Abstract
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.
Highlights
Extrathymic CD4+CD8α+ double-positive T cells are a mature T cell subpopulation distinct from conventional CD4+ single-positive T helper and CD8α+ sp cytotoxic T cells known to occur in different species, e.g. swine, humans, monkeys, mice, rats, and chicken [1,2,3,4,5,6,7,8,9]
Our analysis revealed significant differences concerning the CD4+CD8α+ dp T cell frequencies depending on the organ, with highest frequencies in Peyer’s patches (PP) (1.6% on average) and lowest in tracheobronchial lymph node (tLN) (0.2% on average) (Fig 1B and 1C)
It is known that canine CD4+CD8α+ dp T cells of the peripheral blood form a heterogeneous cell population which can be divided into three different subsets, namely CD4brightCD8αdim, CD4brightCD8αbright, and CD4dimCD8αbright [15]
Summary
Extrathymic CD4+CD8α+ double-positive (dp) T cells are a mature T cell subpopulation distinct from conventional CD4+ single-positive (sp) T helper and CD8α+ sp cytotoxic T cells known to occur in different species, e.g. swine, humans, monkeys, mice, rats, and chicken [1,2,3,4,5,6,7,8,9]. In contrast to CD8αβ, CD8αα does not work as a TCR co-receptor, but was shown to negatively regulate the activation of T cells [17] Another unique feature of the total CD4+CD8α+ dp T cell subpopulation of PBMC is their significantly higher expression level of CD25 and of MHC-II in comparison to their CD4+ and CD8α+ sp counterparts, suggesting a high level of activation and an important immunological potential [15]. Still the developmental origin and the function of canine CD4+CD8α+ dp T cells in vivo have not yet been clarified For this purpose, reliable reference data in a homogeneous group of healthy animals including a comprehensive phenotypical and functional characterization from blood, and from different lymphatic and non-lymphatic organs are required. The data will provide the basis for further functional analyses to elucidate the in vivo role of CD4+CD8α+ dp T cells in dogs
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