MHC Class I Ligands of Rhesus Macaque Killer Cell Ig-like Receptors

Abstract

Definition of MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology, and transplantation. To provide a more complete foundation for studying NK cell responses, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Macaca mulatta (Mamu)-A, -B, -E, -F, -I, and -AG alleles. KIR-MHC class I interactions were identified by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. Ligands for 12 KIRs of previously unknown specificity were identified that fell into three general categories: interactions with multiple Mamu-Bw4 molecules, interactions with Mamu-A-related molecules, including allotypes of Mamu-AG and the hybrid Mamu-B*045:03 molecule, or interactions with Mamu-A1*012:01. Whereas most KIRs found to interact with Mamu-Bw4 are inhibitory, most of the KIRs that interact with Mamu-AG are activating. The KIRs that recognize Mamu-A1*012:01 belong to a phylogenetically distinct group of macaque KIRs with a 3-aa deletion in the D0 domain that is also present in human KIR3DL1/S1 and KIR3DL2. This study more than doubles the number of rhesus macaque KIRs with defined MHC class I ligands and identifies interactions with Mamu-AG, -B*045, and -A1*012. These findings support overlapping, but nonredundant, patterns of ligand recognition that reflect extensive functional diversification of these receptors.

FIGURE 1.

Rhesus macaque KIRs and KIR-CD3ζ expression on JNL cells. (A) Alignment of amino acid sequences for the D0, D1 and D2 domains of rhesus macaque KIRs. Shaded sequences correspond to predicted MHC class I contact sites based on the crystal structure of HLA-B*57 in complex with human KIR3DL1 (91). Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code. (B) KIR-CD3ζ-transduced JNL cells were stained with anti-Flag-tag antibody and Near-IR LIVE/DEAD stain. After excluding dead cells, the fluorescence intensity of KIR-CD3ζ (Flag-tag) staining on KIR-CD3ζ-transduced JNL cells (open) was compared to background staining on parental JNL cells (shaded).

FIGURE 2.

KIR3D04*001, KIR3DL10*002, KIR3DL11*012 and KIR3DS04*003 interact with multiple Mamu-Bw4 ligands. (A) KIR3DL04*001-, KIR3DL10*002-, KIR3DL11*012- and KIR3DS04*003-CD3ζ JNL cells were incubated with 721.221 cells expressing the indicated Mamu-B molecules. The bar graphs represent the mean and standard deviation (error bars) of luciferase activity (RLU) from triplicate wells of KIR-CD3ζ JNL cells incubated with Mamu-B + 721.221 cells (blue), parental 721.221 cells (red) and anti-Flag-tag plus anti-mouse antibodies (X-link, gray). Asterisks denote significant differences by one-way ANOVA with Dunnett test (** p=0.0002, and *** p < 0.0001). These results are representative of at least three independent experiments. (B) Parental and Mamu-B+721.221 cells were stained with Near-IR LIVE/DEAD dye, KIR3DL04*001-, KIR3DL10*002-, KIR3DL11*012- and KIR3DS04*003-Fc followed by goat anti-mouse IgG and MHC class I-specific antibody (W6/32).

FIGURE 3.

KIR3DL07*004, KIR3DS01*003, KIR3DS02*004, KIR3DS06*002, KIR3DSw07*001 and KIR3DSw09*003 interact with Mamu-AG and -B*045:03. (A) KIR3DL07*004-, KIR3DS01*003-, KIR3DS02*004-, KIR3DS06*002-, KIR3DSw07*001- and KIR3DSw09*003-CD3ζ JNL cells were incubated with 721.221 cells expressing the indicated Mamu-AG or -B molecules. The bar graphs represent the mean and standard deviation (error bars) of luciferase activity (RLU) from triplicate wells of KIR-CD3ζ JNL cells incubated with Mamu-AG+721.221 cells (purple), Mamu-B+721.221 (blue), parental 721.221 cells (red) and anti-Flag-tag plus anti-mouse antibodies (X-link, gray). Asterisks denote significant differences by one-way ANOVA with Dunnett test (** p < 0.005, and *** p < 0.0001). These results are representative of at least three independent experiments. (B) Parental, Mamu-AG and Mamu-B+721.221 cells were stained with Near-IR LIVE/DEAD dye, KIR3DL07*004-, KIR3DS01*003-, KIR3DS02*004-, KIR3DS06*002-, KIR3DSw07*001- and KIR3DSw09*003-Fc followed by goat anti-mouse IgG and MHC class I-specific antibody (W6/32).

FIGURE 4.

Mamu-B*045:03 has an α1-domain similar to Mamu-A molecules. (A) Amino acid sequence alignment for the α1- and α2-domains of rhesus macaque MHC class I molecules. Residues 77–83 corresponding to the Bw4/Bw6 motif are underlined and predicted KIR contact sites based on the crystal structure of HLA-B*57 in complex with human KIR3DL1 are shaded (91). Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code. (B & C) Phylogenetic trees of nucleotide sequence coding for the α1-α3 domains (B) or the α1 domain (C) of selected Mamu-A, -B and -AG molecules. Neighbor-joining analysis was performed using the MAFFT server (version 7, https://mafft.cbrc.jp/alignment/server/). Bootstrap support of greater than 50% is indicated.

FIGURE 5.

KIR3DLw03*002 and KIR3DL02*004 interact with Mamu-A*012:01. (A) KIR3DLw03*002- and KIR3DL02*004-CD3ζ JNL cells were incubated with 721.221 cells expressing the indicated rhesus macaque MHC class I molecules. The bar graphs represent the mean and standard deviation (error bars) of luciferase activity (RLU) from triplicate wells of KIR-CD3ζ JNL cells incubated with Mamu-A+721.221 (green), nonclassical (purple), parental 721.221 cells (red) and anti-Flag-tag plus anti-mouse antibodies (X-link, gray). Asterisks denote significant differences by one-way ANOVA with Dunnett test (*** p < 0.0001). These results are representative of at least three independent experiments. (B) Parental and Mamu-A+721.221 cells were stained with Near-IR LIVE/DEAD dye, and KIR3DLw03*002- or KIR3DL02*004-Fc followed by goat anti-mouse IgG and MHC class I-specific antibody (W6/32).

FIGURE 6.

Phylogenetic clustering of rhesus macaque KIRs reflect their patterns of ligand recognition. Neighbor-joining analysis of nucleotide sequences coding for the D0-D2 domains of human and rhesus macaque KIRs was performed using the MAFFT server (version 7, https://mafft.cbrc.jp/alignment/server/). Bootstrap support of greater than 50% is indicated. Human KIR3DL1 and KIR3DL2 were used included as an outgroup. Rhesus macaque KIRs are color coded according to their MHC class I ligands: unknown (gray), Mamu-Bw4 (green), Mamu-AG (dark blue), and Mamu-A1*012:01 (purple).