Thymic macrophages consist of two populations with distinct localization and origin

Tissue-resident macrophages are essential for protection from pathogen invasion and maintenance of organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and found out that they express typical tissue-resident macrophage genes yet also exhibit organ-specific features. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor SpiC with these cells. Using shield chimeras, transplantation of embryonic thymuses, and fate mapping, we found that three distinct waves of precursors generate thymic macrophages. Moreover, some of them proliferated in situ. Single-cell RNA sequencing showed that the macrophages in the adult thymus are composed of two populations with distinct localization and origin. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

Cultural Properties of Cryopreserved Thymic Multipotent Stromal Cells and Fetal Skin and Muscle-Derived Cells

The paper provides a comparison of properties of cryopreserved fetal murine multipotent stromal cells (MSCs) of skin-muscular origin and those derived from adult thymus in culture in vitro. Fetal MSCs showed a 30% higher number of average population doublings within 24 hrs, and 41% lower average population doubling time. It was found that the fetal MSCs of the 4th passage had a 39% higher clonogenic activity than the adult thymus-derived ones. Fetal MSCs and those derived from adult thymus differentiated in osteogenic and adipogenic lineages with equal efficiency in special culture media. Fetal and thymus-derived MSCs were characterized by almost the same high ability of contact interaction with thymocytes, and the fibroblast-lymphocyte rosette (FLR) formation. They were far less active in FLR formation with lymph node cells. This indicated the presence of membrane affinity for immature lymphoid cells in both MSC subpopulations. The results showed the fetal MSCs to be significantly different from the adult thymus-derived MSCs by more active kinetics of growth and clonogenic potential. However, both cell subpopulations had virtually the same ability for linear differentiation and showed high activity during contact with immature lymphoid cells. Linear differentiation and the ability to interact with lymphocytes were found to be quite stable properties of MSCs, but a proliferative activity and in vitro colony formation distinguished significantly in different types of MSCs. This can be taken into account when choosing the cells for therapy, research and results assessment.

Radiation inducible MafB gene is required for thymic regeneration

The thymus facilitates mature T cell production by providing a suitable stromal microenvironment. This microenvironment is impaired by radiation and aging which lead to immune system disturbances known as thymic involution. Young adult thymus shows thymic recovery after such involution. Although various genes have been reported for thymocytes and thymic epithelial cells in such processes, the roles of stromal transcription factors in these remain incompletely understood. MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor expressed in thymic stroma and its expression was induced a day after radiation exposure. Hence, the roles of mesenchymal MafB in the process of thymic regeneration offers an intriguing research topic also for radiation biology. The current study investigated whether MafB plays roles in the adult thymus. MafB/green fluorescent protein knock-in mutant (MafB+/GFP) mice showed impaired thymic regeneration after the sublethal irradiation, judged by reduced thymus size, total thymocyte number and medullary complexity. Furthermore, IL4 was induced after irradiation and such induction was reduced in mutant mice. The mutants also displayed signs of accelerated age-related thymic involution. Altogether, these results suggest possible functions of MafB in the processes of thymic recovery after irradiation, and maintenance during aging.

γδ-Thymocyte Maturation and Emigration

The thymus is the site of both αβ and γδ-T cell development. After several unique waves of γδ-T cells are generated in, and exported from, the fetal/neonatal thymus, the adult thymus continues to produce a stream of γδ-T cells throughout life. One intriguing feature of γδ T cell development is the coordination of differentiation and acquisition of effector function within the fetal thymus, however, it is less clear whether this paradigm holds true in adult animals. To investigate the relationship between maturation and time since V(D)J recombination in adult-derived γδ-thymocytes, we used the Rag2pGFP model. Immature (CD24+) γδ-thymocytes expressed high levels of GFP while only a small minority of mature (CD24-) γδ-thymocytes were GFP+. Similarly, most GFP+ γδ-splenocytes were immature, while some were mature. Analysis of γδ-recent thymic emigrants (RTEs) indicated that most γδ-T cell RTEs were CD24+ and GFP+ and adoptive transfer experiments showed that immature γδ-thymocytes could be maintained in the periphery for at least 3 days over which time they matured. With respect to the mature γδ-thymocytes that were GFP-, parabiosis experiments demonstrated that mature γδ-T cells did not recirculate from the periphery. Instead, a population of mature γδ-thymocytes remained resident in the thymus for at least 60 days while mature γδ-thymocytes derived solely from adult hematopoiesis were mostly lost from the thymus within 60 days. Collectively, these data demonstrate two streams of actively developing γδ-T cells in adult mice: an immature subset that quickly leaves the thymus and matures in the periphery, and one that completes maturation within the thymus over a longer period of time. Furthermore, there is a fetal-derived and heterogeneous population of resident γδ-thymocytes of unknown functional importance.

In Pursuit of Adult Progenitors of Thymic Epithelial Cells

Peripheral T cells capable of discriminating between self and non-self antigens are major components of a robust adaptive immune system. The development of self-tolerant T cells is orchestrated by thymic epithelial cells (TECs), which are localized in the thymic cortex (cortical TECs, cTECs) and medulla (medullary TECs, mTECs). cTECs and mTECs are essential for differentiation, proliferation, and positive and negative selection of thymocytes. Recent advances in single-cell RNA-sequencing technology have revealed a previously unknown degree of TEC heterogeneity, but we still lack a clear picture of the identity of TEC progenitors in the adult thymus. In this review, we describe both earlier and recent findings that shed light on features of these elusive adult progenitors in the context of tissue homeostasis, as well as recovery from stress-induced thymic atrophy.

The role of chromatin organizer Satb1 in shaping TCR repertoire in adult thymus

T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V (D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. The previous study showed that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αβ T cells.

Deciphering the Regulatory Landscape of γδ T Cell Development by Single-Cell RNA-Sequencing

SUMMARYRecent studies have established γδ T cells as critical players in a broad range of infections, antitumor surveillance, autoimmune diseases and tissue homeostasis. However, differentiation of γδ T cells in the adult thymus remains poorly understood, due to the rare frequency of this lineage. Here, we infer high-resolution developmental trajectories of this rare population by single-cell RNA-sequencing. We reveal previously unknown subtypes and identify the transcription factor c-MAF as a novel key regulator of IL-17-producing γδ T cell (γδT17) differentiation. c-MAF knockout mice exhibit a complete block in γδT17 differentiation, absence of these cells from peripheral organs, and protection from an autoimmune phenotype in a psoriasis model. Single-cell RNA-sequencing of Sox13 and Rorc knockout mice pinpoints c-MAF as an essential missing link between these lineage-specifying factors. These findings significantly enhance our understanding of γδ T cell ontogeny. Our experimental strategy provides a blueprint for deciphering differentiation of rare cell types.