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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: J Immunol. 2018 Sep 19;201(9):2776–2786. doi: 10.4049/jimmunol.1800494

Diversification of Bw4 Specificity and Recognition of a Non-Classical MHC Class I Molecule Implicated in Maternal-Fetal Tolerance by Killer-Cell Immunoglobulin-Like Receptors of the Rhesus Macaque

Priyankana Banerjee *,#, Moritz Ries *,#, Sanath Janaka *, Andres G Grandea III *, Roger Wiseman ‡, David H O Connor *,‡, Thaddeus G Golos §,‡, David T Evans *,‡
PMCID: PMC6426299  NIHMSID: NIHMS1504996  PMID: 30232137

Abstract

The rhesus macaque is an important animal model for AIDS and other infectious diseases; however, studies to address NK cell function in this species have been limited by the lack of defined ligands for killer cell Ig-like receptors (KIRs). To identify ligands for rhesus macaque KIRs, we adopted a novel approach based on a pair of stable cell lines. NFAT-responsive luciferase reporter cell lines expressing the extracellular domains of macaque KIRs fused to the transmembrane and cytoplasmic domains of CD28 and CD3ζ were incubated with target cells expressing individual MHC class I molecules, and ligand recognition was detected by the MHC class I-dependent upregulation of luciferase. Using this approach, we found that Mamu-KIR3DL01, -KIR3DL06, -KIR3DL08 and –KIR3DSw08 all recognize Mamu-Bw4 molecules, but with varying allotype specificity. In contrast, Mamu-KIR3DL05 recognizes Mamu-A and -A-related molecules, including Mamu-A1*002 and A3*13, Mamu-B*036, the product of a recombinant Mamu-B allele with α1 and α2 domain sequences derived from a MHC-A gene, and Mamu-AG*01, a non-classical molecule expressed on placental trophoblasts that originated from an ancestral duplication of a MHC-A gene. These results reveal an expansion of the lineage II KIRs in macaques that recognize Bw4 ligands, and identify a non-classical molecule implicated in placental development and pregnancy as a ligand for Mamu-KIR3DL05. In addition to offering new insights into KIR-MHC class I co-evolution, these findings provide an important foundation for investigating the role of NK cells in the rhesus macaque as an animal model for infectious diseases and reproductive biology.

Introduction

Natural killer cells provide an important early defense against viruses and tumors by virtue of their ability to respond to infected or malignant cells without prior exposure to antigen. In humans and other primate species, NK cells differentiate healthy cells from unhealthy cells through interactions between two highly polymorphic and rapidly evolving sets of molecules; the killer cell Ig-like receptors (KIRs) expressed on the surface of NK cells and their MHC class I ligands on target cells (1, 2). As reflected by their nomenclature, KIRs typically have two or three extracellular Ig-like domains (2D or 3D) and either long (L) or short (S) cytoplasmic tails. Whereas receptors with long cytoplasmic tails contain a pair of ITIMs that transduce inhibitory signals, KIRs with short cytoplasmic tails are dependent on transmembrane domain interactions with ITAM-containing adaptor molecules such as DAP12 to transduce activating signals. Inhibitory KIRs normally suppress NK cell responses following interactions with MHC class I ligands on the surface of heathy cells; however, if these interactions are disrupted, for instance as a result of MHC class I downregulation by viral infection or deletion of MHC class I genes from tumors, the loss of this inhibition can trigger an NK cell response (3–7). Activating KIRs also interact with MHC class I molecules, and although the precise nature of ligand recognition by these receptors is not as well defined, they are able to sense changes on the surface of unhealthy cells to trigger NK cell activation (2, 8–10).

KIR and HLA class I polymorphisms are associated with differences in the course of infection for a number of viral pathogens and with the outcome of cancer immunotherapy (11–18); however, studies to address the immunological mechanisms underlying these observations have been limited by the lack of a suitable animal model. Mice and other rodent species do not have KIRs, but instead express polymorphic C type lectin-like molecules on the surface of NK cells as MHC class I receptors (2, 19). Moreover, owing to the rapid pace of KIR and MHC class I evolution in primates (20–22), it is not possible to accurately predict the ligands for KIRs in Old World monkeys on the basis of sequence similarity with human receptors.

In contrast to humans, which have three classical HLA class I genes (HLA-A, -B and –C), macaques and other Old World monkeys express an expanded set of polymorphic MHC class I genes that correspond to HLA-A and –B, but they do not have a C locus, since HLA-C represents a relatively recent duplication of a MHC-B gene that occurred after the separation of apes from Old World monkeys (23, 24). MHC class I haplotypes in the rhesus macaque are polygenic, and typically contain two or three Macaca mulatta (Mamu)-A genes and four to eleven expressed Mamu-B genes (25–27). Depending on their transcriptional abundance, these genes can be further classified as ‘major’ or ‘minor’ loci, with products of the major genes exhibiting the greatest polymorphism and most frequent presentation of viral peptides to CD8+ T cells (27). Macaques accordingly lack lineage III KIR that encode KIR2DL/S receptors for HLA-C, but have expanded their lineage II KIR encoding KIR3DL/S receptors for Mamu-A and -B molecules (21, 28–32). Whereas humans have two lineage II KIR genes, KIR3DL1/S1 and KIR3DL2, that encode receptors for HLA-Bw4 and –A3/A11 allotypes, respectively, rhesus macaques have as many as ten lineage II Mamu-KIR3DL genes and nine Mamu-KIR3DS genes (30, 32–34).

In addition to an expanded repertoire of polymorphic Mamu-A and -B genes, rhesus macaques also express several non-classical MHC class I genes, including Mamu-E, -F, -AG and -I. As a consequence of the remarkable conservation of MHC-E and -F in primates, Mamu-E and -F share extensive sequence similarity with HLA-E and -F (35, 36). Mamu-E serves a similar function as HLA-E in the presentation of unconventional pathogen-derived peptides to CD8+ T cells and the regulation of NK cell responses through interactions with heterodimeric CD94/NKG2 receptors (37), which are also well conserved in macaques (38). The function of Mamu-F is less clear. While open conformers of HLA-F were recently identified as ligands for KIR3DS1 and KIR3DL2 (10, 39, 40), similar interactions cannot be inferred for Mamu-F, since macaques do not express KIRs related to these receptors.

Unlike Mamu-E and -F, orthologs of Mamu-AG and -I are not found in humans. Whereas Mamu-AG is the product of an ancestral duplication of a MHC-A gene with counterparts in other species of macaques and Old World monkeys (41), Mamu-I represents a comparatively recent duplication of a MHC-B gene that is unique to macaques (42). Similar to HLA-G, Mamu-AG exhibits limited polymorphism, a truncated cytoplasmic domain, and is only expressed on placental trophoblasts at the maternal-fetal interface (41, 43–45). Because of these features, and inactivation of the Mamu-G gene (46), Mamu-AG is believed to have evolved a similar function as HLA-G in placental development. By comparison, Mamu-I is more broadly expressed and has molecular properties typical of a MHC-B molecule; however, as the product of a fixed gene with limited sequence variation, Mamu-I exhibits features of both classical and non-classical molecules (42).

A few MHC class I ligands have recently been identified for rhesus macaque KIRs. The serendipitous discovery of reagents for staining Mamu-KIR3DL05 and -KIR3DL01 on primary NK cells enabled functional assays that led to the identification of Mamu-A1*002 as a ligand for Mamu-KIR3DL05 (47), and multiple Mamu-Bw4 allotypes as ligands for Mamu-KIR3DL01 (48). Several Mamu-A molecules were also identified as binding partners for Mamu-KIR3DL05, −3DLW03, −3DL11 and 3DS05 by staining MHC class I-transfected cells with KIR-Fc fusion proteins (49). However, ligands for the majority of rhesus macaque KIRs remain undefined. We therefore developed a novel approach based on the co-incubation of reporter cells expressing chimeric KIR-CD28-CD3ζ receptors with target cells expressing defined MHC class I molecules to identify additional KIR ligands. Our results reveal an expansion of the lineage II KIRs in rhesus macaques that recognize Bw4 ligands, and identify Mamu-A and -A-related molecules, including a non-classical molecule implicated in pregnancy success, as ligands for Mamu-KIR3DL05.

Materials and Methods

KIR-CD28-CD3ζ-expressing Jurkat NFAT reporter cell lines

Complementary DNA sequences encoding the leader peptide of Mamu-KIR3DL05*008 (exons 1 and 2, nucleotides 1–63) and the D0, D1, D2 and stem domains of Mamu-KIR3DL01*012, -KIR3DL05*008, -KIR3DL06*001, -KIR3DL08*002, and -KIR3DSw08*006 (exons 3–6, nucleotides 64–1029) were synthesized (Thermo Fisher GeneArt and IDT) and cloned into pQCXIP in-frame with downstream sequences encoding the membrane-spanning and cytoplasmic domains, respectively, of rhesus macaque CD28 (nucleotides 340–660) and CD3ζ (nucleotides 154–501). The resulting retroviral vectors were packaged into VSV G-pseudotyped murine leukemia virus (MLV) particles by co-transfecting GP2–293 cells with each pQCXIP-KIR-CD28-CD3ζ construct and pVSV-G (Clontech Laboratories). Jurkat NFAT luciferase (JNL) cells (Signosis) were incubated overnight with supernatant collected from transfected GP2–293 cells, and two days later, were placed under selection in RPMI medium supplemented with 10% FBS, L-glutamine, penicillin and streptomycin (R10 medium) plus 0.2 mg/ml hygromycin to maintain the luciferase reporter gene and 1 μg/ml puromycin to select for the KIR-CD28-CD3ζ receptor (Invitrogen). After three to six weeks of selection, the surface expression of chimeric KIR-CD28-CD3ζ (KIR-28Z) receptors was confirmed by staining transduced JNL cells with a goat polyclonal antibody to CD28 (R&D Systems) followed by an Alexa647-conjugated rabbit anti-goat antibody (Invitrogen). KIR-28Z JNL cells were maintained in R10 medium containing 0.2 mg/ml hygromycin and 1 μg/ml puromycin. The Genbank accession numbers for rhesus macaque KIR, CD28 and CD3ζ sequences are as follows: Mamu-KIR3DL01*012 (GU112286.1), -KIR3DL04*001:003 (GU112319.1), -KIR3DL05*008 (GU112291.1), -KIR3DL06*001 (EU419056.1), -KIR3DL08*002 (GU112306.1), and -KIR3DSw08*006 (FN424254.1), CD28 (DQ789898) and CD3ζ(DQ437669).

721.221 cell lines expressing rhesus macaque MHC class I molecules

Complementary DNA sequences encoding rhesus macaque MHC class I molecules were cloned into pQCXIP or pQCXIN. The retroviral vectors were packaged into VSV G-pseudotyped MLV particles by co-transfecting GP2–293 cells with pQCXIP or pQCXIN constructs and pVSV-G (Clontech Laboratories). 721.221 cells were incubated overnight with supernatant collected from transfected GP2–293 cells, and two days later, were placed under selection in R10 medium containing 0.4 μg/ml puromycin or 0.5 mg/ml G418 (Calbiochem), depending on the retroviral vector. After three to six weeks of selection, MHC class I surface expression was confirmed by flow cytometry using a PE-conjugated antibody (clone W6/32, Life Technologies). MHC class I-transduced 721.221 cells were maintained in R10 medium containing either 0.4 μg/ml puromycin or 0.5 mg/ml G418, depending on the vector. The Genbank accession numbers for rhesus macaque MHC class I sequences are as follows: Mamu-A1*001:01 (LT908117), -A1*002:01 (LN899621), -A1*004:01:01 (LM607985), -A2*05:02:01 (LN899639), -A1*008:01:01 (LN899628), -A1*011:01 (AJ542579), -A1*012:01 (AF157398.1), -A3*13:03 (AF157401), -B*001:01:02 (LM608018), -B*002:01 (LN851852), -B*005:01 (LM608023), -B*007:01 (U41829.1), -B*008:01 (U41830), -B*015:01 (AM902541.1), -B*017:01:01 (AF199358), -B*022:01 (LN899675), -B*036:01:01 (AJ556886.1), -B*041:01 (LN899682), -B*043:01 (AJ556893), -B*045:03 (LM608047), -B*056:01 (GQ902079.1), -B*065:01 (AJ620416.1), -I*01:01:01 (LT908864), -E*02:01 (U02978), -F*01:01 (LT899414) and -AG*01:01 (U84783.1).

Ligand identification

KIR-CD28-CD3ζ+ JNL cells were incubated overnight with MHC class I-expressing 721.221 cells, or MHC class I-negative parental 721.221 cells, in R10 medium without G418, hygromycin or puromycin at 1:1 effector to target ratios (1×105 JNL cells and 1×105 721.221 cells in 100 μl R10 medium) in triplicate wells of white 96-well plates. For blocking experiments, 721.221 cells expressing Mamu-AG were pre-incubated with a mouse monoclonal antibody to Mamu-AG (clone 25D3) (43) or an isotype control antibody (clone 11513) for 1 hour at 37° C before the addition KIR3DL05–28Z JNL cells. After an overnight incubation, 100 μl of BriteLite Plus luciferase substrate (PerkinElmer) was added to each well, and luciferase activity was measured approximately 2 minutes later using a Victor X4 multiplate reader (PerkinElmer). The fold-induction of luciferase activity was calculated by dividing the mean relative light units (RLU) of luciferase upregulation by the KIR-28Z JNL cells incubated with MHC class I-transduced 721.221 cells by the mean RLU for KIR-28Z JNL cells incubated with MHC class I-deficient parental 721.221 cells.

Statistical analysis

RLU values for KIR-28Z JNL responses to 721.221 cells expressing rhesus macaque MHC class I molecules were analyzed by one-way ANOVA, followed by Dunnett’s test to compare differences in mean RLU responses to MHC class I-expressing 721.221 cells to mean RLU responses to the MHC class I-deficient parental 721.221 cell line as a negative control. For the analysis of responses to the Mamu-B*065:01 variants, mean RLU values in response to 721.221 cells expressing each variant were compared to mean RLU values in response to 721.221 cells expressing the wild-type Mamu-B*065:01 molecule using an unpaired t test. Statistical analyses were performed using GraphPad Prism 6 for Mac OS X version 6.0h.

Results

Recognition of Bw4 ligands by multiple rhesus macaque KIRs

The identification of ligands for Mamu-KIR3DL01 and -KIR3DL05 was facilitated by the availability of reagents for staining these KIRs on primary NK cells (47, 48); however, similar reagents are not available for differentiating other macaque KIRs. We therefore developed a novel assay based on the use of combinations of reporter and target cell lines to identify additional KIR ligands in this species. Jurkat cells containing an NFAT-inducible luciferase reporter gene (JNL cells) were transduced with retroviral vectors expressing the D0, D1, D2 and stem domains of rhesus macaque KIRs fused to the transmembrane and cytoplasmic domains of macaque CD28 and CD3ζ to generate reporter cell lines expressing chimeric KIR-CD28-CD3ζ (KIR-28Z) receptors with the extracellular domains of Mamu-KIR3DL01*012 (KIR3DL01), -KIR3DL05*008 (KIR3DL05), -KIR3DL06*001 (KIR3DL06), -KIR3DL08*002 (KIR3DL08) and - KIR3DSw08*006 (KIR3DSw08) (Fig. 1). Following an overnight incubation with 721.221 cell lines expressing 26 different rhesus macaque MHC class I molecules, ligand recognition was detected by the MHC class I-dependent upregulation of luciferase by the KIR-28Z JNL cells.

FIGURE 1.

FIGURE 1.

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KIR-CD28-CD3ζ chimeric receptors. Sequences encoding the leader peptide of Mamu-KIR3DL05*008 (residues −21−−1, maroon) and the D0, D1, D2 and stem domains of Mamu-KIR3DL01*001, -KIR3DL05*008, -KIR3DL06*001, -KIR3DL08*002, and -KIR3DSw08*006 (residues 1–322, magenta) were synthesized and cloned into pQCXIP in-frame with downstream sequences encoding the membrane-spanning and cytoplasmic domains, respectively, of rhesus macaque CD28 (residues 114–220, purple) and CD3ζ (residues 52–166, blue). The transmembrane region of CD28 is shaded black. The corresponding nucleotide sequences and Genbank accession numbers for each of the domains are specified in the Materials and Methods section.

In accordance with previously identified ligands for Mamu-KIR3DL01 (48), KIR3DL01–28Z JNL cells responded strongly to 721.221 cells expressing Mamu-B*007, and to a lesser extent, to cells expressing Mamu-B*065 and -B*041 (Fig. 2A). These cells also responded to Mamu-B*043 (Fig. 2A). Surprisingly, KIR3DL06-, KIR3DL08- and KIR3DSw08–28Z JNL cells responded to many of the same ligands; however, differences in allotype specificity were noted. Whereas all four KIRs recognized Mamu-B*007, -B*041 and -B*065, other responses were detected that were not shared by these receptors (Figs. 2B–2D). KIR3DL06, KIR3DL08 and KIR3DSw08, but not KIR3DL01, efficiently recognized Mamu-B*022 (Figs. 2B–2D). Moreover, while KIR3DL08–28Z JNL cells did not respond to Mamu-B*043, these cells exhibited a particularly strong response to Mamu-B*002 that was not observed for any of the other KIRs (Fig. 2C). Hence, these results identify an overlapping, but not identical, set of Mamu-B molecules as ligands for KIR3DL01, KIR3DL06, KIR3DL08 and KIR3DSw08.

FIGURE 2.

FIGURE 2.

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Recognition of Mamu-Bw4 ligands by four different rhesus macaque KIRs. JNL cells expressing the extracellular domains of Mamu-KIR3DL01 (A), -KIR3DL06 (B), -KIR3DL08 (C) and -KIR3DSw08 (D) fused to the transmembrane and cytoplasmic domains of macaque CD28 and CD3ζ, respectively, were incubated overnight at a 1:1 E:T ratio with 721.221 cells expressing the indicated rhesus macaque MHC class I molecules, and ligand recognition was detected by the upregulation of luciferase in response to specific MHC class I molecules. Fold-induction was calculated by dividing the average relative light units (RLU) of luciferase activity from triplicate wells of KIR-28Z JNL cells plated with 721.221 cells expressing a given MHC class I molecule by the average RLU of KIR-28Z JNL cells plated with parental 721.221 cells. Bars are color-coded according to products of the classical Mamu-A (red), Mamu-B (blue) and non-classical Mamu-I, -E, -F and -AG (purple) genes. Error bars indicate standard deviations of the mean and asterisks denote significant differences (****p<0.0001, one-way ANOVA with Dunnett’s test). The data shown is representative of results obtained in at least three independent experiments. (E) An alignment showing the predicted amino acid sequences of Mamu-B*002:01, -B*007:01, -B*041:01,-B*065:01, -B*022:01, and -B*043:01 to Mamu-B*017:01. Residues 77–83 corresponding to the Bw4 motif are underlined and predicted KIR contact sites based on the crystal structure of HLA-B*57 in complex with KIR3DL1*001 are shaded (52). Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code.

The magnitude of responses did not, however, correspond to differences in either KIR-28Z expression on JNL cells or MHC class I expression on 721.221 cells. Expression of the chimeric receptors on JNL cells was confirmed by CD28 staining (Fig. 3A), but did not reflect differences in luciferase induction (Fig. 2). The reasons for this are presently unclear, but may reflect variation among the cell lines in the responsiveness of the luciferase reporter gene and/or KIR affinity for MHC class I ligands. Likewise, although MHC class I staining confirmed the presence of rhesus macaque molecules on the surface of 721.221 cells (Fig. 3B), differences in MHC class I expression levels did not correspond to differences in luciferase induction. In this case, it is difficult to differentiate variation in KIR affinity for MHC class I ligands from differences in expression levels. Thus, while MHC class I recognition by individual KIR-28Z JNL cells is internally controlled by measuring the fold-induction of luciferase in response to each of the MHC class I-expressing 721.221 cells relative to MHC class I-deficient parental 721.221 cells, differences in the magnitude of these responses should be interpreted with some caution.

FIGURE 3.

FIGURE 3.

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Surface expression of KIR-28Z receptors on JNL cells and MHC class I molecules on 721.221 cells. (A) KIR-28Z JNL cells were stained with a goat polyclonal antibody to CD28 followed by an Alexa647-conjugated rabbit anti-goat antibody and Near IR live dead stain. (B) 721.221 cell lines expressing the indicated rhesus macaque MHC class I molecules were stained with a PE-conjugated pan-MHC class I-specific monoclonal antibody (W6/32) and Near IR live/dead stain. After excluding dead cells, the fluorescence intensity of staining on the surface of KIR-28Z-expressing JNL cells and MHC class I-expressing 721.221 cells (shaded) was compared, respectively, to staining on the surface of the parental JNL and 721.221 cell lines (open). Flow cytometry data was analyzed using FlowJo 9.9 software.

Mamu-KIR3DL01 was previously shown to recognize MHC class I ligands with a Bw4 motif at residues 77–83 (48). We therefore compared MHC class I sequences in this region to determine if this motif accounted for the similar pattern of ligand recognition by KIR3DL01, KIR3DL06, KIR3DL08 and KIR3DSw08. HLA-Bw4 molecules typically have either an asparagine, an aspartic acid or a serine at position 77, an isoleucine or a threonine at position 80, an alanine or a leucine at position 81, a leucine at position 82 and an arginine at position 83 (N/D/S77I/T80A/L81L82R83) (50, 51). By contrast, HLA-Bw6 molecules are characterized by a serine or an asparagine at position 77, an arginine at position 82 and a glycine at position 83 (S/N77R82G83) (50, 51). Using these definitions, most of the Mamu-A and -B molecules tested could be classified as Bw4 or Bw6 allotypes (Table I). With the exception of Mamu-B*002, all of the molecules identified as KIR ligands contain a Bw4 motif (Table I). Moreover, four of these molecules (Mamu-B*007, -B*022, - B*041 and -B*065) have identical Bw4 sequences (Fig. 2E). These results therefore suggest that KIR3DL01, KIR3DL06, KIR3DL08 and KIR3DSw08 predominantly recognize Mamu-Bw4 molecules.

Table I.

Rhesus macaque MHC class I molecules evaluated for KIR recognition

Molecule 77–83a Motifb Nef↓c Molecule 77–83a Motifb Nef↓c
A1*001:01 NLRTLLR Bw4 Yes B*015:01 NLRTLLR Bw4 Yes
A1*002:01 NLRNLRG Bw6 Yes B*017:01 NLRTALR Bw4 Yes
A1*004:01 ALRNLRG Bw6 Yes B*022:01 NLRTALR Bw4 No
A1*008:01 GLQNLRG Bw6 Yes B*036:01 DLGTLLR Bw4 Yes
A1*011:01 NLRTALR Bw4 Yes B*041:01 NLRTALR Bw4 No
A1*012:01 SLRNLRG Bw6 Yes B*043:01 SLRTLLR Bw4 NT
A2*05:02 NLRTLLR Bw4 Yes B*045:03 NLRTLRG Bw6 Yes
A3*13:03 ALRNLRG Bw6 Yes B*056:01 NLRTALR Bw4 Yes
B*001:01 NLRIALS non Yes B*065:01 NLRTALR Bw4 No
B*002:01 GLGNLRG Bw6 Yes I*01:01 NLRTALR Bw4 No
B*005:01 SLRNLRG Bw6 NT E*02:01 NLETLRG non No
B*007:01 NLRTALR Bw4 No F*01:01 RVALRKL non NT
B*008:01 DLGTLRG non Yes AG*02:01 NLRTLLR Bw4 NT
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a

Amino acid sequence at positions 77–83 of the α1 domain.

b

Predicted motif corresponding to residues residues 77–83; Bw4, Bw6 or neither (non) (50, 51).

c

Susceptibility of cytoplasmic domain sequences to downmodulation by the SIV Nef protein; Yes, No or not tested (NT) (54).

The presence of a Bw4 motif, however, was not sufficient to predict ligand recognition, since several Bw4 molecules, including Mamu-B*015, -B*017, -B*056 and -I*01, were not recognized by any of the KIRs (Table I). Furthermore, KIR3DL08 recognition of Mamu-B*002 (Fig. 2C), a molecule that contains a sequence more similar to a Bw6 motif (Table I), suggests that this KIR is not strictly dependent on contacts with Bw4 residues. While these observations complicate efforts to define ligands for macaque KIRs, they are nonetheless consistent with crystal structures revealing extensive contacts between human KIR and surfaces of the α1- and α2-domains of their HLA class I ligands (52, 53), as well as a mutagenic analysis identifying residues outside of the Bw4 motif that affect recognition by KIR3DL01 (48).

Interestingly, all of the Mamu-Bw4 molecules identified as KIR ligands have distinct cytoplasmic domain sequences from molecules known to present viral peptides to CD8+ T cells. Whereas Mamu-B*008 and -B*017 have cytoplasmic tails that are nearly identical to HLA-B, and are known to restrict SIV-specific CD8+ T cell responses (54–56), Mamu-B*007, -B*022, -B*041, -B*043 and -B*065 have unique cytoplasmic domain sequences and have not been implicated in CD8+ T cell responses (Fig. 2E) (54). Moreover, we previously demonstrated that molecules with cytoplasmic tails identical to Mamu-B*008 or -B*017 are subject to downmodulation by the SIV Nef protein, whereas molecules with cytoplasmic tails matching Mamu-B*007, -B*022, -B*041 or -B*065 are resistant to downmodulation by Nef (Table I) (54). Although the cytoplasmic domain of Mamu-B*043 was not specifically tested, it shares most of the same polymorphisms as Mamu-B*022 (Fig. 2E) and is therefore also expected to be resistant to Nef (54). These observations suggest a possible functional specialization for molecules encoded by different Mamu-B genes, where the products of certain loci play a dominant role in CD8+ T cell responses, while others predominantly serve as KIR ligands to regulate NK cell responses.

To further investigate the Bw4-specificity of KIR3DL01, KIR3DL06, KIR3DL08 and KIR3DSw08, and to determine if these KIRs differ in their dependence on Bw4 residues for ligand recognition, KIR-28Z JNL cells were tested for recognition of 721.221 cells expressing Mamu-B*065 variants in which Bw4 residues 77–83 were replaced, individually or in combination, with the corresponding Bw6 residues at each position (Fig. 4). An additional Mamu-B*065 variant with a glycine-to-glutamic acid change at position 76 (G76E), which was previously shown to abrogate KIR3DL01 recognition of Mamu-B*017 (48), was also tested. KIR3DL01- and KIR3DL06–28Z JNL cells exhibited similar responses to each of the Mamu-B*065 variants. In each case, Mamu-B*065 recognition was reduced to similar levels as the Bw6 control by the G76E and R83G substitutions, partially impaired by T80N and L82R, and unaffected by the N77S and A81L substitution (Fig. 4A & 4B). The pattern of responses differed, however, for KIR3DL08 and KIR3DSw08. The recognition of Mamu-B*065 by KIR3DL08 was fully abrogated by G76E, N77S, and T80N, partially impaired by L82R and R83G, and unaffected by A81L (Fig. 4C). The less stringent dependence of KIR3DL08 on R83 may account for the ability of this receptor to recognize Mamu-B*002 (Fig. 2C). In the case of KIR3DSw08, Mamu-B*065 recognition was completely disrupted or severely attenuated by G76E, N77S, T80N and R83G, but only partially impaired by A81L and L82R (Fig. 4D). KIR3DSw08 recognition of Mamu-Bw4 molecules is consistent with the evolution of this activating KIR from an inhibitory receptor with similar specificity, and with recent evidence for human KIR3DS1 recognition of HLA-Bw4 ligands (9). These results therefore confirm the recognition of Bw4 ligands by KIR3DL01, KIR3DL06, KIR3DL08 and KIR3DSw08, and reveal differences in the influence of specific Bw4 residues on interactions with these receptors.

FIGURE 4.

FIGURE 4.

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Bw4 substitutions differentially affect ligand recognition by Mamu-KIR3DL01, - KIR3DL06, -KIR3DL08 and -KIR3DSw08. KIR3DL01- (A), KIR3DL06- (B), KIR3DL08- (C) and KIR3DSw08- (D) 28Z JNL cells were incubated overnight at a 1:1 E:T ratio with 721.221 cells expressing wild-type Mamu-B*065:01 (Bw4) or Mamu-B*065:01 mutants with the indicated amino acid substitutions, either individually or combined (Bw6), corresponding to Bw6 residues at positions 77–83 in the α1 domain. An additional Mamu-B*065:01 mutant with a glycine-to-glutamate substitution at position 76 (G76E), which corresponds to a polymorphism in Mamu-B*017 previously shown to abrogate recognition by Mamu-KIR3DL01 (48), was also tested. Fold-induction was calculated by dividing the RLU of luciferase activity from triplicate wells of KIR-28Z JNL cells plated with 721.221 cells expressing each Mamu-B*065:01 variant by the average RLU of KIR-28Z JNL cells plated with parental 721.221 cells. Error bars indicate standard deviations of the mean and asterisks denote significant differences relative to the wild-type Mamu-B*065:01 (Bw4) molecule (ns: not significant, *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001, unpaired t test). The data shown is representative of three independent experiments.

KIR3DL05 recognizes Mamu-A and -A-related molecules, including a non-classical molecule expressed in the placenta

Consistent with previous studies identifying Mamu-A1*002 and -A3*13 as ligands for KIR3DL05 (47, 49), KIR3DL05–28Z JNL cells responded strongly to 721.221 cells expressing each of these molecules (Fig. 5A). Further analysis revealed the recognition of two additional molecules by KIR3DL05 that had not previously been identified as ligands for this KIR; Mamu-B*036 and -AG*01. Whereas Mamu-B*036 is the product of a recombinant allele encoding the extracellular domains (exons 1–5) of a MHC-A molecule and the cytoplasmic domain (exons 6–7) of a MHC-B molecule (Fig. 5C), Mamu-AG*01 is the product of an ancestral duplication of a MHC-A gene that is expressed on cytotrophoblasts and villous syncytiotrophoblasts of the placenta (41, 43). As a consequence of their evolutionary origins as MHC-A molecules, all of the ligands for KIR3DL05 share similar sequences in their α1 and α2 domains (Fig. 5C). Unlike the other KIRs, however, the ligands for KIR3DL05 have either Bw4 (Mamu-A1*002 and -A3*13) or Bw6 (Mamu-AG*01 and -B*036) motifs at positions 77–83 (Fig. 5C), consistent with previous mutagenic analyses showing that KIR3DL05 can bind to molecules with either of these motifs (49).

FIGURE 5.

FIGURE 5.

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Mamu-KIR3DL05 recognizes Mamu-A and A-like molecules. (A) KIR3DL05–28Z JNL cells were incubated overnight at a 1:1 E:T ratio with 721.221 cells expressing the indicated classical Mamu-A (red), Mamu-B (blue) and non-classical Mamu-I, -E, -F and -AG (purple) molecules. Fold-induction was calculated by dividing the RLU of luciferase activity for triplicate wells of KIR3DL05–28Z JNL cells plated with MHC class I-expressing 721.221 cells by the average RLU of KIR3DL05–28Z JNL cells plated with MHC class I-negative parental 721.221 cells. Error bars indicate standard deviations of the mean and asterisks denote significant differences (****p<0.0001, one-way ANOVA with Dunnett’s test). The data shown is representative of three independent experiments. (B) KIR3DL05–28Z JNL cells were incubated with 721.221 cells expressing Mamu-AG*01:01 in the presence of increasing concentrations of a monoclonal antibody to Mamu-AG (25D3) or an isotype control antibody (11513). The plotted values represent the average and standard deviation (error bars) of luciferase activity (RLU) in triplicate wells at each antibody concentration. (C) An alignment comparing the predicted amino acid sequences of four different KIR3DL05 ligands; Mamu-A1*002:01, -A3*13:03, -AG*01:01 and -B*036:01. Residues 77–83 corresponding to the Bw6 motif of Mamu-A1*002 are underlined in the consensus sequence. Positions of identity are indicated by periods and amino acid differences are identified by their single-letter code.

To further verify the recognition of Mamu-AG*01 by KIR3DL05, we took advantage of the availability of a monoclonal antibody to Mamu-AG that was previously used to determine its pattern of expression in the placenta and its importance in pregnant rhesus macaques (43, 57). In accordance with KIR3DL05 recognition of Mamu-AG*01, the Mamu-AG-specific antibody 25D3, but not an isotype control antibody (11513), potently inhibited KIR3DL05–28Z JNL responses in a dose dependent manner (Fig. 5B), thereby corroborating Mamu-AG*01 as a ligand for KIR3DL05.

Discussion

Identification of the MHC class I ligands for rhesus macaque KIRs is important for studying NK cell responses in this species as a model for AIDS and other infectious diseases (58–61), and affords valuable insights into the comparative immunogenetics of KIR-MHC class I co-evolution in primates. Although a few MHC class I ligands have recently been identified for macaque KIRs (47–49, 62), molecular interactions for the majority of these receptors remain undefined due in part to the lack of reagents for differentiating KIRs in non-human primates. To identify additional ligands, we therefore developed a novel assay that does not depend on the availability of KIR-specific antibodies. This assay is based on the co-incubation of NFAT-responsive reporter cells expressing chimeric KIR-CD28-CD3ζ receptors with target cells expressing individual MHC class I molecules. Using this approach, we found that Mamu-KIR3DL01, -KIR3DL06, -KIR3DL08 and –KIR3DSw08 recognize Mamu-Bw4 molecules with varying allotype specificity, and that Mamu-KIR3DL05 recognizes Mamu-A and -A-related molecules, including a non-classical molecule implicated in placental development and pregnancy success.

The recognition of Mamu-Bw4 ligands by four different KIRs reveals an expansion of the lineage II KIR genes of rhesus macaques encoding receptors with overlapping Bw4 specificity. Differences in allotype specificity and sensitivity to amino acid substitutions in Bw4 residues further indicate diversification of ligand recognition. The presence of a Bw4 motif was not, however, sufficient to predict ligand recognition, since several molecules with Bw4 sequences at positions 77–83 were not recognized by any of the KIRs. Although this complicates the definition of ligands for macaque KIRs, these results are consistent with structural studies revealing additional α1 and α2 domain contacts with human KIRs (52, 53), and with mutational analyses identifying residues outside of the Bw4/Bw6 motif that affect recognition by human and macaque KIRs (48, 51). Indeed, three amino acid differences at positions 76, 142 and 149 were previously shown to account the recognition of Mamu-B*065, but not Mamu-B*017, by KIR3DL01, despite the presence of identical Bw4 residues in both molecules (48).

Similar to human KIRs, the KIR genes of rhesus macaques are also highly polymorphic. Thirty alleles of KIR3DL01, two alleles of KIR3DL06, thirteen alleles of KIR3DL08, and thirteen alleles of KIR3DSw08 have been identified so far (32, 63). Since polymorphisms in human KIR3DL1 have been shown to affect KIR3DL1 specificity for different allomorphs of HLA-Bw4 (64), and only a single representative allele of each the macaque KIR genes was analyzed, the ligands identified for these KIRs should be interpreted with some caution. Nevertheless, in marked contrast to humans, which only have a single polymorphic KIR3DL1/S1 locus that encodes receptors for HLA-Bw4, rhesus macaques appear to have at least four KIR genes that encode receptors for Mamu-Bw4 ligands. Hence, the picture that emerges is one of expansion of the lineage II KIR genes in macaques, followed by extensive diversification of the genes encoding receptors for Bw4 molecules.

This pattern of KIR evolution mirrors the course of MHC class I evolution in macaques. In contrast to the HLA class I diversity of humans, which reflects high levels of allelic variation at three loci (HLA-A, -B and -C), the MHC class I diversity of macaques is a function of extensive gene duplication with limited allelic variation (25, 27, 65). MHC class I haplotypes of the rhesus macaque typically contain two or three Mamu-A genes and four to eleven Mamu-B genes that can be classified on the basis of transcriptional abundance, and hence predicted protein expression, as either ‘majors’ or ‘minors’ (25–27). Most haplotypes express a single ‘major’ and one or two ‘minor’ Mamu-A genes in combination with two or three ‘major’ and three to six ‘minor’ Mamu-B genes (27).

While all of the Mamu-Bw4 molecules identified as ligands for rhesus macaque KIRs are products of ‘major’ Mamu-B genes, these are not the same molecules known to present viral peptides to CD8+ T cells. Phylogenetic and segregation analyses suggest that the Mamu-Bw4 molecules identified as KIR ligands, including Mamu-B*007, - B*022, -B*041, -B*043 and -B*065, are encoded by distinct genetic loci from molecules such as Mamu-B*008 and -B*017 that present SIV epitopes for recognition by CD8+ T cells (25, 27, 65–67). Furthermore, whereas the Mamu-B molecules that restrict virus-specific CD8+ T cell responses have cytoplasmic domain sequences that are nearly identical to the cytoplasmic tails of HLA-B molecules, with the exception of Mamu-B*002, all of the Mamu-B molecules identified as KIR ligands have distinctive polymorphisms in their cytoplasmic domains. Because these molecules have not been implicated in CD8+ T cell responses, to our knowledge there is currently no information about their capacity to present peptides or affect the course of SIV infection.

We previously demonstrated that similar to HIV-1 Nef, which selectively downmodulates HLA-A and -B, but not HLA-C, thereby reducing the susceptibility of virus-infected cells to killing by CD8+ T cells (5, 6, 68), SIV Nef also selectively downmodulates Mamu-A and -B molecules on the basis of amino acid differences in their cytoplasmic domains (54). As these differences correspond to the same polymorphisms in the cytoplasmic domain that distinguish Mamu-Bw4 molecules that serve as KIR ligands from molecules known to restrict CD8+ T cell responses (54), these observations suggest a functional dichotomy analogous to HLA-B versus HLA-C, where highly polymorphic products of the HLA-B locus responsible for the majority of virus-specific CD8+ T cell responses are subject to downmodulation by HIV-1 Nef, while the comparatively less polymorphic products of the HLA-C locus that play a dominant role in regulating NK cell responses are resistant to Nef-mediated downmodulation.

Mamu-KIR3DL05 recognizes a comparatively broader range of Mamu-A and -A-related molecules with either Bw4 or Bw6 motifs in their α1 domains. The ligands for Mamu-KIR3DL05 include Mamu-A1*002 and -A3*13 (47, 49), Mamu-B*036, which is the product of a recombinant Mamu-B allele that acquired exons 1–5 encoding the extracellular and transmembrane domains of a MHC-A gene, and Mamu-AG*01, a non-classical molecule that arose from an ancestral duplication of a MHC-A gene (41). In the case of Mamu-A1*002, we previously demonstrated that Mamu-KIR3DL05 recognition of this molecule is also strongly influenced by MHC class I-bound peptides (47, 69). SIV peptides were identified that either stabilized or disrupted Mamu-A1*002 interactions with Mamu-KIR3DL05, leading alternately to NK cell inhibition or activation (69). In this regard, the peptide-dependent recognition of Mamu-A1*002 by Mamu-KIR3DL05 is comparable to human KIR3DL2, which recognizes HLA-A*03 and -A*11 molecules in complex with certain viral peptides (70).

The recognition of Mamu-AG*01 by Mamu-KIR3DL05 suggests that this interaction may also participate in placental development. During early stages of human pregnancy, fetal trophoblasts that invade and remodel the decidual spiral arteries of the uterus express HLA-G, which is thought to promote pregnancy success by regulating NK cells and macrophages of the placental decidua (71–73). In lieu of a direct ortholog of HLA-G (Mamu-G is a pseudogene) (46), rhesus macaques express Mamu-AG, which has similar molecular features and tissue distribution as HLA-G (41). Like HLA-G, Mamu-AG exhibits limited polymorphism, a truncated cytoplasmic domain, and is developmentally expressed on cytotrophoblasts and syncytiotrophopblasts at the maternal-fetal interface (41, 43). KIR2DL4 and ILT-2, which have been identified as receptors for HLA-G (72, 74, 75), are also expressed in the macaque decidua (76, 77); however, there is currently no evidence for the recognition of Mamu-AG by these receptors. To our knowledge, Mamu-KIR3DL05 is therefore the first and only receptor to be identified for Mamu-AG. Nevertheless, since the gene frequency of Mamu-KIR3DL05 in rhesus macaques is 40–60% (33, 47), this KIR is not expressed in all animals. Thus, while Mamu-KIR3DL05 may facilitate pregnancy by contributing to the functional redundancy of NK cell inhibition in the placenta, it is probably not the only receptor for Mamu-AG.

In conclusion, the present study identifies an overlapping, but non-redundant, set of Mamu-Bw4 molecules as ligands for Mamu-KIR3DL01, -KIR3DL06, -KIR3DL08 and -KIR3DSw08, and four different Mamu-A and A-related molecules as ligands for Mamu-KIR3DL05. These findings are consistent with the expansion of the lineage II KIR genes of macaques in concert with extensive duplication of their MHC-A and -B genes (32). While this stands in contrast to only two lineage II KIR genes and the expansion of the lineage III KIRs encoding KIR2DL/S receptors for HLA-C in humans, similarities in ligand recognition suggest a functional correspondence between macaque and human KIRs. Indeed, KIR recognition of Bw4 ligands by both species indicates that this specificity has been conserved since humans and macaques shared a common ancestor approximately 25 million years ago (78); however, unlike humans, which only have a single KIR3DL1/S1 gene that encodes receptors for HLA-Bw4, Bw4 recognition has been diversified in rhesus macaques to include the products of at least four different genes. By comparison, although the specificity of Mamu-KIR3DL05 does not directly correspond to any human KIR, the preferential recognition of Mamu-A molecules by this receptor, and the influence of viral peptides on interactions with Mamu-A1*002 (47, 69), suggest that Mamu-KIR3DL05 may be functionally analogous to human KIR3DL2. The recognition of Mamu-AG by Mamu-KIR3DL05 further suggests that this inhibitory KIR may also play a role during pregnancy. Together, these observations afford a better understanding of the comparative immunology of KIR-MHC class I interactions in humans versus macaques and provide an important foundation for studying NK cell biology in the rhesus macaque as a model for infectious disease and reproductive biology.

Acknowledgments

This work was supported by Public Health Service Grants AI095098, AI098485 and AI121135 (to DTE), RR021745 (to DHO) and OD011106 (to the WNPRC). DTE is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.

This research was supported by Public Health Service grants AI095098, AI098485 and AI121135 to DTE, and RR021745 to DHO. Additional support was provided by PHS grant OD011106 to the WNPRC. DTE is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.

Abbreviations used in this article:

KIR

killer-cell Ig-like receptor

Mamu

Macaca mulatta

KIR-28Z

KIR-CD28-CD3ζ

JNL

Jurkat NFAT luciferase

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