Early studies indicate that rats may have a repertoire of MHC class Ib–reactive Ly49 stimulatory receptors capable of mounting memory-like NK cell alloresponses. In this article, we provide molecular and functional evidence for this assumption. Pairs of Ly49 receptors with sequence similarities in the lectin-like domains, but with opposing signaling functions, showed specificity for ligands with class Ia–like structural features encoded from the first telomeric MHC class Ib gene cluster, RT1-CE, which is syntenic with the H2-D/H2-L/H2-Q cluster in mice. The activating Ly49s4 receptor and its inhibitory counterparts, Ly49i4 and Ly49i3, reacted with all allelic variants of RT1-U, whereas Ly49s5 and Ly49i5 were specific for RT1-Eu. NK cell cytolytic responses were predictably activated and inhibited, and potent in vivo NK alloresponses were induced by repeated MHC class Ib alloimmunizations. Additional Ly49–class Ib interactions, including RT1-Cl with the Ly49s4/Ly49i4/Ly49i3 group of receptors, were characterized using overexpressed receptor/ligand pairs, in vitro functional assays, and limited mutational analyses. Obvious, as well as subtle, Ly49–class Ib interactions led to ligand-induced receptor calibration and NK subset expansions in vivo. Together, these studies suggest that in vivo NK alloresponses are controlled by pleomorphic Ly49–class Ib interactions, some of which may not be easily detectable in vitro.

Natural killer cells are innate immune lymphocytes that respond to infection and malignancy, and they regulate placentation and reproductive fitness. NK cell responses are controlled by arrays of activating and inhibitory receptors. In accordance with the “missing-self” hypothesis, NK cells discriminate normal “self”-cells from foreign or abnormal cells, predominantly through inhibitory NK cell receptors that interrupt cellular activation in the presence of self–MHC class I ligands (1). Although inhibitory receptors for MHC are a fundamental feature of NK cells, activating receptors for MHC ligands have also been identified. The NK cell receptors that recognize polymorphic MHC class I molecules are encoded by polymorphic and rapidly evolving gene families that contribute to the diversity and target cell repertoire of NK cells. Among different mammalian species, analogous MHC-recognition functions are often performed by structurally unrelated glycoproteins. For instance, NK cell recognition of polymorphic MHC class I ligands is performed by killer cell Ig-like receptors (KIRs) in humans, higher primates, and cattle but by lectin-like members of the Ly49 family in rodents and horses (2, 3). These species-specific differences in the evolution of KIR and Ly49 emphasize the evolutionary plasticity and versatility of NKR families, and they suggest that the differential evolution of KIR and Ly49 gene families may be determined, in part, by pressures unique to the environmental niche of each species.

Among MHC-binding NKRs, inhibitory function is conferred by an ITIM motif in the cytoplasmic tail, and activating function is affected by a positively charged transmembrane residue that recruits stimulatory signaling adaptors, such as DAP12 (4, 5). In contrast to humans and mice, in which inhibitory NK alloresponses predominate, early genetic and functional studies pointed to the existence of powerful stimulatory NK cell responses directed against MHC-encoded ligands in rats (69). A phylogenetic analysis of KIR and Ly49 genes suggests that activating variants evolve from inhibitory receptors by species-specific evolutionary events (10). In the rat, several clusters of Ly49 genes containing activating and inhibitory members, as well as “transitional” variants containing activating and inhibitory features, appear to have expanded in a species-specific fashion (11).

To establish the physiologic role of activating NK cell alloreceptors, we have undertaken an extensive functional analysis of select rat Ly49 receptors and ligands encoded within the rat MHC, RT1. We show that the recognition of nonclassical MHC class Ib molecules is a generalizable feature of activating rat Ly49 receptors. These ligands are encoded within the first class I gene cluster in the telomeric part of the rat MHC, termed RT1-CE, and situated in a position syntenic to the H2-D/H2-L/H2-Q cluster in the mouse. The RT1-CE cluster contains an expanded array of nonclassical class Ib genes, which show extensive genomic plasticity based on the “presence or absence” of genes, rather than nucleotide substitutions at the allele level. Phylogenetic analyses have shown that RT1-CE genes are more related to each other and to the classical RT1-A genes than to the H2-K, H2-D, H2-L, and H2-Q genes in mice. Sharing of several unique rat-specific genomic features among RT1-CE and RT1-A genes suggests independent evolution following speciation of rat and mouse as a reflection of the unique challenges encountered in their respective environmental niches (12). Unlike classical RT1-A molecules, which are expressed on nearly all nucleated cells and restrict T cell functions, RT1-CE proteins typically do not present antigenic peptides to T cells (13, 14). Moreover, RT1-CE molecules are normally expressed at low levels, but their cell surface expression may be upregulated during inflammation or infections with specific pathogens, such as Listeria monocytogenes (15, 16). In this manner, the upregulated expression of class Ib molecules might serve as a danger signal promoting NK cell–mediated immune surveillance during pathologic conditions. These findings point to the species-specific evolutionary expansion of activating Ly49 receptors and their polymorphic nonclassical RT1-CE ligands in rats. These paired NK cell receptor–ligand systems likely constitute a novel rat innate immune surveillance mechanism involving an expanded repertoire of activating receptors that have evolved in parallel with a unique array of polymorphic nonclassical MHC class Ib ligands.

MHC congenic and intra-MHC recombinant rat strains PVG-RT7b (PVG.7B) [RT1c or c], PVG.1N (n), PVG.1LLEW (l), PVG.1LF344 (lv1), PVG.1U (u), and PVG.R23 (RT1-Au-B/Da-CE/N/M av1 [class Ia–class II–class Ib] abbreviated u-a-av1) were bred in Oslo. BN (n), F344 (lv1), and congenitally athymic nude rats (c) with the Han/rnu mutation (HsdHan: RNU-Foxn1rnu/Foxn1+) were from Harlan UK (Bicester, U.K.). Rats were used at 8–12 wk of age. Animal experiments were conducted in compliance with “The Norwegian Regulations on Animal Experimentation” and were approved by the institutional veterinarian with delegated authority from the Norwegian Animal Research Authority. Intraperitoneal injections were performed under neuroleptanalgesia with fentanyl citrate and fluanisone (Hypnorm; VetaPharma, Leeds, U.K.).

A modification of a previously described immunization protocol was used (17). Rats (congenitally athymic nude or F344) were injected i.p. once weekly for a total of 4 wk with 107 mononuclear splenocytes from PVG.1U or PVG.1N rats or with irradiated YB2/0 cells transfected with RT1-Un (YB.Un) or control wild-type YB2/0 cells. Peritoneal cells were retrieved with cold PBS 3 d after the last injection. Mononuclear cells were obtained by centrifugation on Lymphoprep. DAR13+ and DAR13 IL-2–activated NK cells were generated as described (18). Results are presented as the proportions of the indicated cell subpopulations in individual rats. Statistical differences between groups were evaluated with a Student t test based on the individual values.

Peritoneal cells or Ly49-transfected RNK-16 cells were used as effector cells against the indicated target cells in a 4-h [51Cr]-release assay, as previously described (6). Results are presented as mean values of triplicates, with bars representing 1 SD. Statistical difference was evaluated with a Student t test using the triplicate values.

For the reporter assay, 5 × 104 BWZ reporter cells were incubated with 2.5 × 104 stimulator cells in a total volume of 200 μl in U-bottom 96-well plates. One microgram of mAb DAR13 or FLY5, together with the FcR+ P815 cells, or 3 μM ionomycin was added for positive control. Cells were incubated for 24 h at 37°C in RPMI 1640, 1% FCS, and 10 ng/ml PMA; washed; lysed with 0.125% Nonidet P-40 in PBS containing MgCl, 2-ME, and Chlorophenol Red-β-D-galactopyranoside reagent; and incubated for 1–3 h at 37°C or for up to 24 h at room temperature before determination of OD. Results are presented as mean values of triplicates (A595–A650), and bars represent 1 SD. Statistical differences were evaluated in individual experiments with a Student t test using the triplicates values.

A total of 1–10 × 105 cells was labeled with different combinations of the following conjugated mAbs: biotinylated DAR13 (anti-Ly49s3/Ly49i3/Ly49s4/Ly49i4) (18, 19), FLY5 (anti-Ly49i5/Ly49s5) (20), STOK2 (anti-Ly49i2) (21), STOK9 (anti-KLRH1) (22), or WEN23 (anti-NKp46) (23); Alexa Fluor–conjugated 3.2.3 (anti-NKR-P1A/B) or STOK27 (anti–NKR-P1B (24); and PE- or FITC-conjugated G4.18 (anti-CD3, Pharmingen) or 10/78 (anti–NKR-P1A/B, Pharmingen). Biotinylated mAbs were revealed by PE-Cy5–conjugated streptavidin, and cells were analyzed on a FACSCalibur.

Total RNA was isolated from Con A lymphoblasts from the different MHC-congenic strains on a PVG background or from the R2 macrophage cell line (d haplotype) and reverse transcribed to cDNA. Gene-specific primers used for the amplification by Pwo DNA polymerase (Roche) are shown in Supplemental Table I. The identities of receptor and ligand genes were confirmed by the sequencing of >10 clones per isolate on an ABI3730 sequencer. cDNA for RT1-EU, RT1-Au, and rat β2m was kindly provided by Dr. E. Joly. MHC class I genes containing an N-terminal FLAG tag were subcloned into the EMCV.SRα expression vector and used for stable transfection of the rat YB2/0 cell line. For MHC class I transfection of mouse NSO cells, we used the pMX Puro vector (puromycin resistance), with or without rat β2m, using a picornavirus-derived coexpression strategy (from the foot-and-mouth-disease virus; F2A) inducing a balanced expression of two partner chains (25). This is based on transfection of a single open reading frame with cleavage of the polypeptide chain at the translational stage by ribosomal “skipping” (the core recognition site of a “2A peptide skipping motif” is underlined, and the cleavage site is marked: QTLNFDLLKLAGDVESNPG^P). RNK-16 cells were transfected with full-length Ly49 receptor constructs using pMX Puro vector and a Neon electroporation system for RNK-16 (26). 293T cells were transfected with full-length Ly49 receptors, with or without rat DAP12, using pMX Puro vector and FuGene (Roche) and the F2A coexpression strategy, as above. BW5147 cells expressing the LacZ gene under the control of 3XNFAT-1 promoter (BWZ) (from N. Shastri, University of California, Berkeley) were transfected with a construct encoding parts of a reversed ζ-chain, together with most of the Ly49 molecule, using pMX Puro vector. MHC reporter cells were generated using a fusion construct containing the extracellular part of RT1-Eu or RT1-Un, the transmembrane part of rat CD8, and the intracellular part of rat ζ-chain, together with rat β2m, using the F2A coexpression strategy.

NK cells from the peritoneal cavity of alloimmunized F344 rats were enriched with WEN23-coated microbeads, stained with appropriate Abs, and subjected to sorting, separating DAR13+ and DAR13 cells, using a BD FACStar (BD Biosciences). cDNA was generated from total RNA. Allele-specific FAM-TAMRA–conjugated primers (Supplemental Table I)were used for Ly49i3, Ly49s3, Ly49i4, and Ly49s4 expression, with CD45 used as an endogenous control. Real-time PCR was performed using an ABI 7900HT analyzer. Relative expressions were calibrated based on 1/216 of CD45 expression level. Results are presented as mean values of triplicates, and bars represent 1 SD. Statistical differences were evaluated with a Student t test using the triplicates values.

A map of the rat NK gene complex and the MHC in the reference n haplotype is depicted in Fig. 1. The receptors investigated in this study are encoded by a chromosomal cluster of rat Ly49 genes (block II) that are closely related phylogenetically. This species-specific clustering suggests relatively recent expansion and homogenization events following speciation of rat and mouse (11). Previous studies using subsets of IL-2–activated NK cells and Con A lymphoblast target cells have shown that the Ly49s3 and/or Ly49s4 receptor reacts with a class Ib allodeterminant expressed by most RT1 haplotypes from many rat strains, whereas the Ly49s5 receptor displays a narrower class Ib specificity (1820). Ly49s4 has close sequence homology with Ly49s3, as well as with the inhibitory receptors Ly49i4 and Ly49i3, whereas Ly49s5 and Ly49i5 constitute a separate group. Select receptor–ligand interactions were investigated in more detail with a panel of Ly49 transfectants of BWZ reporter cells (Supplemental Fig. 1A). In agreement with previous work, the Ly49s4 reporter reacted with lymphoblast stimulator cells from a panel of MHC congenic PVG rats expressing the n, av1, lv1, and c, but not the u or l RT1, haplotypes. The same was true for Ly49i4, whereas Ly49i3 showed weak or negative responses to av1, lv1, and c. In contrast, Ly49s5 reporter cells specifically reacted with u stimulator cells (Fig. 2). The Ly49s3 and Ly49i5 reporters failed to react with any of the stimulator cells; however, as discussed below, the BWZ reporter may lack the sensitivity necessary to detect all relevant receptor–ligand interactions.

FIGURE 1.

Schematic maps of the rat NKC and MHC (RT1) gene complexes. (A) The Ly49 region is located in the telomeric part of the rat NKC and contains three major blocks of Ly49 genes in the reference n haplotype. Block II and III encode inhibitory (i) and activating (s) receptors, whereas block I contains uncharacterized inhibitory and bifunctional (si) receptors. The Ly49 receptors characterized in this study are primarily encoded from block II, but members from block III were also included. Ly49 receptors with defined MHC ligands are marked with an asterisk (*). The Ly49s3 gene is missing in the n haplotype, and its location is not known. (B) RT1 class I gene clusters are shaded in gray. Classical class Ia molecules are encoded by the RT1-A cluster, which is located centromeric to the class II/III regions. The RT1-CE cluster is in the same genomic location as the H2-D/L/Q cluster in the mouse and contains 16 class I genes (CE1–CE16) in the reference n haplotype; functional genes are shown in black.

FIGURE 1.

Schematic maps of the rat NKC and MHC (RT1) gene complexes. (A) The Ly49 region is located in the telomeric part of the rat NKC and contains three major blocks of Ly49 genes in the reference n haplotype. Block II and III encode inhibitory (i) and activating (s) receptors, whereas block I contains uncharacterized inhibitory and bifunctional (si) receptors. The Ly49 receptors characterized in this study are primarily encoded from block II, but members from block III were also included. Ly49 receptors with defined MHC ligands are marked with an asterisk (*). The Ly49s3 gene is missing in the n haplotype, and its location is not known. (B) RT1 class I gene clusters are shaded in gray. Classical class Ia molecules are encoded by the RT1-A cluster, which is located centromeric to the class II/III regions. The RT1-CE cluster is in the same genomic location as the H2-D/L/Q cluster in the mouse and contains 16 class I genes (CE1–CE16) in the reference n haplotype; functional genes are shown in black.

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FIGURE 2.

MHC specificity of Ly49 reporter cells. (A and B) Ly49 reporter cells were generated by stable Ly49 transfection of the BWZ reporter line expressing the LacZ gene under the control of NFAT promoters (e.g., Ly49i3, BWZ.i3; Ly49s3, BWZ.s3). Reporter cells were coincubated with Con A–activated lymphoblasts from a panel of MHC congenic PVG strains expressing the n, av1, lv1, c, l, and u rat MHC (RT1) haplotypes. Reporter cells stimulated by ionomycin (io.) or by a combination of a cross-linking mAb [DAR13 in (A) and FLY5 in (B)] and FcR-expressing P815 cells (+) are positive controls. Reporter cells incubated with culture medium only (−) are negative controls. Data are representative of at least three independent experiments. *p < 0.01.

FIGURE 2.

MHC specificity of Ly49 reporter cells. (A and B) Ly49 reporter cells were generated by stable Ly49 transfection of the BWZ reporter line expressing the LacZ gene under the control of NFAT promoters (e.g., Ly49i3, BWZ.i3; Ly49s3, BWZ.s3). Reporter cells were coincubated with Con A–activated lymphoblasts from a panel of MHC congenic PVG strains expressing the n, av1, lv1, c, l, and u rat MHC (RT1) haplotypes. Reporter cells stimulated by ionomycin (io.) or by a combination of a cross-linking mAb [DAR13 in (A) and FLY5 in (B)] and FcR-expressing P815 cells (+) are positive controls. Reporter cells incubated with culture medium only (−) are negative controls. Data are representative of at least three independent experiments. *p < 0.01.

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We searched for ligand(s) for the Ly49s4/Ly49i4/Ly49s3/Ly49i3 group of receptors within the n RT1 haplotype, in which the full genomic sequence is available. We focused on the first telomeric class Ib cluster RT1-CE based on earlier data that mapped an activating NK allodeterminant to a class Ib deletion within the LEW.1LM1 mutant rat strain (6). All 13 functional RT1-CE members, together with the classical class Ia genes RT1-A1n and RT1-A2n, were stably transfected into the YB2/0 rat lymphoma line (Table I, Supplemental Fig. 1B). The most distal RT1-CE member, RT1-CE16, elicited a brisk response by the Ly49s4, Ly49i4, and Ly49i3 reporters but no response by Ly49s3 (Fig. 3A). The protein encoded by RT1-CE16 corresponds to a previously identified molecule, known alternatively as RT1-Un (27), and addition of the RT1-Un–reactive mAb AAS6 specifically blocked these responses (Fig. 3B).

Table I.
Rat MHC molecules, expression levels, and mAb staining profiles
AnnotationsMHC ClusterAccession NumberExpression in YB 2/0 (anti-FLAG)OX18 mAbAAS1 mAbAAS5 mAbAAS6 mAb8G10 mAb
A1n RT1-A MG963095 NA +++ +++ − +++ − 
A2n RT1-A MG963096 +++ ++ − − − − 
A3n RT1-A MG963097 ND − − − − 
CE1 RT1-CE MG963098 ++ − − − − 
CE2 RT1-CE MG963099 ++ ++ − − +/− − 
CE3 RT1-CE MG963100 − − − − − 
CE4 RT1-CE MG963101 − − − − − 
CE5 RT1-CE MG963102 +/− − − − − − 
CE7 RT1-CE MG963103 − − − − − 
CE10 RT1-CE MG963104 +/− +/− − − − − 
CE11 RT1-CE MG963105 ND − − − − − 
CE12 RT1-CE MG963106 ++ ++ − − − − 
CE13 RT1-CE MG963107 − − − − − 
CE14 RT1-CE MG963108 ++ ++ − − − − 
CE15 RT1-CE MG963109 − − − − − 
CE16/Un RT1-CE MG963110 ++ ++ ++ ++ ++ − 
Uc/d RT1-CE MG963112 − 
Ulv1 RT1-CE MG963113 − 
Uav1 RT1-CE MG963111 − 
Au RT1-A X82106 +++ +++ − − − − 
Eu RT1-CE AJ306619 − − − − 
Cl RT1-CE X70066.1 ND ND ND ND − 
AnnotationsMHC ClusterAccession NumberExpression in YB 2/0 (anti-FLAG)OX18 mAbAAS1 mAbAAS5 mAbAAS6 mAb8G10 mAb
A1n RT1-A MG963095 NA +++ +++ − +++ − 
A2n RT1-A MG963096 +++ ++ − − − − 
A3n RT1-A MG963097 ND − − − − 
CE1 RT1-CE MG963098 ++ − − − − 
CE2 RT1-CE MG963099 ++ ++ − − +/− − 
CE3 RT1-CE MG963100 − − − − − 
CE4 RT1-CE MG963101 − − − − − 
CE5 RT1-CE MG963102 +/− − − − − − 
CE7 RT1-CE MG963103 − − − − − 
CE10 RT1-CE MG963104 +/− +/− − − − − 
CE11 RT1-CE MG963105 ND − − − − − 
CE12 RT1-CE MG963106 ++ ++ − − − − 
CE13 RT1-CE MG963107 − − − − − 
CE14 RT1-CE MG963108 ++ ++ − − − − 
CE15 RT1-CE MG963109 − − − − − 
CE16/Un RT1-CE MG963110 ++ ++ ++ ++ ++ − 
Uc/d RT1-CE MG963112 − 
Ulv1 RT1-CE MG963113 − 
Uav1 RT1-CE MG963111 − 
Au RT1-A X82106 +++ +++ − − − − 
Eu RT1-CE AJ306619 − − − − 
Cl RT1-CE X70066.1 ND ND ND ND − 

FLAG-tagged MHC class Ia and Ib molecules (A1n was not FLAG tagged) were transfected into the rat YB2/0 cell line (expressing rat β2m) and tested for expression levels with anti-FLAG or different MHC-reactive mAbs. Functional RT1-CE genes were cloned from BN strain rats expressing the reference n haplotype and are numbered according to their positioning in this class Ib gene cluster (RT1CE6, RT1-CE8, and RT1-CE9 are pseudogenes and were not studied). The presumed alleles of RT1-CE16/Un were cloned from the c, d, lv1, and av1 haplotypes and are designated Uc/d, Ulv1, and Uav1 (Uc and Ud were identical at the DNA level). cDNA clones of Au, Eu, and C1 (kindly provided by E. Joly, Toulouse, France) were subcloned into relevant expression constructs. Note that the broadly reactive mouse anti-rat mAb OX18 failed to stain several of the RT1-CE molecules in BN and the Eu molecule. The AAS1, AAS5, and AAS6 alloreactive mAbs reacted with all alleles of RT1-U but not with the other RT1-CE molecules tested (CE1–15) or with RT1-Eu.

The sequences can be accessed at https://www.ncbi.nlm.nih.gov/nuccore/.

NA, not applicable. −, negative; +/−, borderline; +, weak; ++, intermediate; +++, strongly positive.

FIGURE 3.

RT1-Un (RT1-CE16) is a ligand for the Ly49s4/Ly49i4/Ly49s3/Ly49i3 group of receptors. (A) Ly49s4/Ly49i4/Ly49s3/Ly49i3 reporter cells were coincubated with YB2/0 stimulator cells stably transfected with different MHC class Ia (A1n, A2n) or class Ib (CE1-CE16) molecules from the n haplotype. Stimulation with YB2/0 wild-type cells (YB) and ionomycin (io.) are negative and positive controls, respectively. (B) Blocking of the specific response induced by the RT1-CE16/Un transfectant by addition of 1 μg of mAb AAS6 (anti-CE16/RT1-U). Purified mAb LOV8 (anti-CDw93) was added as isotype control (Ctr.). (C) Testing of a reciprocal RT1-Un reporter cell against 293T stimulator cells stably transfected with Ly49i3, Ly49s3, Ly49i4, or Ly49s4. The response could be blocked with mAb DAR13 binding these four Ly49 receptors. mAb FLY5 was used as isotype control (Ctr.). (D) Comparison of wild-type (BWZ.s3) and point-mutated Ly49s3 reporter (Ser85Cys in the stem region; BWZ.s3-S85C) against YB2/0.RT1-Un (YB.Un) stimulator cells. (E) Comparison of a reciprocal RT1-Un reporter cell against 293T stimulator cells transfected with wild-type Ly49s3 (s3) and the point-mutated variant (Ser85Cys in the stem region; s3-S85C). Data are representative of at least three independent experiments, *p < 0.01. ns, not significant.

FIGURE 3.

RT1-Un (RT1-CE16) is a ligand for the Ly49s4/Ly49i4/Ly49s3/Ly49i3 group of receptors. (A) Ly49s4/Ly49i4/Ly49s3/Ly49i3 reporter cells were coincubated with YB2/0 stimulator cells stably transfected with different MHC class Ia (A1n, A2n) or class Ib (CE1-CE16) molecules from the n haplotype. Stimulation with YB2/0 wild-type cells (YB) and ionomycin (io.) are negative and positive controls, respectively. (B) Blocking of the specific response induced by the RT1-CE16/Un transfectant by addition of 1 μg of mAb AAS6 (anti-CE16/RT1-U). Purified mAb LOV8 (anti-CDw93) was added as isotype control (Ctr.). (C) Testing of a reciprocal RT1-Un reporter cell against 293T stimulator cells stably transfected with Ly49i3, Ly49s3, Ly49i4, or Ly49s4. The response could be blocked with mAb DAR13 binding these four Ly49 receptors. mAb FLY5 was used as isotype control (Ctr.). (D) Comparison of wild-type (BWZ.s3) and point-mutated Ly49s3 reporter (Ser85Cys in the stem region; BWZ.s3-S85C) against YB2/0.RT1-Un (YB.Un) stimulator cells. (E) Comparison of a reciprocal RT1-Un reporter cell against 293T stimulator cells transfected with wild-type Ly49s3 (s3) and the point-mutated variant (Ser85Cys in the stem region; s3-S85C). Data are representative of at least three independent experiments, *p < 0.01. ns, not significant.

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The failure of the Ly49s3 reporter to react with the RT1-Un stimulator cell was unexpected because we previously showed that the surface expression of this receptor is strongly downmodulated in n haplotype rats, suggesting ligand-induced Ly49s3 receptor downregulation in this haplotype (19). We repeated these experiments with a separate GFP reporter cell line, yielding similar results (data not shown), so this negative Ly49s3-triggering result was not due to a signaling defect in BWZ reporter cells. We also tested a reciprocal reporter expressing a chimeric RT1-Un/CD3zeta receptor, together with rat β2m, against Ly49-transfected 293T cells as stimulator cells (Supplemental Fig. 1A, 1C). Reciprocal reporters can be informative about successful receptor–ligand interactions per se but not about the functional consequences of such interactions in NK cells. Consistent with the Ly49 reporter experiments, the BWZ.RT1-Un (BWZ-Un) ligand reporter specifically reacted with Ly49s4, Ly49i4, and Ly49i3, but not with Ly49s3, stimulator cells (Fig. 3C).

We also performed limited site–directed mutagenesis of Ly49s3 by converting Ser in the stem region to the consensus Cys, which is presumably involved in homodimerization of Ly49 molecules. Interestingly, this Ser85Cys Ly49s3 reporter displayed a weak response to RT1-Un stimulator cells but not against other MHC transfectants tested (Fig. 3D, data not shown). This interaction was confirmed with the reverse BWZ.Un reporter (Fig. 3E). These results suggest that Ly49s3 has an inherent capacity for MHC binding, consistent with the in vivo calibration effect mentioned above, but that the serine in the stem region of Ly49s3 likely lowers the MHC-binding affinity out of the range of detection in our reporter assays.

The RT1-U molecule is present in a relatively broad range of haplotypes and exhibits considerable allelic polymorphisms that can be discriminated by alloantibodies and alloreactive T cells (27). To test whether the Ly49s4/Ly49i4/Ly49i3 receptors could distinguish between allelic variants of RT1-U, we isolated and stably expressed RT1-U molecules from four additional haplotypes encoding three variants at the protein level (Fig. 4A, Supplemental Fig. 1B). We were unable to isolate RT1-U transcripts from the two NK ligand–negative MHC haplotypes u and l (refer to Fig. 2A), suggesting that the RT1-U locus is nonfunctional or deleted in these two haplotypes. This conclusion is in line with the previous finding that u or l cells do not stain with a panel of RT1-U–specific alloantibodies (27) and supports a gain-and-loss paradigm of genomic plasticity within this part of the rat MHC (28).

FIGURE 4.

All cloned variants of RT1-U function as ligands for Ly49s4/Ly49i4/Ly49i3. (A) Amino acid sequence of the different allelic variants of RT1-U used in this study (i.e., Un [corresponds with CE16 in the n haplotype], Uav1, Ulv1, and Uc/d and RT1-Cl). (B) Testing of the response of Ly49s4/Ly49i4/Ly49i3 reporter cells against YB2/0 stimulator cells stably transfected with the different RT1-U alleles. Reporter cells incubated with wild-type YB2/0 cells (YB) or with ionomycin (io.) are negative and positive controls, respectively. (C) Cytolytic activity of DAR13+ IL-2–activated NK cells generated from spleens of normal PVG and F344 rats was tested against Con A–activated lymphoblast targets from the MHC congenic strains PVG.1N (n), PVG.LV1 (lv1), and PVG (c), in the presence of blocking quantities of mAb DAR13 (+) or the isotype-control mAb FLY5 (−). Results are shown for the 50:1 E:T ratio. Data are representative of at least three independent experiments. *p < 0.01. ns, not significant.

FIGURE 4.

All cloned variants of RT1-U function as ligands for Ly49s4/Ly49i4/Ly49i3. (A) Amino acid sequence of the different allelic variants of RT1-U used in this study (i.e., Un [corresponds with CE16 in the n haplotype], Uav1, Ulv1, and Uc/d and RT1-Cl). (B) Testing of the response of Ly49s4/Ly49i4/Ly49i3 reporter cells against YB2/0 stimulator cells stably transfected with the different RT1-U alleles. Reporter cells incubated with wild-type YB2/0 cells (YB) or with ionomycin (io.) are negative and positive controls, respectively. (C) Cytolytic activity of DAR13+ IL-2–activated NK cells generated from spleens of normal PVG and F344 rats was tested against Con A–activated lymphoblast targets from the MHC congenic strains PVG.1N (n), PVG.LV1 (lv1), and PVG (c), in the presence of blocking quantities of mAb DAR13 (+) or the isotype-control mAb FLY5 (−). Results are shown for the 50:1 E:T ratio. Data are representative of at least three independent experiments. *p < 0.01. ns, not significant.

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The Ly49s4 and Ly49i4 reporters displayed brisk responses against YB2/0 cells expressing any of the RT1-U variants (i.e., Un, Uav1, Ulv1, and Uc). Ly49i3 reporter cells showed weaker responses (Fig. 4B). It is unknown whether these ligands are encoded by a single locus or by two duplicated loci (U1 and U2) (27); the former is more likely, because we have not been able to isolate more than one variant from any single haplotype (data not shown).

We then investigated the functions of the stimulatory Ly49s3/Ly49s4 and the inhibitory Ly49i3/Ly49i4 receptors in IL-2–activated splenic NK cells from two rat strains with divergent NK gene complexes. The function of the inhibitory variants was assessed in the F344 strain, in which the activating variants are missing (Ly49s3) or mutated to nonfunction (Ly49s4) (11, 19). As shown in Fig. 4C, Ly49i3/Ly49i4+ NK cells from F344 (lv1) rats failed to react with Con A blast target cells of the n, lv1, and c haplotypes, but cytolysis was induced by addition of the anti-Ly49s3 mAb DAR13, which also binds the Ly49s4/Ly49i4/Ly49i3 receptors (19). This is consistent with the “missing self” hypothesis and blocking of an inhibitory signal mediated by Ly49i3/Ly49i4 in F344 NK cells. In contrast, PVG (c) rat NK cells, which also express the activating Ly49s3 and Ly49s4 receptors, showed brisk lysis of allogeneic n and lv1 targets, while sparing syngeneic c targets. Cytolysis was partially or completely blocked by the addition of mAb DAR13, in line with previous data (18). Ligand-negative u targets were effectively lysed by the F344 and PVG effector cells, and the addition of mAb DAR13 had no significant effect (data not shown).

The RT1-Eu class Ib molecule resembles RT1-U, in that it is class Ia–like and is encoded within the first telomeric class Ib cluster, RT1-CE. It has been identified as an activating MHC ligand for rat NK cells, but its cognate receptor still awaits identification (17, 20). Previous genomic data have shown that the u haplotype is divergent from the reference n haplotype in the proximal part of the RT1-CE cluster, with no class I genes detected within 20 kb of the Bat1 gene, where CE1 is located in the n haplotype (28). The rest of the RT1-CE cluster is most likely very different, because we have not been able to clone RT1-U or other functional RT1-CEn genes from u strain cells (data not shown). We stably expressed RT1-Eu in NSO mouse myeloma cells in the presence or absence of rat β2m (Fig. 5A). Ly49s5 reporter cells reacted specifically with RT1-Eu stimulator cells in a rat β2m-dependent manner, whereas the class Ia molecule RT1-Au was not recognized. The Ly49s5–RT1-Eu reaction was blocked by the addition of the anti–RT1-Eu mAb 8G10 (Fig. 5B). The Ly49i5 reporter failed to respond to RT1-Eu stimulator cells. This was not because Ly49i5 fails to recognize RT1-Eu; rather, the activation threshold of the reporter was not reached. A reciprocal BWZ.Eu reporter, in which the ζ-chain–coupled RT1-Eu molecule is expressed at a high level on the cell surface (Fig. 5A), showed brisk responses to Ly49s5- and Ly49i5-transfected 293T stimulator cells, and these responses could be blocked by addition of the anti-Ly49s5/Ly49i5 mAb FLY5 (Fig. 5C).

FIGURE 5.

RT1-Eu is a ligand for the Ly49s5/Ly49i5 pair of structurally related receptors. (A) Single-color flow staining with anti-FLAG mAb M2 of stable RT1-Eu transfectants in NSO cells, with and without rat β2m (rβ2m) and BWZ with rβ2m. (B) Testing of the response of Ly49s5/Ly49i5 reporter cells against mouse myeloma NSO cells stably transfected with the class Ia molecule RT1-Au (Au) and class Ib molecule RT1-Eu (Eu) in the presence (+β2m) or absence of rβ2m. The response of the Ly49s5 reporter against Eu together with rβ2m was blocked by addition of the anti-Eu mAb 8G10 but not a control mAb (AAS5). (C) Testing of a reciprocal RT1-Eu reporter cell (BWZ.Eu) against 293T stimulator cells stably transfected with Ly49s5 or Ly49i5. The response could be blocked with mAb FLY5 reacting with these two Ly49 receptors. mAb DAR13 was used as isotype control (Ctr.). (D) Cytolytic activity of RNK cells stably transfected with Ly49i5 (RNK.Ly49i5) and Ly49s5 (RNK.Ly49s5) against Eu-transfected NSO or BWZ target cells in a [51Cr]-release assay, in the presence of blocking quantities of anti-Ly49s5/Ly49i5 mAb FLY5 or the isotype-control (Ctr.) mAb DAR13. Data are representative of at least three independent experiments, *p < 0.01.

FIGURE 5.

RT1-Eu is a ligand for the Ly49s5/Ly49i5 pair of structurally related receptors. (A) Single-color flow staining with anti-FLAG mAb M2 of stable RT1-Eu transfectants in NSO cells, with and without rat β2m (rβ2m) and BWZ with rβ2m. (B) Testing of the response of Ly49s5/Ly49i5 reporter cells against mouse myeloma NSO cells stably transfected with the class Ia molecule RT1-Au (Au) and class Ib molecule RT1-Eu (Eu) in the presence (+β2m) or absence of rβ2m. The response of the Ly49s5 reporter against Eu together with rβ2m was blocked by addition of the anti-Eu mAb 8G10 but not a control mAb (AAS5). (C) Testing of a reciprocal RT1-Eu reporter cell (BWZ.Eu) against 293T stimulator cells stably transfected with Ly49s5 or Ly49i5. The response could be blocked with mAb FLY5 reacting with these two Ly49 receptors. mAb DAR13 was used as isotype control (Ctr.). (D) Cytolytic activity of RNK cells stably transfected with Ly49i5 (RNK.Ly49i5) and Ly49s5 (RNK.Ly49s5) against Eu-transfected NSO or BWZ target cells in a [51Cr]-release assay, in the presence of blocking quantities of anti-Ly49s5/Ly49i5 mAb FLY5 or the isotype-control (Ctr.) mAb DAR13. Data are representative of at least three independent experiments, *p < 0.01.

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The function of the Ly49s5/Ly49i5 receptors in NK cells was assessed by stable transfection of the RNK-16 NK cell line (Supplemental Fig. 1C). In line with the reporter data, RNK-16.Ly49s5 cells efficiently killed NSO and BWZ cells expressing RT1-Eu together with rat β2m, and lysis could be blocked by the addition of mAb FLY5. In contrast, RT1-Eu mediated weak inhibition of RNK-16.Ly49i5 cells but only when it was expressed at high levels in the BWZ line (Fig. 5D). It should be noted that this inhibition was considerably weaker than that previously observed using IL-2–activated splenic Ly49i5+ PVG NK cells and u haplotype Con A blasts as target cells (20), again indicating that the reporter system and the RNK-16 assay have limitations in their sensitivities.

The activation potential of Ly49 receptors in vivo was evaluated by their ability to recruit and/or expand alloreactive NK cell subsets in the peritoneal cavity upon repeated alloimmunizations. Inbred athymic nude rats (c) were immunized once weekly (four times total) with mononuclear splenocytes from MHC allogeneic strains expressing the n or u haplotype. Peritoneal exudate cells were retrieved 1 wk after the last injection. Following alloimmunization, we observed a massive recruitment of leukocytes, including NK cells, to the peritoneum (data not shown). Immunization with u and n cells led to a preferential expansion of FLY5+ (anti-Ly49s5/Ly49i5) and DAR13+ (anti-Ly49s4/Ly49i4/Ly49s3/Ly49i3) NK cells, respectively, although both subsets were heavily expanded by either stimulus compared with control rats receiving PBS (Fig. 6A). Ly49i2, which is an inhibitory receptor for a class Ia molecule (RT1-A1c) in the strain used for immunization (21, 29), was also markedly expanded (data not shown). The marked coclustering of different Ly49 receptors following alloimmunization was expected, because rat NK cells express more than one Ly49 receptor, and most Ly49 receptors are expressed within a common subpopulation of NK cells lacking the NKR-P1B receptor (24, 30). Concordantly, the NKR-P1B+ subset (relative to the Ly49+ subset) contracted following alloimmunization (Fig. 6A). The in vivo skewing of NK subsets appeared to be limited to the peritoneal cavity, because no skewing effects were observed among splenic NK cells (data not shown).

FIGURE 6.

Specific adaptation of the NK cell repertoire to repeated alloimmunizations in vivo. (A) Congenitally athymic nude rats (c haplotype) were immunized i.p. once weekly for a total of 4 wk with mononuclear splenocytes from PVG.1U (u) or PVG.1N (n) rats. Nude rats receiving PBS only were used as negative controls (Ctr.). Peritoneal cells were retrieved 1 wk after the last injection and analyzed by flow cytometry, gating on CD3NKR-P1A+ NK cells. Data are representative of four experiments; the percentage of positive cells is given. (B) Cytolytic activity of ex vivo peritoneal cells from nude rats immunized with PVG.1U (ImmU) or PVG.1N (ImmN) or nonimmunized nude controls (Ctr) against lymphoblast target cells from PVG.1U (u), PVG.1N (n), and PVG.R23 (u-a-av1) rats. Data are representative of at least three independent experiments. *p < 0.01.

FIGURE 6.

Specific adaptation of the NK cell repertoire to repeated alloimmunizations in vivo. (A) Congenitally athymic nude rats (c haplotype) were immunized i.p. once weekly for a total of 4 wk with mononuclear splenocytes from PVG.1U (u) or PVG.1N (n) rats. Nude rats receiving PBS only were used as negative controls (Ctr.). Peritoneal cells were retrieved 1 wk after the last injection and analyzed by flow cytometry, gating on CD3NKR-P1A+ NK cells. Data are representative of four experiments; the percentage of positive cells is given. (B) Cytolytic activity of ex vivo peritoneal cells from nude rats immunized with PVG.1U (ImmU) or PVG.1N (ImmN) or nonimmunized nude controls (Ctr) against lymphoblast target cells from PVG.1U (u), PVG.1N (n), and PVG.R23 (u-a-av1) rats. Data are representative of at least three independent experiments. *p < 0.01.

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The in vivo–induced NK cells were also tested for cytotoxicity against normal lymphoblast targets from informative donor strains. Cytolytic activity against u target cells was strongly enhanced following immunization with u cells and much more so than against third-party n targets or the intra-MHC recombinant u-a-av1 (RT1-Au-B/Da-CEav1), which expresses u in the RT1-A region. This suggests that this response was not directed to a classical class Ia molecule (compare the three panels of Fig. 6B). Conversely, NK cell alloreactivities against targets from haplotypes n and u and intra-MHC congenic u-a-av1 lymphoblasts were all strongly enhanced upon immunization with n cells (Fig. 6B). Taken together, these results suggest that the observed NK alloresponses were directed against RT1-CE class Ib ligands and that the key components of these responses may vary considerably based upon the Ly49 receptor repertoire of the induced NK cells.

To investigate, in detail, single receptor–ligand interaction(s) in the induction of alloreactive NK cells in vivo, we immunized the RT1-U–negative strain PVG.1U with an RT1-Un–transfected cell line YB2/0 (YB.Un). This line is identical to the recipient strain PVG.1U in the MHC (both u haplotype), thus avoiding T cell alloresponses against major class Ia/class II mismatches that might potentially override the stimulatory effects on NK cells. Repeated i.p. immunizations led to a marked accumulation of NK cells compared with immunization with YB2.0 wild-type cells or PBS only. There was a marked expansion of DAR13+ (Ly49s4/Ly49i4/Ly49s3/Ly49i3) NK cells and a comparable contraction of NKR-P1B+ NK cells (Fig. 7A, 7B). T cells and NK-T cells were similarly expanded by YB.Un and YB2/0 control cells (Fig. 7A).

FIGURE 7.

Allostimulation with an RT1-Un transfectant induces highly alloreactive Ly49s4/Ly49i4/Ly49s3/Ly49i3+ NK cells in vivo. (A) PVG.1U rats were immunized i.p. once weekly, for a total of 4 wk, with RT1-Un–transfected YB2/0 cells (YB.Un), wild-type YB2/0 cells (YB 2/0), or PBS only. Proportions of lymphocytes, T cells (CD3+), NK-T cells (CD3+NKR-P1+), NK cells (CD3NKR-P1A+), DAR13+ NK cells, and NKR-P1B+ NK cells were evaluated by flow cytometry; data for individual rats (n = 4–13) are given for each group. *p < 0.01. (B) Two-dimensional dot plots showing the two dominant DAR13+ and NKR-P1B+ NK subsets in the three experimental groups. (C) Cytolytic activity of ex vivo peritoneal cells from immunized (YB.Un) versus control (Ctr.) PVG.1U rats against Con A–activated lymphoblast target cells from PVG.1N (n), PVG.R23 (u-a-av1), or syngeneic PVG.1U (u) rats. Data are representative of at least three independent experiments. *p < 0.01. (D) Quantitative PCR of Ly49s4, Ly49i4, and Ly49s3 expression among DAR13+ sorted NK cells from PVG.1U rats immunized with transfected YB.Un cells compared with untransfected YB2/0 cells as control [YB (Ctr.)]. *p < 0.05. ns, not significant.

FIGURE 7.

Allostimulation with an RT1-Un transfectant induces highly alloreactive Ly49s4/Ly49i4/Ly49s3/Ly49i3+ NK cells in vivo. (A) PVG.1U rats were immunized i.p. once weekly, for a total of 4 wk, with RT1-Un–transfected YB2/0 cells (YB.Un), wild-type YB2/0 cells (YB 2/0), or PBS only. Proportions of lymphocytes, T cells (CD3+), NK-T cells (CD3+NKR-P1+), NK cells (CD3NKR-P1A+), DAR13+ NK cells, and NKR-P1B+ NK cells were evaluated by flow cytometry; data for individual rats (n = 4–13) are given for each group. *p < 0.01. (B) Two-dimensional dot plots showing the two dominant DAR13+ and NKR-P1B+ NK subsets in the three experimental groups. (C) Cytolytic activity of ex vivo peritoneal cells from immunized (YB.Un) versus control (Ctr.) PVG.1U rats against Con A–activated lymphoblast target cells from PVG.1N (n), PVG.R23 (u-a-av1), or syngeneic PVG.1U (u) rats. Data are representative of at least three independent experiments. *p < 0.01. (D) Quantitative PCR of Ly49s4, Ly49i4, and Ly49s3 expression among DAR13+ sorted NK cells from PVG.1U rats immunized with transfected YB.Un cells compared with untransfected YB2/0 cells as control [YB (Ctr.)]. *p < 0.05. ns, not significant.

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YB.Un stimulator cells induced a strong NK cell cytolytic alloresponse (Fig. 7C). Peritoneal NK cells efficiently killed RT1-Un–expressing PVG.1N (n) lymphoblasts while sparing syngeneic PVG.1U (u) targets. Target cells from the intra-MHC recombinant strain PVG.R23 (u-a-av1) were also effectively lysed, consistent with recognition of the n and av1 alleles of RT1-U (refer to Fig. 4B).

We predicted that RT1-Un allostimulation would induce a preferential expansion of Ly49s4/Ly49s3-expressing cells and not Ly49i4/Ly49i3-expressing cells. This would parallel our previous observation that the inhibitory receptor Ly49i5 leads to contraction of the Ly49i5+ NK cell subset upon allostimulation of BN strain rats with cells expressing its cognate Eu ligand (20). It is not possible to distinguish among the Ly49s4/Ly49i4/Ly49s3/Ly49i3 receptors with our current mAbs, so we performed quantitative PCR to measure their relative contribution to the receptor pool among DAR13+ cells from immunized rats. Interestingly, transcripts for the two stimulatory variants (Ly49s3 and Ly49s4) increased following stimulation with YB.Un cells compared with YB2/0 control cells, and transcripts for Ly49i4 diminished slightly (Fig. 7D). We were unable to assess Ly49i3, because we have never been able to amplify Ly49i3 from PVG strain rats (data not shown), suggesting that this gene is nonfunctional or missing in the PVG NKC genome. These data show that there is a specific increase in Ly49s4 and Ly49s3 transcripts as a result of immunization with RT1-Un.

Early studies of the LEW.1LM1 mutant strain, with a genomic deletion in the RT1-CE cluster (including the polymorphic RT1-Cl molecule) (6), prompted us to investigate RT1-Cl as an alloactivating ligand. Our panel of Ly49 reporter cells failed to trigger against l haplotype cells (Fig. 1), from which the LEW.1LM1 mutant is derived. To increase the sensitivity, we generated an RT1-Cl reverse reporter in the BWZ line overexpressing ζ-chain–coupled RT1-Cl and tested it against a panel of Ly49-transfected 293T stimulator cells (the activating Ly49 variants cotransfected with DAP12 to facilitate receptor expression). As shown in Fig. 8A, the BWZ.Cl reporter showed brisk responses against Ly49i3, Ly49i4, and Ly49s4, as well as the Ly49s3 Ser85Cys mutant, whereas several other Ly49 receptors, including native Ly49s3, were negative. To verify that this result is not due to reporter artifact, we investigated whether RT1-Cl–transfected target cells could selectively expand relevant NK cell subsets in vivo. This was indeed the case, because RT1-Cl–negative PVG1.U rats repeatedly immunized i.p. with YB.Cl cells showed a marked expansion and contraction of DAR13+ and NKR-P1B+ subsets, respectively, compared with rats injected with YB2/0 control cells (Fig. 8B). Furthermore, the Ly49s4/Ly49i4/Ly49s3/Ly49i3 group of receptors was downmodulated in NK cells from l haplotype rats compared with u haplotype rats, as detected by reduced staining with the anti-Ly49s4/Ly49i4/Ly49s3/Ly49i3 mAb DAR13 (Fig. 8C), a clear indication of the presence of cognate ligand(s) in the l haplotype (18, 19). Using the Ly49s3-specific mAb STOK6, we were able to show that even native Ly49s3 was downmodulated in l haplotype strains (Fig. 8C). These experiments are a further indication that in vitro assays may not always be sufficiently sensitive to detect some relevant in vivo receptor–ligand interactions. Based on these data and the aforementioned LEW.1LM1 class Ib deletion data, it is likely that the haplotype 1–encoded alloactivating ligand is indeed RT1-Cl. It should also be noted that Ly49s4/Ly49i4/Ly49s3/Ly49i3 downmodulation was particularly pronounced in n haplotype NK cells (Fig. 8C), in agreement with previous studies (18, 19). As can be deduced from Supplemental Fig. 1B, their common cognate ligand RT1-Un is expressed at particularly high levels, leading to predictably stronger receptor downmodulation.

FIGURE 8.

Recognition of RT1-Cl by the Ly49s3/Ly49s4/Ly49i3/Ly49i4 group of receptors. (A) Testing of a reciprocal BWZ.RT1-Cl reporter cell against 293T stimulator cells stably transfected with various Ly49 receptors (the activating variants cotransfected with DAP12). Data are representative of at least three independent experiments. (B) PVG.1U rats were immunized i.p. once weekly, for a total of 4 wk, with RT1-Cl–transfected YB2/0 cells (YB.Cl), wild-type YB 2/0 cells, or PBS only. Two-dimensional dot plots show the distribution of the two dominant Ly49s3+ and NKR-P1B+ NK subsets. (C) Downmodulation of Ly49s3/Ly49s4/Ly49i3/Ly49i4 receptors on ex vivo splenic NK cells in n and l haplotype rats, but not in u haplotype rats, as detected with the anti-Ly49s3/Ly49s4/Ly49i3/Ly49i4 mAb DAR13 and the Ly49s3-specific alloantibody STOK6. Mean fluorescence intensity values are given. *p < 0.01.

FIGURE 8.

Recognition of RT1-Cl by the Ly49s3/Ly49s4/Ly49i3/Ly49i4 group of receptors. (A) Testing of a reciprocal BWZ.RT1-Cl reporter cell against 293T stimulator cells stably transfected with various Ly49 receptors (the activating variants cotransfected with DAP12). Data are representative of at least three independent experiments. (B) PVG.1U rats were immunized i.p. once weekly, for a total of 4 wk, with RT1-Cl–transfected YB2/0 cells (YB.Cl), wild-type YB 2/0 cells, or PBS only. Two-dimensional dot plots show the distribution of the two dominant Ly49s3+ and NKR-P1B+ NK subsets. (C) Downmodulation of Ly49s3/Ly49s4/Ly49i3/Ly49i4 receptors on ex vivo splenic NK cells in n and l haplotype rats, but not in u haplotype rats, as detected with the anti-Ly49s3/Ly49s4/Ly49i3/Ly49i4 mAb DAR13 and the Ly49s3-specific alloantibody STOK6. Mean fluorescence intensity values are given. *p < 0.01.

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Unlike inhibitory mouse Ly49 receptors, which primarily bind classical MHC Ia molecules, we show in this article that activating rat Ly49 receptors react with three polymorphic nonclassical class Ib RT1-CE proteins: RT1-U (also termed CE16 in the reference n haplotype), RT1-Eu, and RT1-Cl. These ligands are encoded from the first telomeric class Ib gene cluster, RT1-CE, which shares structural features with classical class Ia genes, RT1-A, and are located in the same position as H2-D/H2-L/H2-Q in the mouse. Expansion of activating receptors for class Ib–encoded ligands may have driven the parallel evolution of inhibitory receptors for similar ligands, such as the inhibitory receptors Ly49i3, Ly49i4, and Ly49i5, which bind the same RT1-CE ligands as their activating counterparts. The Ly49s4/Ly49i4/Ly49s3/Ly49i3 receptors react with RT1-Cl, as well as with all known allelic variants of RT1-U present in a broad range of haplotypes (n, lv1, av1, c, and d). Ly49s5/Ly49i5 are specific for RT1-Eu in the u haplotype. We also show that potent NK alloresponses can be induced in vivo by repeated alloimmunizations with allogeneic cells or class Ib transfectants.

Early studies in the rat have suggested that alloreactive NK cells may have adaptive features. Alloimmunization of BN rats with spleen cells from WF (u) rats led to expansion of allospecific NK cells (31, 32) that selectively killed target cells derived from the immunizing rat strain. An increased percentage of activated NK cells was observed in the peritoneum and spleen of BN rats immunized i.p. This was caused by an increased proliferation of NK cells at both sites (33). The heightened NK alloresponse in BN rats was directed against the nonclassical MHC I molecule RT1-Eu (17), as shown by in vitro cytotoxic assays. This finding was in accordance with our early data showing that rat NK cell alloreactivity is controlled mainly by molecules encoded within the nonclassical MHC class Ib region (6). In this study, we show that the activating Ly49s5 receptor recognizes RT1-Eu and may potently induce in vivo alloresponses in PVG rats. However, this receptor is nonfunctional in BN rats, suggesting that another activating Ly49 receptor, yet to be defined, is responsible for the previously described adaptive NK alloresponse in the BN strain (20). We also show that Ly49s4, and possibly Ly49s3, recognizes the MHC class Ib molecule RT1-U and evokes potent in vivo responses in PVG rats. Alloimmunization of PVG rats with cells expressing RT1-Eu or RT1-U led to expansion and enhanced cytotoxicity of Ly49s5+ and Ly49s3/Ly49s4+ NK cell subsets, respectively. In contrast to the aforementioned experiments in BN rats, we observed expansion of peritoneal, but not splenic, alloreactive NK cells in PVG rats, suggesting that the immunization-induced expansion of alloreactive NK cells may be more localized in PVG rats. In a separate set of experiments, we have been able to confirm marked skewing in the distribution of NK cell subsets in the spleen upon i.p. alloimmunization of BN strain rats (L. Kveberg and J.T. Vaage, unpublished observations), in line with the original data referred to above (17, 32, 33). Together, these findings show that activating Ly49 receptors can play an important role in shaping the NK cell repertoire and may induce strong in vivo responses with some adaptive features. The mouse-activating Ly49H receptor has been shown to evoke adaptive NK cell responses during mouse CMV infection in vivo (34). After an expansion and contraction phase, memory Ly49H+ NK cells reside in the animals for months and can mediate specific protective immunity (34). Expansion and differentiation of Ly49D+ mouse NK cells have also been studied upon alloantigen stimulation of mice (35). These studies showed that activating Ly49 receptors have the capacity to induce adaptive immune responses in mouse NK cells. The adaptive features of alloreactive rat NK cells resemble those of mouse Ly49H and Ly49D responses after CMV or alloantigen challenge. However, we do not know how long these enhanced responses persist, and we cannot assume that activating NK alloresponses exhibit strict memory features in the rat.

Other evolutionary pressures are evident from an analysis of ligand specificities of MHC-binding receptors in rats. Not all activating rat Ly49 receptors have identifiable RT1-CE–encoded ligands. Despite its structural similarity to Ly49s4/Ly49i4/Ly49i3, a single amino acid difference in the extracellular domain of Ly49s3 (compared with Ly49s4) appears to significantly reduce or abrogate the binding of this receptor to RT1-encoded ligands in vitro. However, this might be an in vitro artifact, because the expression of Ly49s3 is clearly downmodulated in the presence of its ligand in vivo (19), suggesting that Ly49s3 is functional in vivo. The functional attenuation of stimulatory innate immune receptors against self-MHC might be evolutionarily advantageous, because some high-affinity activating self-receptors may promote excessive inflammation, autoimmunity, or anergy due to immune exhaustion.

A careful analysis of our data and previously published studies suggests that the binding of rat and mouse Ly49 receptors to their respective MHC class I ligands is controlled by orthologous structural constraints. The first cocrystallization studies of mouse Ly49A and its ligand H-2Dd showed that Ly49 molecules had two potential interaction sites with MHC class I molecules. Site 1 was found at one end of the peptide-binding groove, whereas site 2 was below the peptide-binding groove where the Ly49 molecule makes contact with the α1/α2, α3, and β2m domains. Later studies have shown that site 2 is more important; studies in the rat with Ly49i2 and its ligand RT1-A1c have supported this view. It has been shown that the P2 anchor amino acid (Pro/Val) and its associated B pocket are especially important. It has been proposed that changes in these alter the conformation of solvent-exposed residues at site 2 that are important for Ly49 binding (36, 37). Interestingly, when we generated RT1-Eu transfectants in mouse NS0 cells expressing the mouse β2m, target cells were not recognized by BWZ Ly49s5 reporter cells. However, when NS0.Eu cells coexpressed rat β2m, they were recognized by Ly49s5 (Fig. 5B), despite the fact that both transfectants expressed similar levels of RT1-Eu. The requirement for rat β2m was confirmed in the RT1-U interactions, when NS0–RT1-Un transfectants without rat β2m were recognized to a much lower degree or not at all by reporter cells (Ly49s4/Ly49i4/Ly49i3, data not shown). These findings support the notion that site 2 is important for optimal binding of rat Ly49 receptors, but these interactions are likely sensitive to minimal differences in ligand structure

All rat Ly49 receptors that have been functionally characterized belong to the same phylogenetic clade, which is significantly expanded within the second cluster of rat Ly49 genes (11). This cluster includes the Ly49s4/Ly49i4 /Ly49i3 and Ly49s5/Ly49i5 molecules, as well as the inhibitory Ly49i2 receptor. Ly49s3 likely also maps to this cluster, but its gene is missing in the n haplotype. Two additional activating receptors in this cluster, Ly49s2 and Ly49s6, as well as the activating receptors Ly49s1, Ly49s7, and Ly49s8 in another phylogenetic clade remain functionally uncharacterized, and their ligands are not known. In contrast to activating KIRs in humans and Ly49 in mice, our data suggest that the recognition of nonclassical RT1-CE–encoded ligands by activating Ly49 receptors may be a generalizable feature of rat NK cells. Moreover, our reporter data demonstrate that reporter activation by RT1-CE proteins is exquisitely sensitive to changes in ligand density on target cells and to subtle structural changes in specific receptors or their ligands. The telomeric part of the rat MHC contains several clusters of class Ib genes in addition to RT1-CE, with RT1-N being homologous to H2-T and RT1-M being homologous to H2-M in mice. In this context, it is interesting that the mouse class Ib molecules H2-Q10 and H2-M3 function as ligands for the Ly49C and Ly49A inhibitory receptors, respectively (38, 39). The mouse MHC does not contain formal orthologs of the RT1-CE genes located in the first class Ib gene cluster (refer to Fig. 1B), and this cluster does not contain direct H2-D/L orthologs. The number of RT1-CE genes varies remarkably among different rat MHC haplotypes. However, the NK ligands defined in this article, RT1-U, RT1-Eu, and RT1-Cl, display some class Ia–like structural features and contribute significantly to haplotype diversity (28). Although polymorphic rat RT1-A molecules and mouse H2-K/D/L molecules are expressed on all nucleated cells, nonclassical class Ib proteins are normally expressed at low levels and do not typically serve as restriction elements for cytotoxic T cells (14, 27). Therefore, the rat RT1-CE cluster contains an array of polymorphic nonclassical MHC molecules with unique features. Although they are normally expressed at low levels, their expression may be upregulated by inflammatory cytokines or by specific pathologic conditions. We propose that rats have selectively coevolved an expanded array of activating Ly49 receptors and class Ib RT1-CE ligands, which together have evolved to serve as a uniquely calibrated immune sensing system for inflamed, infected, or otherwise abnormal target cells.

Bent Rolstad died in 2015, but he was involved and very enthusiastic about this project, which addressed receptor–ligand interactions, explaining early findings of NK cell–mediated allogeneic lymphocyte cytotoxicity dating back to the 1970s. He was an inspirational leader and is deeply missed.

This work was supported by the Norwegian Cancer Society and the South-Eastern Norway Regional Health Authority. K.-Z.D. received a fellowship from the Norwegian Cancer Society during the early phase of this project. J.C.R. received support from National Institutes of Health Grants R01 AI 083113 and P30 DK 02673 (University of California, San Francisco Liver Center) and the U.S. Department of Veterans Affairs.

The sequences presented in this article have been submitted to Genbank under accession numbers MG963095–MG963113.

The online version of this article contains supplemental material.

Abbreviations used in this article:

KIR

killer cell Ig-like receptor.

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The authors have no financial conflicts of interest.

Supplementary data