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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.

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