Canine peripheral blood CD4⁺CD8⁺ double-positive Tcell subpopulations exhibit distinct Tcell phenotypes and effector...

Canine peripheral blood CD4⁺CD8α⁺ double-positive (dp) Tcells represent a small population of T lymphocytes that are not only derived from single-positive (sp) CD4⁺ but also from CD8⁺ Tcells. Dp Tcells can be subdivided in three different subpopulations according to their expression levels of CD4 and CD8α, i.e. the CD4^dimCD8α^bright, CD4^brightCD8α^bright, and the CD4^brightCD8α^dim subsets. Previously we could show that total canine peripheral CD4⁺CD8α⁺ dp Tcells have features of activated Tcells. Here we demonstrate that the individual dp Tcell subsets distinctly differ in their activation status and phenotype. Thus, among the dp Tcell subsets the CD4^dimCD8α^bright subpopulation has the lowest and the CD4^brightCD8α^bright subset the highest frequency of CD25⁺ cells pointing to the CD4^brightCD8α^bright subset with the highest activation status. Consistent with that, analysis of CD44 vs. CD62L expression demonstrates that the CD4^brightCD8α^bright subset contains the highest frequencies of effector-memory Tcells (T_EM). Upon in vitro stimulation, the CD4^brightCD8α^bright and CD4^dimCD8α^bright dp Tcell subsets show the highest frequencies of IFN-γ producing cells. Expression of granzyme B was found exclusively in the CD4^dimCD8α^bright dp Tcell subset indicating cytotoxic potential of this dp Tcell subset. Furthermore, T-bet expression was restricted to the CD4^dimCD8α^bright subset, while Foxp3⁺ regulatory Tcells were detected in the CD4^brightCD8α^dim dp Tcell subset. In conclusion, canine dp Tcells are phenotypically and functionally heterogeneous consistent with the diverse characteristics of their single-positive CD4⁺ and CD8⁺ Tcell progenitors. The data provide the basis for future in vivo studies to elucidate the role of individual dp Tcell subsets in host defense and/or immune pathological diseases of dogs.