Canine tissue-associated CD4 ⁺ CD8α ⁺ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential

Canine CD4 ⁺ CD8α ⁺ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4 ⁺ or CD8α ⁺ single-positive (sp) T cells. In this study, we investigated CD4 ⁺ CD8α ⁺ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer’s patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a ^- CD4 ⁺ CD8α ⁺ dp T cell population in all analyzed organs, with highest frequencies within Peyer’s patches. Constitutive expression of the activation marker CD25 is a feature of many CD4 ⁺ CD8α ⁺ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4 ⁺ CD8α ⁺ dp T cells is FoxP3 ⁺ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4 ⁺ CD8α ⁺ dp intraepithelial lymphocytes. In addition, a fraction of CD4 ⁺ CD8α ⁺ dp intraepithelial lymphocytes and of mesenteric lymph node CD4 ⁺ CD8α ⁺ dp T cells is TCRγδ ⁺ . However, the main T cell receptor of all tissue-associated CD4 ⁺ CD8α ⁺ dp T cells could be identified as TCRαβ. Interestingly, the majority of the CD4 ⁺ CD8α ⁺ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α ⁺ sp T cells, and CD4 ⁺ CD8α ⁺ dp thymocytes which are mainly CD8αβ ⁺ . The presented data provide the basis for a functional analysis of tissue-specific CD4 ⁺ CD8α ⁺ dp T cells to elucidate their role in health and disease of dogs.