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Keywords:

  • NK cell;
  • KIR2DL4;
  • HLA-G;
  • Reproductive immunology;
  • Human

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

A central issue of reproductive immunology in mammals is why a semi-allogeneic embryo is not rejected by the pregnant mother. This is particularly intriguing since, in different species, the early pregnant uterus is infiltrated by numerous maternal lymphocytes, predominantly NK cells. The human NK cell receptor KIR2DL4 has been implicated in the maternal tolerance to the embryo due to its recognition of HLA-G, a non-classical MHC molecule expressed preferentially in the placenta. Killer cell Ig-like receptors (KIR) are believed to participate in the natural immunity to infection and tumors, but KIR2DL4 has unique structural, functional and genetic features that could confer it a different role. However, we demonstrate here that the KIR2DL4:HLA-G interaction is not essential for human reproduction by showing that a multiparous woman lacks a KIR2DL4 gene.

Abbreviations:
KIR:

Killer cell Ig-like receptor

PCR-SSP:

PCR with sequence-specific primers

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Mammalian pregnancy resembles some aspects of tissue engraftment, since embryos are genetically dissimilar from their mothers and could theoretically induce an allogeneic response. Furthermore, the early pregnant uterus of different mammalian species homes numerous maternal lymphocytes, predominantly a specific subset of NK cells, that could reject the embryo unless they are tolerized byyet to be understood mechanisms 14. In humans, maternal immune tolerance to the conceptus could be related to the unusual expression profile of MHC class I molecules in the placental extravillous cytotrophoblast. This tissue does not express those molecules that induce strong graft rejection (HLA-A and -B), but a unique combination of HLA-C, -E and -G molecules more effective in modulating NK cell function than in stimulating T lymphocytes 1. Among the latter MHC molecules, HLA-G is unique because of its restricted tissue distribution and its capacity to interact with different NK cell receptors, of which only KIR2DL4 (CD158d) has been reported to be specific for HLA-G 512. These data have inspired the hypothesis that the recognition of HLA-G by KIR2DL4 could exert an important function in the maternal-fetal interface 5, 1114.

KIR2DL4 belongs to the family of killer cell Ig-like receptors (KIR), believed to participate in the natural immunity to infection and tumors 1518. However, KIR2DL4 is the only MHC receptor whose gene is transcribed in all NK cells, in contrast with the clonal distribution seen in all other KIR, in LILRB1 and in CD94:NKG2 heterodimers 17, 1921. The structure of KIR2DL4 is also divergent and transmits a complex signal to NK cells: it inhibits their cytotoxicity but induces secretion of IFN-γ 14, 2224, a cytokine that contributes to placental development in mice 25. Finally, whereas other KIR genes are represented in only some individuals from a few primate species, KIR2DL4 has orthologs in all those species and is remarkably conserved in different humans 18, 26, 27.

The aforementioned distinctive features suggest that KIR2DL4 could exert a unique function, possibly different from those of other KIR. However, we provide clear-cut evidence in this report that lack of KIR2DL4 does not compromise severely human fertility and health.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 An unusual KIR haplotype lacking central "framework" genes: 3DP1, 2DL4 and 3DL/S1

We investigated the degree of conservation of the KIR2DL4 gene in different populations by analyzing its presence in DNA of 304 individuals of three ethnic groups, including 134 Spanish caucasoids, 74 Spanish Gypsy caucasoids and 96 Bubi blacks from the Bioko Island (Equatorial Guinea) 28, 29, using the PCR with sequence-specific primers (PCR-SSP) method 30. The KIR2DL4 gene was detected in all samples except for one Bubi woman, GB11, whose genomic DNA failed to amplify with the KIR2DL4-specific primers. Complete KIR genotyping by PCR-SSP showed GB11 to have the 2DL2, 2DL5, 3DL2, 3DL3, 2DS1, 2DS2 and 2DS5 genes; and to lack 2DL1, 2DL3, 2DL4, 3DL1, 2DS3, 2DS4, 3DS1, 2DP1 and 3DP1 (Fig. 1). The absence of 3DP1 and both of 3DL1 and 3DS1 is remarkable because, like 2DL4, these are considered "framework" genes due to their ubiquitous presence in different KIR haplotypes 26, 3040. KIR2DL4, 3DP1 and 3DL1/3DS1 are contiguous genes in the central region of the KIR gene cluster, 2DL4 being in the middle of 3DP1 (centromeric) and 3DL1/ 3DS1 (telomeric) 33, 34. Thus, GB11 appears to be homozygous for a KIR haplotype that bears a deletion comprising from 3DP1 through 3DL1/3DS1. Alternatively, she could be heterozygous for KIR haplotypes that share the latter deletion and differ in other regions of the KIR gene cluster (thus lacking additional genes).

Existence of similar haplotypes in two South Asian individuals has recently been proposed 38, but the evidence supporting the deletion of KIR2DL4 was so far based on the negative results obtained with a limited PCR-SSP assay and two oligonucleotide probes (38 and this study), which could also be explained by unrecognized polymorphisms of this gene. To rule out this possibility, we explored further the presence of KIR2DL4 in the genome of GB11 using seven additional PCR primer pairs, as detailed in Sect. 4; in none of those reactions was a KIR2DL4 product amplified. Of particular relevance were the results obtained with primers F153 and R939 (Fig. 2), which recognize sequences conserved in all KIR genes and pseudogenes but KIR2DL5 32. In most DNA samples, those primers amplify a ∼3.8-kb segment, derived from exons (and pseudoexons) 3–5 of most KIR2D and KIR3D genes; and a shorter product (∼1.4 kb) from KIR2DL4, which lacks a region homologous to exon 4 of other KIR genes. By contrast, only the longer fragment was amplified from genomic DNA of GB11, strongly supporting the absence of KIR2DL4 in this donor. The absence of 3DP1 and 3DL1 in GB11 was also confirmed with PCR primers different from those used in regular typing (not shown).

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Figure 1.  KIR genotyping of GB11 (upper panel) fails to detect genes and pseudogenes conserved in most humans. Genotyping of a donor having all KIR genes and pseudogenes is shown in the lower panel. Gene order in the gel does not reflect that in the chromosome. I.c.: internal control band. Marker: 1-kb ladder (Life Technologies, Paisley, GB).

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thumbnail image

Figure 2.  PCR with primers for sequences conserved in exons 3 and 5 of most KIR genes supports the absence of a KIR2DL4 gene in the genome of GB11. The ∼1.4-kb band corresponds to KIR2DL4; the ∼3.8-kb band corresponds to the other KIR genes and pseudogenes (except KIR2DL5, which is not recognized by the reverse primer). Marker: 1-kb ladder.

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2.2 Southern blotting reveals a reduced KIR gene content in the genome of GB11

To further confirm the reduced KIR gene complement of donor GB11, we used the Southern blot method, whose results are less likely to be influenced by sequence polymorphisms. Genomic DNA of GB11 was digested with the restriction endonuclease HindIII and hybridized with KIR cDNA probes, which showed a simplified banding pattern in comparison with those of donors with more common KIR genotypes (Fig. 3, left panel). The latter donors produced patterns similar to those reported in previous studies 26: eight relatively conserved bands (B–I, Fig. 3) with sizes ranging ∼6–20 kb; and a fragment longer than 24 kb (band A) that is observed only in donors having KIR2DL5 (a gene that lacks all the HindIII recognition sites seen in other KIR). All these fragments roughly match predictions based on the HindIII restriction maps of sequenced KIR haplotypes (Fig. 3, right panel).

Only five of the eight conserved HindIII bands (C, D, E, F and I) and the KIR2DL5-associated fragment (A) were revealed in GB11, whereas three other bands with relative sizes of ∼20, ∼10.5 and ∼7.5 kb (B, G and H, respectively) were absent (Fig. 3). Furthermore, whilst other donors had ∼17-kb bands (C) with enhanced intensity due to superposition of two or more DNA fragments of similar lengths, band C was weaker in GB11 (Fig. 3), suggesting the presence of a single fragment.

The lengths of the fragments missing in the Southern blot of GB11 sum up to ∼55 kb. Considering that the KIR gene cluster spans only ∼150 kb and that KIR genes have an average size of ∼12 kb 33, 34, this indicates that the KIR gene content is considerably reduced in the genome of GB11. Furthermore, the HindIII bands observed in the Southern blot of GB11 correlate precisely with the fragments predicted by comparison of her apparent KIR genotype with a sequenced KIR haplotype (Fig. 3, right).

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Figure 3.  Southern blotting analysis reveals reduced KIR gene content in the genome of GB11 and is consistent with a deletion affecting the central region of the KIR gene cluster. Left panel: HindIII-digested DNA of GB11 and four other donors, hybridized with KIR cDNA probes. Right panel: HindIII fragments deduced from the nucleotide sequences of three genomic clones isolated from two different KIR haplotypes (AC006293, AC011501 and AL133414) 33, 34, the latter of which resembles more closely those of GB11. A hypothetical deletion mechanism for the generation of the KIR haplotypes of GB11 is proposed below clone AL133414. KIR gene order in GB11 has not been determined experimentally, except for 2DL2 and 2DL5, found to be contiguous in at least one chromosome using a previously reported method 51. Sizes (in kb) and possible correspondence with bands seen in Southern blotting (labeled with letters A–I) are shown over each HindIII segment of both haplotypes; fragments of clone AL133414 apparently shared by GB11 are boxed. The sizes of fragments containing only non-coding sequence or small exons are not indicated since they are not revealed by the cDNA probes. KIR genotypes: GB11: 2DL2, 2DL5, 3DL2–3, 2DS1–2, 2DS5; controls 1 and 2: 2DL1, 2DL3–4, 3DL1–3, 2DS4, 2DP1, 3DP1-ex2; control 3: 2DL1, 2DL3–5, 3DL1–3, 2DS1, 2DS4–5, 3DS1, 2DP1, 3DP1-ex2; control 4: 2DL1–5, 3DL2–3, 2DS1–3, 2DS5, 3DS1, 2DP1, 3DP1-ex2. Markers: lambda DNA, digested with HindIII, and 1-kb ladder.

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2.3 Recognition of HLA-G by KIR2DL4 is not essential for reproduction

The results of PCR and Southern blot studies we have presented demonstrate that donor GB11 lacks KIR2DL4 and other common KIR genes. Moreover, the HLA class I alleles of GB11 (HLA-A*30; B*42(Bw6),*1503(Bw6); Cw*0202Lys80,*17Lys80) provide no known ligand for the inhibitory KIR encoded in her genome (KIR2DL2 and KIR3DL2), but only for the activating receptor KIR2DS1. Unfortunately, we could not explore in vitro the consequences of this rare genotype on the phenotype and function of NK cells, since the only available material was genomic DNA and the donor was inaccessible for rebleeding. However, GB11 is an adult woman who carried five successful pregnancies and suffered a single miscarriage. Furthermore, GB11 appeared to have a normally functioning immune system, as she had suffered neither opportunistic nor recurrent infections, nor clinical manifestations suggestive of immune deficiency or dysregulation. Her historyof infections included: onchocerciasis, a cutaneous filariasis hyperendemic in her living area (Bioko Island, Equatorial Guinea) 41; vaginosis by Gardnerella vaginalis; and a cleared infection by hepatitis B virus (HBsAg-negative, anti-HBs- and anti-HBc-positive). Previously performed leukocyte counts were normal in absolute and relative numbers, except for 18% eosinophils (1134 cells/μl); tuberculin skin test was positive; and quantitation of serum Ig showed moderate increase of IgG (2480 mg/dl), with normal IgM (275 mg/dl) and IgA (90 mg/dl) concentrations.

3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The ubiquitous transcription of the KIR2DL4 gene in the NK cell clones of all analyzed individuals and its conservation in evolution suggest that the product of this gene could essentially contribute to the biology of human NK cells, a view further supported by its distinct structure and signaling properties 11, 14, 16, 17, 22, 23. Preferential expression of the KIR2DL4 ligand, HLA-G, in the placenta pointed to a possible participation of KIR2DL4 in the poorly understood functionof uterine NK cells in the maternal-fetal interface. However, we have shown here that a woman reached the reproductive age and had several successful pregnancies despite lacking both a KIR2DL4 geneand HLA ligands for her inhibitory KIR due to an unusual genotype, which excludes an essential and unique function of KIR2DL4 in human reproduction and immunity.

Previous description of adult individuals homozygous for mutations that prevent expression of HLA-G1 raised concern on the role of this MHC molecule in promoting maternal-fetal tolerance 42, 43. It was subsequently proposed that other HLA-G isoforms produced by alternative splicing of the mutant allele could be recognized by KIR2DL4 and inhibit maternal NK cells 44. Our findings indicate that, even if such is the case, those interactions do not need to be claimed to explain the survival of embryos lacking HLA-G1, as the absenceof KIR2DL4 in maternal NK cells does not pose a major threat to their development. It must be emphasized, however, that the deletion of either KIR2DL4 or HLA-G could have more subtle effects on NK cell function and reproduction.

Redundancy of NK cell receptors for MHC could compensate or attenuate the effect of the loss of the KIR2DL4 function. For instance, HLA-G and, perhaps, HLA-C can be recognized directly by uterine NK cells through LILRB1; in addition, the leader peptides of those MHC molecules can be presented by HLA-E to NK cell clones expressing CD94/NKG2 heterodimers 9, 10, 4547. However, none of the aforementioned receptors has signaling properties or distributions similar to those reported for KIR2DL4 11, 14, 2224. By contrast, KIR3DL3, whose ligand and functionality are unknown, shares with KIR2DL4 having a single ITIM and being well conserved in different individuals of several primate species 3234, 4850. Therefore, although the structures of these two receptors are otherwise divergent, this convergent feature makes it worth considering whether they might substitute for some of each other's functions. Alternatively, the lack of a gross effect of the unusual KIR genotype reported here on fertility could support the view 6 that HLA-G and its receptors may exert functions that are unrelated to the maternal tolerance to the fetus.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Analysis of KIR genes by PCR and Southern blot

Genotyping of KIR2DL4 and other KIR genes was performed using the PCR-SSP method 30. The possible presence of KIR2DL4, KIR3DP1 and KIR3DL1 in the genome of GB11 was further explored by PCR using additional primer combinations: F153/R939, F153/R933 (AGCCGAAGCATCTGTAGG), F121 (TTCTTGGACCAGAGTGTG)/R137, F121/Rc401 (TGTGACCATGATCACCAG), F121/R933, Ftgc157/Rc401, and Ftgc157/R933 for KIR2DL4; F153/Rt667 and Ft550 (CAGAGAGGGGAAGTTTAAT)/R939 for KIR3DP1; and Ft124 (GGTCCAGAGGGCCGGT)/R380 (TTGCTGGGTGCCGACCA) and Fcon187 (CGCTGTGGTGCCTCGA)/Rt697 (CCACGATGTCCAGGGGA) for KIR3DL1. The primers whose sequences are not shown have been described elsewhere 30, 32.

Southern blot analysis was performed by standard methods 26 using HindIII and a mixture of KIR3DL1 and KIR2DL4 cDNA probes. Analysis of the physical linkage between the KIR2DL2 and KIR2DL5 genes was performed as previously described, using PCR primers F1450 and LRcon364 51.

4.2 Genotyping of HLA class I genes

The HLA type of GB11 had been determined previously by both serology and DNA methods 29, 52. The asparagine-80/lysine-80 dimorphism of HLA-C was analyzed byPCR-SSO (RELI HLA-C test, Dynal).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

We thank Marcelo Pando and Heather G. Shilling (Dept. of Structural Biology, Stanford University) for kindly providing cDNA probes, and Rosario Solís for help in Southern blot studies. This work was supported by grant FIS-01/0381 from the Instituto de Salud Carlos III. Natalia Gómez-Lozano is a resident of the BIR program (Ministerio de Sanidad y Consumo).

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