The dog is an important companion animal and, moreover, increasingly recognized as a model for human disease. In this regard, it is essential to intensify basic research on the canine immune system which is still incompletely understood. The focus of our investigations is on newly discovered canine non-conventional T cells. Their function in immune defence and immunopathology will be studied.

Two exciting groups of non-conventional T cells are part of the canine immune system: (1) CD4+CD8+ double-positive T cells and (2) CD4-CD8- double-negative T cells. Their function is subject of current research. Photo:...

Non-conventional T-cells of the dog

CD4 and CD8 double-positive T-cells of the dog

Lymphocytes, the cells of adaptive immunity, include B and T lymphocytes. While the B lymphocytes belong to the humoral (lat. humor = fluid) defence because they produce antibodies and release them into the blood, the T cells take on various tasks within the cell-mediated immunity. Conventional T cell receptor (TCR)aβ T cells can generally be divided into two subpopulations: CD4+ T cells and CD8+ T cells, i.e. cells that express either the CD4 or CD8 receptor (cluster of differentiation 4 or 8) on their surface. Conventional T cell receptor (TCR)aβ T cells CD4+ T-cells are also called T-helper cells because they contribute to the activation of macrophages, to the differentiation of B- to plasma cells, or to neutrophil recruitment by interacting with antigen-presenting cells and secreting various cytokines/transmitters. A subpopulation of the CD4+ T-helper cells (so-called regulatory T-cells) counteracts an overshooting of the immune system. CD8+ T-cells are known as cytotoxic T-cells and their function is to eliminate the body's own cells when they are virally infected or mutated into tumor cells.

It has long been assumed that T cells expressing both the CD4 and CD8 co-receptors on their surface, so-called CD4+CD8+ double-positive (dp) T cells, occur exclusively as immature developmental stages in the thymus. In the meantime, mature dp T cells have also been detected outside the thymus in various species: In humans for the first time by Blue et al. 1986 (1), also in pigs (2), chickens (3), monkeys (4), rats (5), and mice (6).  The detection in the peripheral blood of dogs was successful in the context of a research project from the Institute of Immunology of the Veterinary Medicine Faculty Leipzig (7).

TCRγδ T cells represent a separate lineage of non-conventional T cells characterized by a CD4-CD8α- double-negative (dn) phenotype. Based on early studies in comparative immunology, “γδ T cell high” species with frequencies of 15-50% (e.g., swine, sheep, cattle, chicken) and “γδ T cell low” species with frequencies less than 10% of circulating cells (man, mouse) were identified (Review: (8)). Studies on canine non-conventional lymphocyte subsets later characterized the dog as a “γδ T cell low” species with only about 1-2% of lymphocytes in the periphery expressing the TCRγδ (9). As shown recently by our group, about 40% of the small canine TCRγδ T cell population harbours a CD4-CD8α- dn phenotype (10). Thus, in healthy dogs, TCRγδ dn T cells constitute a small population of about 0.5% of peripheral lymphocytes.

Of note, in the same study, we identified a surprisingly high frequency of non-conventional TCRαβ CD4-CD8α- dn T cells in healthy dogs (10).

References:

(1) Blue ML, J Immunol. 1986 Aug 15;137(4):1202-7
(2) Saalmüller A, Eur J Immunol. 1987 Sep;17(9):1297-301
(3) Luhtala M, Eur J Immunol. 1997 Jan;27(1):189-93
(4) Akari H, Int Immunol. 1997 Apr;9(4):591-7
(5) Takimoto H, Eur J Immunol. 1992 Jan;22(1):159-64
(6) Mosley RL, Int Immunol. 1990;2(4):361-5
(7) Schütze N, Vet Microbiol. 2009 Jun 12;137(3-4):260-7
(8) Holderness J, Annu Rev Anim Biosci. 2013;1:99-124
(9) Faldyna M, Vet Immunol Immunopathol. 2004;104(3-4):239-47
(10) Rabiger FV, Front Immunol. 2019;10:2748

Proposed and approved projects:

DFG:

  • "Phänotypische und funktionelle Analyse caniner peripherer CD4+CD8+ doppelt-positiver T-Zellen" (Projekt: 173176068 )

T cells of dogs

Further studies

Studies by the Institute of Immunology have shown that this T-cell subpopulation of peripheral blood can arise from both CD4+ T-helper cells and CD8+ cytotoxic T-cells  (1-2). In addition, a large proportion is constitutively activated (3) and produces large amounts of the cytokine interferon-γ (IFN-γ) (4). IFN-γ is particularly important for the cellular immune response, for example in viral infections or tumor diseases. These properties make canine CD4+CD8+ dp T cells particularly interesting because they could be used therapeutically in the future. The Institute of Immunology was also able to show that canine CD4+CD8+ dp T cells of peripheral blood have not only effector function but also regulatory potential (4). This gives dp T cells a heterogeneous character that could be both protective and harmful to the body. Accordingly, it is not yet clear what role dp T-cells play in diseases such as autoimmune diseases or infections. To get to the bottom of this question, a subsequent study investigated the distribution of CD4+CD8+ dp T cells in different organs of healthy dogs. It was found that CD4+CD8+ dp T cells are found in mucosal organs such as the intestine or lungs as typical entry ports for infectious agents (5). The results of previous studies in healthy animals form the basis for current studies aimed at further understanding the physiological function of canine CD4+CD8+ dp T cells and highlighting their involvement in various diseases (e.g. inflammatory bowel disease in dogs), which may contribute to the development of alternative therapeutic strategies.
A summary on canine CD4+CD8+ dp T cells of peripheral blood can be found in the review of Buttlar et al. 2015 (6).

Proposed and approved projects:

DFG:

  • "Phenotypic and functional analysis of canine peripheral CD4+CD8+ double-positive T cells " (Project: 173176068)

Publications:

(1) Schütze N, Vet Microbiol. 2009 Jun 12;137(3-4):260-7
(2) Bismarck D, Vet Immunol Immunopathol. 2014 Dec 15;162(3-4):72-82
(3) Bismarck D, Vet Immunol Immunopathol. 2012 Oct 15;149(3-4):157-66
(4) Rothe K, Vet Immunol Immunopathol. 2017 Mar;185:48-56
(5) Rabiger FV, PLoS One. 2019 Mar 13;14(3):e0213597
(6) von Buttlar H, Vet Immunol Immunopathol. 2015 Dec 15;168(3-4):169-7

Cooperations:

Persons involved:

Dr. Maria Eschke
Anett Grohs

Alumni:

Dr. Heiner von Buttlar
Dr. Doris Bismarck
Dr. Friederike Rabiger
Dr. Kathrin Rothe

 

Further studies

Remarkably, up to 15% of canine TCRaβ T cells exhibit a CD4-CD8α double-negative (dn) phenotype in peripheral blood as well as in lymphoid and non-lymphoid organs (Rabiger et al., 2019).

Furthermore, our systematic phenotypic analysis revealed unique features of canine TCRαβ+ dn T cells, compared with their less numerous human (1) and murine (2) counterparts. In dogs, we identified three interesting subsets showing (a) a FoxP3+ regulatory T cell (Treg)-like, (b) a GATA-3+ T helper 2 cell (Th2)-like and (c) a FoxP3+ GATA‑3+ hybrid Treg-like phenotype (3). We now want to functionally characterise these interesting subsets of non-conventional TCRβ dn T cells, as they may play an important role in immune homeostasis and inflammation.

For that purpose, state-of-the-art methods of immunology and molecular biology are applied: Selected marker molecules are investigated by flow cytometry and Prime-FlowTM RNA assay. In addition, TCRaβ dn T cells will be isolated by fluorescence-activated cell sorting (FACS) for single-cell RNA sequencing (scRNA-seq). This will identify further specific molecules of Treg-like, hybrid Treg-like and Th2-like dn T cells. In addition, this analysis will provide further important insights into the heterogeneity of TCRaβ dn T cells. Finally, the hypothesis that Treg-like dn T cells have a restricted TCR repertoire will be investigated using 5' rapid amplification of cDNA ends (RACE) and next generation sequencing (NGS).

While the small population of canine TCRγδ dn T cells, in contrast to TCRaβ dn T cells,  is FoxP3-negative, a substantial portion also express GATA-3 (3). Therefore, a possible involvement of TCRγδ dn T cells in Th2-associated immune responses will also be nvestigated.

Proposed and approved projects:

DFG:

Publications:

(1) Fischer K, Blood. 2005;105(7):2828-35
(2) Zhang ZX, Nat Med. 2000;6(7):782-9
(3) Rabiger FV, Front Immunol. 2019;10:2748

Cooperations:

Persons involved:

Dr. Maria Eschke
Anett Grohs
Martina Protschka

Alumni:

Dr. Friederike Rabiger

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