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Tracking γδ T cell development and TCRγδ proximal signalling

Periodic Reporting for period 1 - DevoSignGammaDelta (Tracking γδ T cell development and TCRγδ proximal signalling)

Okres sprawozdawczy: 2018-09-01 do 2020-08-31

What is the problem/issue being addressed?

T cells and B cells, the two main components of adaptive immunity, express highly diverse antigen receptors generated by somatic gene rearrangement during their development. T cell development is restricted to the specialized environment of the thymus. Two types of T cells are conserved throughout evolution and across species: alphabeta and gammadelta T cells. In the thymus, alphabeta T cells develop through discrete steps from co-receptor CD4-CD8- (double negative, DN) T-cell receptor (TCR)- via CD4+CD8+ thymocytes into mature CD4+ or CD8+ single positive TCRalphabeta+ T cells, which populate the peripheral lymphoid tissues and are restricted to recognize antigens as peptides bound to major histocompartibility complex (pMHC). gammadelta T cells develop in the thymus alongside alphabeta T cells, but rearrange a distinct TCR (TCRgammadelta) that is not restricted by classical MHC. Successive waves of gammadelta T cells develop in the fetal and later in the adult thymus and different waves are associated with variable TCR-Vgamma segment usage, functional differentiation and localization in different tissues. Unique antigen-specificities of gammadelta T cells, their high clonal frequency and pre-activated differentiation status allows for their rapid, innate-like responses and confers them non-redundant roles in immune responses to infections and tumours.

Why is it important for society?

gammadelta T cells can effectively kill tumour cells and provide IFNgamma-mediated protective responses against cancer. Still, in some cases they can promote tumour growth via IL-17A production. Together with the recent advancements in T cell-based immunotherapy for cancer, gammadelta T cells are becoming an attractive tool for clinical treatments. Still, our knowledge of gammadelta T cells biology and development is rather limited and requires investigation to improve fine-tuning and manipulation for their clinical use.

What are the overall objectives?

This research project aims to elucidate the developmental trajectories and requirements of different gammadelta T cell subsets. To this end, we investigate i) the lineage relationship from uncommitted thymic progenitor cells to mature gammadelta T cell effector subsets producing either IL-17 (named gd17) or IFNgamma (named gdIFN), ii) the impact of TCRgammadelta signalling during thymic development and iii) the role of on candidate genes differentially expressed between gd17 and gdIFN cells and their role in gammadelta T cell subsets development.
We investigated the lineage relationships from uncommitted DN2 thymic progenitors to mature gammadelta T cell subsets, namely the two gammadelta T cells effector subsets producing either gd17 or gdIFN cells. To this end we applied cellular barcoding of progenitors, tracking of purified progenitor subsets and TCR sequencing. We found evidence that, unlike gdIFN cells, progenitors of gd17 cells do not develop via the DN2 stage, but rather originate from DN1 cells. This supports a novel hypothesis (Spidale, Kang, Immunity 2018) that gd17 cells originate from a progenitor population present in early life exclusively and undergo a different developmental trajectory compared to common T cell development.

We established a protocol to stimulate immature CD24+ gammadelta T cells from fetal thymi in vitro to measure consequences of TCR activation. These studies demonstrated that forced TCR ligation of fetal gammadelta T cells induced reduction of mitochondrial membrane potential and a metabolic shift from oxidative phosphorylation (in the unstimulated gammadelta progenitors) to aerobic glycolysis in gammadelta thymocytes that commit to the gdIFN pathway. This suggested that the different metabolic profiles associated with the gd17 and gdIFN phenotypes are already established in the thymus most probably following TCR-mediated “selection” (Lopes et al., Nature Immunology in press).

We hypothesised that TCR signalling might qualitatively differ between gd17 and gdIFN cells. To identify candidate genes differentially expressed in the two gammadelta T cells effector subsets, we compared the transcript levels of signalling mediators and transcription factors downstream of TCR and cytokine receptors in gd17 and gdIFN subsets using transcriptome data. The identified candidate genes were further investigated for their roles in the development of gammadelta T cells. Using different loss- and gain-of-function approaches in vitro and in vivo, we further analysed the impact of selected candidate genes on i) T cell development, ii) gd17 and gdIFN commitment and iii) cytokine production.
During the project, we studied murine gammadelta T cell development and the impact of TCR signalling and genes involved in signalling events. We combined analyses of transcriptome data, elaborated in vitro techniques and the use of novel genetic mouse models. This allowed us to study developmental pathways in the fetal thymus and to test the impact of selected candidate genes on gammadelta T cell development. We found that TCR signalling during thymic development induced a metabolic shift associated with the gdIFN phenotype. The results obtained during the course of the action will lead to a better understanding of the impact of TCR signalling on gammadelta T cell subset development and how to manipulate this process.

By tracking subpopulations of gammadelta thymocytes throughout ontogeny (from fetal to adult development), we identified a novel gammadelta T cell population that produces IFNgamma but not IL-17 and develops mainly in the perinatal period. These enigmatic gdIFN cells were further investigated during the course of the action. We characterized their developmental requirements and kinetics, established them as a stable perinatal gammadelta T cell subset and a preferential target in the development of leukemia. Given the functional and ontogenic characteristics of this novel gdIFN subset, we anticipate it may play important roles in anti-viral or anti-parasitic defense in early life.

We will explore the possibilities to translate these novel findings from the murine to the human immune system with the ultimate goal to test its potential for immunotherapy.
Thymic development of gammadelta T cells during mouse ontogeny.