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. 2011 Jun 7;12:295.
doi: 10.1186/1471-2164-12-295.

Characterization of Killer Immunoglobulin-Like Receptor Genetics and Comprehensive Genotyping by Pyrosequencing in Rhesus Macaques

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Free PMC article

Characterization of Killer Immunoglobulin-Like Receptor Genetics and Comprehensive Genotyping by Pyrosequencing in Rhesus Macaques

Anna J Moreland et al. BMC Genomics. .
Free PMC article

Abstract

Background: Human killer immunoglobulin-like receptors (KIRs) play a critical role in governing the immune response to neoplastic and infectious disease. Rhesus macaques serve as important animal models for many human diseases in which KIRs are implicated; however, the study of KIR activity in this model is hindered by incomplete characterization of KIR genetics.

Results: Here we present a characterization of KIR genetics in rhesus macaques (Macaca mulatta). We conducted a survey of KIRs in this species, identifying 47 novel full-length KIR sequences. Using this expanded sequence library to build upon previous work, we present evidence supporting the existence of 22 Mamu-KIR genes, providing a framework within which to describe macaque KIRs. We also developed a novel pyrosequencing-based technique for KIR genotyping. This method provides both comprehensive KIR genotype and frequency estimates of transcript level, with implications for the study of KIRs in all species.

Conclusions: The results of this study significantly improve our understanding of macaque KIR genetic organization and diversity, with implications for the study of many human diseases that use macaques as a model. The ability to obtain comprehensive KIR genotypes is of basic importance for the study of KIRs, and can easily be adapted to other species. Together these findings both advance the field of macaque KIRs and facilitate future research into the role of KIRs in human disease.

Figures

Figure 1
Figure 1
Rhesus macaque lineage II KIR alleles form 19 distinct groups. Neighbor-joining and parsimony analysis was performed using predicted amino-acid sequences for the dataset comprising the lineage II sequences obtained in this study. The neighbor-joining tree is shown, which was not significantly different from the parsimony tree [data not shown]. Bootstrap support of greater than 50% is indicated. Representative hominoid lineage II KIR were used as an outgroup. For each gene, the numbers in parentheses indicate the total number of alleles discovered in this study compared with the total number known.
Figure 2
Figure 2
Domain shuffling has acted to form rhesus macaque KIR genes. The results of a domain-by-domain phylogenetic analysis are shown schematically. Predicted amino-acid consensus sequences for each of the genes were used to form both neighbor-joining and parsimony trees as described in the Methods section. The sequences encoding the three extracellular Ig-like domains were analyzed separately. Boxes are colored for the D0, D1, and D2 domains where the grouping was supported by >50% bootstrap support in the phylogenetic analyses. Boxes that are not completely colored represent cases where support of >50% was only found in the neighbor-joining analysis. Long cytoplasmic tails are colored red and short tails are colored green. The domains colored white were not resolved into any group in the analysis and should not be interpreted as being closely related to each other. The stem, transmembrane and cytoplasmic tails were grouped as either long or short by inspection of the alignment. D0, D1, and D2 denote the Ig-like domains. ST/TM/CYT denotes the stem, transmembrane, and cytoplasmic tail domains.
Figure 3
Figure 3
KIR 454 Titanium Amplicon Primer Design. A) Schematic representation of macaque KIR3D and KIR2D molecules showing domain structure. B) Variability plot for a cDNA sequence alignment of all published KIR3DL alleles. PCR primer sites are indicated along with the region amplified by PCR. Note that 454 sequencing reads span the D1, D2, and stem (ST) regions. The signal sequence (SS), D0, transmembrane region (T), and cytoplasmic region (C) are not amplified by our PCR primers.
Figure 4
Figure 4
Outline of KIR genotyping strategy. The input material is isolated RNA, which will represent all KIRs transcribed by the subject. PCR is performed using conserved KIR-specific primers that add adapter tags. The PCR products are pyrosequenced, producing 1000s of reads, with each read representing a single input molecule. These reads are compared against a reference database of macaque KIR sequences. This analysis produces a list of all KIRs detected per subject, including the relative frequency of each KIR.
Figure 5
Figure 5
Differential KIR expression between subjects with a common KIR haplotype. A) KIR genotypes obtained by pyrosequencing are shown for three rhesus macaque half-siblings. The relative expression level of each detected KIR allele is shown. Striped bars indicate alleles present on their shared KIR haplotype. The number of pyrosequencing reads is shown to the left of each graph. B) For r95061, the KIR alleles detected by cDNA cloning are shown. A total of 101 clones were examined. If indicated, the allele detected is listed. A plus sign indicates the genotyping resolution obtained by cloning is identical to the resolution obtained by pyrosequencing.
Figure 6
Figure 6
Pyrosequencing produces reproducible frequency estimates for KIR transcripts. Pyrosequencing results from four animals are shown. Per animal, two independent cell preparations were performed, and two independent PCRs were performed per cell preparation. For each replicate, the expression of each KIR is graphed as a percentage of total reads. Bars represent the average and standard deviation among replicates. For animal 225-97 (asterisk), only one cell pellet was available, so only two data points are shown.
Figure 7
Figure 7
Novel rhesus macaque KIR sequences share homology with cynomolgus macaque KIRs. Amino acid differences between the consensus sequences for the lineage II Mamu-KIR genes, KIRnov03, KIRnov04 and the Mafa-KIR sequence EU419113 are shown. The amino-acid position is indicated above the amino-acid sequences. The positions in the novel sequences that vary from the consensus are highlighted. Residues predicted to be involved in MHC-binding or alpha-helix loops are indicated below the sequences with B or L respectively. Black shading indicates residues for which no sequence coverage was available.
Figure 8
Figure 8
Frequency and relative expression of KIR genes in the rhesus macaque cohort. A) Y-axis indicates the percentage of animals within the cohort (n = 69) that express the indicated KIR gene. Genes not listed were not present in any animal within our cohort. Mamu-KIR2DL04 was excluded since it is not amplified by our pyrosequencing amplicon. B) Graph illustrates the percent of total pyrosequencing reads per animal (n = 61) for each KIR gene. Averages and SEM are represented by error bars. Each animal included had at least 100 sequencing reads. Genotyping results representing less than 1% of total reads in an animal were excluded to mitigate the influence of PCR artifacts. Ambiguous reads matching more than one KIR gene and splice variants were also excluded.
Figure 9
Figure 9
Novel rhesus macaque KIR haplotypes. KIR genes are indicated along the top axis. The identity of the allele is indicated within the schematic boxes if it was determined. Because data were generated from cDNA expression, only expressed KIRs are shown, and the physical map of gene order is arbitrary. Brackets indicate gene duplication. Since Mamu-KIR2DL04 cannot be amplified by our pyrosequencing amplicon, dotted boxes indicate haplotypes in which a false negative typing for this locus is possible.

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References

    1. Lanier LL. Evolutionary struggles between NK cells and viruses. Nat Rev Immunol. 2008;8(4):259–268. doi: 10.1038/nri2276. - DOI - PMC - PubMed
    1. Gardiner CM. Killer cell immunoglobulin-like receptors on NK cells: the how, where and why. Int J Immunogenet. 2008;35(1):1–8. - PubMed
    1. Parham P. MHC class I molecules and KIRs in human history, health and survival. Nat Rev Immunol. 2005;5(3):201–214. doi: 10.1038/nri1570. - DOI - PubMed
    1. Beck JC, Wagner JE, DeFor TE, Brunstein CG, Schleiss MR, Young JA, Weisdorf DH, Cooley S, Miller JS, Verneris MR. Impact of cytomegalovirus (CMV) reactivation after umbilical cord blood transplantation. Biol Blood Marrow Transplant. 2010;16(2):215–222. doi: 10.1016/j.bbmt.2009.09.019. - DOI - PMC - PubMed
    1. Miller JS, Cooley S, Parham P, Farag SS, Verneris MR, McQueen KL, Guethlein LA, Trachtenberg EA, Haagenson M, Horowitz MM. et al. Missing KIR ligands are associated with less relapse and increased graft-versus-host disease (GVHD) following unrelated donor allogeneic HCT. Blood. 2007;109(11):5058–5061. doi: 10.1182/blood-2007-01-065383. - DOI - PMC - PubMed

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