How to develop IL-17-producing γδ T cells.

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See also: Original Article by Zarin et al. γδ T cells are an evolutionary conserved branch of the immune system and the first T cells to be generated in the developing mouse embryo. While αβ T cells are the predominant T cell subset in lymphoid organs, γδ T cells can constitute up to 50% of all T cells in tissues. Both αβ and γδ T cell subsets develop in the thymus, whereas αβ T cells egress as naïve cells that only aquire effector function upon activation in the periphery, a substantial fraction of γδ T cells exit the thymus precommitted to cytokine production. In particular, thymic γδ T cells can already display interferon-γ (IFN-γ) or interleukin-17 (IL-17) effector phenotypes,1, 2 which are important for tissue homeostasis, infection, autoimmunity and cancer (reviewed in Papotto et al.3). Starting with Rag2-deficient fetal liver cells, the authors generated early T cell progenitors of the double-negative 3 (DN3) stage in an OP9-DL1 co-culture system. These TCR-deficient DN3 cells were transduced with the KN6 transgenic γδ TCR with known ligand specificity. The OP9 stromal cell line expresses both weak and strong KN6 TCR ligands, which hampers assessment of the role of TCR signaling strength on KN6 TCR-expressing T cell differentiation. Therefore, Zarin et al. first produced stromal cells solely expressing the weak KN6 TCR ligand T10, and then transduced them with the strong KN6 TCR ligand T22 and/or the Notch ligand Delta-like 4 (Dll4), and the soluble cytokines were added to the co-cultures. Using this well-defined in vitro system, Zarin et al. could effectively manipulate each of the three types of signaling. Expression of a TCR ligand alone was sufficient for IFN-γ+ γδ T cell generation; additional expression of the Notch ligand Dll4 enhanced the number of cells produced as well as the fraction of IFN-γ+ γδ T cells, while it was insufficient to induce IL-17+ γδ T cells. Interestingly, upon addition of the cytokines, IL-1β, IL-21 and IL-23, IL-17+ γδ T cells developed alongside the IFN-γ producers. Although IL-17+ γδ T cell development was favored by weak TCR signaling, stronger TCR signaling did not prevent their generation when the cytokines were available, unlike in other experimental systems.7 Dissecting the effect of the individual cytokines on IL-17+ γδ thymocyte differentiation, the authors found that IL-1β (exacerbated by IL-21) led to the generation of IL-17 producers but only in the presence of Notch ligand and favored by weak TCR ligand expression. Importantly, blocking IL-1β, IL-21, and IL-23 cytokine receptors in fetal thymic organ cultures (FTOCs) resulted in a massive loss of IL-17+ γδ T cells, suggesting an important physiological role for these cytokines in thymic γδ T cell development (Figure 1). Thus, basal expression of these cytokines in the thymus may preprogram IL-17+ γδ T cells that will then respond en masse to their abundance at inflammatory sites.9 In the current study, γδ T cells expressing the KN6 transgenic γδ TCR were assessed. It is worth noting that the KN6 TCR was originally cloned from an IFN-γ+ γδ T cell and it remains unclear whether the original progenitor also generated IL-17+ progeny during thymic development. Moreover, the transduced DN3 cells originated from fetal liver cells cultured on OP9-DL1 stroma, which may constitute different progenitors to bona fide DN3 thymocytes. Furthermore, a previous study suggested that the branching out of IL-17+ γδ T cells occurs at the earlier DN2 stage, whereas DN3 cells are more prone to develop into IFN-γ+ γδ T cells.10 Thus, precursor–product relationships should be further characterized in independent experimental systems. Overall, the study by Zarin et al. provides interesting novel insights on the developmental potential of DN3 progenitors and indicates that they retain the capability to develop into IL-17+ γδ T cells under certain environmental conditions where cytokines, especially IL-1β, play a major role. Future studies on the actual cytokine microenvironment and requirements in fetal and adult thymi should translate the current findings into an in vivo setting, thus confirming that these cytokines play a key developmental role to add to their well-established importance for IL-17+ γδ T cell activation, differentiation and expansion under inflammatory conditions.3, 9