A novel system of polymorphic and diverse NK cell receptors in primates

Abstract

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

Figure 1. Comparison of human and grey mouse lemur LRC, NKC, and MHC genomic regions.

Ψ denotes pseudogene (criteria based on: premature stop codons, frameshift deletions/insertions, absence of essential coding/non-coding parts). (A) Sequenced BAC clones (GenBank accession no. CR974412, CR974436, CR974413) of the LRC. Open rectangles denote KIR genes, filled rectangles all other genes. Arrows point to corresponding KIR genes. No gene designations are shown for human KIR haplotypes. (B) Sequenced BAC clone contig of the NKC that constitute a complete haplotype. Open rectangles denote NKG2 genes, grey rectangles CD94 genes, and filled rectangles all other genes. The order of BAC clones in this CD94 to Ly49L genomic interval is: 50I2, 96C7, 146L20, and 1E15 (haplotype 1: GenBank accession no. FP236838). A second contig of BAC clones includes the CD94-1-NKG2-8 interval represented by clones 481C2, 492D19, 489H5, and 222F7 (not shown) and constitutes a second haplotype (GenBank accession no. FP236834). (C) Sequenced BAC clones (GenBank accession no. AB480748, FP236831, FP236832, FP236833, FP236839) of the MHC class I gene regions. Class I genes of BAC clone CH257-465D12 that could not be assigned to an already known gene or allele, are named according to their position on the BAC clone (numbers in parentheses). Putative alleles Mimu-W01 and Mimu-W04 are indicated. Open rectangles denote MHC class I genes, grey rectangles MHC class II genes, and filled rectangles all other genes.

Figure 2. Chromosomal localisation of lemur MHC and NK cell receptor genes.

FISH using mouse lemur BACs containing MHC class I and class II genes reveals a split of class I genes to chromosome 6 (GNL, TRIM39, TRIM26, ZNRD1, MOG, BAT1, MIC, POU5F1) and to chromosome 26 (ANKRD11, Mimu-W01, Mimu-W04) of Microcebus murinus. Nomenclature of M.murinus and M.coquereli chromosomes are according to Ref. The same split is shown for Mirza coquereli: ZNRD1 and MOG hybridise to chromosome 6 and ANKRD11, Mimu-W01, and Mimu-W04 to chromosome 26. All tested MHC class II region genes (TAP2, DQ, DR, BTNL2) map to chromosome 6 in Microcebus murinus and Mirza coquereli, respectively. Note that in both species secondary signals appear scattered along the X chromosome. In Mirza coquereli, KIR-including BAC CD257-37C10 hybridises to a small acrocentric chromosome not identical with chromosome 26 and to the X chromosome, and NKG2-including BAC CH257-96C7 maps to chromosome 7, which is orthologous to human chromosome 12. Note that further signals appear on the chromosome X.

Figure 3. Major diversification of CD94 and NKG2 occurred in “lower” primates after their separation with “higher” primates.

Phylogenetic analyses of primate CD94 (A) and NKG2 (B) C-type lectin-like domain sequences and NKG2 stalk, transmembrane, cytoplasmic regions (C). The tree topology obtained with the Neighbor-Joining (NJ) analysis was used for the display (with a midpoint rooting) and numbers at nodes indicate support obtained for Bayesian, parsimony, and NJ methods (from top to bottom). Support is shown if Bayesian posterior probability (PP) ≥ 88% and other methods bootstrap proportion (BP) ≥ 50% (at least 2 methods). Filled circles at nodes indicate PP > 95% and BP ≥ 80%. *: BP < 50 or PP < 80. Mimu, Microcebus murinus; Vava, Varecia variegata. A maximum-likelihood analysis was also performed, and the maximum-likelihood bootstrap support is indicated in parenthesis for two nodes.

Figure 4. “Lower” primates lack a strict ortholog or functional equivalent of “higher” primate MHC-E.

Phylogenetic analyses of MHC class I genes with complete coding sequences (A), peptide binding domain (B), peptide binding domain excluding peptide binding residues (C) and peptide binding residues only (D). A phylogenetic analysis of MHC class I genes with complete coding sequences excluding the peptide binding residues was also conducted and the results are presented in Figure S5. Analysis and display are as described for Figure 3. Numbers as gene names for MHC class I are the same as in Figure 1C. Non-primate sequences included are mouse (H2) and rat (RT1). Peptide binding residues were defined according to Bjorkman et al. . Patr, Pan troglodytes; Gogo, Gorilla gorilla; Hyla, Hylobates lar; Saoe, Saguinus oedipus; Sasc, Saimiri sciureus; Mamu, Macaca mulatta; Mimu, Microcebus murinus; Aotr, Aotus trivirgatus; Caja, Callithrix jacchus; Pipi, Pithecia pithecia.

Figure 5. Natural selection diversified “lower” primate CD94 and NKG2 sequences and particularly targeted the residues interacting with the peptide presented by MHC class I ligands.

(A) Positively selected sites of ‘lower’ primates CD94/NKG2 superimposed on the three-dimensional structure of the human CD94/NKG2A/HLA-E complex ,. The PDB file 3cdg was used and represented with PyMOL . (B) Top panel: CD94/NKG2 residues known to interact with MHC class I-bound peptide (based on human CD94/NKG2A/HLA-E). Positively selected residues in ‘lower’ primates are shown in red. Peptide positions are indicated. Residue in italics indicates allelic substitutions were found at this site. Bottom panel: ribbon diagram focused on the region where the interaction between CD94-NKG2A and HLA-E occurs; side chains are displayed.

Figure 6. Co-immunoprecipitation of various combinations of grey mouse lemur CD94 and NKG2 expression constructs.

CD94 constructs fused at the NH2 terminus with GFP and NKG2 molecules tagged at the COOH terminus with the V5 tag were transiently transfected into 293T cells. Analyses were performed 24h upon transfection. Representatives of at least three independent experiments are shown. Differential degree of glycosylation gives rise to multiple bands. In the lower panel deglycosylation with PNGase F is demonstrated exemplarily for inhibitory receptor NKG2-3.

Figure 7. COOH terminally tagged NKG2 (V5 tag) and CD94 (FLAG tag) molecules were transiently transfected into 293T cells.

Analyses were performed 24 h upon transfection. Representatives of at least three independent experiments are shown. Numbers indicate the percentage of cells in each quadrant. Cells in the upper right quadrant show V5- and FLAG-double-positive cells. (A) CD94 and NKG2 cell surface expression without co-expression of DAP12. (B) Enhancement of NKG2-2 and NKG2-5 expression at the cell surface by co-expression of myc-tagged DAP12.

Figure 8. Model of NK cell receptor evolution in primates.