Date Published: March 13, 2019
Publisher: Public Library of Science
Author(s): Friederike V. Rabiger, Doris Bismarck, Martina Protschka, Gabriele Köhler, Peter F. Moore, Mathias Büttner, Heiner von Buttlar, Gottfried Alber, Maria Eschke, Jose C. Crispin.
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.
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 [1–9]. Canine CD4+CD8α+ dp T cells within peripheral blood mononuclear cells (PBMC) were first described around ten years ago [10–13]. To date, our group was able to characterize this unconventional T cell subpopulation in peripheral blood as mature T cell receptor (TCR) αβ+CD1a- effector memory T cells [14,15]. CD4+CD8α+ dp T cells can develop from both, CD4+ sp as well as from CD8α+ sp T cells upon in vitro stimulation, but CD4+ sp T cells are the more potent progenitors . Furthermore, canine CD4+CD8α+ dp T cells of the peripheral blood can be divided into three different subsets, i.e. CD4dimCD8αbright, CD4brightCD8αbright, and CD4brightCD8αdim, which differ in phenotype and functional features. The CD4dimCD8αbright subset expresses the CD8αβ heterodimer, whereas most cells of the other two subsets express the unconventional CD8αα homodimer [14,15]. In contrast to CD8αβ, CD8αα does not work as a TCR co-receptor, but was shown to negatively regulate the activation of T cells .
Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a heterogeneous effector memory T cell subpopulation suggesting an important role in immune response by their constitutively high activation [14,15]. Here we provide the first systematic characterization of CD4+CD8α+ dp T cells in different tissues within one homogeneous group of healthy Beagle dogs. This reveals insight into potential induction and/or effector sites of this unique T cell subpopulation and is a prerequisite for subsequent functional analyses. Our comprehensive study shows that highest frequencies of CD4+CD8α+ dp T cells are present in Peyer’s patches (PP) compared to tracheobronchial and mesenteric lymph nodes (LN), spleen, intraepithelial lymphocytes of the small intestine (IEL), and lung. Former reports about canine CD4+CD8α+ dp T cells in LN [24,38], bone marrow , spleen , and IEL  are limited by either a low number  or by heterogeneity in breed, age, and/or health status of dogs [24,38,39]. Moreover, a detailed characterization of these cells was still missing. We could demonstrate that the majority of CD4+CD8α+ dp T cells in dogs is TCRαβ+, and, interestingly, up to 96% on average (IEL, PP) express the unconventional CD8αα homodimer. CD8αα was formerly thought to be a functional homolog of the MHC class I binding TCR co-receptor CD8αβ . However, the CD8β chain has been identified as key molecule for CD8-dependent TCR function . Transient co-expression of CD8αα on CD8αβ+ T cells, in contrast, results in decrease of TCR signal transduction inducing a higher activation threshold proportional to the expression level of CD8αα. Nevertheless, increased antigen stimulation can overcome the CD8αα repressor function [17,42]. CD4+CD8α+ dp T cells are characterized by high frequencies of CD25 expression, the IL-2 receptor α-chain (IL-2Rα) which constitutes the high-affinity IL-2 receptor in combination with IL-2Rβ and IL-2Rγ. The high-affinity IL-2 receptor enables rapid and effective proliferation of T cells . As CD8αα has been shown to repress T cell activation [17,42], it might thus contribute to adequate induction of CD4+CD8α+ dp T cell effector functions only upon high antigenic stimulation and thereby prevent excessive immune responses. However, the function of CD8αα on CD4+ single-positive (sp) T cells has not yet been clarified and needs further investigation.