Howard T. Petrie

Maintenance of T Cell Specification and Differentiation Requires Recurrent Notch Receptor–Ligand Interactions

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.

T cell receptor β chain gene rearrangement and selection during thymocyte development in adult mice

The identification is made in normal mice of the stages in T cell development at which the rearranged beta chain of the T cell receptor (TCR) is utilized to promote T cell maturation, independent of the TCR alpha chain. In addition, evidence is provided that utilization of beta chains in T cell progenitors does not preclude differentiation to TCR gamma delta + T cells. This is consistent with the view that an initial consequence of beta chain expression by early thymocytes is clonal expansion, increasing the size of the pool of useful precursors. This allows the proposal to be made that allelic exclusion may be a byproduct of cell cycle regulation during early thymocyte differentiation, which may in turn explain why the efficiency of allelic exclusion varies at different TCR or immunoglobulin loci.

Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here we show that CXCL12/CXCR4 signaling is absolutely required for proper localization of early progenitors into the cortex and thus for successful steady state differentiation. All early progenitors in the thymus express CXCR4, and its ligand (CXCL12) is expressed only by stromal cells in the cortex, where early progenitors are found. Early progenitors migrate in response to CXCL12 in vitro, while thymus-specific deletion of CXCR4 in vivo results in failed cortical localization and developmental arrest. These findings indicate a crucial and nonredundant role for CXCR4 in facilitating localization of early lymphoid progenitors to tissue regions of the thymus, where lineage commitment and proliferation are controlled.

Thymic T Cell Development and Progenitor Localization Depend on CCR7

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus. Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4−CD8−) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Cell migration and the control of post-natal T-cell lymphopoiesis in the thymus

Similar to all haematopoietic lineages, T cells must be replenished throughout life — a process that is the main function of the thymus. New progenitors are recruited to leave the bloodstream and enter the thymus, then to migrate in a defined pattern within the thymus during differentiation, and finally to return to the blood after maturation. Thereby, directional migration is intrinsically linked to all stages of T-cell differentiation. This review focuses on what is known and what is unknown about the signals that support this migration process in the mouse model of post-natal thymocyte differentiation.

Regulation of Thymus Size by Competition for Stromal Niches among Early T Cell Progenitors

Thymic T cell production is characterized by differentiating waves of non-self-renewing, bone marrow-derived progenitors. The factors constraining new progenitor recruitment, intrathymic precursor expansion, and thymus size remain enigmatic, but are believed to be controlled by a feedback loop responding to lymphoid cellularity and competition for stromal niches. In this study, we show that competition for stromal niches does occur, but is solely limited to cells at the early CD4−8− precursor stages of differentiation. The overall size of the organ is determined both by this limitation on early precursor expansion, and by a second, cell-intrinsic limit on expansion of progenitor cells transiting to the CD4+8+ stage. Together with asymmetric use of marrow-derived progenitors to reconstitute the intrathymic pool, these processes facilitate continuous generation of new T cells while maintaining a relatively stable organ size.

Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin β4 cytoplasmic domain

The cytoplasmic domain of the integrin β4 subunit mediates both association with the hemidesmosomal cytoskeleton and recruitment of the signaling adaptor protein Shc. To examine the significance of these interactions during development, we have generated mice carrying a targeted deletion of the β4 cytoplasmic domain. Analysis of homozygous mutant mice indicates that the tail‐less α6β4 binds efficiently to laminin 5, but is unable to integrate with the cytoskeleton. Accordingly, these mice display extensive epidermal detachment at birth and die immmediately thereafter from a syndrome resembling the human disease junctional epidermolysis bullosa with pyloric atresia (PA‐JEB). In addition, we find a significant proliferative defect. Specifically, the number of precursor cells in the intestinal epithelium, which remains adherent to the basement membrane, and in intact areas of the skin is reduced, and post‐mitotic enterocytes display increased levels of the cyclin‐dependent kinase inhibitor p27Kip. These findings indicate that the interactions mediated by the β4 tail are crucial for stable adhesion of stratified epithelia to the basement membrane and for proper cell‐cycle control in the proliferative compartments of both stratified and simple epithelia.

Kinetics of Steady-state Differentiation and Mapping of Intrathymic-signaling Environments by Stem Cell Transplantation in Nonirradiated Mice

Upon thymus entry, thymic-homing progenitors undergo distinct phases of differentiation as they migrate through the cortex to the capsule, suggesting that the signals that induce these differentiation steps may be stratified in corresponding cortical regions. To better define these regions, we transplanted purified stem cells into nonirradiated congenic recipients and followed their differentiation with respect to both tissue location and time. The earliest progenitors (DN1) remained confined to a very narrow region of the cortex for about the first 10 d of intrathymic residence; this region virtually overlaps the sites of thymic entry, suggesting that DN1 cells move very little during this lengthy period of proliferation and lineage commitment. Movement out of this region into the deeper cortex is asynchronous, and corresponds to the appearance of DN2 cells. Differentiation to the DN3 stage correlates with movement across the midpoint of the cortex, indicating that stromal signals that induce functions such as TCR gene rearrangement reside mainly in the outer half of the cortex. The minimum time to reach the capsule, and thus transit to the DP stage, is ∼13 d, with the average time a few days longer. These findings reveal for the first time the kinetics of steady-state progenitor differentiation in the thymus, as well as defining the boundaries of cortical regions that support different phases of the differentiation process. We also show that the first lineage-positive progeny of transplanted stem cells to appear in the thymus are dendritic cells in the medulla, suggesting that each new wave of new T cell production is preceded by a wave of regulatory cells that home to the medulla and ensure efficient tolerance and selection.

In-frame TCR δ gene rearrangements play a critical role in the αβ/γδ T cell lineage decision

Abstract Using a quantitative multiprobe Southern blot analysis, we demonstrate the surprising result that a significant proportion of αβ T cells and thymocytes retain T cell receptor δ locus sequences. A substantial portion of the retained δ locus is In a fully V-to-D-to-J rearranged configuration and 20% of these δ rearrangements are functional, significantly less than the 33% predicted for random gene rearrangements. Our observations are In conflict with the idea that αβ and γδ T cells derive from distinct precursors and suggest that commitment of a common precursor to the γδ lineage depends upon expression of a γδ T cell receptor. We propose that the Intrathymic T cell lineage decision is determined by a competition between the production of functional γδ and β-pre-T cell receptor complexes.

Harnessing of the nucleosome-remodeling-deacetylase complex controls lymphocyte development and prevents leukemogenesis

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage–determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell–specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.