The thymus provides a nurturing environment for the differentiation and selection of T cells, a process orchestrated by their interaction with multiple thymic cell types. We used single-cell RNA sequencing to create a cell census of the human thymus across the life span and to reconstruct T cell differentiation trajectories and T cell receptor (TCR) recombination kinetics. Using this approach, we identified and located in situ CD8αα+ T cell populations, thymic fibroblast subtypes, and activated dendritic cell states. In addition, we reveal a bias in TCR recombination and selection, which is attributed to genomic position and the kinetics of lineage commitment. Taken together, our data provide a comprehensive atlas of the human thymus across the life span with new insights into human T cell development.

SummaryMacrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.

The first checkpoint in T cell development, beta selection, has remained incompletely characterized for lack of specific surface markers. We show that CD27 is upregulated in DN3 thymocytes initiating beta selection, concomitant with intracellular TCR-beta expression. Clonal analysis determined that CD27high DN3 cells generate CD4+CD8+ progeny with more than 90% efficiency, faster and more efficiently than the CD27low majority. CD27 upregulation also occurs in gammadelta-selected DN3 thymocytes in TCR-beta-/- mice and in IL2-GFP transgenic reporter mice where GFP marks the earliest emerging TCR-gammadelta cells from DN3 thymocytes. With CD27 to distinguish pre- and postselection DN3 cells, a detailed gene expression analysis defined regulatory changes associated with checkpoint arrest, with beta selection, and with gammadelta selection. gammadelta selection induces higher CD5, Egr, and Runx3 expression as compared to beta selection, but it triggers less proliferation. Our results also reveal differences in Notch/Delta dependence at the earliest stages of divergence between developing alphabeta and gammadelta T-lineage cells.

Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types1. Its activity is controlled by the multi-subunit γ–secretase complex (γSE) complex2. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations3. Here we identify novel somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) resulted in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue.

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer1,2. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males3. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is significantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.