificity of several KIR allotypes has been investigated using various assays. Our initial study with the multiplex bead-binding assay showed that KIR2DL2*001–Fc recognized HLA-C2 allotypes with higher avidity than its allotypic variant KIR2DL3*001–Fc (Moesta et al., 2008). A cellular cytotoxicity assay subsequently showed that KIR2DL2*001, but not KIR2DL3*001 effectively inhibited lysis when incubated with HLA class I deficient 221 cells transfected with the HLA-C2 allotype, HLA-C*04:01 (Moesta et al., 2008). Another group investigated a second allotypic variant, KIR2DL3*005 (Frazier et al., 2013) using a similar multiplex assay. They showed that KIR2DL3*005–Fc bound HLA-C1 allotypes with higher avidity than KIR2DL3*001–Fc. This result was concordant with a cellular assay in which NK cells expressing either 2DL3*005 or 2DL3*001 respectively were incubated with 221 cells expressing the C1 bearing allotype HLA-C*03:04. Natural killer cells expressing 2DL3*005 exhibited a more potent inhibitory signal than those transfected with 2DL3*001 (Frazier et al., 2013). A third type of assay, surface plasmon resonance, confirmed that the 2DL3*005 variant bound with greater avidity than the 2DL3*001 variant to the HLA-C1 allotype HLA-C*03:04 (Frazier et al., 2013).

In summary, we have developed a simplified method for the production of KIR–Fc and designed an assay that tests their binding to 97 HLA class I allotypes simultaneously. The assay is easy to perform and correlates well with more complicated experimental techniques such as cellular cytotoxicity and surface plasmon resonance that have traditionally been used to determine the avidity and specificity of KIR for HLA class I ligands.

Section snippets

Materials and methods

KIR–Fc fusion protein generation.

This section describes the generation of a DNA insert, flanked by restriction sites, that encodes the first 224 amino acids of the KIR2D of interest and the Fc region of human IgG1 (Fig. 1A). This insert is first cloned into the pAcGP67a transfer vector (Fig. 1B). Subsequently it is co-transfected with linearized baculovirus into insect cells.

Results and discussion

The purpose of this protocol is to provide a simplified method with which to produce and test the reactivity of soluble two-domain KIR–Fc fusion proteins. We chose to use an insect cell expression system because most post-translational modifications are made correctly in insect cells and the system is scalable, allowing production of large quantities of soluble recombinant protein in a comparatively short time. We have sought to reduce variability in final protein yield by implementing a series

Conclusions

We have described the production of KIR–Fc fusion proteins in an insect cell expression system and their interaction in a multiplex binding assay with a panel of 97 HLA class I allotypes. KIR–Fc production in insect cells is relatively simple, allowing production of large amounts of recombinant protein in around 20 days. The assay is sensitive enough to discriminate between single amino acid substitutions in the extracellular domains of the KIR molecule and has, as a result, greatly facilitated

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