Kinetics of steady-state differentiation and mapping of intrathymic-signaling environments by stem cell transplantation in nonirradiated mice

Abstract

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 approximately 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.

Figure 1.

Kinetics of progenitor differentiation in the steady-state thymus. Progenitor cells were purified and injected into nonirradiated CD45-congenic recipients. At various intervals, recipient mice were killed and the developmental stage of intrathymic progeny derived from transplanted cells was determined. Differentiation through stages of development that correlate with outward migration through the cortex (i.e., CD4⁻8⁻ cells) is shown. Days 7, 8, and 19 represent single experiments; in all other cases data points represent mean ± SE for two or more recipients.

Figure 2.

Intrathymic localization of T cell progenitors at critical transitions during the early lymphopoietic process. To determine the relative tissue range for each stage of T progenitor differentiation, the progeny of transplanted cells (CD45.2, red) were localized at time points corresponding to key developmental transitions, as defined by the data in Fig. 1. Pan-cytokeratin staining (green) and nuclear counterstain (DAPI, blue) were included to distinguish the boundaries of cortical and medullary regions. Quantitation of numerous such experiments is presented in Fig. 3. All panels are 200× original magnification.

Figure 3.

Quantitative assessment of kinetics of movement between signaling regions in the cortex. The cortex was divided equally into quartiles, with 1 being nearest to the medulla and 4 nearest the capsule. The number of stem cell-derived progeny in each quartile of transverse sections (see Fig. 2) was determined at various intervals; sufficient fields were analyzed until at least 100 donor cells were counted.

Figure 4.

Donor-derived dendritic cells appear in the thymic medulla long before mature T cells. (a) The location of donor-derived progeny (CD45.2, red) at day 15 of differentiation; the dashed white line indicates the junction of cortical and medullary compartments. Note that donor-derived cells in the medulla are not lymphoid in morphology. In panel b, costaining with a dendritic cell marker (CD11c) is shown in green. Panels c and d show CD45.2 (donor) and CD11c (dendritic) stains separately; note that CD11c staining is largely absent from the cortex, but present on cells with dendritic morphology in the medulla. Panel e shows the same image as panel a, but with the CD11c staining subtracted from the CD45 staining; all donor-derived medullary cells disappear, revealing that such donor-derived medullary cells are of the dendritic lineage. Panel f shows a higher magnification view of CD45 X CD11c staining. Sections were costained with DAPI (blue). Original magnifications: a–e, 200×; f, 400×.