Development of ferret immune repertoire reference resources and single-cell-based high-throughput profiling assays

Abstract

Domestic ferrets (Mustela putorius furo) are important for modeling human respiratory diseases. However, ferret B and T cell receptors have not been completely identified or annotated, limiting immune repertoire studies. Here, we performed long-read transcriptome sequencing of ferret splenocyte and lymph node samples to obtain over 120,000 high-quality full-length immunoglobin (Ig) and T cell receptor (TCR) transcripts. We constructed a complete reference set of the constant regions of ferret Ig and TCR isotypes and chain types. We also systematically annotated germline Ig and TCR variable (V), diversity (D), joining (J), and constant (C) genes on a recent ferret reference genome assembly. We designed new ferret-specific immune repertoire profiling assays by targeting positions in constant regions without allelic diversity across 11 ferret genome assemblies and experimentally validated them using a commercially compatible single-cell-based platform. These improved resources and assays will enable future studies to fully capture ferret immune repertoire diversity.IMPORTANCEDomestic ferrets (Mustela putorius furo) are an increasingly common model organism to study human respiratory diseases such as influenza infections. However, researchers lack ferret-specific reagents and resources to study the immune system and immune response in ferrets. In this study, we developed comprehensive ferret immune repertoire reference resources and assays, which will enable more accurate analyses of the ferret immune system in the future.

Keywords: VDJ annotation; ferret; immune repertoire; reference resource development; repertoire profiling; single-cell sequencing.

Fig 1

Genomic organization of ferret Ig regions. (A) Heavy chains and associated V, D, and J genes; (B) Kappa light chains and associated V and J genes; and (C) Lambda light chains and associated V and J genes. Constant regions are annotated in orange, V regions are in blue, D regions are in yellow, J regions are in red, and surrogate light chain components VpreB and λ5 are shown in gray. The orientation of each gene is indicated with an arrow. Kappa annotations are on the sense strand of the reference contig but depicted on the antisense strand in (C). Genome positions relative to ferret reference genome assembly GCA_011764305.2, contigs JAADYL010000038.1 (heavy chain locus), JAADYL010000784.1 (light chain lambda locus), and JAADYL010000091.1 (light chain kappa locus) are indicated below the bar. See Fig. S1 for additional details.

Fig 2

Genomic organization of ferret TCR region. (A) TRA associated V, D, and J genes; (B) TRB and associated V, D, and J genes; (C) TRG and associated V and J genes; and (D) TRD and associated V and J genes. Constant regions are annotated in orange, V regions are in blue, D regions are in yellow, and J regions are in red. The orientation of each gene is indicated with an arrow. Genome positions relative to ferret reference genome assembly GCA_011764305.2, contigs JAADYL010000821.1 (TCR alpha and delta chain locus), and JAADYL010000772.1 (TCR beta and gamma chain locus) are indicated below the bar. See Fig. S1 for additional details.

Fig 3

Validation of ferret-specific primers. (A) Individual ferret Ig- and TCR-specific primers were used to amplify the corresponding genes of the C-region genes from cDNAs isolated from a pool of three PBMC samples. Corresponding forward primers that were upstream but still within the C regions were designed solely for the purposes of this assay. PCR amplicon products of such reactions were analyzed on a 1% agarose gel to confirm the amplification specificity. Gel image on the left: primers for the first enrichment reactions. Gel image on the right: primers for the second enrichment reactions. On each gel, image lanes 1, 9, and 14 are 100 bp ladder. Lanes for Igs are labeled as IGHA (A), IGHD (D), IGHE (E), IGHG (G), IGHM (M), IGK (K), and IGL (L). Lanes for TRs are labeled as TRA (A), TRB (B), TRD (D), and TRG (G). (B) Recovery of ferret V(D)J transcripts using the single cell-based V(D)J assays with the ferret C-region specific primers. The tiled plot indicates the total number of ferret V(D)J transcript contigs with respective ferret primer and C region matches recovered from the ferret PBMC and splenocyte samples. See also Fig. S4.

Fig 4

Validation of ferret single-cell gene expression and immune repertoire profiling assay. (A) UMAP plots of the (left to right) assignment of the cell cluster, Ig chains, TCR chains in the ferret PBMC sample. (B) Same as (A) but for the splenocyte sample. (C) Ig and TCR pairing efficiencies of the cell clusters in the PBMC sample. Percentages and numbers indicate the frequency of B and T cells with paired chains (VDJ, VJ), VDJ-only, VJ-only, or no recovered chains. (D) Same as (C) for the splenocyte sample. (E) IGH isotype usage of B cell clusters in the splenocyte sample shown in (D).(F) Expressions of selected B cell development-related marker genes in the individual cell clusters of the splenocyte sample. The cell type of cell clusters in PBMC (A) and splenocyte (B) samples was collectively annotated based on the detection of Ig and TCR transcripts, SingleR-based cell type prediction, and manual review of selected canonical cell markers. The list of canonical markers used: common T cell marker: CD3D, CD3D, CD3G; common B cell marker: CD79A, CD79B, CD19; neutrophil in PBMC: S100A8, S100A9; monocyte: S100A4, LOC101672794 (PYCARD), LOC101687145 (FCN1); plasma cell: JCHAIN, TNFRSF17, MZB1; pre-B cell: surrogate light chains VPREB1 (LOC101692964) and IGLL5 (LOC101692660); pro-B cell: EBF1, DNTT; myelocyte/ immature neutrophil: MMP9, MMP8, NCF1, NCF2, NCF4; and NK cell: NCR3, NCR1 (LOC101670751). n/a in (B) indicates the cell type was not annotated due to the total number of cells (20) in cluster #12 was extremely small. For additional details, see Fig. S5 for the cell type prediction and Table S4 for differentially expressed genes in each cell cluster.