The nature of early T lineage progenitors in the thymus or bone marrow remains controversial. Here we assess lineage capacity and proliferative potential among five distinct components of the earliest intrathymic stage (DN1, CD25(-)44(+)). All of these express one or more hemato-lymphoid lineage markers. All can produce T lineage cells, but only two of them display kinetics of differentiation, proliferative capacity, and other traits consistent with being canonical T progenitors. The latter also appeared limited to producing cells of the T or NK lineages, while B lineage potential derived mainly from the other, less typical T progenitors. In addition to precisely defining canonical early progenitors in the thymus, this work reconciles conflicting results from numerous groups by showing that multiple progenitors with a DN1 phenotype home to the thymus and make T cells, but possess different proliferative potentials and lineage capacities.
Cellular differentiation is a complex process involving integrated signals for lineage specification, proliferation, endowment of functional capacity, and survival or cell death. During embryogenesis, spatially discrete environments regulating these processes are established during the growth of tissue mass, a process that also results in temporal separation of developmental events. In tissues that undergo steady-state postnatal differentiation, another means for inducing spatial and temporal separation of developmental cues must be established. Here we show that in the postnatal thymus, this is achieved by inducing blood-borne precursors to enter the organ in a narrow region of the perimedullary cortex, followed by outward migration across the cortex before accumulation in the subcapsular zone. Notably, blood precursors do not transmigrate the cortex in an undifferentiated state, but rather undergo progressive developmental changes during this process, such that defined precursor stages appear in distinct cortical regions. Identification of these cortical regions, together with existing knowledge regarding the genetic potential of the corresponding lymphoid precursors, sets operational boundaries for stromal environments that are likely to induce these differentiative events. We conclude that active cell migration between morphologically similar but functionally distinct stromal regions is an integral component regulating differentiation and homeostasis in the steady-state thymus.
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.
The pre-TCR complex (TCRβ-pre-TCRα chain (pTα)), first expressed in a fraction of CD8−4−CD44−25+ (DN3) cells, is believed to facilitate or enable an efficient transition from the CD8−4− double-negative (DN) to the CD8+4+ double-positive (DP) developmental stage. Subsequent to pre-TCR expression, DN3 thymocytes receive survival, proliferation, and differentiation signals, although it is still unclear which of these outcomes are directly induced by the pre-TCR. To address this issue, we generated mice bearing a range of pTα transgene copy number under the transcriptional control of the p56lck proximal promoter. All lines exhibited increased DN3 cycling, accelerated DN3/4 transition, and improved DN4 survival. However, the high copy number lines also showed a selective reduction in thymic cellularity due to increased apoptosis of DP thymocytes, which could be reversed by the ectopic expression of Bcl-2. Our results suggest that transgenic pTα likely caused apoptosis of DP thymocytes due to competitive decrease in surface TCRαβ formation. These results highlight the critical importance of precise temporal and stoichiometric regulation of pre-TCR and TCR component expression.
We provide evidence that thymocytes receive signals from the thymic microenvironment which regulate the protein kinase C (PKC) signaling pathway. Thus, phorbol 12-myristate 13-acetate (PMA) causes a PKC-dependent down-regulation of CD4 expression and induces apoptosis in isolated thymocytes but has little effect on thymocytes maintained within intact thymic lobes or in reaggregate lobes containing purified thymocytes with either thymic or non-thymic stromal cells. Moreover, compact pellets of thymocytes alone are protected from the effects of PMA. This protection is maintained when the compacted thymocytes are rigorously depleted of MHC class II-expressing cells. We conclude that signals arising from thymocyte-thymocyte contact control the utilization of the PKC cascade. These observations have implications for thymocyte signaling in general as well as for the interpretation of studies carried out on thymocyte suspensions.
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