Construction of a 3D brain extracellular matrix model to study the interaction between microglia and T cells in co-culture.

Neurodegenerative disorders are characterised by the activation of brain-resident microglia cells and by the infiltration of peripheral T cells. However, their interplay in disease has not been clarified yet. It is difficult to investigate complex cellular dynamics in living animals, and simple two-dimensional (2D) cell culture models do not resemble the soft 3D structure of brain tissue. Therefore, we developed a biomimetic 3D in vitro culture system for co-cultivation of microglia and T cells. As the activation and/or migration of immune cells in the brain might be affected by components of the extracellular matrix, defined 3D fibrillar collagen I-based matrices were constructed and modified with hyaluronan and/or chondroitin sulphate, resembling aspects of brain extracellular matrix. Murine microglia and spleen-derived T cells were cultured alone or in co-culture on the constructed matrices. Microglia exhibited in vivo-like morphology and T cells showed enhanced survival when co-cultured with microglia or to a minor degree in the presence of glia-conditioned medium. The open and porous fibrillar structure of the matrix allowed for cell invasion and direct cell-cell interaction, with stronger invasion of T cells. Both cell types showed no dependence on the matrix modifications. Microglia could be activated on the matrices by lipopolysaccharide resulting in interleukin-6 and tumour necrosis factor-α secretion. The findings herein indicate that biomimetic 3D matrices allow for co-cultivation and activation of primary microglia and T cells and provide useful tools to study their interaction in vitro.

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

The role of conventional TCRαß+CD4+ or TCRαß+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαß 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 TCRαß+ 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 TCRαß+CD4+ sp, and almost half of the frequency of TCRαß+CD8α+ sp T cells (i.e., ~15% of all TCRαß+ T cells). Among TCRαß+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+TCRαß+CD4-CD8α- dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαß+CD4-CD8α- dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαß+CD4-CD8α- dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3-TCRαß+CD4-CD8α- dn T cells was even higher as compared with TCRαß+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, TCRαß+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 TCRαß+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.

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 TCRαß. 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 CD8αß+. 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.