Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

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.

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.

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.

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.

Foxn1 Protein Expression in the Developing, Aging, and Regenerating Thymus.

The forkhead box N1 (Foxn1) protein is the key regulator of thymic epithelial cell (TEC) development, yet how Foxn1 functions remains largely unknown. All mature TECs arise from Foxn1-expressing progenitors/immature TECs and it is widely assumed that TECs as a whole are defined by Foxn1 expression. However, data on the Foxn1 protein are virtually lacking. In this study, we developed novel tools to visualize Foxn1 protein expression at single-cell resolution. We generated Foxn1 knock-in mice expressing a C-terminal hemagglutinin-tagged Foxn1 protein, and a cytometry-grade monoclonal anti-Foxn1 Ab. We evaluated Foxn1 expression patterns in TEC subsets and its dynamics during normal thymus development, aging, injury, and regeneration. Upon challenges, upregulation of Foxn1 was a common feature of thymus regeneration, but the timing of Foxn1 expression changed and the responding TEC subsets depended on the type of treatment. Whereas dexamethasone and recombinant human fibroblast growth factor 7 promoted expansion of Foxn1(+)Ly51(+)CD80(-) TECs, castration led to expansion of Foxn1(+)Ly51(-)CD80(+) TECs. Collectively, Foxn1 expression is highly heterogeneous in the normal thymus, with large fractions of Foxn1(low) or Foxn1(-) TECs accumulating with age. Furthermore, Foxn1 expression is responsive to perturbations.

Cell competition is a tumour suppressor mechanism in the thymus.

Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

In vivo fate mapping identifies pre-TCRα expression as an intra- and extrathymic, but not prethymic, marker of T lymphopoiesis.

Expression of the pre-T cell receptor α (pTα) gene has been exploited in previous studies as a molecular marker to identify tiny cell populations in bone marrow (BM) and blood that were suggested to contain physiologically relevant thymus settling progenitors (TSPs). But to what extent these cells genuinely contribute to thymopoiesis has remained obscure. We have generated a novel pTα(iCre) knockin mouse line and performed lineage-tracing experiments to precisely quantitate the contribution of pTα-expressing progenitors to distinct differentiation pathways and to the genealogy of mature hematopoietic cells under physiological in vivo conditions. Using these mice in combination with fluorescent reporter strains, we observe highly consistent labeling patterns that identify pTα expression as a faithful molecular marker of T lineage commitment. Specifically, the fate of pTα-expressing progenitors was found to include all αß and most γδ T cells but, in contrast to previous assumptions, to exclude B, NK, and thymic dendritic cells. Although we could detect small numbers of T cell progenitors with a history of pTα expression in BM and blood, our data clearly exclude these populations as physiologically important precursors of thymopoiesis and indicate that they instead belong to a pathway of T cell maturation previously defined as extrathymic.

Thymus-autonomous T cell development in the absence of progenitor import.

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell-deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2(-/-)γ(c)(-/-)Kit(W/Wv) mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2(-/-)γ(c)(-/-)Kit(W/Wv) hosts, γ(c)-mediated signals alone played a key role in the competition between thymus-resident and bone marrow-derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

Ltbetar signaling does not regulate Aire-dependent transcripts in medullary thymic epithelial cells.

Thymic medullary epithelial cells (mTECs) play a major role in central tolerance induction by expressing tissue-specific Ags (TSAs). The expression of a subset of TSAs in mTECs is under the control of Aire (autoimmune regulator). Humans defective for AIRE develop a syndrome characterized by autoimmune disease in several endocrine glands. Aire has been proposed to be regulated by lymphotoxin beta receptor (Ltbetar) signaling and there is evidence that, additionally, Aire-independent transcripts may be regulated by this pathway. Given the potential clinical importance of Aire regulation in mTECs for the control of autoimmunity, we investigated the relation between Ltbetar signaling and TSA expression by whole genome transcriptome analysis. In this study, we show that the absence of Ltbetar has no effect on the expression of Aire and Aire-dependent TSAs. Also, the lack of Ltbetar signaling does not disturb regulatory T cells or the distribution of dendritic cells in the thymus. However, mTECs in Ltbetar-deficient mice show an aberrant distribution within the thymic medulla with disruption of their three-dimensional architecture. This is predicted to impair the interaction between mTECs and thymocytes as shown by the reduced surface uptake of MHCII by mature thymocytes in Ltbetar-deficient mice. We propose that the physiological medullary architecture ensures negative-selection by supporting lympho-epithelial interaction through a large epithelial cell surface distributed evenly across the medulla.

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development.

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

Bone marrow-derived hemopoietic precursors commit to the T cell lineage only after arrival in the thymic microenvironment.

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.