γδTCR-dependent and independent differentiation of innate lymphoid cells

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ABSTRACT Innate lymphoid cells (ILCs) reside in tissues and act as sentinels to a variety of environmental cues from nutrients, neuropeptides, microbiota to pathogens. They are heterogeneous populations with diverse functions and developmental origins. We have previously suggested that in addition to the bone marrow, the thymus can contribute to ILC pools in peripheral tissues based on findings that lung ILC2s contain rearranged T cell receptor genes, likely due to the divergence of developing thymocytes into the ILC fate. Consistently, T cell precursors from the thymus can differentiate into ILC2s in vitro. Single cell RNA sequencing studies detected a heterogeneous population of ILC precursors in the blood, which is greatly diminished in athymic nude mice, suggesting the involvement of the thymus. These cells possess CD3 proteins in the cytoplasm but not on the surface, thus making intracellular CD3 (icCD3) a marker for thymus-derived ILCs. Indeed, icCD3+ ILC subsets have been detected in the lung and small intestine. Interestingly, the production of icCD3+ ILC1s and to a lesser extent, ILC3s depends on Tcrd but that of icCD3+ ILC2s does not. This prompted us to propose that ILC differentiation in the thymus involves Tcrd-dependent and independent mechanisms. The purpose of this application is to study how thymus-derived ILCs are generated and to investigate the functions of these cells. There are three aims. Aim1 will test the hypothesis that developing T cells with non-productive TCR gene rearrangement contributes to multipotent ILC precursors and immature γδ T cells gives rise to ILC1- or ILC3- biased precursors after down-regulation of their TCRs. We will employ approaches such as in vitro differentiation cultures and analyses of TCR gene rearrangement events. Aim2 will characterize icCD3+ ILCs in the small intestine by examining the age-dependent differentiation of ILC1s and ILC2 in relationship to T cell development and changes in gut microbiota, and by comparing the type 1 responses of icCD3+ ILC1s to those of conventional ILC1s and NK cells as well as γδ T cells. Aim3 will investigate the interplay of type 2 and type 3 immune responses and compare the behaviors of icCD3+ ILC2s to their icCD3- counterparts. Taken together, these studies may make conceptual advancement in developmental immunology and shed light on the contribution of thymus-derived ILCs to age-related differences in immunity.