The Role of Environmental Cues in Gamma-Delta T-Cell Lineage Choice and Functional Programming

The thymic landscape provides an array of spatially and temporally controlled cues that guide the development of functionally distinct effector immune cells. This thesis focuses on how signals downstream of the T cell receptor (TCR), Notch and critical cytokine receptors, dictate lineage choice between αβ and γδ T cells and, furthermore, can combine to play a role in programming γδ T cell effector function. The signal strength hypothesis posits that the quantity of the signal downstream of the TCR can direct lineage choice through the variable accumulation of inhibitor of DNA (deoxyribonucleic acid) binding 3 (Id3) proteins downstream of the TCR-induced ERK (extracellular signal-regulated kinase)- early growth response (Egr) pathway. To test this hypothesis, we assessed the differentiation outcome of Rag2-/- (Recombination-activating gene) DN3 (double negative 3) progenitors transduced to co-express both TCR and γδ TCRs, followed by co-culture with cells expressing a weak or strong binding γδ TCR ligand in the presence or absence of Notch ligands. In the presence of Notch and weak γδ ligands, a percentage of progenitors expressing only the γδ TCR matured towards the CD4+ CD8+ double positive (DP) stage of T cell development. Importantly, progenitors co-expressing TCR and γδ TCR cultured under the same conditions developed towards the γδ lineage as a result of higher accumulation of Id3 downstream of each of the two receptors, hence lending further support for the signal strength hypothesis. With this in mind, we set out to describe the role of these and other signals necessary for the development of functional γδ T cell subsets as defined by differential cytokine production. To this end, Rag2-/- DN3 progenitors were transduced with a transgenic KN6 γδ TCR, and co-cultured on various stromal cells engineered to express Notch, KN6 TCR ligands and supplemented with combinations of cytokines, such as IL-1β (Interluekin-1 β), IL-7, IL-21 and IL-23. Our results showed that specific combinations of these signals are required to program IFN-γ (Interferon-γ), IL-17, and IL-22 producing KN6 γδ T cell subsets. More specifically, I established that weak but not absent TCR-ligand interactions are required for γδ17 development, and Notch enables the survival of γδ T cells in an apparent inflammatory milieu. This was shown to be relevant in the fetal thymus as blockade of IL-1β, IL-21 and IL-23 in hanging drop cultures resulted in the absence of γδ17 cells. This work provides a framework for the integration of signals downstream of the Notch, TCR, and cytokine receptors as they lead to the development of γδ T cell functional subsets that can play a fundamental role in regulating health and disease.Ph.D