Distinct Features of Canine Non-conventional CD4−CD8α− Double-Negative TCRαβ+ vs. TCRγδ+ T Cells

Abstract

The role of conventional TCRαβ+CD4+ or TCRαβ+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαβ or TCRγδ but lacking CD4 and CD8α expression [i.e., CD4−CD8α− double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCRγδ. In contrast, TCRγδ+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCRαβ+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCRαβ+CD4+ sp, and almost half of the frequency of TCRαβ+CD8α+ sp T cells (i.e., ~15% of all TCRαβ+ T cells). Among TCRαβ+CD4−CD8α− dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCRαβ+CD4−CD8α− dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαβ+CD4−CD8α− dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαβ+CD4−CD8α− dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3−TCRαβ+CD4−CD8α− dn T cells was even higher as compared with TCRαβ+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCRγδ+CD4−CD8α− dn T cells also expressed substantial proportions of GATA-3. In addition, TCRαβ+CD4−CD8α− dn T cells produced IFN-γ and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCRαβ+CD4−CD8α− dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-γ or IL-17A and (ii) a small TCRγδ+CD4−CD8α− dn T cell subset also expressing GATA-3 without production of IFN-γ or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

T Cell Immune Profiles of Blood and Tumor in Dogs Diagnosed With Malignant Melanoma

“…In contrast to reports for frequencies of healthy human CD25+ CD4+ T cell ( 68 , 69 ), our studies revealed a higher frequency of this subset within both healthy controls and patients which proved to include both FoxP3- as well as FoxP3+ cell populations. Other reports describing CD25+ cell frequencies within the CD4+ T cell subset in healthy dogs also revealed similar or higher frequencies compared to our current studies ( 44 , 70 , 71 ). Our results also revealed increased frequencies of the CD25+ cells for both CD4+ and CD4-CD8- T cell subsets in canine melanoma patients compared to healthy dog controls.…”

“…Data generated from these studies of canine melanoma patients afforded an opportunity to analyze different canine T cell subsets and subpopulations for specific T cell markers based on healthy control dogs. An interesting CD4-CD8- CD3+ T cell subset was reported and characterized previously to express regulatory markers including FoxP3 and CD25 ( 44 ). Our results for 20 healthy control dogs also show FoxP3 expression by CD4-CD8- T cells although frequencies of FoxP3+ cells for this subset are lower compared to CD4+ cells with a difference trending for significance ( p = 0.052) ( Figure 8A ).…”

“…Representative gating strategies are shown for characterizing frequencies of T cell (CD3+) subsets including CD4+, CD8+, and CD4-CD8- cells ( Figure 1A ) and frequencies of regulatory and activated populations within each T cell subset ( Figure 1B ). The CD4-CD8- T cell subset was included in analyses for different T cell markers as a population of interest based on a recent report ( 44 ) and was consistently observed in all patient and control blood samples. Based on these gating strategies different T cell subsets were interrogated for FoxP3, CD25, Ki67, and granzyme B to distinguish regulatory, activated, and putative quiescent subsets.…”

“…However, analysis of healthy dog controls within these studies allowed this type of analysis for canine T cells. The CD4-CD8- T cell subset in dogs was described previously as an activated subset that demonstrates significantly higher frequencies of CD25+ cells ( 44 ) compared to CD4+ and CD8+ T cells. This finding was also observed in our studies with a large range of values for frequencies of CD25+ cells within the CD4-CD8- T cell subset, but with a median frequency well above that of CD4+ T cells.…”

Peripheral blood lymphocyte subtypes in dogs with different stages of myxomatous mitral valve disease

“…Furthermore, it may be of interest that the percentage of DN T lymphocytes found both in cardiac patients and healthy dogs in our study was much higher than in healthy mice and humans, which typically have <5% of DN T‐cells 37,38 . Two studies in healthy dogs 39,40 also found a high percentage of DN T lymphocytes in peripheral blood, approximately 10% in 1 study 39 and 15% in another study 40 . The reason for this species‐specific difference remains to be investigated.…”