Characterization of the ferret TRB locus guided by V, D, J, and C gene expression analysis
The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.
KeywordsT cell receptor TRB locus Ferret (Mustela putorius furo) IMGT Comparative genomics Immune repertoire sequencing
The ferret (Mustela putorius furo) is an important mammalian model species to study human respiratory infections. Ferret infection models are well suited to study the pathogenicity and transmissibility of Coronaviruses (SARS), Pneumoviridae (RSV) and Orthomyxoviruses that include human and avian influenza viruses (Enkirch and von Messling 2015; Oh and Hurt 2016). Ferrets are an attractive mammalian model species for these infections since ferrets and humans share similar lung physiology, and most notably a very similar (viral) receptor distribution throughout the respiratory tract (Belser et al. 2011; van Riel et al. 2006). A significant drawback of the ferret model is a lack of ferret specific reagents for detailed studies of the host immune response to these pathogens. Nevertheless, the use of the ferret model has increased over the years, and its usage, with the recent publication of the ferret (draft) genome (Peng et al. 2014), is likely to increase even further. Currently, little is known about the T cell receptor (TCR) repertoire of the ferret, limiting the options to monitor the immune response of ferrets to experimental infections with influenza virus and other pathogens.
TCRs mediate recognition of peptide antigens presented to T lymphocytes via the peptide-MHC complex (Davis and Bjorkman 1988). Conventional TCRs are αβ or γδ heterodimers that are formed by somatic rearrangement of Variable (V), Diversity (D), and Joining (J) gene segments for the β and δ chains, and V and J gene segments for the α and γ chains (Davis and Bjorkman 1988). Although the ratio between αβ and γδ T cell subsets is not known for the ferret, the αβ T cells are much more common than γδ T cells in both human and dog (Mineccia et al. 2012). The β chain (at least in humans) tends to interact more closely with the peptide antigen than the α chain (Glanville et al. 2017), making the TRB locus the most interesting first candidate to annotate in detail.
In this study, we annotate the expressed V, D, J, and C genes in the ferret TRB locus by combining genomic information from the locus with HTS of the ferret TRB repertoire. We find that the TRB locus of the ferret has a similar structure to that of other mammalian TRB loci, such as mouse and human (Glusman et al. 2001), bovine (Connelley et al. 2009), dog (Mineccia et al. 2012), and rabbit (Antonacci et al. 2014): a library of V genes, followed by two (or three in bovine) D-J-C clusters. Each cluster consists of one D gene, six or seven (six in ferret) J genes, and a single C gene. The D-J-C clusters are followed by a V gene with an inverted transcriptional orientiation. We also performed a phylogenetic analysis, showing that the ferret V and J genes are indeed most closely related to those of the dog. The ferret locus is small like that of the dog, about 300 Kb, and has a (largely) conserved synteny with the dog TRB locus. Our annotation of the ferret TRB locus will enable detailed studies of T cell responses to support research on novel or improved antiviral strategies for influenza and other viral infections employing the ferret as a model organism. All TRB genes identified in our analysis (expressed or not) have been approved by the IMGT/WHO-IUIS nomenclature committee.
Materials and methods
The ferret genomic scaffolds (GL896904.1 and GL897291.1) representing the TRB locus were retrieved from Genbank (ferret whole genome shotgun sequence Mus-PutFur 1.0, ref 5) guided by sequence homology with the dog TRB locus (chro- mosome:CanFam3.1:16:6706526:7027700:1). The blast algorithm (Altschul et al. 1990) and Mauve (Darling et al. 2004) software were applied to align the ferret genome scaffolds with the dog genome sequence.
Four surplus cryopreserved healthy control blood samples were obtained from an influenza vaccination-challenge study (Bodewes et al. 2010). The control blood samples originated from four 6 to 12 months old healthy outbred female ferrets.
RNA isolation and 5′-RACE
Template switch primer
Step-out primer 1
Step-out primer 2
TRB transcript sequence analysis
The resulting TCR amplicons were subjected to high-throughput sequencing ac- cording to the instructions of the manufacturers using the Ovation Low Complexity Sequencing System kit from NuGEN (San Carlos, CA, USA) and the Illumina MiSeq or the Hiseq2500 platforms both using indexed paired end 300 cycle runs. All sequence reads having the same UMI were collapsed into consensus sequences using the RTCR pipeline (Gerritsen et al. 2016). The BLAST+ (Camacho et al. 2009) and exonerate (Slater and Birney 2005) (version 2.2.0) software were used to align the consensus sequences with the ferret genomic scaffolds to identify V, D, and J genes. After describing the V and J sequences in the ferret TRB locus, the RTCR pipeline was used to annotate the sequences, and perform additional error correction.
Identification of expressed ferret TRB V, D, J, and C genes
Interestingly, in the functional V gene TRBV19, we discovered a non-functional splice variant (see Figure S1 for an example), leading to a frameshift and the loss of the FR1 and CDR1 regions of the V-EXON. This splice variant occurs on average in more than 25% of the sequence reads that align to TRBV19 in each of the four ferrets. To be able to detect this non-functional variant, sequence reads should extend nearly 200 bp into the V-EXON region. If reads do not extend this far into the V gene, this may lead to an overestimation of the expression level of V genes having such non-functional splice variants. To this end repertoire sequencing experiments should use reads that are long enough to detect the splice variants, or perform a bias correction based on dedicated experiments quantifying the proportion of non-functional splice variants in the ferret population.
The ferret TRBD1 and TRBD2 genes are 12 bp and 15 bp long, respectively, and both genes are productively read in all 3 reading frames (Fig. 3a). The ferret J genes are between 44 and 53 bp long and conserve the FGXG motif, required for a functional J gene. The only exception is TRBJ1-4, classified as an ORF because it has a noncanonical J-MOTIF (“FASG”; Fig. 3b) identical to the homologous gene, TRBJ1-4, in the dog. Both TRBJ2-1 and TRBJ2-5 are also classified as an ORF due to having a noncanonical J-NONAMER. Although TRBJ1-3 is a pseudogene (because of a stop codon), it can still lead to functional transcripts by VDJ recombination, as it is used in about 3% of the TCRB clonotypes (Fig. 6b). Like other mammalian species such as human, mouse, dog, and rabbit (Antonacci et al. 2014), the ferret TRBC genes consist of 4 exons each (Fig. 4). The TRBC genes of the ferret are identical to each other for the first 2 exons (EX1 and EX2), and differ by only 2 nt in EX3 and also by 2 nt in EX4. The FG loop is one amino acid longer than the longest TRBC FG loop described by IMGT (Lefranc et al. 2005). We extended the numbering of the FG loop to accommodate the additional amino acid (Fig. 4). Both TRBC genes appear to be functional, having proper acceptor and donor splice sites for each exon, not containing any stop codon or frameshifts.
Phylogenetic analysis of the ferret TRBV and TRBJ genes
Analysis of the ferret TRBV and TRBJ usage
We combined HTS and genome analysis to describe the (expressed) T cell receptor genes in the TRB locus of the ferret. The genomic organization of the ferret TRB locus is very similar to that described in other mammals such as human, mouse, dog, and rabbit (Mineccia et al. 2012; Antonacci et al. 2014): the locus is flanked by MOXD2 and EPHB6 at the 5′ and 3′ ends, respectively, and consists of a library of V genes followed by two D-J-C clusters, followed by a V gene, which is non-functional in the ferret, with an inverted transcriptional orientation. Thus, the ferret confirms the strong organizational conservation of mammalian TRB loci. The ferret and dog TRB loci are closely related, because the ferret and the dog are in the same mammalian order, the Carnivora. Like in the dog, the ferret TRB locus is relatively small (300 Kb) and both contain about 20 functional TRBV genes. The ferret also expresses TRBV and TRBJ genes that contain stop codons, which nonetheless lead to functional transcripts, because these stop codons are deleted during VDJ recombination.
The ferret TRB locus is represented by two scaffolds of the draft genome assembly of the ferret as indicated in Fig. 1. Since, one, the highly conserved synteny with the dog TRB locus (any genomic sub-region of the dog TRB locus has a ferret counterpart on either of the two contigs), and two, the facing ends of the two scaffolds (Fig. 1, top line) display considerable sequence homology (> 1400 nt, data not shown), all ferret TRB VDJ genes are most likely contained in the current genome build. It is to be expected that the two scaffolds will be connected in a future build of the ferret genome when additional sequence information is available to complete genome regions encompassing gene families like the TRB locus that are particularly difficult to assemble correctly.
As previously described (Mineccia et al. 2012), the CDR3 length distribution is highly conserved, which in this study is also confirmed as the ferret and the human have nearly identical CDR3 length distributions (Supplemental Figure S2). Despite the relatively low number of V genes in the TRB locus of the ferret, the ferret repertoire is highly diverse as there is hardly any overlap in TCRβ chains between the ferrets (Supplemental Figure S3 and S4). Within ferrets the TCRβ chain overlap between samples is about 50%, probably reflecting the presence of memory clonotypes in the blood that have expanded due to antigen stimulation.
Although the ferret is an important animal model in research on respiratory infections, its adaptive immune responses were until now poorly characterized (Enkirch and von Messling 2015). Our characterization of expressed TRB genes in the ferret paves the way for detailed analysis of the cellular immune responses of ferrets in health and disease.
BG, AA, and FZB were supported by the VIRGO consortium funded by the Netherlands Genomics Initiative and the Dutch Government (FES0908). RdB and AP were supported by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement 317040 (QuanTI). AP was also supported by the Netherlands Organization for Scientific Research (NWO) VENI grant agreement 016.178.027.
- Antonacci R, Giannico F, Ciccarese S, Mas-sari S (2014. ISSN 1432-1211) Genomic characteristics of the T cell receptor (TRB) locus in the rabbit (Oryctolagus cuniculus) revealed by comparative and phylogenetic analyses. Immunogenetics 66:255–266. https://doi.org/10.1007/s00251-013-0754-1 CrossRefPubMedGoogle Scholar
- Bodewes R, Kreijtz JHCM, van Amerongen G, Geelhoed-Mieras MM, Verburgh RJ, Heldens JGM, Bedwell J, van den Brand JMA, Kuiken T, van Baalen CA, Fouchier RAM, Osterhaus ADME, Rimmelzwaan GF (2010. ISSN 1098-5514) A single immunization with CoVaccine HT-adjuvanted H5N1 influenza virus vaccine induces protective cellular and humoral immune responses in ferrets. J Virol 84:7943–7952. https://doi.org/10.1128/JVI.00549-10 CrossRefPubMedPubMedCentralGoogle Scholar
- Glanville J, Huang H, Nau A, Hatton O, Wagar LE, Rubelt F, Ji X, Han A, Krams SM, Pettus C, Haas N, Lindestam Arlehamn CS, Sette A, Boyd SD, Scriba TJ, Martinez OM, Davis MM (2017. ISSN 1476-4687) Identifying specificity groups in the T cell receptor repertoire. Nature 547:94–98. https://doi.org/10.1038/nature22976 CrossRefPubMedPubMedCentralGoogle Scholar
- Lane J, Duroux P, Lefranc M-P (2010. ISSN 1471-2105) From IMGT-ONTOLOGY to IMGT/LIGMotif: the IMGT standardized approach for immunoglobulin and T cell receptor gene identification and description in large genomic sequences. BMC bioinformatics 11:223. https://doi.org/10.1186/1471-2105-11-223 CrossRefPubMedCentralGoogle Scholar
- Lefranc M-P, Pommié C, Ruiz M, Giudicelli V, Foulquier E, Truong L, Thouvenin-Contet V, Lefranc G (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental and comparative immunology 27:55–77 ISSN 0145-305XCrossRefGoogle Scholar
- Lefranc M-P, Pommié C, Kaas Q, Duprat E, Bosc N, Guiraudou D, Jean C, Ruiz M, Da Piédade I, Rouard M, Foulquier E, Thouvenin V, Lefranc G (2005. ISSN 0145-305X) IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Dev Comp Immunol 29:185–203. https://doi.org/10.1016/j.dci.2004.07.003 CrossRefGoogle Scholar
- Mamedov IZ, Britanova OV, Zvyagin IV, Turchaninova MA, Bolotin DA, Putintseva EV, Lebedev YB, Chudakov DM (2013. ISSN 1664-3224) Preparing unbiased T-cell receptor and antibody cDNA libraries for the deep next generation sequencing profiling. Frontiers in immunology 4:456. https://doi.org/10.3389/fimmu.2013.00456 CrossRefPubMedPubMedCentralGoogle Scholar
- Mineccia M, Massari S, Linguiti G, Ceci L, Ci-ccarese S, Antonacci R (2012. ISSN 1879-0089) New insight into the genomic structure of dog T cell receptor beta (TRB) locus inferred from expression analysis. Developmen-tal and comparative immunology 37:279–293. https://doi.org/10.1016/j.dci.2012.03.010 CrossRefGoogle Scholar
- Peng X, Alföldi J, Gori K, Eisfeld AJ, Tyler SR, Tisoncik-Go J-n, Brawand D, Law GL, Skunca N, Hatta M, Gasper DJ, Kelly SM, Chang J, Thomas MJ, John-son J, Berlin AM, Lara M, Russell P, Swofford R, Turner-Maier J, Young S, Hourlier T, Aken B, Searle S, Sun X, Yi Y, Suresh M, Tumpey TM, Siepel A, Wisely SM, Dessimoz C, Kawaoka Y, Birren BW, Lindblad-Toh K, Di Palma F, Engelhardt JF, Palermo RE, Katze MG (2014. ISSN 1546-1696) The draft genome sequence of the ferret (Mustela putorius furo) facilitates study of human respiratory disease. Nature biotechnology 32:1250–1255. https://doi.org/10.1038/nbt.3079 CrossRefPubMedPubMedCentralGoogle Scholar
- Andrew Rambaut (2009), FigTree v1.4.4. <http://tree.bio.ed.ac.uk/software/figtree/>.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.