Upon reaching the mature heat stable antigen (HSA)^low thymic developmental stage, CD1d-restricted Vα14-Jα18 thymocytes undergo a well-characterized sequence of expansion and differentiation steps that lead to the peripheral interleukin-4/interferon-γ–producing NKT phenotype. However, their more immature HSA^high precursors have remained elusive, and it has been difficult to determine unambiguously whether NKT cells originate from a CD4⁺CD8⁺ double-positive (DP) stage, and when the CD4⁺ and CD4⁻CD8⁻ double-negative (DN) NKT subsets are formed. Here, we have used a CD1d tetramer-based enrichment strategy to physically identify HSA^high precursors in thymuses of newborn mice, including an elusive DP^low stage and a CD4⁺ stage, which were present at a frequency of ∼10⁻⁶. These HSA^high DP and CD4⁺ stages appeared to be nondividing, and already exhibited the same Vβ8 bias that characterizes mature NKT cells. This implied that the massive expansion of NKT cells is separated temporally from positive selection, but faithfully amplifies the selected TCR repertoire. Furthermore, we found that, unlike the DN γδ T cells, the DN NKT cells did not originate from a pTα-independent pathway bypassing the DP stage, but instead were produced during a short window of time from the conversion of a fraction of HSA^low NK1.1^neg CD4 cells. These findings identify the HSA^high CD4⁺ stage as a potential branchpoint between NKT and conventional T lineages and between the CD4 and DN NKT sublineages.

We explored in this study the status and potential role of IL-17-producing iNKT cells (iNKT17) in type 1 diabetes (T1D) by analyzing these cells in patients with T1D, and in NOD mice, a mouse model for T1D. Our analysis in mice showed an increase of iNKT17 cells in NOD vs control C57BL/6 mice, partly due to a better survival of these cells in the periphery. We also found a higher frequency of these cells in autoimmune-targeted organs with the occurrence of diabetes, suggesting their implication in the disease development. In humans, though absent in fresh PMBCs, iNKT17 cells are detected in vitro with a higher frequency in T1D patients compared to control subjects in the presence of the proinflammatory cytokine IL-1β, known to contribute to diabetes occurrence. These IL-1β-stimulated iNKT cells from T1D patients keep their potential to produce IFN-γ, a cytokine that drives islet β-cell destruction, but not IL-4, with a reverse picture observed in healthy volunteers. On the whole, our results argue in favour of a potential role of IL-17-producing iNKT cells in T1D and suggest that inflammation in T1D patients could induce a Th1/Th17 cytokine secretion profile in iNKT cells promoting disease development.

The transcription factor promyelocytic leukemia zinc finger (PLZF) is encoded by the BTB domain–containing 16 (Zbtb16) gene. Its repressor function regulates specific transcriptional programs. During the development of invariant NKT cells, PLZF is expressed and directs their effector program, but the detailed mechanisms underlying PLZF regulation of multistage NKT cell developmental program are not well understood. This study investigated the role of acetylation-induced PLZF activation on NKT cell development by analyzing mice expressing a mutant form of PLZF mimicking constitutive acetylation (PLZF^ON) mice. NKT populations in PLZF^ON mice were reduced in proportion and numbers of cells, and the cells present were blocked at the transition from developmental stage 1 to stage 2. NKT cell subset differentiation was also altered, with T-bet⁺ NKT1 and RORγt⁺ NKT17 subsets dramatically reduced and the emergence of a T-bet⁻RORγt⁻ NKT cell subset with features of cells in early developmental stages rather than mature NKT2 cells. Preliminary analysis of DNA methylation patterns suggested that activated PLZF acts on the DNA methylation signature to regulate NKT cells’ entry into the early stages of development while repressing maturation. In wild-type NKT cells, deacetylation of PLZF is possible, allowing subsequent NKT cell differentiation. Interestingly, development of other innate lymphoid and myeloid cells that are dependent on PLZF for their generation is not altered in PLZF^ON mice, highlighting lineage-specific regulation. Overall, we propose that specific epigenetic control of PLZF through acetylation levels is required to regulate normal NKT cell differentiation. Key Points Constitutive acetylation of PLZF (PLZF^ON) blocks NKT cell development. PLZF^ON might act on the DNA methylation signature to control gene expression. PLZF acetylation levels regulate normal NKT cell differentiation.

Almost 20 years ago, CD1d tetramers were developed to track invariant natural killer T (NKT) cells based on their specificity, and to define developmental steps during which differentiation markers and functional features are progressively acquired from early NKT cell precursor to fully mature NKT cell subsets. Based on these findings, a linear developmental model was proposed and subsequently used by all studies investigating the specific role of factors that control NKT cell development. More recently, based on intracellular staining patterns of lineage‐specific transcription factors such as T‐bet, GATA‐3, promyelocytic leukemia zinc finger and RORγt, a lineage differentiation model was proposed for NKT cell development. Currently, studies on NKT cells development present lineage differentiation model data in addition to the linear maturation model. In the perspective presented here, we discuss current knowledge relating to NKT cell developmental models and particularly focus on the approaches and strategies, some of which appear nebulous, used to define NKT cell developmental stages and subsets.

Following positive selection, NKT cell precursors enter an “NK-like” program and progress from an NK^– to an NK⁺ maturational stage to give rise to NKT1 cells. Maturation takes place in the thymus or after emigration of NK^– NKT cells to the periphery. In this study, we followed the fate of injected NKT cells at the NK^– stage of their development in the thymus of a series of mice with differential CD1d expression. Our results indicate that CD1d-expressing cortical thymocytes, and not epithelial cells, macrophages, or dendritic cells, are necessary and sufficient to promote the maturation of thymic NKT1 cells. Migration out of the thymus of NK^– NKT cells occurred in the absence of CD1d expression, however, CD1d expression is required for maturation in peripheral organs. We also found that the natural ligand Isoglobotriosylceramide (iGb3), and the cysteine protease Cathepsin L, both localizing with CD1d in the endosomal compartment and crucial for NKT cell positive selection, are also required for NK^– to NK⁺ NKT cell transition. Overall, our study indicates that the maturational transition of NKT cells require continuous TCR/CD1d interactions and suggest that these interactions occur in the thymic cortex where DP cortical thymocytes are located. We thus concluded that key components necessary for positive selection of NKT cells are also required for subsequent maturation.