Canine peripheral non-conventional TCRαβ+ CD4-CD8α- double-negative T cells show T helper 2-like and regulatory properties

The dog is an important companion animal and also serves as model species for human diseases. Given the central role of T cells in immune responses, a basic understanding of canine conventional T cell receptor (TCR)αβ+ T cells, comprising CD4+ single-positive (sp) T helper (Th) and CD8α+ sp cytotoxic T cell subsets, is available. However, characterization of canine non-conventional TCRαβ+ CD4+CD8α+ double-positive (dp) and TCRαβ+ CD4−CD8α− double-negative (dn) T cells is limited. In this study, we performed a comprehensive analysis of canine dp and dn T cells in comparison with their conventional counterparts. TCRαβ+ T cells from peripheral blood of healthy dogs were sorted according to their CD4/CD8α phenotype into four populations (i.e. CD4+ sp, CD8α+ sp, dp, and dn) and selected surface markers, transcription factors and effector molecules were analyzed ex vivo and after in vitro stimulation by RT-qPCR. Novel characteristics of canine dp T cells were identified, expanding the previously characterized Th1-like phenotype to Th17-like and Th2-like properties. Overall, mRNA expression of various Th cell-associated cytokines (i.e. IFNG, IL17A, IL4, IL13) in dp T cells upon stimulation highlights their versatile immunological potential. Furthermore, we demonstrated that the CD4-CD8α- dn phenotype is stable during in vitro stimulation. Strikingly, dn T cells were found to express highest mRNA levels of type 2 effector cytokines (IL4, IL5, and IL13) upon stimulation. Their strong ability to produce IL-4 was confirmed at the protein level. Upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn than in the conventional CD4+ sp population. Constitutive transcription of IL1RL1 (encoding IL-33Rα) further supports Th2-like properties within the dn T cell population. These data point to a role of dn T cells in type 2 immunity. In addition, the high potential of dn T cells to transcribe the gene encoding the co-inhibitory receptor CTLA-4 and to produce the inhibitory cytokine IL-10 indicates putative immunosuppressive capacity of this population. In summary, this study reveals important novel aspects of canine non-conventional T cells providing the basis for further studies on their effector and/or regulatory functions to elucidate their role in health and disease.


Introduction
Dogs develop a number of spontaneous immune-mediated disorders, such as autoimmune diseases (1) and allergies (2), as well as cancers (3).Similarities to these complex diseases in humans make the dog particularly interesting as an animal model.Given the importance of T cells in health and disease, comprehensive knowledge of canine T cells is essential to elucidate pathogenesis mechanisms, to develop new treatment strategies, and to appropriately use dogs as a model for these strategies.
Besides the conventional TCRab + T cells expressing either CD4 or CD8a as co-receptor (i.e.CD4 + single-positive (sp) and CD8a + sp T cells), dogs harbor non-conventional TCRab + CD4 + CD8a + double-positive (dp) (4) and TCRab + CD4 -CD8a -double-negative (dn) T cells (5).Whereas conventional cytotoxic CD8 T cells mainly express the CD8ab heterodimer, canine non-conventional dp T cells predominantly express the CD8aa homodimer (6, 7).Due to their special co-receptor equipment, dp T cells possess unique immunological potential.In humans, dp T cells have been shown to be functionally relevant in cancer, infections and autoimmune diseases (8,9), while in mice, they have been found to accumulate at the site of injection following subcutaneous vaccination (10).In dogs, increased frequencies of CD4 + CD8a + dp T cells were reported after infection with Ehrlichia chaffeensis (11) and in the context of canine leishmaniasis (12).As shown by our group, mature extrathymic TCRab + dp T cells comprise about 2% of circulating T cells in healthy dogs and are characterized by an activated phenotype indicated by expression of CD25 and MHC-II (6, 13).Furthermore, consistent with their effector memory phenotype, these cells have a strong IFN-g production capability (6, 7).
Whereas dn T cells account for only 1-3% of all T cells in the peripheral blood of mice and humans (14,15), in dogs, approximately 15% of all circulating TCRab + T cells are doublenegative (5).This substantial population of non-conventional T cells is characterized by a remarkably high expression of CD25.CD25 is either co-expressed with FoxP3 (reminiscent of a regulatory phenotype), or without FoxP3 (reminiscent of an effector phenotype).In addition, subsets expressing IFN-g, GATA-3, or IL-17A suggest properties of conventional type 1 T helper (Th1), Th2, and Th17 cells, respectively (5).
The recently reported increase of dn T cells after allergen desensitization, as well as the greatly increased frequency of dp T cells in dogs with adverse food reactions (16) indicates that canine non-conventional T cells can be involved in immune responses in vivo.However, knowledge about these cells is still limited.
To gain deeper insight into their phenotype(s) and functional potential, we performed a comprehensive ex vivo and in vitro analysis of canine peripheral blood dp and dn T cells in comparison with their conventional CD4 + sp and CD8a + sp counterparts.
While dp T cells have versatile immunological potential, our results point to a stable dn phenotype and highlight a remarkable Th2-like potential of dn T cells.Furthermore, their high ability to express inhibitory molecules indicates putative immunosuppressive capacity of canine dn T cells.

Isolation of canine peripheral blood mononuclear cells
PBMC were isolated by density gradient centrifugation.Briefly, blood was diluted with phosphate buffered saline (PBS) at a ratio of 1:1, layered above Biocoll Separating Solution (density 1077 g/l, Biochrom AG, Berlin, Germafny) and centrifuged at 500g for 30 min at room temperature (minimal acceleration and braking).PBMC at the interphase were harvested into PBS and centrifuged at 500g for 10 min at RT.After another washing step with PBS, remaining erythrocytes were lysed by incubation in 150 mM NH 4 Cl, 8 mM KHCO 3 , 2 mM EDTA (pH 7.0) for 5 min at RT.The reaction was stopped by addition of PBS containing 3% fetal bovine serum (FBS, Thermo Fisher Scientific, Carlsbad, USA; and PAN-Biotech, Aidenbach, Germany).After washing with PBS, PBMC were counted in Trypan blue (Sigma-Aldrich, Taufkirchen, Germany) using a hemocytometer (Laboroptik, Lancing, UK).

Fluorescence-activated cell sorting of conventional and non-conventional TCRab + T cell subpopulations
All incubation steps performed to stain cells for fluorescenceactivated cell sorting were performed for 15-20 min on ice in the dark.First, PBMC were incubated with the fixable viability dye eFluor 780 (Thermo Fisher Scientific, Carlsbad, USA) according to the manufacturer's protocol to discriminate viable from dead cells.The subsequent staining steps were preceded by washing with PBS, 3% FCS.To block binding of Fc receptors, cells were preincubated for 5 min with a mixture of heat-inactivated normal serum derived from dog and rat (each 15% in PBS) before addition of the anti-canine TCRab (clone CA15.8G7) hybridoma supernatant (Table 1) and further incubation.In the next incubation step, a PerCP/Cy5.5conjugatedgoat-anti-mouse IgG secondary antibody (Biolegend, San Diego, USA) was used for detection of TCRab.Finally, PBMC were incubated with FITC-conjugated anti-canine CD4 (clone YKIX302.9) and APC-conjugated CD8a (clone YCATE55.9)antibodies (Table 1).Stained cells were applied over a 30 µm filter (Sysmex, Norderstedt, Germany) and immediately sorted using a BD FACSAria ™ III Cell Sorter (Becton Dickinson, Heidelberg, Germany).The sorting strategy is shown in Figure 1A.After exclusion of dead cells and doublets, TCRab + T cells from the lymphocyte gate were selected and CD4 + single-positive (sp), CD8a + sp, CD4 bright CD8a bright double-positive (dp), and CD4 -CD8a -double-negative (dn) T cell subpopulations were sorted with a purity >99% (Re-analysis using the FlowJo ™ 10 software (Treestar Inc., Ashland, OR, USA).

Stimulation of sorted TCRab + T cell subpopulations and whole PBMC
Isolated TCRab + T cell subpopulations or whole PBMC were seeded at a density of 5x10 5 per well in IMDM medium with L-Glutamine and 25 mM HEPES (PAA Laboratories, Cölbe, Germany) supplemented with 10% FBS (Thermo Fisher Scientific, Carlsbad, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (both purchased from PAA Laboratories) into 96-well round bottom cell culture plates (Techno Plastic Products AG, Trasadingen, Switzerland) and rested over night at 37°C and 5% CO 2 .Then, cells were polyclonally stimulated for 5 hours at 37°C with phorbol myristate acetate and Ionomycin (P/I) (both: 0.22 µg/ ml; Sigma-Aldrich, Taufkirchen, Germany).Medium incubation (M) served as negative control.For subsequent intracellular cytokine staining, 5 µg/ml Brefeldin A (BrefA) (Enzo Life Sciences, New York, USA) was added to the culture.

RNA isolation, reverse transcription and real-time PCR analysis
Total RNA from sorted TCRab + T cell subpopulations was isolated using 1 ml RNA-Solv ® Reagent (Omega Bio-tek, Norcross, USA) following manufacturer instructions.To increase the RNA yield, 2 µl glycogen (Thermo Fisher Scientific, Vilnius, Lithuania) were added per sample.The concentration of the RNA was determined spectrophotometrically (NanoDrop, PEQLAB, Erlangen).DNA was digested with one unit DNase (Thermo Fisher Scientific)/µg RNA for 30 min at 37°C.For isolation of poly(A)+ RNA, an additional ethanol precipitation was conducted with 1/8 volume RNase-free 3 M NaOAc, pH 5.7.Reverse transcription was performed using the 'High Capacity cDNA Reverse Transcription Kit' (Applied Biosystems, Foster City, USA), containing RiboLock RNase inhibitor (Thermo Fisher Scientific), and a mixture of oligo (dT)18 (Thermo Fisher Scientific) and random hexamer primers in a PTC-200 (MJ Research, Watertown, USA).Specific primers were designed using the primer-BLAST Software (National Center for Biotechnology Information, Bethesda, USA) and synthesized by Metabion (Planegg, Germany) (Table 2).The primers for IL2RA (CD25), IL4, and IL5 were commercially available (Bio-Rad, Munich, Germany).Real-time PCR with SYBR ® Green detection was performed using the iTaq Universal SYBR ® Green Supermix (Bio-Rad, Munich, Germany) and a CFX Connect real time PCR detection system (Bio-Rad).Specificity was tested by melting curve analysis and an agarose gel electrophoresis (LE agarose, Biozym, Hess.Oldendorf; Germany).Only primers resulting in a single peak in melting curve analysis and a single band with the expected fragment size in gel electrophoresis were used.RT-qPCR efficiencies were calculated with the CFX Maestro Software version 2.2 (Bio-Rad) from standard curves generated by serial dilutions of appropriate cDNA samples (2-fold dilutions, 8 point-curve, conducted in duplicates).The efficiency of all reactions ranged between 90 and 110%.Beta-2-microglobulin (B2M) and succinate dehydrogenase subunit A (SDHA), were used as reference genes of different functional classes with stability verified using the CFX Maestro software based on the GeNorm algorithm (18) (Supplementary Table 1).The same amount of cDNA was used for analysis of expression of the respective reference gene and target genes from the same sample.Relative quantification of the transcripts was done by the 2 (-DCt) method (19,20).Results obtained with B2M as reference gene are shown in Figures 1-4, those normalized to SDHA in Supplementary Figures 1-4.

Analysis of canine TCRab + T cell subpopulations by flow cytometry
Viability-dye and surface staining of stimulated canine PBMC were essentially performed as described in paragraph 2.3, except for using flow cytometry buffer (3% FBS, 0.1% sodium azide in PBS) instead of PBS/3% FBS.Primary antibodies used are listed in Table 1.For intracellular staining of IL-4, the FoxP3 staining kit (Thermo Fisher Scientific) was used according to the manufacturer's protocol.For intracellular detection of IL-10, alternatively, cells were fixed with 2% paraformaldehyde (Serva, Heidelberg, Germany) for 15 minutes at 4°C and subsequently permeabilized using 0.5% saponin (w/v, Serva) in flow cytometry buffer.After permeabilization, blocking of Fc receptors was performed by incubation with a mixture of rat, mouse and dog serum (each 15% in PBS) for 10 minutes at room temperature.Cells were incubated for 30 minutes at room temperature with the monoclonal antibodies specific for the selected cytokines (Table 1).Finally, cells were incubated for 20 minutes with PE-  Cy7-conjugated streptavidin to detect IL-4 and IL-10, respectively.Data were acquired with a BD LSR Fortessa (Becton Dickinson, Heidelberg, Germany).FlowJo 10.7.1 software (FlowJo, LLC, Ashland, OR, USA) was used for analysis.Gates were set using appropriate controls: unstimulated controls, and fluorescence minus one (FMO) controls, which include all specific antibodies of the staining panel, except the one of interest, which is replaced by its isotype control.

Statistical analysis
Statistical analysis was performed using Graph Pad Prism 10.2.0 software (San Diego, CA, USA).Depending on normal distribution determined using the Shapiro-Wilk test, data sets are presented with the mean or median.Statistical analysis was performed using One-way ANOVA with Tukey's post-test or Kruskal-Wallis test with Dunn's post-test to compare for differences between T cell subpopulations.Additionally, significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of medium (M) versus the PMA/ionomycin-stimulated (P/I) equivalent using the unpaired t test or Mann-Whitney test (two-tailed).The level of confidence for significance is shown in figure legends.

RT-qPCR analysis of sorted canine conventional and non-conventional TCRab + T cell subpopulations demonstrates stability of the CD4 -CD8a - double-negative phenotype upon activation
For a detailed investigation, non-conventional TCRab + CD4 + CD8a + double-positive (dp) and CD4 -CD8a -double-negative (dn) T cells were compared to conventional CD4 + and CD8a + singlepositive (sp) T cells.All four subpopulations were isolated with high purity (>99%) from peripheral blood of healthy Beagle dogs by fluorescence-activated cell sorting (Figure 1A) and gene expression was analyzed by reverse transcription-quantitative PCR (RT-qPCR).Noteworthy, a very high transcriptional activity of the dp T cell subset was observed.Despite a >20x lower cell yield of the dp T cell subset compared to conventional T cell subpopulations, the resulting amount of RNA was similar in all groups (Table 3), which allowed comparison of gene expression between the populations.
The transcription of selected key markers was analyzed ex vivo (Figures 1B-D; Supplementary Figures 1B-D) and after stimulation

Canine CD4 + CD8a + double-positive and particularly CD4 -CD8a -doublenegative T cells have remarkable T helper 2-like features
To test for a potential role of non-conventional dn and dp T cells in type 2 immune reactions, characteristic features of conventional Th2 cells, including mRNA expression of genes encoding the master transcription factor GATA-3, the key surface  , E). Intriguingly, dn T cells showed highest stimulation-induced expression levels of both inhibitory molecules, which even exceeded those of conventional CD4 + T cells (Figures 4D, E; Supplementary Figures 4D, E).Taken together, these data point to a putative regulatory role of canine non-conventional dn T cells.

Canine CD4 -CD8a -double-negative T cells are potent IL-4 and IL-10 producers
The capacity of dp and dn T cells to produce IL-4 and IL-10 was analyzed at the protein level by flow cytometry.Analysis of IFN-g by intracellular cytokine staining (ICS) served as a positive control.As expected (7), IFN-g was detected in all TCRab + T cell populations analyzed after 5 h of PMA/Iono stimulation, with highest frequencies in dp and CD8a + T cells, followed by CD4 + sp and dn T cells (Figure 5A).Specific detection of IL-4 and IL-10 by ICS upon PMA/Iono stimulation was confirmed using several controls, including unstimulated and fluorescence minus one (FMO) controls.CD8a + sp T cells showed almost no IL-4/IL-10 expression after stimulation (Figures 5, 6).
As expected, the Th1 and Th2 effector cytokines, IFN-g and IL-4, are barely co-produced (Figure 5A).Polyclonal activationinduced IL-4 expression was detected in CD4 + sp T cells, and especially in dn T cells.Of note, the frequency of IL-4 + cells was higher in the dn than in the CD4 + sp population (Figures 5A, C).Furthermore, IL-4 + dn and CD4 + sp T cells exhibit an activated phenotype, indicated by CD25 expression (Figures 5B, D).
Production of the immunosuppressive cytokine IL-10 was significantly induced in dn T cells and CD4 + sp (but not in CD8a + sp and dp T cells) in response to PMA/Ionomycin stimulation (Figures 6A-C).As expected, the majority of dn T cells producing IL-10 are CD25 + reminiscent of a T regulatory phenotype (Figure 6D).
Taken together, ICS analysis confirmed the high ability of dn T cells to produce IL-4 and IL-10, further supporting a potential role of these non-conventional T cells in type 2 and regulatory immune responses.

Discussion
Despite indications of their in vivo relevance, e.g. in the context of desensitization of dogs with food hypersensitivity (16), canine non-conventional dp and dn T cells have been poorly characterized.To gain deeper insight into their phenotype(s) and potential function(s), in this study, peripheral blood non-conventional dp and dn T cells were comprehensively analyzed ex vivo and in vitro upon stimulation in comparison to their conventional CD4 and CD8a + sp counterparts.Besides identification of novel characteristics of canine dp T cells (e.g.Th17 potential), three remarkable novel features of dn T cells were revealed, i.e. (1) the CD4 -CD8a -dn phenotype appears to be stable during in vitro stimulation, (2) dn T cells have a high ability to express Th2associated factors, and in addition to a putative role in type 2 immunity, (3) canine dn T cells seem to have immunosuppressive capacity indicated by strong expression of regulatory molecules.
A role of canine dn T cells in type 2 immunity was hypothesized based on the previous finding that a significant proportion of these cells express GATA-3 (5), the master transcription factor of conventional Th2 cells (25,26).To better assess a potential type 2 phenotype of canine dn T cells here, characteristic features of Th2 cells including the ability to transcribe the genes encoding IL-4, IL-5, and IL-13 were investigated.These effector cytokines are associated with distinct functions in vivo: IL-4 is critical for induction of IgE production, IL-5 mediates recruitment of eosinophils, and IL-13 induces goblet cell hyperplasia, mucus production, and smooth muscle contraction (27).In line with GATA3 expression, dn T cells showed highest stimulationinduced mRNA expression of IL4, IL5, and IL13.Their strong ability to produce IL-4 was confirmed at the protein level.Noteworthy, upon stimulation, the percentage of IL-4-producing cells was even higher in the non-conventional dn compared to the conventional CD4 + sp population.This supports an important role of canine GATA-3 + dn T cells in type 2 immunity, like anti-parasite responses and/or allergy.A potential involvement of murine TCRab + dn T cells in type 2 immune responses has been suggested in a single study, showing that murine splenic TCRab + dn T cells are able to produce high amounts of IL-4 upon activation (28).Interestingly, TCRab + dn T cells of healthy humans hardly secrete IL-4 and only some IL-5 upon stimulation (14).The distinct type 2 phenotype of canine non-conventional dn T cells identified in this study was further supported by their constitutive transcription of the gene encoding the IL-33Ra.IL-33 is a well-known key mediator to promote Th2-associated immunity exerting its biological effects via the IL-33R (29).Thus, the impact of IL-33 on the effector functions of canine dn T cells will be an interesting aspect to further investigate in future experiments.Noteworthy, some features of Th2 cells, i.e. transcription of GATA3 and stimulation-dependent up-regulation of IL-4 and IL13 expression were also identified in non-conventional dp T cells in the present study.Similar to human allergic disease, canine allergy is characterized by increased expression of the type 2 cytokines IL-4, IL-5 and IL-13, e.g. in dogs with atopic dermatitis (30-32).In light of our present in vitro data it will be interesting to evaluate whether canine dn T cells are a major cellular source of type 2 cytokines also in vivo and contribute to the pathogenesis of allergic disease.
Besides a potential role in type 2 immunity, canine nonconventional dn T cells are likely involved in immune regulation: In accordance with expression of FoxP3 already shown previously by us (5), we demonstrated a high capacity of dn T cells to transcribe the co-inhibitory receptor CTLA-4 and to produce the immunosuppressive cytokine IL-10 upon stimulation here.Interestingly, the increase of peripheral blood dn T cells in dogs with adverse food reaction after specific immunotherapy could be based on an immunoregulatory role of dn T cells in vivo (16).However, FoxP3 expression as well as immunosuppressive effector molecules were not analyzed in this study.Pinheiro et al., already suspected but did not analyze a possible regulatory function of canine FoxP3 + dn T cells (33).Noteworthy, albeit murine and human dn T cells lack FoxP3 expression (14,(34)(35)(36), they were shown to control immune responses both in vitro and in vivo and thus have been termed dn regulatory T cells (14,(37)(38)(39)(40). Therefore, it can be speculated that in dogs both, FoxP3 + and FoxP3 - regulatory T cells exist.Their actual suppressive capacity needs to be assessed in vitro and in vivo in future experiments.Noteworthy, in canine dp T cells, in contrast to their conventional CD4 + sp and dn counterparts, FOXP3 mRNA expression is not associated with constitutive transcription of IL10.Additionally, only weak induction of IL10 transcription comparably low as in the CD8a + sp population and no upregulation of CTLA4 mRNA expression was observed in dp T cells upon stimulation.Whereas FoxP3 is a distinct marker of murine regulatory CD4 + T cells, in humans, activated nonregulatory CD4 + T cells were shown to also express FoxP3 albeit transiently, and at lower levels than human regulatory CD4 + T cells (41,42).In future experiments, the investigation of further regulatory effector molecules and mechanisms will shed light onto the actual regulatory capacity of dp T cells.
The diverse immunological potential of dp T cells was further highlighted in this study showing induction of mRNA expression of various T helper cell-associated cytokines (i.e., IFNG, IL17A, IL4, IL13) upon stimulation.The increased transcriptional activity of canine dp T cells shown here represents an important prerequisite for future single-cell RNA sequencing analyses, that could shed light on their potential polyfunctionality, i.e. their ability to produce combinations of cytokines at the single-cell level.Furthermore, scRNA-seq including TCR repertoire analysis, recently established for total canine TCRab + T cells by our group (43), should be applied to both, purified dp and dn T cells in future experiments as this could provide initial insights into a potential biased recognition of antigens.
Taken together, the data presented here reveal new insights into phenotypic features and potential functions of canine dp and dn T cells and provide the basis for future in vitro and in vivo studies to elucidate their role in host defense and immunopathological diseases of dogs.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding author.

1
FIGURE 1 Gene expression analysis of purified canine peripheral conventional and non-conventional TCRab + T cell subpopulations reveals stability of the CD4 -CD8a -double-negative phenotype after PMA/Ionomycin stimulation.(A) Strategy for isolation of indicated CD4/CD8a TCRab + subpopulations (sp: single-positive, dp: double-positive, dn: double-negative) by fluorescence-activated cell sorting and the purity (percentages) of the sorted populations are shown (representative data of one dog).(B-G) These populations were analyzed by RT-qPCR.Expression of indicated genes ex vivo (B-D) and after 5 h of medium (M) incubation or stimulation with PMA/ionomycin (P/I) (E-G) defines the sp/dp/dn phenotype of sorted T cell subpopulations.Note that the expression of ThPOK (which is known to be associated with the gene program of CD4 T helper cells) and Runx3 (which establishes the gene program characteristic of cytotoxic CD8 T cells) is significantly higher in CD4 + CD8a + dp T cells compared to CD8a + sp and CD4 + T cells, respectively.(D, G).Each dot represents one individual dog analyzed separately in independent experiments (n=4 or 5).Depending on normal distribution, data sets are presented with the mean or median (horizontal bars).Statistical differences between subpopulations are marked with lines.Additionally, in (E-G), statistical significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of M versus the P/I equivalent (colored asterisks without lines).(* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).Blue asterisks in (B) indicate significantly increased expression of CD8A in the purified CD8A sp population compared to all other populations.B2M, Beta-2-Microglobulin (reference gene for normalization).

2 3
FIGURE 2 Canine CD4 + CD8a + double-positive T cells comprise T helper (Th) 1 and Th17 expression properties.(A-D) Sorted TCRab + T cell subpopulations, as shown in Figure 1A, were analyzed for expression of indicated genes ex vivo (A, B) or after 5 h of medium (M) incubation or stimulation with PMA/ ionomycin (P/I) (C, D) (sp, single-positive; dp, double-positive; dn, double-negative).Each dot represents one individual dog analyzed separately in independent experiments (n=5).Depending on normal distribution, data sets are presented with the mean or median.Statistical differences between subpopulations are marked with lines.Additionally, in (C, D), statistical significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of M versus the P/I equivalent (colored asterisks without lines).(** p < 0.01; *** p < 0.001; **** p < 0.0001).

4
FIGURE 4 Constitutive expression of key regulatory markers and stimulation-induced upregulation of inhibitory effector molecules indicate immunosuppressive potential of CD4 -CD8a -double-negative T cells.(A-E) TCRab + T cell subpopulations sorted as shown in Figure 1A were analyzed for expression of indicated Treg-related genes ex vivo (A-C) or after 5 h of medium (M) incubation or stimulation with PMA/ionomycin (P/I) (D, E) (sp, single-positive; dp, double-positive; dn, double-negative).Each dot represents one individual dog analyzed separately in independent experiments (n=5).Depending on normal distribution, data sets are presented with the mean or median.Statistical differences between subpopulations are marked with lines.Additionally, in (D, E), statistical significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of M versus the P/I equivalent (colored asterisks without lines).(* p < 0.05; ** p < 0.01; **** p < 0.0001).

5
FIGURE 5 Canine CD4 -CD8a -double-negative T cells are potent IL-4 producers upon PMA/Ionomycin stimulation.(A-D) Whole PBMC of five dogs analyzed in three independent experiments were incubated for 5 h in medium (M) or stimulated with PMA/ionomycin (P/I) in the presence of Brefeldin A and intracellular cytokine staining was performed.(A) Representative zebra plots illustrating IL-4 and IFN-g expression by canine CD4 + single-positive (sp), CD8a + sp, CD4 + CD8a + double-positive (dp) and CD4 -CD8a -double-negative (dn) T cell subpopulations gated as in Figure 1A.Appropriate cytokine gates were set using unstimulated control (medium), and fluorescence minus one (FMO) controls.The dn population shows the highest frequency of IL-4-producing cells upon stimulation.(B) Representative zebra diagrams show that upon stimulation, IL-4 is primarily produced by dn T cells with an activated CD25 + phenotype.(C) Quantification of IL-4 + cells within the indicated TCRab + subpopulations after 5 h of medium (M) incubation or stimulation with PMA/ionomycin (P/I).Horizontal bars indicate median values.Statistical differences between subpopulations are marked with lines.Additionally, statistical significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of M versus the P/I equivalent (colored asterisks without lines) (* p < 0.05; ** p < 0.01; *** p < 0.001).(D) The frequency of CD25 + cells within the IL-4 - and IL-4 + fraction of P/I-stimulated CD4 + sp and dn populations was quantified.The horizontal bars indicate mean values.(* p < 0.05; **** p < 0.0001).(CD8a + sp and dp populations were excluded from this analysis due to limiting absolute numbers of IL-4 + cells).
FIGURE 6Canine CD4 -CD8a -double-negative T cells produce IL-10 upon stimulation (A) Whole PBMC were incubated for 5 h in medium (M) or stimulated with PMA/ionomycin (P/I) in the presence of Brefeldin A and intracellular cytokine staining was performed.(A) Representative zebra plots illustrating IL-10 and IFN-g expression by canine CD4 + single-positive (sp), CD8a + sp, CD4 + CD8a + double-positive (dp) and CD4 + CD8a + double-negative (dn) T cell subpopulations gated as in Figure1A.Appropriate cytokine gates were set using the unstimulated control (medium).Dn T cells show the highest frequency of IL-10-producing cells.(B) IL-10 producers within dn T cells are mainly CD25 + .(C) Quantification of IL-10 + cells within the indicated TCRab + subpopulations after 5 h of medium (M) incubation or stimulation with PMA/ionomycin (P/I) (n=5).Horizontal bars indicate median values.Statistical differences between subpopulations are marked with lines.Additionally, statistical significance of stimulation-induced effects was calculated for each subpopulation by direct comparison of M versus the P/I equivalent (colored asterisks without lines) (* p < 0.05; ** p < 0.01).(D) The frequency of CD25 + cells was quantified within the IL-10 -and IL-10 + fraction of P/I-stimulated CD4 + sp and dn populations with significant induction of IL-10.Horizontal bars indicate mean values.(* p < 0.05; ** p < 0.01, **** p < 0.0001).

TABLE 1
Primary antibodies used for fluorescence-activated cell sorting and flow cytometry analysis.

TABLE 2
Primers used for real-time PCR.

TABLE 3
RNA yield from sorted T cell populations: The low cell number of dp T cells is compensated by their high transcriptional activity.