Phenotypic characterisation of regulatory T cells in dogs reveals signature transcripts conserved in humans and mice

Regulatory T cells (Tregs) are a double-edged regulator of the immune system. Aberrations of Tregs correlate with pathogenesis of inflammatory, autoimmune and neoplastic disorders. Phenotypically and functionally distinct subsets of Tregs have been identified in humans and mice on the basis of their extensive portfolios of monoclonal antibodies (mAb) against Treg surface antigens. As an important veterinary species, dogs are increasingly recognised as an excellent model for many human diseases. However, insightful study of canine Tregs has been restrained by the limited availability of mAb. We therefore set out to characterise CD4+CD25high T cells isolated ex vivo from healthy dogs and showed that they possess a regulatory phenotype, function, and transcriptomic signature that resembles those of human and murine Tregs. By launching a cross-species comparison, we unveiled a conserved transcriptomic signature of Tregs and identified that transcript hip1 may have implications in Treg function.


Isolation of peripheral blood mononuclear cells.
Mononuclear cells were isolated from the peripheral blood by density gradient centrifugation, using Histopaque ® -1077 (Sigma-Aldrich, Dorset, UK). Blood was diluted by an equal volume of phosphate-buffered saline (PBS; Sigma-Aldrich) with 2% v/v fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). The diluted blood was then layered onto an equal volume of Histopaque, before centrifugation at 400 g for 30 minutes at room temperature with minimal acceleration and braking. The purified peripheral blood mononuclear cells (PBMCs) were washed twice in PBS with 10% v/v FBS by centrifuging at 600 g for five minutes at 4 °C. After washing, cells were re-suspended in PBS with 10% v/v FBS, and counted using a haemocytometer before flow cytometric analysis. Dead cells were excluded by trypan blue staining.
In vitro suppression assay. CD4 + CD25 high and CD4 + CD25 − T cells sorted from the peripheral blood of healthy dogs were immediately re-suspended in complete culture medium (RPMI-1640 complemented with 10% v/v FBS, 10 mM HEPES, 100 μg/mL streptomycin, 100 U/mL penicillin and 0.5 mM β-mercaptoethanol; all reagents from Sigma-Aldrich). The responder T (Tresp) cell population (CD4 + CD25 − ) was stained with CellTrace ™ violet proliferation dye according to the manufacturer's instructions (Thermo Fisher Scientific), and seeded into a 96-well plate at a density of 1-5 × 10 4 cells per well. The suppressor cell population (CD4 + CD25 high ) was co-cultured with Tresp cells at a ratio (Treg:Tresp) of 1:1 and/or 1:2. A population of autologous CD5 − CD11b + monocytes at a proportion of 1/5 of that of Tresp cells were also seeded into each well, as APCs. The mixed cell culture contained a total volume of 200 μL with 2.5 μg/mL concanavalin A (ConA) (Sigma-Aldrich) and was incubated for 96 hours at 37 °C, with 5% CO 2. Three control groups were set up in the same fashion, including un-stimulated Tresp alone, stimulated Tresp alone and CD4 + CD25 − co-cultured with Tresp. RNA extraction. CD4 + CD25 high and CD4 + CD25 − T cells sorted from the peripheral blood of five healthy dogs were immediately re-suspended in RNA Bee (AMS Biotechnology, Abingdon, UK) at a density of 2 × 10 6 cells/mL. Two hundred microlitres of chloroform (Sigma-Aldrich) per millilitre of RNA Bee suspension were added, before thorough admixture, transfer to a 2 mL MaXtract High Density tube (QIAGEN, Hilden, Germany), and incubation on ice for three minutes. The tube was then centrifuged at 12,000 g for 15 minutes at 4 °C. After centrifugation, the upper aqueous layer was carefully transferred to a 1.5 mL DNase/RNase-free Eppendorf Tube ® (Eppendorf, Stevenage, UK), before being mixed completely with an equal volume of 100% ethanol (Sigma-Aldrich). The mixture was then transferred into a Zymo-Spin ™ IC column on top of a collection tube and centrifuged according to the manufacturer's instructions (Direct-zol ™ RNA MicroPrep Kit, Zymo Research, Read processing and expression quantification. Sequencing reads were trimmed using Skewer (version 0.1.125) to remove the adapter and anchor sequences added during library construction and sequencing. Trimmed transcript reads were mapped to the canine genome, CanFam3.1 (Ensembl Genes, release 91), using HISAT2 (version 2.0.0-beta). The uniquely mapped read pairs were quantified using featureCounts (version 1.5.0), and annotated using the same canine genomic data. Mapping metrics were generated using Picard Tools (version 1.92). The metrics and variants for assessing read distribution, biotype distribution and mapped transcripts were generated using R packages (version 3.4.2) with in-house scripts. Read counts were all converted to transcripts per million (TPM) to normalise sequencing depth and gene lengths.

Differential expression analysis.
Transcripts differentially expressed between canine CD4 + CD25 high and CD4 + CD25 − T cells were identified using Bioconductor package edgeR (Bioconductor version 3.6), with fold change (FC) values and statistical significance, the latter of which was represented by false discovery rate (FDR). R version 3.4.2 was used to conduct principal component analysis (PCA) and volcano plots.

Ingenuity pathway analysis. Differentially expressed transcripts (FDR < 0.05) with FC and FDR values
were input into the software Ingenuity Pathway Analysis (IPA; Ingenuity Systems Inc., Redwood City, CA, USA) to identify biological pathways affected by the altered expression of these transcripts (|Z| score ≥ 2).

Reverse transcription and quantitative PCR.
Purified total RNA was converted to cDNA by performing reverse transcription (RT), using the Precision nanoScript ™ 2 Reverse Transcription Kit (Primerdesign, Southampton, UK). One reaction of 20 μL volume in total contained RNA template (up to 2 μg), combined Oligo (dT) and random nonamer primers, nanoScript ™ 2 Buffer, dNTP mix, nanoScript ™ 2 enzyme and RNase/DNase free water. The reaction included an annealing step of 65 °C for five minutes, then immediate cooling on ice, followed by an extension step at room temperature for five minutes and 42 °C for 20 minutes, then 75 °C for 10 minutes. The abundance of transcripts of interest was then measured by quantitative (q) PCR, using cDNA as reaction template, according to the manufacturer's instructions. Primers specific to each transcript were all from the Taqman ® Gene Expression Assays (GEAs) (Thermo Fisher Scientific), targeting fam129a (Cf02724989_ Two reference transcripts, ubc encoding CG11624-PA, isoform A and sdha encoding succinate dehydrogenase flavoprotein subunit, were selected following validation by means of the Primerdesign Dog geNorm ™ Kit. The relative expression of the target transcript was calculated using Pfaffl's model 39 as below: Interspecies comparisons. To compare the transcriptomic profiles of canine CD4 + CD25 high T cells across species with those of human and murine Tregs, published resources were used. The selected human and murine studies 22,40 used different analytical methods from those in this study, but were the most comprehensive in the literature and conducted on freshly isolated Tregs in comparison to CD4 + CD25 − T cells. Raw transcriptomic data of the published human and murine studies were analysed following the same pipeline as for canine CD4 + CD25 high T cells, with respective genomic information. The data were processed using the web-based bioinformatics platform Galaxy 41 . Similarity scores were calculated using R OrderedList 42 (version 1.48.0), to determine the number of shared transcripts between two species in the first n consensus transcripts, which were ordered by differential expression FC values. A similarity score was yielded, in which transcripts received higher weight the closer they were to the top or bottom end of the ordered list. Similarity scores for n = 100, 150, 200, 300, 400, 500 and 750 transcripts were reported, respectively. Statistical significance was assessed for each of the similarity scores, by comparing with a null distribution generated by randomly scrambling the order of the transcripts. Statistical analysis. Summary data are shown as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA, USA).

Results
Freshly isolated canine CD4 + CD25 high T cells are enriched for FoxP3. To test the hypothesis that freshly isolated canine CD4 + CD25 + T cells have a regulatory phenotype, PBMCs of 11 healthy dogs were labelled with a mAb panel incorporating all markers of canine Tregs to date. When the CD25 gate was moved upwards to incorporate increasing CD25 expression per cell, from the highest 5% to the highest 0.2%, the proportion of FoxP3 + cells significantly increased from 36.89 ± 2.79% to 74.07 ± 4.81%, suggesting that ex vivo CD4 + CD25 high T cells were enriched for FoxP3 (Fig. 1a,b).
The top 1% of CD4 + CD25 + T cells were selected for subsequent phenotypic characterisation, balancing the enrichment for FoxP3 (61.59 ± 4.76%) with the need to isolate sufficient numbers. The proportional expression of FoxP3 in CD4 + CD25 high T cells was compared to CD4 + CD25 − cells of the same dogs, the latter selected by gating the 20% of CD4 + T cells showing the lowest CD25 expression. FoxP3 + cells in the CD25 high fraction were gated in two ways, making a comparison with either the corresponding isotype control or the paired CD25 − population (a negative biological control). The two gating methods yielded similar results: CD25 high T cells had significantly greater FoxP3 expression than CD25 − T cells from the same dogs (Fig. 1c). showing that proportional expression of FoxP3 increased with increasing CD25 expression by CD4 + T cells from the highest 5% to the highest 0.5% of one healthy dog (all CD4 + CD25 + T cells in this figure were analysed as CD45 + CD5 + CD8 -CD4 + CD25 + , following a cascaded gating strategy). (b) Scatter dot plot summarising the increasing proportional expression of FoxP3 (mean ± SEM) among CD4 + T cells of 11 healthy dogs, with increasing CD25 expression from the highest 5% to the highest 0.2%. (c) Summary scatter dot plot comparing the higher proportional expression of FoxP3 in top 1% of CD25 high cells, in which gating was determined by the corresponding isotype control (iso) or biological negative control (bio; CD25 − ). No significant difference was found in CD25 high cells between the two gating methods. Statistical significance in (b,c) was analysed by one-way ANOVA, followed by Dunn's multiple comparisons test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). (2019) 9:13478 | https://doi.org/10.1038/s41598-019-50065-8 www.nature.com/scientificreports www.nature.com/scientificreports/ Freshly isolated canine CD4 + CD25 high t cells are suppressive in vitro. Freshly isolated CD4 + CD25 high T cells suppressed conventional CD4 + CD25 − T cell proliferation, as indicated by reduced cell divisions at a ratio of 1:1 or 1:2 in the presence of autologous monocytes (CD5 -CD11b + ) and ConA (Fig. 2). Our findings therefore confirmed the suppressive function of ex vivo canine CD4 + CD25 high T cells. Given their regulatory phenotype and function, we then hypothesised that canine CD4 + CD25 high T cells have a transcriptomic profile characteristic of Tregs.
Ingenuity pathway analysis of canine CD4 + CD25 high T cells. Pathway analysis further consolidated functional annotations of the CD25 high T cell expression signature in comparison to CD25 − T cells, which identified three pathways associated with development and function of Tregs to be activated, namely phospholipase C signalling, p38-mitogen activated protein kinase (MAPK) signalling and cell cycle regulation (Fig. 3c).

A Treg-specific expression signature is conserved in humans, mice and dogs. We compared
Treg-specific transcriptomic signatures between species using similarity scores, which revealed a resemblance of canine CD4 + CD25 high T cells to both human and murine Tregs for the top 100 most differentially expressed www.nature.com/scientificreports www.nature.com/scientificreports/ transcripts (Fig. 4a). Of interest, human and murine Tregs showed no significant similarity ( Supplementary  Fig. S1). Thirty-one transcripts highly enriched in Tregs (FC > 2) were consensus in all three species (Fig. 4b).

Discussion
We have shown that canine CD4 + CD25 high T cells isolated ex vivo have the transcriptomic signature of Tregs, reconciling with their regulatory phenotype and function. Moreover, the transcriptomic signature of canine CD4 + CD25 high T cells resembled those of human and murine Tregs, consistent with our view that they represent Tregs. Apart from FoxP3 and other Treg signature molecules, we found that the canine CD4 + CD25 high T cells expressed transcripts encoding transcription factors specific to pro-inflammatory T helper (Th) cells in greater abundance than CD4 + CD25 − T cells. For instance, CD25 high T cells preferentially expressed gata3 and irf4 of Th2 cells [51][52][53] and, batf, ikzf3, ikzf4 and rorα of Th17 cells [54][55][56][57] . A trivial explanation of this phenomenon was enrichment of effector Th cells within the CD25 high T cells, which were not exclusively FoxP3 + and likely to be contaminated by Th cells. Healthy dogs are exposed to environmental antigens at mucosal surfaces on a continuous basis, with subsequent polarisation of a proportion of the local T cells and escape of these cells into the peripheral blood. An alternative explanation was that some of the peripheral Tregs themselves expressed Th-specific transcription factors, as has been previously documented 25,[58][59][60][61][62] . The CD4 + CD25 high T cells also expressed a number of homing receptor transcripts at greater abundance than the CD4 + CD25 − T cells. For instance, CD25 high T cells preferentially expressed Th2-associated chemokine receptor transcripts ccr3, ccr4 and ccr8 [63][64][65] , in line with the greater expression of Th2 transcription factor transcripts gata3 and irf4. Other chemokine receptors enriched in canine CD25 high T cells are expressed by human and murine Tregs resident in various tissues and organs, i.e. CXCR6 and CCR3 in adipose tissue 66 , CCR2, CCR5 and CXCR3 in pancreas 67 , CCR4 in skin 68,69 and, CCR2, CCR5 and CCR8 in muscle 70 . In contrast, CD25 high T cells expressed three transcripts encoding naïve T cell homing molecules CD62L (L-selectin), CCR7 and IL7R [71][72][73][74] in lower abundance. Trafficking of Tregs to peripheral lymphoid and non-lymphoid niches is critical to their functions in homeostasis, autoimmune disease and cancer in humans and mice, and expression of homing receptors may vary with developmental stage and target locations of Tregs 68,70,[75][76][77][78][79][80] . Single-cell RNA-seq would be required to distinguish whether these differential expression patterns were attributable to contaminant Th cells or to bona fide Tregs. Nevertheless, these data raise the intriguing possibility of ectopic expression of Th-specific transcripts by Tregs in dogs, as in other species: for instance, human Tregs isolated ex vivo from healthy donors express gata3 and ccr4 of Th2 cells 25 , and murine Tregs incorporate irf4 to suppress Th2 response 58 .
Pathways associated with the development and function of canine Tregs were identified in our dataset. A cascade of signal transduction pathways is engaged upstream and downstream of FoxP3, dedicating Tregs to lineage-specific commitment [81][82][83][84][85][86][87][88] . Phospholipase C signalling is a critical transduction pathway downstream of TCR activation in Tregs, and its defect causes profound autoimmune lesions in mice 89 . The dominant mediator phospholipase C produces secondary messenger molecules 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) [90][91][92] . IP 3 activates calcium flux, which then triggers the transcription factor nuclear factor of activated T cells (NFAT) to interact with FoxP3 89,91 . DAG functions in a cascade upstream of p38-MAPK signalling, which regulates the cell cycle and is indispensable in the induction of anergy and maintenance of Treg suppressive function 93 . The upregulation of phospholipase C, p38-MAPK and cell cycle regulation pathways in canine Tregs accords with these observations.
We interrogated expression signatures of Tregs across species, reasoning that similarity of transcripts would speak to their core function in Tregs. Canine Tregs resembled both human and murine Tregs, yielding 31 common differentially expressed transcripts. More than half of the 31 consensus transcripts encode Treg-specific molecules, indicative of interspecies conservation of Treg signature. Of the 12 transcripts not hitherto related to Tregs, hip1 has potential immunoregulatory relevance. Hip1 is a serine hydrolase protein embedded in cell envelopes of Mycobacterium tuberculosis, which reside intracellularly in macrophages and dendritic cells (DCs) of the host, evading immune responses by impeding functions of these primary APCs using Hip1 [94][95][96][97] . First, M. tuberculosis deactivates Toll-like receptor 2 and MyD88-dependent pathways via Hip1, reducing activation and cytokine production of macrophages and DCs 94,96 . Second, M. tuberculosis disrupts interactions between CD4 + T cells and APCs through GroEL2, a product of Hip1 hydrolysis 95,97 . Therefore, Hip1 may be another mechanism by which Tregs negatively modulate APCs. Fam129a and Alpha actinin-4 encoded by fam129a and actn4 inhibit cell apoptosis 98,99 , and Cathepsin Z, encoded by ctsz, promotes angiogenesis and metastasis 100,101 . These three proteins could potentially be blocked by specific mAb to attenuate the number and function of Tregs in the cancer microenvironment. The remaining eight transcripts are involved in T cell activation: protein products of cadm1, frmd4b, lmna, anxa2, galm, pou2f2, csf1 and ptprj may directly or indirectly enhance TCR signalling or interaction of T cells with APCs 102-109 . Single cell RNA-seq would be required to further explore the significance of these transcripts to Tregs, along with confirmation of differential expression of their protein products and their role in suppressive function, if any.
In conclusion, we have characterised the phenotype, function, and transcriptomic signature of canine Tregs. We have delineated a core set of 31 transcripts that show differential expression by the Tregs of three mammalian species, including humans. More than half of these transcripts have been previously associated with Tregs in mice and humans. However, 12 transcripts have hitherto not been associated with Tregs in any species, prompting further questions about their role in this cellular context. This comparative approach is a powerful tool in generating hypotheses that may yield fresh mechanistic insights or novel immunotherapeutic targets in this important, yet elusive, area of immunology.

Data Availability
Raw and processed canine RNA-seq data of this study have been deposited to Gene Expression Omnibus (GEO), accession number GSE132068.