Thomas Barthlott

TGF-beta signaling in thymic epithelial cells regulates thymic involution and postirradiation reconstitution.

The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cells capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.

MicroRNAs Control the Maintenance of Thymic Epithelia and Their Competence for T Lineage Commitment and Thymocyte Selection

Thymic epithelial cells provide unique cues for the lifelong selection and differentiation of a repertoire of functionally diverse T cells. Rendered microRNA (miRNA) deficient, these stromal cells in the mouse lose their capacity to instruct the commitment of hematopoietic precursors to a T cell fate, to effect thymocyte positive selection, and to achieve promiscuous gene expression required for central tolerance induction. Over time, the microenvironment created by miRNA-deficient thymic epithelia assumes the cellular composition and structure of peripheral lymphoid tissue, where thympoiesis fails to be supported. These findings emphasize a global role for miRNA in the maintenance and function of the thymic epithelial cell scaffold and establish a novel mechanism how these cells control peripheral tissue Ag expression to prompt central immunological tolerance.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

Signaling by kinases downstream of the Hippo homolog mediates thymocyte migration. Sending thymocytes into action MST1, the mammalian homolog of Hippo, plays a role in apoptosis and cellular proliferation by activating the kinase LATS, which inhibits the transcriptional coactivator YAP; however, MST1 also functions independently of LATS and YAP in T cell adhesion and migration. Tang et al. generated mice with a T cell–specific deficiency in both isoforms of the LATS-related kinase NDR. These mice had reduced numbers of naïve T cells in the periphery because mature thymocytes were trapped in the thymus. Chemoattractants stimulated actin polymerization and the migration of thymocytes in an MST1- and NDR-dependent manner, suggesting that the NDRs act downstream of MST1 to mediate thymocyte egress. The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell–specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Maintenance of a normal thymic microenvironment and T-cell homeostasis require Smad4-mediated signaling in thymic epithelial cells

Signals mediated by the transforming growth factor-beta superfamily of growth factors have been implicated in thymic epithelial cell (TEC) differentiation, homeostasis, and function, but a direct reliance on these signals has not been established. Here we demonstrate that a block in canonical transforming growth factor-beta signaling by the loss of Smad4 expression in TECs leads to qualitative changes in TEC function and a progressively disorganized thymic microenvironment. Moreover, the number of thymus resident early T-lineage progenitors is severely reduced in the absence of Smad4 expression in TECs and directly correlates with extensive thymic and peripheral lymphopenia. Our observations hence place Smad4 within the signaling events in TECs that determine total thymus cellularity by controlling the number of early T-lineage progenitors.

A subpopulation of CD103 pos ICOS pos Treg cells occurs at high frequency in lymphopenic mice and represents a lymph node specific differentiation stage

Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self‐reactive, chronic, and homeostatic T‐cell responses. Here, we report that the increase in Treg‐cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103posICOSpos Treg cells. Though present in the thymus, CD103posICOSpos Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103posICOSpos Treg cells is not affected by the absence of IL‐7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103posICOSpos Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103posICOSpos phenotype represents a LN‐specific differentiation stage arrived at by several other Treg‐cell subsets. Thus, tissue‐specific cues determine the overall potency of the peripheral Treg‐cell pool by shaping its subset composition.

Differential Response of Mouse Thymic Epithelial Cell Types to Ionizing Radiation-Induced DNA Damage

Thymic epithelial cells (TECs) are the main components of the thymic stroma that support and control T-cell development. Preparative regimens using DNA-damaging agents, such as total body irradiation and/or chemotherapeutic drugs, that are necessary prior to bone marrow transplantation (BMT) have profound deleterious effects on the hematopoietic system, including the thymic stroma, which may be one of the main causes for the prolonged periods of T-cell deficiency and the inefficient T cell reconstitution that are common following BMT. The DNA damage response (DDR) is a complex signaling network that allows cells to respond to all sorts of genotoxic insults. Hypoxia is known to modulate the DDR and play a role affecting the survival capacity of different cell types. In this study, we have characterized in detail the DDR of cortical and medullary TEC lines and their response to ionizing radiation, as well as the effects of hypoxia on their DDR. Although both mTECs and cTECs display relatively high radio-resistance, mTEC cells have an increased survival capacity to ionizing radiation (IR)-induced DNA damage, and hypoxia specifically decreases the radio-resistance of mTECs by upregulating the expression of the pro-apoptotic factor Bim. Analysis of the expression of TEC functional factors by primary mouse TECs showed a marked decrease of highly important genes for TEC function and confirmed cTECs as the most affected cell type by IR. These findings have important implications for improving the outcomes of BMT and promoting successful T cell reconstitution.

Violation of the 12/23 rule of genomic V(D)J recombination is common in lymphocytes

V(D)J genomic recombination joins single gene segments to encode an extensive repertoire of antigen receptor specificities in T and B lymphocytes. This process initiates with double-stranded breaks adjacent to conserved recombination signal sequences that contain either 12- or 23-nucleotide spacer regions. Only recombination between signal sequences with unequal spacers results in productive coding genes, a phenomenon known as the 12/23 rule. Here we present two novel genomic tools that allow the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues using second-generation sequencing. Further, we provide strong evidence that the 12/23 rule of genomic recombination is frequently violated under physiological conditions, resulting in unanticipated hybrid recombinations in ∼10% of Tcra excision circles. Hence, we demonstrate that strict adherence to the 12/23 rule is intrinsic neither to recombination signal sequences nor to the catalytic process of recombination and propose that nonclassical excision circles are liberated during the formation of antigen receptor diversity.