Functional analysis of canine non-conventional TCRαβ+ CD4-CD8α- double-negative T cells

• Applicant: Dr. Maria Eschke
• Subject Area: Veterinary Medical Science
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 469323442

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 role of conventional TCRαβ+CD4+ and TCRαβ+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, in a recent study, we identified a surprisingly high frequency of non-conventional TCRαβ+ CD4−CD8α− double-negative (dn) T cells in peripheral blood and tissues of healthy dogs. Furthermore, our systematic phenotypic analysis revealed unique features of canine TCRαβ+ dn T cells, compared with their less numerous human and murine counterparts, with subsets showing a FoxP3+ regulatory T cell (Treg)-like and a GATA-3+ T helper 2 cell (Th2)-like phenotype. Interestingly, the majority of FoxP3+ Treg-like dn T cells in peripheral blood of dogs co-expresses GATA-3.The aim of this project is a functional characterization of canine non-conventional TCRαβ+ dn T cells focussing on the identified FoxP3+(GATA-3+) Treg-like and FoxP3-GATA-3+ Th2-like subsets which may play important roles during immune homeostasis and inflammation. Based on initial evidence for the regulatory activity of canine Treg-like dn T cells, the underlying mechanisms of suppression will be studied. I will test the hypothesis that co-expression of GATA-3 in FoxP3+ Treg-like dn T cells is associated with superior suppressive capacity. Furthermore, two opposing hypotheses regarding the impact of FoxP3+GATA-3+ Treg-like dn T cells on Th2 immune responses, i.e. selective suppression or promotion, will be investigated. To test for the potential role of Th2-like dn T cells in type 2 immunity, characteristic features of conventional Th2 cells such as production of IL-4, IL-5, and IL 13 as well as their IL-33 dependency will be analysed. Selected subset-specific key markers, regulatory and effector molecules will be characterised in the proposed project by flow cytometry and PrimeFlow RNA assay. Moreover, to identify novel candidate molecules expressed specifically by Treg-like and Th2-like dn T cells, transcriptome analysis by 5’ single-cell RNA sequencing will be done. In addition, unbiased high-throughput sequencing of the expressed TCRαβ repertoire of sort-purified FoxP3+(GATA-3+) Treg-like and FoxP3- GATA-3+ Th2-like dn T cells will be used to test for the hypothesis that Treg-like dn T cells express a restricted TCR repertoire.In light of high interspecies diversity of non-conventional T cells CD4-CD8α- dn T cells, it is important to unveil species-specific characteristics. Thus, the data generated in this project will contribute to a better understanding of canine T cell biology and comparative immunology.

• DFG Programme: Research Grants