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.

Peptidylprolyl isomerase C (Ppic) regulates invariant Natural Killer T cell (iNKT) differentiation in mice.

Peptidyl-prolyl cis-trans isomerase C (Ppic) is expressed in several bone marrow (BM) hematopoietic progenitors and in T-cell precursors. Since the expression profile of Ppic in the hematoimmune system was suggestive that it could play a role in hematopoiesis and/or T lymphocyte differentiation, we sought to test that hypothesis in vivo. Specifically, we generated a Ppic-deficient mouse model by targeting the endogenous locus by CRISPR/Cas9 and tested the requirement of Ppic in hematopoiesis. Several immune cell lineages covering BM progenitors, lymphocyte precursors, as well as mature cells at the periphery were analyzed. While most lineages were unaffected, invariant NKT (iNKT) cells were reduced in percentage and absolute cell numbers in the Ppic-deficient thymus. This affected the most mature stages in the thymus, S2 and S3, and the phenotype was maintained at the periphery. Additionally, immature transitional T1 and T2 B lymphocytes were increased in the Ppic-deficient spleen, but the phenotype was lost in mature B lymphocytes. In sum, our data show that Ppic is dispensable for myeloid cells, platelets, erythrocytes, αß, and γδ T lymphocytes in vivo in the steady state, while being involved in B- and iNKT cell differentiation.

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 in hematopoietic cells: Quality control in homeostasis and its role in leukemia.

Cell competition contributes to optimal organ function by promoting tissue homogeneity. In the hematopoietic system, cell competition has been described in two distinct cell populations: in hematopoietic stem cells, and in differentiating T lymphocytes, or thymocytes. In hematopoietic stem cells, cell competition was studied in the context of mild irradiation, whereby the levels of p53 determined the outcome of the cellular interactions and the cells with lower p53 were in advantage. In the thymus, cell competition was addressed in thymus transplantation experiments, and found to be a homeostatic process that contributes to thymus turnover. Cell competition in the thymus depends on the capacity of T lymphocyte precursors to respond to interleukin 7 (IL-7). Failed cell competition permitted thymocyte self-renewal and autonomous thymopoiesis for several weeks, that culminated with leukemia onset. Beyond the work addressing cell competition in these cells, we discuss current hypotheses and observations that could be explained by cell competition. These include the clonal dynamics of hematopoietic stem cells in the ageing organism and initiation 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.

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.

EpiLog: A software for the logical modelling of epithelial dynamics.

Cellular responses are governed by regulatory networks subject to external signals from surrounding cells and to other micro-environmental cues. The logical (Boolean or multi-valued)  framework proved well suited to study such processes at the cellular level, by specifying qualitative models of involved signalling pathways and gene regulatory networks.  Here, we describe and illustrate the main features of EpiLog, a computational tool that implements an extension of the logical framework to the tissue level. EpiLog defines a collection of hexagonal cells over a 2D grid, which embodies a mono-layer epithelium. Basically, it defines a cellular automaton in which cell behaviours are driven by associated logical models subject to external signals.  EpiLog is freely available on the web at http://epilog-tool.org. It is implemented in Java (version ≥1.7 required) and the source code is provided at https://github.com/epilog-tool/epilog under a GNU General Public License v3.0.