A conserved transcriptional program for MAIT cells across mammalian evolution.

Mucosal-associated invariant T (MAIT) cells harbor evolutionarily conserved TCRs, suggesting important functions. As human and mouse MAIT functional programs appear distinct, the evolutionarily conserved MAIT functional features remain unidentified. Using species-specific tetramers coupled to single-cell RNA sequencing, we characterized MAIT cell development in six species spanning 110 million years of evolution. Cross-species analyses revealed conserved transcriptional events underlying MAIT cell maturation, marked by ZBTB16 induction in all species. MAIT cells in human, sheep, cattle, and opossum acquired a shared type-1/17 transcriptional program, reflecting ancestral features. This program was also acquired by human iNKT cells, indicating common differentiation for innate-like T cells. Distinct type-1 and type-17 MAIT subsets developed in rodents, including pet mice and genetically diverse mouse strains. However, MAIT cells further matured in mouse intestines to acquire a remarkably conserved program characterized by concomitant expression of type-1, type-17, cytotoxicity, and tissue-repair genes. Altogether, the study provides a unifying view of the transcriptional features of innate-like T cells across evolution.

IL-7 Receptor Drives Early T Lineage Progenitor Expansion.

IL-7 and IL-7R are essential for T lymphocyte differentiation by driving proliferation and survival of specific developmental stages. Although early T lineage progenitors (ETPs), the most immature thymocyte population known, have a history of IL-7R expression, it is unclear whether IL-7R is required at this stage. In this study, we show that mice lacking IL-7 or IL-7R have a marked loss of ETPs that results mostly from a cell-autonomous defect in proliferation and survival, although no changes were detected in Bcl2 protein levels. Furthermore, a fraction of ETPs responded to IL-7 stimulation ex vivo by phosphorylating Stat5, and IL-7R was enriched in the most immature Flt3+Ccr9+ ETPs. Consistently, IL-7 promoted the expansion of Flt3+ but not Flt3- ETPs on OP9-DLL4 cocultures, without affecting differentiation at either stage. Taken together, our data show that IL-7/IL-7R is necessary following thymus seeding by promoting proliferation and survival of the most immature thymocytes.

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the ß-selection checkpoint.

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the ß-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.

Cell Competition, the Kinetics of Thymopoiesis, and Thymus Cellularity Are Regulated by Double-Negative 2 to 3 Early Thymocytes.

Cell competition in the thymus is a homeostatic process that drives turnover. If the process is impaired, thymopoiesis can be autonomously maintained for several weeks, but this causes leukemia. We aimed to understand the effect of cell competition on thymopoiesis, identify the cells involved, and determine how the process is regulated. Using thymus transplantation experiments, we found that cell competition occurs within the double-negative 2 (DN2) and 3 early (DN3e) thymocytes and inhibits thymus autonomy. Furthermore, the expansion of DN2b is regulated by a negative feedback loop that is imposed by double-positive thymocytes and determines the kinetics of thymopoiesis. This feedback loop affects the cell cycle duration of DN2b, in a response controlled by interleukin 7 availability. Altogether, we show that thymocytes do not merely follow a pre-determined path if provided with the correct signals. Instead, thymopoiesis dynamically integrates cell-autonomous and non-cell-autonomous aspects that fine-tune normal thymus function.

T Cell Acute Lymphoblastic Leukemia as a Consequence of Thymus Autonomy.

Thymus autonomy is the capacity of the thymus to maintain T lymphocyte development and export independently of bone marrow contribution. Prolonging thymus autonomy was shown to be permissive to the development of T cell acute lymphoblastic leukemia (T-ALL), similar to the human disease. In this study, performing thymus transplantation experiments in mice, we report that thymus autonomy can occur in several experimental conditions, and all are permissive to T-ALL. We show that wild type thymi maintain their function of T lymphocyte production upon transplantation into recipients with several genotypes (and corresponding phenotypic differences), i.e., Rag2 - / - γc - / -, γc - / -, Rag2 - / - IL-7rα - / -, and IL-7rα - / - We found that the cellularity of the thymus grafts is influenced exclusively by the genotype of the host, i.e., IL-7rα-/- versus γc -/- Nonetheless, the difference in cellularity detected in thymus autonomy bore no impact on onset, incidence, immunophenotype, or pathologic condition of T-ALL. In all tested conditions, T-ALL reached an incidence of 80%, demonstrating that thymus autonomy bears a high risk of leukemia. We also analyzed the microbiota composition of the recipients and their genetic background, but none of the differences found influenced the development of T-ALL. Taken together, our data support that IL-7 drives cellular turnover non-cell autonomously, which is required for prevention of T-ALL. We found no influence for T-ALL in the specific combination of the genotypic mutations tested (including the developmental block caused by Rag deficiency), in microbiota composition, or minor differences in the genetic background of the strains.

Thymus autonomy as a prelude to leukemia.

Cell competition in the thymus promotes turnover and functions as a tumor suppressor by inhibiting leukemia. Using thymus transplantation experiments, we have shown that the presence of T lymphocyte precursors, recently seeding the thymus, promotes the clearance of precursors with a longer time of thymus residency. If cell competition is impaired and no cells seed the thymus, the organ is capable of sustaining T lymphocyte production, a state termed thymus autonomy. However, we observed consistently that prolonged autonomy is permissive to the emergence of T cell acute lymphoblastic leukemia (T-ALL). This resembled the onset of T-ALL in patients treated by gene therapy for X-linked severe combined immunodeficiency (SCID-X1). Following treatment, thymus activity was established, with T lymphocyte production, although no bone marrow contribution was detected. However, some patients developed T-ALL. The favored explanation for malignant transformation was considered to be genotoxicity due to integration of the retroviral vector next to oncogenes, thereby activating them ectopically. Although plausible, we consider an alternative, mutually nonexclusive explanation: that any condition enabling prolonged thymus autonomy will promote leukemogenesis. In support of this view, two independent studies have recently shown that the efficacy of reconstitution of the bone marrow in the context of SCID-X1 dramatically influences the outcome of treatment, and that lymphoid malignancies emerge following transplantation of a small number of healthy progenitors. Here, we discuss the most recent data in light of our own studies in thymopoiesis and the conditions that trigger malignant transformation of thymocytes in various experimental and clinical settings.