Project description:We report the identification of immature thymic CD4(-),CD8(-) double-negative (DN)1e cells with the NKT cell lineage potential. We also analyzed the gene expression profiles of DN1e thymocytes compared with those of mature thymic NKT cell developmental stages termed NKT stage-1, 2, and -3, which are characterized by differential expression levels of NK1.1 and CD44 antigens in C57BL/6 mouse strain. Next generation sequencing of total transcriptomes using total RNA isolated from FACS sorted ex vivo thymic DN1eP (Lin-/CD44+/CD25-/CD24low/CD5+/CD27+/Ly108-/CXCR3+) fraction, and mature thymic alphaGalCer-loaded CD1d dimer+TCRbeta+ NKT cell developmental stage-1 (CD44-/NK1.1-), stage-2 (CD44+/NK1.1-), and stage-3 (CD44+/NK1.1+) cells.
Project description:We report the identification of immature thymic CD4(-),CD8(-) double-negative (DN)1e cells with the NKT cell lineage potential. We also analyzed the gene expression profiles of DN1e thymocytes compared with those of mature thymic NKT cell developmental stages termed NKT stage-1, 2, and -3, which are characterized by differential expression levels of NK1.1 and CD44 antigens in C57BL/6J mouse strain.
Project description:We report the application of single-molecule-based sequencing technology for mapping the Egr2 transcriptional program in developing thymic NKT. We found that Egr2 controls the induction of genes required for NKT development. Examination of developing NKT cells and thymocytes receiving a strong TCR signal in vivo by injecting 500ug anti-TCRb antibody.
Project description:Semi-invariant natural killer T (NKT) cells are thymus-derived innate lymphocytes that modulate microbial and tumour immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learnt regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-xL expression. Additionally, IL-15 regulated thymic developmental stage 2 (ST2) to ST3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic ST3 NKT cells. The loss of IL15-dependent T-bet expression resulted in poor expression of IFN-γ and several NK cell receptors in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-xL, and effector differentiation, which is regulated by T-bet. Gene expression was measured in NKT cells sorted from pooled thymi of wild-type (3 replicates) or IL-15 deficient (2 replicates) mice.
