Expression Tissue

The Journal of Immunology

Killer Cell Ig-Like Receptor and Leukocyte Ig-Like Receptor Transgenic Mice Exhibit Tissue- and Cell-Speciﬁc Transgene Expression1

Danny Belkin,* Michaela Torkar,* Chiwen Chang,* Roland Barten,* Mauro Tolaini,† Anja Haude,* Rachel Allen,* Michael J. Wilson,* Dimitris Kioussis,† and John Trowsdale2*

To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufﬁcient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice. The Journal of Immunology, 2003, 171: 3056Ð3063.

atural killer cells are involved in immune responses to transmembrane domain. Associated transmembrane adapter pro- tumor cells and to viral, bacterial, and parasitic infec- teins carry an immunotyrosine-based activation motif for signal N tions (1, 2). Attack by NK cells is regulated by a com- transduction. Inhibitory type KIR and LILR, characterized by a bination of signals from inhibitory and stimulatory receptors on the long cytoplasmic tail-bearing immunotyrosine-based inhibitory cell surface. MHC class I-specific inhibitory receptors are the best motif, have been shown to inhibit cell activation of monocytes, B understood of these and include members of the human killer cell cells, NK cells, and T cells (8Ð11). 3 Ig-like receptor (KIR) family, their functional homologues the Different KIR gene products can be detected on overlapping sets murine Ly49 family, and the NKG2/CD94 receptors (present in of human NK cells (12, 13). Each NK clone expresses at least one both species) (3, 4). Additional immune receptors such as the leu- inhibitory receptor. Variegated patterns of expression suggest a kocyte Ig-like receptors (LILR, also known as Ig-like transcripts stochastic mechanism of KIR expression (14). KIR expression is (ILT), LIR, or CD85), and the murine paired Ig-like receptors are likely to be controlled by highly homologous upstream promoter closely related to KIRs in structure and presumably also in func- sequences (with the exception of KIR2DL4, which possesses a tion. Both KIRs and LILRs are encoded within the leukocyte re- more divergent promoter and is constitutively expressed in all NK ceptor complex (LRC) on human chromosome 19q13.42, a region clones) (5). It has been demonstrated that the frequency with which of the genome that exhibits marked inter- and intraspecies varia- cells express a combination of Ly49 receptors is a product of their tion (5Ð7). individual frequencies, leading to the proposal of the “product KIR and LILR receptors are members of the Ig superfamily and rule” (15). Higher-order, chromatin-related regulation also seems exist in both activating and inhibitory isoforms. The activating to be involved, as there appears to be a correlation between meth- type, characterized by a short cytoplasmic tail, associate with ylation upstream of the transcriptional start site and reduced KIR adapter proteins via a positively charged arginine residue in their expression (16). MHC class I molecules also exert an effect on the repertoire of NK receptor expression. An example of this was seen *Immunology Division, Department of Pathology, University of Cambridge, Cam- in MHC class I-deficient mice, which showed higher frequencies † bridge, United Kingdom; and Division of Molecular Immunology, National Institute of NK cells coexpressing different inhibitory Ly49 receptors when for Medical Research, London, United Kingdom compared with normal mice, indicating that the interaction of Ly49 Received for publication November 20, 2002. Accepted for publication July 3, 2003. receptors with their MHC class I ligands shape the receptor rep- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ertoire by limiting receptor coexpression (17, 18). This effect is with 18 U.S.C. Section 1734 solely to indicate this fact. consistent with a sequential regulated expression model, which 1 This work was supported by the Wellcome Trust and the Medical Research Council. proposes that the MHC reactivity of receptors expressed early 2 Address correspondence and reprint requests to Dr. John Trowsdale, Immunology in the sequence determines whether other receptors will be sub- Division, Department of Pathology, University of Cambridge, Tennis Court Road, sequently expressed. This happens to the extent that even MHC Cambridge CB2 1QP, U.K. E-mail address: [email protected] haplotypes, which did not detectably bind certain Ly49 recep- 3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; LILR, leukocyte Ig-like receptor; FISH, fluorescence in situ hybridization; LRC, leukocyte receptor tors, impacted coexpression of these receptors with other Ly49 complex; PAC, P1 artificial chromosome. molecules (15, 17Ð20).

Many LILR-related sequences have been described to date. be addressed. Previous work generated KIR2DL3 transgenic mice, Most are transmembrane glycoproteins with an extracellular, li- and used them to investigate KIR function and the possible affects gand-binding region composed of either two or four Ig domains. the ligand for this KIR, HLA-Cw3, exerts on KIR expression (32). LILR-mediated immune modulation may affect cells of both the This study showed that KIRs can be expressed without the need innate and adaptive immune systems. LILRB1 (ILT2/LIR1) shows for the presence of their cognate human ligands, MHC class I. an exceptionally wide expression pattern and can be found on the The use of the H-2kb promoter in this transgene conferred an ϩ majority of CD14 myelomonocytic cells and B cells in addition expression pattern that differed markedly from the one observed to NK and T cell subsets (21, 22). LILRA2 (ILT1/LIR7) and in humans. LILRB3 (ILT5/LIR3) are expressed by myeloid cells, granulo- In this study we describe the generation of two lines of LILR trans- cytes, and subsets of NK or T cells (21, 23), while LILRB4 (ILT3/ genic mice, and one line of KIR transgenic mice, using genomic con- LIR5) is expressed only on myeloid cells (8). cDNA studies indi- structs from the LRC. Official KIR and LILR nomenclature is de- cate that LILRB5 (LIR8) may be the only NK cell specific LILR scribed at http://www.gene.ucl.ac.uk/nomenclature/genefamily.shtml. (24). ILT7, ILT8 and LILRA1 (LIR6) are expressed in monocytes but not on B, T, or NK cells (24, 25). Little is known about the Materials and Methods control of LILR gene expression, although there is preliminary Transgene production evidence that some LILRs show similar clonotypic expression patterns to KIRs (21). The P1 artificial chromosome (PAC) clones 1060P11 and 52N12 (con- structed using the vector pCYPAC2 in the Escherichia coli host strain The ligands that have been identified for LILRB1, LILRB2 DH10B) were used for the creation of transgenic mice (Fig. 1) (33). For (ILT4/LIR2), and LILRA1 are all HLA class I molecules. LILRB1 large scale, genomic DNA-free purification of PAC DNA, cesium chloride and LILRB2 recognize classical, nonclassical, and viral ortho- gradients and a Qiagen Large Construct kit (Valencia, CA) were used. The logues of HLA class I (10, 21, 22, 26). LILRB2 can also recognize microinjection fragments were isolated from the vector by restriction with free H chain forms of HLA class I. LILRA1 has been shown to the restriction enzymes NgoMIV and NotI followed by centrifugation through a 10Ð25% sodium chloride gradient. Fractions were collected and recognize HLA B27 but does not recognize the nonclassical analyzed by pulse field gel electrophoresis to assess for purity of insert HLA-F protein (27) (R. L. Allen and E. Lepin, unpublished ob- from both vector and E. coli DNA. servations), so it appears to have more restricted specificity. Insert DNA was diluted, precipitated with ethanol, then resuspended ϫ Activation of monocytes through receptors such as Fc␥RIII and twice in 0.25 Tris-taurin-EDTA. Before microinjection, DNA was passed ␥ through an Elutip minicolumn (Schleicher and Scheull, Keene, NH) fol- Fc RII can be inhibited by coligation of inhibitory LILR (8, 10). lowing the manufacturer’s instructions. Eluted DNA was precipitated with The inhibitory process is mediated by Src homology 2-containing ethanol then resuspended in 10 mM Tris-HCl, 2 mM EDTA, pH7.4 at a phosphatases 1 and 2 (28). Activating LILRs use a similar signal- concentration of 5Ð10 ␮g/ml and then microinjected (43). ing mechanism to the Fc␣R. LILRA2, an activating member of the Monoclonal Abs LILR family, associates with the common Fc␥ chain (25, 29). It is therefore likely that the signaling machinery in rodents and hu- Anti-CD158a/h-FITC (HP-3E4), anti-CD158b-FITC (CH-L), anti-Ly49A/ mans is conserved (24, 25), which could allow correct signaling D-PE, anti-NK1.1-PE, anti-CD19-PE, anti-CD3-PE, DX5-PE, and DX5- Biotin, as well as Streptavidin-PerCP were purchased from BD PharMin- through exogenous LILRs in transgenic mice. gen (San Diego, CA). The anti-FcR mAb 2.4G2 (34) has been previously A recently proposed mechanism governing the sequential ex- described. Anti-LILRB1 (HP-F1) was a gift by Dr. M. Colonna and anti- pression of LRC-encoded receptors suggests that NK cell precur- LILRB4 (ZM3.8) was a gift from Dr. M. Lopez-Botet. sors in the bone marrow are initially unresponsive to self MHC, Flow cytometry expressing the broadly self-specific LILRB1 and CD94/NKG2A receptors, and later begin expressing different KIRs, which stim- PBLs, splenocytes, and thymocytes from transgenic and nontransgenic ulate the proliferation and survival of individual clones to form the mice were preincubated with mouse serum and mAb to block Fc receptors. KIR/LILR expression on NK, T, and B cells was determined by staining repertoire of mature peripheral NK cells (30). Similar results have with anti-CD158a/h (which detects KIR2DL1/2DS1, but as only the latter been shown for peripheral effector CD8 T cells, which initially is present on the PAC used to generate these mice only KIR2DS1 will be express LILRB1 and later show KIR expression in the same detected by this Ab), anti-CD158b (detecting KIR2DL2/2DS2), anti- clones. These KIRϩ cells survive activation-induced cell death and LILRA1, anti-LILRB1, and anti-LILRB4 in conjunction with anti-NK1.1, anti-CD4, -CD8, -CD19, and DX5. Comparison with Ly49 expression was become long-term memory T cells (31). conducted using anti-Ly49A/D-PE. Although KIRs have been better studied than LILRs with re- spect to ligands, immune modulation, and developmental regula- Fluorescence in situ hybridization (FISH) tion, these issues are not well understood in either receptor family. Chromosome spreads were obtained from mouse spleens after culturing in In vivo models would provide a system to allow such problems to 20 mg/ml LPS (Sigma-Aldrich, St. Louis, MO) for 40 h. The 1060P11 and

FIGURE 1. The LRC contains members of the Ig-SF receptor superfamily including the KIRs and LILRs. The genomic fragments contained in 1060P11 and 52N12, the PAC clones used to create the transgenics, are designated. (Ⅺ), Pseudogenes. 3058 KIR AND LILR TRANSGENIC MICE

52N12 transgenes were digoxigenin-labeled using a digoxigenin-nick Analysis of transgene expression translation mix (Roche Diagnostics, Mannheim, Germany) and mixed with salmon sperm and mouse cot-1 DNA (Life Technologies, Rockville, MD) To detect transgene expression, we analyzed Ab binding to cells before hybridization. Signal was detected using anti-digoxigenin Ab with collected from transgenic animals. Cy3-conjugated secondary reagent (Stratech Scientific, Cambridgshire, LILR mice. mAbs were available for both LILRB1 and LILRB4 U.K.). Images were captured using a Photometrics CCD camera (Tucson, AZ) on a Zeiss Axioskop microscope (Don Mills, Ontario, Canada). Im- receptors. To examine the LILR expression pattern in the LILR ages were pseudocoloured and merged using SmartCapture software (Vy- transgenics, we stained spleen cells with LILRB1- or LILRB4- sis, Chicago, IL). specific Abs, and this confirmed transgene expression in vivo. All mice carrying the transgene showed consistent cell surface expres- RT-PCR sion of LILRB1 on B cells, as ascertained by FACS (Fig. 2A). To Livers and spleen from transgenic and nontransgenic mice were used for determine which cell types express the transgene, splenocytes were RNA preparation using a Qiagen Rneasy kit. RT-PCR was performed on costained with Abs against mouse CD19 for B cells and NK1.1 as ϩ RNA isolated from tissue or sorted NK1.1 cells using a Superscript One- an NK cell marker. Furthermore, we compared LILRB1 and step RT-PCR kit (Invitrogen, San Diego, CA) or an Omniscript RT kit LILRB4 expression patterns in the transgenic mice to those seen (Qiagen), respectively. KIR-specific and mouse GAPDH primers were used. for human PBLs, using Abs against human CD56 for NK cells and CD19 for B cells. These stainings show that almost all CD19ϩ B Generation of LILRA1-specific mAb cells in transgenic mouse spleen expressed LILRB1 (Fig. 2B), sim- Analysis of LILRA1 expression in this and in previous studies has been ilar to the expression pattern observed in human blood (Fig. 2C). hampered by the lack of a specific Ab. Recombinant LILRA1-Fc fusion Costaining with LILRB1 and NK1.1 indicated that the majority of protein was produced using a rat myeloma-based expression system that NK cells also expressed the transgene (Fig. 2B), in contrast with had been established by Drs. Kathryn Armour and Mike Clark (Department human peripheral blood NK cells that showed no such expression of Pathology, University of Cambridge, Cambridge, U.K.), who kindly (Fig. 2C). LILRB4 expression was detected on a high proportion of provided reagents and expertise. Protein (100 mg) was injected s.c. into rats ϩ and spleens removed for fusions. Hybridoma supernatants were first CD19 transgenic spleen cells (Fig. 2B), whereas in human blood it screened with the LILRA1/IgG Ag by ELISA. To exclude Abs for the IgG is expressed only on a small percentage of B cells (Fig. 2C). part of the molecule, positive hybridoma supernatants were further tested Thymocytes were also isolated from transgenic and nontrans- using a LILRB4/IgG Ag. Finally, Ab specificity was tested on a panel of genic littermates. Ab staining confirmed that thymic T cell popu- LILR transfectants. mAb that bound LILRA1 specifically were selected for further studies. A single hybridoma-produced clone, which showed high lations in the transgenics displayed normal CD4:CD8 ratios (data affinity for LILRA1 and low cross-reactivity with other members of the not shown). Thymocytes were then analyzed for the presence of LILR family, was grown and the purified mAb was used in subsequent LILRB1 and LILRB4 using appropriate mAbs. LILRB1 was ex- experiments. pressed at low levels in thymocytes whereas LILRB4 could not be detected on any thymocyte population (Fig. 2D). Results KIR mice. To assess KIR expression in the KIR transgenic Generation of KIR and LILR transgenic lines founder mice, PBLs were examined for CD158b (KIR2DL2/ Transgenic mice were generated using the recombinant constructs 2DS2) expression, staining with anti-CD158b and DX5 (which indicated in Fig. 1. The PAC 52N12 encodes three functional reacts with NK and small T cell subpopulations) mAbs (Fig. 3A). ϩ ϩ LILR genes: LILRB1, LILRB4, and LILRA1 in addition to two Costaining with anti-CD158b and DX5, a CD158b DX5 popu- potential pseudogenes and partial KIR3DL3 and LILRA2 loci. The lation was observed in three of eight founder mice examined PAC 1060P11 contains a genomic fragment within which ten KIR (KIR5, -8, -9), suggesting that the transgenes are expressed by NK genes are encoded, of which one (KIR3DL2) was cleaved during the and T cell subsets in the transgenic mice. Subsequent RT-PCR DNA production process and one is a pseudogene (KIR3DP1/KIRX). experiments established that KIR2DL2, but not KIR2DS2, was Following egg injections with LILR DNA, nine mice were born being expressed in KIR5 mice (see below). Positive staining with one of which was transgene positive, whereas of 32 born after KIR the anti-CD158b Ab will therefore be referred to as detecting DNA injection, nine mice were positive for the transgene and con- KIR2DL2. tained varying transgene copy numbers (data not shown). Founders The KIR5 line was studied in further detail (Fig. 3B): to estab- from both sets of transgenics were crossed with CBA/Ca ϫ lish the tissue specificity and type of cells that show KIR expres-

C57BL/6 F1 nontransgenic mice. sion, PBLs and splenocytes from KIR5 offspring were double- The one LILR founder mouse was found by FISH to have three stained with anti-CD158a/h or b in conjunction with anti-CD19 independent transgene integration sites. These sites were segre- and anti-CD8. Both PBLs and splenocytes showed similar expres- gated in subsequent generations and led to the establishment of sion patterns and percentages of KIR2DL2 expressing cells, but no two LILR transgenic lines. significant expression was observed on thymocytes (data not All nine KIR founders were bred and six transmitted the trans- shown). KIR2DL2 expression in the transgenic mice was observed gene (the rest either did not breed or did not transmit). Of those only on DX5ϩ cells. An apparent KIR2DS1ϩ population, observed that transmitted the transgene, one (KIR5) showed KIR expression in all mice, was the result of nonspecific binding to mouse B cells by flow cytometry (see below), and this line was chosen for further (data not shown). No KIR2DS1ϩ NK or T cell population was examination and subsequently bred to homozygosity. Two of the observed. founders (KIR3 and KIR9) showed KIR2DL4 expression by RT- To examine how KIR expression correlates with the expression PCR (see below). of the functional homologues of KIR in mice, namely the Ly49 To determine whether the chromosomal integration site of the receptors, dual color analysis using anti-CD158b and anti- transgene was causing repression of transgene expression, KIR Ly49A/D was conducted. This showed similar results in both transgene integration in the founders that did not show KIR ex- PBLs and splenocytes, in which three distinct populations were pression was examined by FISH. All examined KIR lines showed seen: KIR2DL2ϩLy49A/DϪ, KIR2DL2ϪLy49A/Dϩ, and double a single integration site, which varied in its location in different positive KIR2DL2ϩLy49A/Dϩ. lines; both long-arm and peritelomeric locations were observed To examine whether T cells constitute a subpopulation of the (data not shown). KIRϩDX5ϩ population in KIR5 mice, costaining of splenocytes The Journal of Immunology 3059

FIGURE 2. Transgene expression in LILR transgenic mice. A, LILRB1 expression in three transgenic mice is shown compared with a nontransgenic B10 control mouse. B, Overexpression of LILRB1 and LILRB4 in transgenic mouse B and NK cells: spleen staining with anti-LILRB1. C, LILRB1 and LILRB4 expression in human PBLs. D, LILRB1 and LILRB4 expression in transgenic thymocytes, compared with a nontransgenic littermate. with anti-CD158b, anti-CD3, and DX5 was performed. This showed mic immunotyrosine-based inhibitory motif, suggesting inhibitory a small proportion of DX5ϩKIR2DL2ϩ cells to be CD3ϩ, indicating function, and a positively charged amino acid in the transmem- that a small percentage of KIRϩ cells are T cells (Fig. 3C). brane region, a feature typical of activating KIR (35). KIR2DL4 RT-PCR was conducted on NK1.1ϩ spleen cells from KIR5 possesses a unique 14-Kb region upstream of its promoter, and mice to examine which additional KIRs were being expressed in unlike other clonally distributed KIRs, is transcribed by all KIRϩ the transgenic mice (Fig. 4A). Transcripts were detected for NK and T cell clones. To assess KIR2DL4 expression in the dif- KIR2DL2, 2DL4, 3DS1, and 2DL5, but not for KIR3DL3, 2DS2, ferent KIR founders, we performed RT-PCR on RNA isolated 2DS5, 2DS1, and 3DL2. from the liver and spleen of a nontransgenic, KIR2DL2 expressing transgenic founder (KIR9), and a KIR2DL2 nonexpressing trans- KIR2DL4 expression variation genic founder (KIR3), using KIR2DL4 RNA primers. Expression KIR2DL4 is a KIR family member that shares structural features of KIR2DL4 was observed at the RNA level in both lines (Fig. with both activating and inhibitory receptors. It bears a cytoplas- 4B). Interestingly, KIR2DL4 RNA levels were much higher in the 3060 KIR AND LILR TRANSGENIC MICE

FIGURE 3. KIR2DL2 expression in KIR transgenic mice. A, PBLs from eight transgenic founders were stained with anti-CD158b (detecting KIR2DL2) and DX5 (a marker for NK cells and a subset of T cells). B, PBL and splenocytes from KIR5 mice and nontransgenic littermates were stained with anti-CD158b and costained with anti-CD8, CD19, Ly49A/D, and DX5 Abs, showing cell and tissue speciﬁcity of KIR2DL2 expression in KIR transgenic mice. C, PBL from KIR5 homozygote and nontransgenic mice were stained with anti-CD158b, anti-CD3, and DX5. A small proportion of DX5ϩKIR2DL2ϩ cells were also CD3ϩ. mouse that did not express KIR2DL2. Only a low level was readily available from humans. Various tissue sections were pre- present in spleen and liver from the mouse that exhibited high pared from transgenic mice and stained for LILRA1 expression. levels of KIR2DL2 expression (KIR9). Using the new Ab, LILRA1 protein was detected in spleen sec- In conclusion, KIR expression in KIR transgenic mice was re- tions, in a ring of cells surrounding B cell areas, and in lymph stricted to those cells that normally express the genes in humans. nodes (Fig. 5). No LILRA1 expression was observed in thymus Expression of both inhibitory and activating KIRs was observed, (data not shown). although not all KIRs contained in the 1060P11 PAC clone were expressed. Discussion LILRA1 expression in vivo KIR and LILR transgene expression pattern Screening of human PBLs with the anti-LILRA1 Ab failed to iden- In the LILR mice we have examined so far, LILRB1 was ex- tify any cell type expressing LILRA1. Similar results were previ- pressed at high levels on almost all B and NK cells, while thymo- ously observed for LILRA2 (36). Transgenic mice provide an op- cytes expressed LILRB1 at much lower levels. When compared portunity to study receptor expression in tissue samples that are not with LILRB1 expression in humans (8, 9, 22), the expression is The Journal of Immunology 3061

expression in a tissue- and cell type-specific pattern in mice. In- terestingly, the KIR expression pattern in these transgenic mice appears consistent with the variegated expression pattern observed in humans. The KIR expressing subset of both PBLs and splenocytes in the transgenic mice was independent of but overlapped with the frac- tion that expressed Ly49A/D receptors. The presence of a population expressing both human and murine receptors confirms that each receptor locus is expressed in an independent manner that leads to the observed variegated expression pattern. Future experiments examining transgenic KIR expression during mouse ontog- eny will shed further light on the expression mechanisms as well as on the effects these two receptor types have upon each other. Flow cytometry experiments showed that KIR2DL2 expression in KIR transgenic mice is limited to a proportion of NK cells and T cells, consistent with a variegated expression pattern. This expression pattern could stem from transgene position effect variegation (37), although this is unlikely because transgenic mice generated using large constructs (such as Y1 artificial chromosomes or PACs) are less likely to be influenced by transgene position effect variegation (38). The variegation observed in these mice may thus be due to an intrinsic stochastic mechanism that affects cis-regulatory elements and leads to a naturally variegated KIR expression pattern in NK cells. This indicates that the sequences bringing about this expression pattern are included in the 160-Kb genomic transgene fragment. A similar conclusion can be reached regarding the LILR transgene, which brings about cell-specific expression in the LILR transgenics. These findings are even more significant when considering the fact that these transgenes originate from a different species, i.e., the KIRs and LILRs are expressed despite the fact that their natural ligand is lacking in mice—demonstrating that the presence of human MHC class I is not an essential requirement for KIR or LILR expression. RT-PCR experiments detected transcripts for both inhibitory and a single activating KIR. A surprising observation was the lack of transcripts for additional activating KIRs, namely KIR2DS1, FIGURE 4. KIR expression in KIR transgenic mice. A, Expression in 2DS2 and 2DS5. It is possible that expression in the KIR gene NK1.1ϩ cells from KIR5 transgenic mice spleen. RT-PCR was performed complex favors those genes proximal to KIR2DL4 although this on (I) cDNA from transgenic mice (II) positive controls from PCR using cDNA from decidual human NK cells or cloned genes. Lane 1, KIR3DL3 requires further investigation. (700 bp); lane 2, KIR2DS2 (290 bp): lane 3, KIR2DL2 (370 bp); lane 4, FISH experiments performed on splenocytes from LILR trans- KIR2DL4 (1100 bp); lane 5, KIR3DS1 (900 bp); lane 6, KIR2DL5 (200 genic mice showed LILR transgene insertion sites on three differ- bp); lane 7, KIR2DS5 (330 bp); lane 8, KIR2DS1 (330 bp); lane 9, ent chromosomes, at different locations upon these chromosomes KIR3DL2 (800 bp). The control lane in this case was negative for (in the long arm, located close to the telomere, or close to the KIR2DS5. B, KIR2DL4 expression in nontransgenic, KIR2DL2Ϫ trans- centromere). All LILR transgenic mice that have been analyzed ϩ genic founder (KIR3), and KIR2DL2 transgenic founder (KIR9) mice. showed LILRB1 expression on all B cells. Thus, the transgene RT-PCR was performed on RNA samples isolated from the liver and does not appear to be subject to repressive position effects. It is spleen of the three mice, using KIR2DL4 and GAPDH primers. KIR2DL4 worth noting that the LILR and KIR gene clusters are naturally expression can be seen in both types of transgenic mice. located near the telomere on human chromosome 19. Telomeres are heterochromatic and can induce variegated gene repression in yeast, Drosophila, and possibly mammals (39Ð41). This suggests observed in the expected cell types. LILRB4, which is expressed that for appropriate regulation of LILR and KIR genes there may be mainly on human myelomonocytic cells but is also found in B cells a requirement for a powerful chromatin opening function to con- at very low levels, was expressed at a relatively low level on most tend with this potentially repressive chromosomal environment. B cells in the transgenic mice (8). The frequency of transgene expression in the KIR mice was Using spleen section stainings, LILRA1 expression was ob- lower than in humans, both in heterozygote and homozygote trans- served in a ring of cells surrounding B cell areas. Little is known genic mice. This may be explained either by the lack of expres- about the cellular expression of the LILRA1 receptor in humans. sion-driving enhancer regions or an LCR from the KIR transgene, Additional experiments using our novel Ab should help clarify this by the transgene insertion site being close to a heterochromatin- and indicate whether these mice will prove an effective model for rich region, by the lack of immune challenges during the devel- studying this activating receptor. opment of the KIR transgenics, or by a combination of these fac- In the KIR transgenic mice, KIRs were expressed on NK and T tors. As large construct-transgenes seem to be less prone to cell subsets in spleen and PBLs, demonstrating that endogenous positional effects, the second explanation seems less likely, and in regulatory elements within the KIR region are sufficient to drive addition to this the high percentages of expression observed from 3062 KIR AND LILR TRANSGENIC MICE

FIGURE 5. LILRA1 expression in LILR transgenic mice: tissue sections from LILR transgenics and nontransgenic littermate were stained with LILRA1 specific mAb 3/70.5. Spleen sections show a ring of positive stained cells in the transgenic animal (D) but not in the control animal (A) or with the isotype control (B and E). The stained cells form a ring or sheath apparently around B cell areas stained with CD19 (C and F), which may indicate that LILRA1 is expressed on tissue macrophages. A different LILRA1-specific mAb, 3/17.5, was tested to confirm these results (G). Some cells in lymph nodes do show LILRA1 expression (H), which is not seen using an isotype control (I).

all three transgene insertion sites in the LILR mice point to the first overexpression of KIR on all lymphocytes. We intend to examine explanation. Thus, the existence of a putative LCR in the 52N12 the possible effect of MHC class I on KIR expression in our trans- construct region but not in the 1060P11 region may provide a genic model that more closely mimics the human situation by simple explanation for the differences between the observed ex- crossing the KIR transgenics with HLA transgenics. pression frequencies. In addition, the FISH experiments performed Although KIR2DL2 expression was observed only in three of on the nonexpressing KIR transgenic founders showed varying the nine transgenic founders that were created, RT-PCR results single integration sites in these mice, of both heterochromatic and show that KIR2DL4 is expressed both in one of the founder mice nonheterochromatic nature. This finding would indicate that the that does show KIR2DL2 expression, as well as on a mouse that reduced expression percentage is less likely to be directly caused does not express these KIRs. This suggests that flanking this gene by a heterochromatinous integration site, and points toward the are genetic elements that may allow KIR2DL4 to be expressed importance of an LCR in pushing expression to normal even if the entire transgene has integrated into a heterochromatic frequencies. region that prevents expression of, at least, KIR2DL2. The region An alternative factor that could be relevant to the expression 14 Kb upstream of KIR2DL4 is unique, and the receptor has been frequency observed is the lack of MHC class I, the natural ligand shown to be expressed in all KIRϩ human NK and T cell clones. to the KIRs. Theoretically, this could cause a disturbance in the This, and the fact that KIR2DL4 is one of the three “framework” cellular education process of KIR-positive cells, by not allowing KIR genes present in all haplotypes observed in humans and chim- KIRϩ cell proliferation, which may be brought about by the MHC panzees (42), points to an important conserved function. class I-KIR interaction. Cambiaggi et al. (32) generated In summary, the pattern of KIR and LILR expression we have KIR2DL3 ϫ HLA-Cw3 double transgenic mice, and their results observed in the transgenic mice indicates that the genomic con- showed no effect on KIR expression by the presence of its HLA structs containing these gene families are sufficient to drive an ligand. As mentioned before, this model exhibited a significant expression pattern similar to the one seen in humans for at least The Journal of Immunology 3063 part of the KIR genes contained on the 1060P11 PAC clone. The 18. Salcedo, M., A. D. Diehl, M. Y. Olsson-Alheim, J. Sundback, L. Van Kaer, transgene expression pattern of those genes that were expressed K. Karre, and H. G. Ljunggren. 1997. Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J. Immunol. seems to be encoded within the DNA sequences and is indepen- 158:3174. dent of the presence of the ligands for these receptor families. The 19. Roth, C., J. R. Carlyle, H. Takizawa, and D. H. Raulet. 2000. Clonal acquisition of inhibitory Ly49 receptors on developing NK cells is successively restricted and KIR receptor family also seems to possess an inherent ability for regulated by stromal class I MHC. Immunity 13:143. variegated expression. 20. Hanke, T., H. Takizawa, and D. H. Raulet. 2001. MHC-dependent shaping of the The availability of these mice permits the developmental ex- inhibitory Ly49 receptor repertoire on NK cells: evidence for a regulated sequen- pression of LILRs and KIRs to be studied, the ways in which their tial model. Eur. J. Immunol. 31:3370. 21. Colonna, M. 1997. Specificity and function of immunoglobulin superfamily NK expression is regulated in response to infections, as well as their in cell inhibitory and stimulatory receptors. Immunol. Rev. 155:127. vivo effects on disease susceptibility. 22. Cosman, D., N. Fanger, L. Borges, M. Kubin, W. Chin, L. Peterson, and M. L. Hsu. 1997. A novel immunoglobulin superfamily receptor for cellular and viral MHC class I molecules. Immunity 7:273. Acknowledgments 23. Nakajima, H., J. Samaridis, L. Angman, and M. Colonna. 1999. 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