International Immunology

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International Immunology, Vol. 14, No. 3, 331-338, March 2002
© 2002 Japanese Society for Immunology

Functional demonstration of intrathymic binding sites and microvascular gates for prothymocytes in irradiated mice

Deborah L. Foss, Elina Donskoy and Irving Goldschneider

Department of Pathology, School of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3105, USA

Correspondence to: I. Goldschneider; E-mail: igoldsch{at}neuron.uchc.edu

	Abstract

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Quantitative intrathymic (i.t.) and i.v. adoptive transfer assays for prothymocytes show strict log dose saturation kinetics, consistent with a finite number of i.t. binding sites (microenvironmental niches). This inference is supported here by demonstration of competitive antagonism obeying one-on-one receptor occupancy kinetics during the establishment of thymic chimerism in irradiated adult mice. The results of primary and secondary transfer experiments suggested that hematogenous precursors (i) enter specific i.t. niches between 4 and 24 h after injection, (ii) compete reversibly with subsequently introduced precursors, (iii) establish unsurmountable competition within 5–7 days, (iv) mature through the initial stages of thymocytopoiesis preceding proliferative expansion, and (v) vacate the niches between 7 and 14 days after entry. The results also suggested that, as in non-irradiated mice, prothymocyte importation in irradiated mice is a gated phenomenon. Gate closure was indicated by the inability of i.v.-, but not i.t.-, injected bone marrow (BM) cells to induce thymic chimerism when administered 7–14 days after a primary injection and gate opening by the ability of i.v.-injected BM cells to induce thymic chimerism in competition with circulating host prothymocytes. Gate closing was log dose-responsive and could be induced in individual thymic lobes by unilateral i.t. injection, whereas gate opening, which occurs bilaterally, was not initiated until most of the niches for prothymocytes had been vacated. We therefore posit the existence of a series of associated microvascular gates and microenvironmental niches that act in concert to regulate prothymocyte importation and early thymocyte differentiation.

Keywords: bone marrow, lymphoid migration, lymphoid organization, thymus

	Introduction

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We have previously demonstrated that thymocytopoiesis in non-myeloablated adult mice (1), as in fetal and neonatal mice (2–5), is maintained by the importation of hematogenous thymocyte precursors (prothymocytes). More recently, we have demonstrated that the importation of prothymocytes in normal adult mice is a gated phenomenon (6), again as in fetal and neonatal mice. Gate opening had a periodicity of ~30 days, lasted 7–10 days and extended beyond the onset of physiological thymic involution. Hence, although the adult thymus no longer processes developmentally discrete waves of precursors, the underlying pattern of importation of prothymocytes that is established in the fetus appears to be retained throughout post-natal life.

Other investigators have suggested that a combination of chemotactic, endothelial cell adhesion and extracellular matrix (ECM)-dependent mechanisms are involved in the directed migration (homing) of prothymocytes to the thymus (7–9). This has been especially well documented in the gated importation of waves of prothymocytes during embryonic/larval and neonatal/post-metamorphic development in birds, mice and frogs (10–12). It has also been demonstrated that thymic epithelial cells, fibroblasts, macrophages and dendritic cells, along with ECM proteins and adhesion molecules, participate in the formation of a series of intrathymic (i.t.) microenvironmental niches for double-positive (CD4⁺CD8⁺) and single-positive (CD4⁺CD8^- or CD4^-CD8⁺) thymocytes (e.g. 13–16). However, evidence for the existence of niches for prothymocytes and triple-negative (CD3^-CD4^-CD8^-) thymocytes has been difficult to obtain due to the small numbers of prothymocytes imported by the thymus (17–20) and the ensuing lag period of thymocytopoiesis (21–23). Nonetheless, the existence of such niches has been suggested by the retention of prothymocytes in the inner cortex of the thymus for ~1 week after importation (18), apparently in contact with epithelial cells (7).

In our description of prothymocyte gating in non-myeloablated mice (6), we observed that the induction of thymic chimerism by i.t. injection of bone marrow (BM) cells was cyclical, whereas that by i.v. injection was periodic. These differential kinetics suggested that the receptive period for the importation of hematogenous prothymocytes (open gate) coincided with the maximal availability of microenvironmental niches and the refractory period (closed gate) with the progressive emptying of these niches. However, due to the asynchronicity of gating within cohorts of non-myeloablated mice, it was not possible to formally document by competitive antagonism the existence of specific i.t. binding sites for prothymocytes.

We therefore have conducted such studies in radioablated mice, using our quantitative i.v. and i.t. BM adoptive transfer systems for thymocytopoiesis (24). These assays have been utilized primarily to trace the developmental potentials of prethymic and i.t. lymphoid precursor cell subsets (reviewed in 25). However, as both assays obey classical log dose saturation kinetics (24), it seemed likely that, when used competitively, they might also provide insights into the microenvironmental regulation of importation and differentiation of prothymocytes.

This is confirmed here by the detection of one-on-one receptor occupancy kinetics, which strongly supports the existence of a finite number of specific binding sites (which we equate operationally with microenvironmental niches) for thymocyte precursors. Furthermore, differential timing of receptivity and refractivity for i.v.- and i.t.-injected thymocyte precursors indicated that, as in non-ablated mice (6), the importation of prothymocytes is a gated phenomenon in radioablated mice. In addition, the results of dose–response, time–response and secondary transfer experiments suggested that, when occupied by prothymocytes, signals from these niches induce the closure of associated microvascular gates and regulate the initial stages of thymocyte commitment and differentiation.

Methods

Animals
Ly5 congenic C57BL/6NCR(B6) mice (4–6 weeks old), obtained from NCI (Charles River, Frederick, MD), were used throughout. Cohorts of male and female animals and Ly5.1 and Ly5.2 donors and recipients were tested, with no differences being noted. Cell transfer was carried out in sex-matched combinations only. Animals were maintained on commercial mouse chow and water ad libitum in the Center for Laboratory Animal Care, University of Connecticut Health Center.

Preparation of cell suspensions
BM cell suspensions were prepared by flushing the marrow from the tibia and femur with cold RPMI 1640 (Gibco, Grand Island, NY) supplemented with Na₂HCO₃ (2 mg/ml) and 1% HEPES (1.5 M), as described (24). Repeated gentle pipetting further dispersed the cells, which were then washed in cold medium and centrifuged at 4°C for 5 min at 1500 r.p.m. Thymocytes and peripheral lymph node (LN) cells were separated from the stroma by gently pressing the tissues through a stainless steel screen (50 mesh), followed by washing in cold medium. The cells were counted on a Z1 Coulter counter (Coulter, Hialeah, FL). The modified enzymatic digestion technique of Wu et al. (26) was used in some experiments to optimize recovery of i.t. precursors. Briefly, thymus lobes were diced into small fragments, suspended in 10 ml RPMI 1640 plus 2% FCS adjusted to mouse osmolality (0.38 g/ml NaCl in 100 ml of RPMI 1640 supplemented with HEPES buffer, pH 7.2) and centrifuged at 4°C for 6 min at 1000 r.p.m. The washed cells and fragments were then digested for 25 min at 22°C with continuous agitation in 15 ml RPMI 1640 plus 2% FCS containing 1 mg/ml type II collagenase B and 0.02 mg/ml grade II bovine pancreatic DNase I (Boehringer Mannheim, Mannheim, Germany) to which EDTA (1.2 ml, 0.1 M EDTA, pH 7.2) was added for the final 5 min. The tissue fragments were passed through a stainless-steel sieve and the resulting cell suspension was washed by centrifugation in RPMI.

I.t. adoptive transfer assay for prothymocytes
Recipient mice received 6 Gy total body irradiation (0.97 Gy/min) from a ¹³⁷Cs source (Gamma Cell 40 Irradiator; Atomic Energy of Canada, Ottawa, Canada) at the times indicated prior to BM cell injection. After anesthesia (ketamine/acepromazine), the thymus was surgically exposed and the indicated number of cells injected into the anterior superior portion of each lobe (10 µl/site) using a 1-ml syringe (with attached 28-gauge needle) mounted on a Tridek Stepper (Indicon, Brookfield Center, CT), as described (24). The skin incision was closed with Nexaband Liquid (Veterinary Products, Phoenix, AZ). Control mice were injected with unfractionated normal thymocytes or lymph node (LN) cells or RPMI alone. In some experiments, thymocytes, obtained at timed intervals from primary (1°) recipients of BM were injected i.t. alone or mixed with BM cells into secondary (2°) recipients to measure relative reconstituting and competitive activities.

I.v. adoptive transfer assay for prothymocytes
The indicated number of BM cells suspended in 0.5 ml of RPMI was injected through a 28-gauge needle into the lateral tail veins of irradiated (6 Gy) unanesthetized recipient mice. Control mice were injected with unfractionated normal thymocytes or LN cells or RPMI alone.

Flow immunocytometric (FCM) analysis of thymic chimerism
Peak levels (%) of thymic chimerism, observed 28 days after BM cell transfer, were determined by FCM analysis (FACScan; Becton Dickinson, Sunnyvale, CA) after development for immunofluorescence with anti-Ly5.1 and anti-Ly5.2 mAb (Jackson Laboratory, Bar Harbor, ME). Dead cells and non-lymphoid cells were excluded from the analysis by gating for forward and side angle light scatter, and 10,000 viable cells were collected in each file. Specificity and sensitivity of staining were controlled by checkerboard analyses against normal Ly5.1 and Ly5.2 thymocytes and purposeful mixtures thereof. The percentage of positive cells was determined by overlaying the fluorescence histogram with the negative control profile and using the intersection as the cut-off point. In studies of competitive antagonism, percent inhibition of thymocyte chimerism was calculated according to the formula: [(expected number of thymocytes – observed number of thymocytes)/expected number of thymocytes]x100, in which the expected number of donor- (or host-) origin thymocytes was derived from the respective i.v. or i.t. dose–response curves obtained in the absence of antagonist.

	Results

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Reversible competitive antagonism during the initiation of thymocytopoiesis
In preliminary experiments, injection of graded mixtures (1:9 to 9:1) of Ly5.1 and Ly5.2 BM cells i.v. or i.t. into sublethally irradiated Ly5.1 or Ly5.2 recipients, whether at saturating, subsaturating or supersaturating doses, generated proportional levels of thymic chimerism when harvested 28 days later. Proportional chimerism was also observed when similar ratios of Ly5.1 and Ly5.2 BM cells were injected sequentially i.v. and/or i.t. at 1–3 h intervals (data not shown). However, by 4 h, this random in vivo mixing of sequentially injected cells was replaced by competitive antagonism, presumably for i.t. binding sites supportive of prothymocyte engraftment.

Hence, as shown in Fig. 1(A), i.v. or i.t. injection of graded doses of host-allotype (Ly5.1) BM cells linearly reduced the number of donor-allotype (Ly5.2) thymocytes generated by a saturating dose (20x10⁶ i.v.; 2.5x10⁶ i.t.) of BM cells injected 4 h later. In both instances, the maximal degree of inhibition was ~50%. Furthermore, the slopes of the dose–competition curves approximated one-half of those of the corresponding dose–response curves for Ly5.2 BM cells (insert) and each set of curves shared a common x-intercept. In contrast, the degree of inhibition was essentially constant (mean % = 50.2 ± 2.2 i.t. and 40.0 ± 2.1 i.v.) when Ly5.1 chimerism was plotted as the dependent variable in response to saturating doses of Ly5.2 BM cells (Fig. 1B).

View larger version (29K): [in this window] [in a new window]   Fig. 1. Competitive antagonism of thymic chimerism as a function of dose of BM cells. Dose–competition curves (solid lines) were established by injecting sublethally irradiated Ly5.1 mice i.t. () or i.v. () with graded doses of Ly5.1 BM cells followed 4 h later with saturating doses of Ly5.2 BM cells injected by the opposite route (2.5x106 i.t. or 20x106 i.v.). The total numbers of Ly5.2 (A) and Ly5.1 (B) thymocytes present 28 days later were compared with those generated by the corresponding number of BM cells in the respective i.v. and i.t. dose–response curves in the absence of antagonist (insert, dashed lines). Results are expressed as the percent decrease (inhibition) in the mean numbers of thymocytes generated in experimental versus control animals. Each point indicates the mean of four or five animals. Lines of best fit and intercepts are determined by linear regression analysis. Representative experiment (one of three).

 
Similar results were obtained when the interval between injections was extended to 24 h; the order of administration of graded and saturating doses of BM was reversed, and both injections were given by the same route (data not shown). Furthermore, the total number of thymocytes generated remained constant during these permutations and dose combinations (mean ± SD = 172 ± 29x10⁶).

Hence, as defined by Schild regression analysis (27), the results appeared to obey simple one-on-one receptor occupancy kinetics between agonist and antagonist of equal potency. This in turn suggested that, at all dose levels tested, the Ly5.1 BM cells competed reversibly for a finite number of i.t. binding sites with an equivalent number of precursor cells in the saturating dose of Ly5.2 BM. The specificity of binding was further demonstrated by the inability of saturating doses of Ly5.1 LN cells and unfractionated thymocytes, both of which are poor sources of thymocyte precursors (24,28,29) to decrease thymocytopoiesis induced by Ly5.2 BM cells (data not shown).

Unsurmountable competitive antagonism during the initiation of thymocytopoiesis
Extended timed competition experiments using saturating doses of both Ly5.1 and Ly5.2 BM cells revealed that the thymus becomes progressively more refractory to the establishment of chimerism between days 1 and 4 after the initial injection (Fig. 2). Both i.v.- and i.t.-injected Ly5.2 BM cells were equally affected. This suggested that the occupation of i.t. binding sites by thymocyte precursors becomes progressively more stable after 24 h, such that saturating doses of Ly5.2 BM cells administered ~4 days after the injection of Ly5.1 BM cells can no longer surmount this antagonism.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Competitive antagonism of thymic chimerism as a function of time between i.v. and/or i.t. injection of BM cells. Sublethally irradiated Ly5.1 mice were injected i.v. or i.t. with a saturating dose of Ly5.1 BM cells and 1–5 days later groups of four to six of these mice were reinjected with a saturating dose of Ly5.2 BM cells by the same or opposite route: (A) i.v./i.v., (B) i.t./i.v. and (C) i.v./i.t. injections. The numbers of Ly5.2 thymocytes present 28 days later were compared with those generated in irradiated mice injected with a saturating dose of Ly5.2 BM cells at timed intervals after an initial sham injection (buffer only). The lines of best fit were determined by linear regression analysis. The slopes for (A), (B) and (C) were 15.2, 11.4 and 17.8, and the projected x-intercepts at 100% inhibition were 4.8, 5.1 and 4.8 days respectively. Representative experiment (one of three).

 
The occupation of i.t. binding sites for ~1 week was also suggested by the continued ability of thymocytes obtained between 1 and 7 days after i.v. injection of Ly5.1 BM cells (but not from control mice) to inhibit the induction of thymic chimerism by Ly5.2 BM cells in secondary recipients (Fig. 3 solid line). Interestingly, the continued presence of competitive activity was associated with a linear decrease in recoverable thymocyte reconstituting activity, as judged by the ability of thymus cells from primary recipients to initiate thymocytopoiesis when transferred i.t. into secondary recipients (Fig. 3 dashed line). Comparable results were obtained after enzymatic dissociation of thymocytes (data not shown), suggesting that the absence of reconstitution potential was not due to the failure of mechanical dissociation alone to recover the active population. Conversely, during week 2, thymocytes from the primary recipients regained their thymocyte reconstituting activity, but lost their ability to compete for binding sites in secondary recipients. These latter events coincided temporally with the evacuation of i.t. niches in the primary recipients (see results below).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Recovery of thymocyte reconstituting and prothymocyte competitive activities as a function of time after i.v. or i.t. injection of BM cells. Sublethally irradiated Ly5.1 mice were injected i.t. with a saturating dose of Ly5.2 BM cells or i.v. with a saturating dose of Ly5.1 BM cells. At timed intervals, whole thymus cell suspensions from the primary recipients of Ly5.2 BM cells were injected i.t. into secondary Ly5.1 recipients to measure thymocyte reconstituting activity (), whereas those from the recipients of Ly5.1 BM cells were mixed with a saturating dose of Ly5.2 BM cells before i.t. injection to measure competitive antagonism of thymic chimerism (). The numbers of Ly5.2 thymocytes present in the secondary recipients 28 days later were determined by FCM analysis. Results were compared with those obtained in recipients injected with Ly5.2 BM cells only or with Ly5.2 BM cells mixed with thymocytes from sham-injected primary recipients (buffer only). Data at each time point indicate the means of three to five animals. Representative experiment (one of two).

 
Gated importation of blood-borne prothymocytes
As shown in Fig. 4 (solid line), the thymus of primary recipients remained refractory to the induction of chimerism by i.v.-injected BM cells for an additional 7–10 days after the establishment of unsurmountable antagonism. However, during this same period (days 7–14), the thymus became increasingly receptive to the induction of chimerism by i.t.-injected BM cells (Fig. 4 dashed line). Shortly thereafter, a cycle of partial receptivity to i.v.-injected BM cells (days 17–24) and partial refractivity to i.t.-injected BM cells (days 14–28) occurred.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Demonstration of prothymocyte gating by differential induction of thymic chimerism in recipients of i.v.- and i.t.-injected BM cells. Sublethally irradiated Ly5.1 mice were injected i.v. or i.t. with a saturating dose of Ly5.1 BM cells. Between 1 and 28 days later, groups of four or five of these mice were injected i.t. () or i.v. () respectively with a saturating dose of Ly5.2 BM cells. The numbers of Ly5.2 thymocytes present 28 days later were compared with those generated in irradiated mice injected i.t. or i.v. with Ly5.2 BM cells at timed intervals after an initial sham injection (buffer only). Representative experiment (one of two).

 
These results suggested that, by day 7 after the initial injection of BM, closure of a vascular gate prevents the further importation of hematogenous precursors for an additional 7–10 days. The results further suggest that, during this time, the i.t. binding sites for prothymocytes are progressively vacated (Fig. 4 dashed line), prothymocyte competitive activity is lost (Fig. 3 dashed line) and a wave of thymocyte reconstituting activity is generated (Fig. 3 solid line). Thereafter donor-origin precursors are able to enter the reopened gate and occupy niches in competition with circulating prothymocytes from the regenerating host BM (18).

Regulation of prothymocyte gating
The preceding results suggested that gate opening occurs in a threshold-dependent fashion after most i.t. niches for prothymocytes have been vacated. In contrast, as shown in Fig. 5 gate closure was rigorously dose-responsive over a 100- to 200-fold range (extrapolated from 0 to 100% inhibition) of BM cells injected i.v. or i.t. 10 days previously. Furthermore, both the slopes and x-intercepts of the dose–inhibition curves for gate closure closely approximated those of the corresponding dose–response curves for thymocytopoiesis (Fig. 1A insert).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Dose-responsiveness of gate closing. Groups of five sublethally irradiated Ly5.1 mice were injected i.t. () or i.v. () with the indicated doses of Ly5.1 BM cells, and reinjected i.v. 10 days later with a saturating dose of Ly5.2 BM cells. The numbers of Ly5.2 thymocytes present 28 days later were compared with those generated in irradiated mice injected i.v. with Ly5.2 BM cells 10 days after an initial sham injection (buffer only).

 
In addition to being dose-responsive, gate closure appeared to be regulated entirely within the thymus itself. This was shown by split inoculation of contralateral thymic lobes in individual mice, one lobe receiving a saturating dose of BM and the opposite lobe buffer only. Upon subsequent challenge with an i.v. injection of BM 10 days later, only the thymic lobe that initially had received BM was refractory to the induction of thymic chimerism (mean % inhibition = 71.0 ± 21.2).

Discussion

In the present study, thymus-homing cells in unfractionated BM displayed the ability to compete in a saturatable fashion for representation in the i.t. precursor cell pool of sublethally irradiated recipients. The onset of reversible competitive antagonism within 4 h of i.v. (or i.t.) injection is consistent with previous reports of rapid importation of purified hematogenous thymocyte progenitors (19,30). Of special importance, the precursors in donor BM were able to compete with recently imported endogenous precursors from host blood (Fig. 4 days 17–24), thereby operationally identifying them as prothymocytes. This inference was reinforced by the inability of unfractionated LN cells and thymocytes, both of which are poor sources of prothymocytes (24,28,29), to inhibit the induction of thymocytopoiesis by BM cells. Furthermore, the demonstration of one-on-one binding kinetics suggests that the thymus-seeking precursor population in BM is relatively homogeneous, even though BM cells other than to those that generate thymocytes may indirectly influence the kinetics of migration or importation (e.g. 31). Hence, the combination of competitive antagonism, saturation kinetics and cell specificity strongly supports the existence of a finite number of binding sites for prothymocytes. However, because the agonists and antagonists in these reactions are cells, we envision the binding sites as being microenvironmental niches. This seems especially likely, as cognitive associations with stromal cells and ECM appear to be essential for the earliest stages of thymocyte development (15,32).

It could, of course, be argued that prothymocytes compete for limiting numbers of regulatory cells or cytokines rather than for specific microenvironmental niches. However, this distinction may be largely semantic, as such cells and factors are likely to be components of such niches. Similarly, reports that a single hematogenous thymic progenitor can regenerate the full complement of thymocytes in a single lobe (16,33–36) and that reproducible reconstitution of the thymus occurs after i.t. injection of five purified hemopoietic stem cells (33) might appear to challenge the notion of a finite number of i.t. niches for prothymocytes. However, these phenomena are more likely to be explained by the enhanced proliferative potential of extremely immature precursors, and the inverse relationship between cell dose and burst size (reviewed in 37). Nonetheless, cell purification and immunohistological studies will be required to establish the precise nature and number of the i.t. binding sites for prothymocytes (see Discussion). In addition, it will be important to determine whether the subset(s) of BM cells that competitively occupies i.t. niches in radioablated recipients is the same as that which occupies niches is non-ablated recipients (6,38).

Three stages of competition for prothymocyte binding sites were distinguished by sequential injections of BM cells. The first stage (3 h after injection) consists of in vivo mixing of Ly5.1 and Ly5.2 BM cells followed by random competition for unoccupied niches. The observed thymic chimerism precisely reflects the ratio of competing cells. In retrospect, this probably was the major type of competition observed by Kadish and Basch (20) after i.v. injections of spleen and BM cells into irradiated recipients. The second stage (maximal between 4 and 24 h after injection, but extending for ~5 days) consists of competition for reversibly occupied niches (Figs 1 and 2). This kind of competition results in an ~50% displacement of bound prothymocytes after challenge. A previous example may have been provided by Scollay et al. (39) after sequential i.v. injections of BM cells. The third stage (increasing between days 1 and 5, maximal between days 5 and 7, and decreasing between days 7 and 14 after injection) consists of unsurmountable competition, presumably for stably occupied niches (Figs 2 and 4). Hence, in their aggregate, the results indicate that most if not all i.t. niches for prothymocytes are filled within 24 h of i.v. injection and remain occupied for a minimum of 1 week and a maximum of 2 weeks.

Although the reversible stage of competition theoretically could occur at i.t. vascular binding sites rather than within the thymic parenchyma itself, this is improbable given the observed competition between i.v.- and i.t.-injected BM cells. Similarly, it is unlikely that the unsurmountable stage of competition is due to negative feedback rather then physical occupation of niches, as the injection of subsaturating doses of BM cells does not affect subsequent access to unoccupied niches (Fig. 5 and unpublished observations). Furthermore, as thymocyte competitive activity reaches saturating levels by 24 h (Fig. 3), and thymocyte reconstituting activity decreases precipitously between days 1 and 4, it is also unlikely that the apparent transition from reversible to unsurmountable competition is due instead to continued importation of prothymocytes over a 4- to 5-day period.

The 2-week wave-like pattern of filling, occupation and emptying of i.t. niches observed in Fig. 4 corresponds roughly to the lag period of thymocytopoiesis in irradiated recipients (19,24,29). During the first week, the increasingly stable binding of prothymocytes to niches (Fig. 2) correlates temporally with the reported transient `disappearance' of double-negative (DN) thymocytes (25) and the loss of reconstitution potential (Fig. 3) correlates with the onset of thymocyte differentiation (30). During the second week, the restoration of reconstitution potential (Fig. 3) correlates temporally with the `reappearance' of DN thymocytes (25) and the loss of competitive activity with their commitment to thymocytopoiesis (40). We have demonstrated elsewhere (29 and unpublished observations) that the loss of detectable donor-origin thymocyte reconstituting activity during week 1 is due in part to the co-transfer of radioresistant i.t. host-origin precursors, which may occupy the same microenvironmental niches as do prothymocytes (35), and that the reappearance of proliferative potential between days 7 and 10 correlates with the maturation of these radioresistant precursors. Conversely, the loss of competitive activity between days 7 and 10 (Fig. 3) suggests that, at this time, developing thymocytes lose their ability to reoccupy niches for prothymocytes. This correlates precisely with the time when these cells begin to vacate the niches in the primary host (Fig. 4).

These kinetics and developmental hallmarks suggest that the i.t. niches for prothymocytes regulate the transition of pro-T1 (CD44⁺CD25^-) to pro-T2 (CD44⁺CD25⁺) thymocytes (5,25,32,41–43). This inference is directly supported by recent immunohistological studies of thymic chimerism in both non-ablated mice (38) and radioablated mice (H. Petrie, Memorial Sloan Kettering Cancer Center, NY, pers. commun.). The experiments in non-ablated recipients indicated that recently imported, as well as resident, pro-T1 cells are concentrated in the perimedullary cortical region and that the pro-T2 cells move outward from this region towards the supcapsular zone. The latter experiments indicated that, on day 14 after irradiation, donor-origin c-kit⁺ DN thymocytes (characteristic of pro-T1 and pro-T2 cells) mainly reside in the corticomedullary and inner cortical regions of the thymus, whereas c-kit^- DN thymocytes (pro-T3 and pro-T4 cells) mainly reside in the subcapsular zone.

In addition to providing evidence for the existence of i.t. niches for prothymocytes, the present results also support the existence of microvascular gates. Gate closure (refractory period) was demonstrated by the ability of i.t.- but not i.v.-injected BM cells to induce thymic chimerism when administered 7–14 days after a primary injection of BM cells (Fig. 4), and gate opening (receptive period) by the ability of i.v.- as well as i.t.-injected BM cells to induce thymic chimerism when administered between days 14 and 24. Gate opening was further documented by the concurrent importation of endogenous prothymocytes from regenerating BM during this time. However, as suggested in Fig. 4 (days 24–28) and described in detail elsewhere (manuscript in preparation), regenerating thymus-seeking BM cells from radioablated mice, unlike their counterparts from non-ablated mice, do not effectively reinduce gate closing.

Importantly, sublethal irradiation does not appear to alter fundamentally the number or function of niches for prothymocytes, as both the doses of BM cells required to achieve maximum thymic chimerism and the total numbers of thymocytes generated are the same as in non-ablated recipients (6). Similarly, assuming first in/first out kinetics, the duration of occupation of niches by individual prothymocytes and their immediate descendants would appear to be similar (i.e. 1–2 weeks) in both models. However, by making these niches simultaneously and almost instantaneously available to i.v.- (and i.t.-) injected prothymocytes, irradiation permits the establishment of thymic chimerism to be quantified more precisely. It also permits more precise analysis of the regulation of gating. For example, gate closure appears to be: (i) dose-responsive, (ii) initiated by i.t. as well as i.v. injection and (iii) induced in a single thymic lobe by unilateral injection.

Based on these observations, we postulate that a series of i.t. microvascular gates exists (~100), each of which is closed consequent to the occupation of an individual microenvironmental niche by an individual prothymocyte. As these gates are almost certainly located within the post-capillary venules (PCVs) near the corticomedullary junction, we further postulate that they are anatomically contiguous to a finite number of specialized niches for prothymocytes in the perimedullary cortex. Such an arrangement is supported by the in situ association of PCV endothelium with the cytoplasmic processes of thymic epithelial cells (44), enhanced binding of pro-T cells to endothelium cultured in the presence of thymic epithelial cells (45) and demonstration of transmigration of purified precursors from the PCVs into the surrounding thymic parenchyma of non-ablated recipients (38).

Gate opening, on the other hand, is not initiated until most of the niches for prothymocytes have been vacated and appears to by synchronized between thymus lobes (6). Furthermore, our ongoing experiments suggest that it is coordinated with the export of a wave of prothymocytes from the BM (unpublished observation). Hence, we suspect that gate opening is regulated by a threshold-dependent signal downstream of the microenvironmental niches for prothymocytes, and that it is associated with both extrathymic and interthymic feedback loops.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant AI-33741.

	Abbreviations

 

BM bone marrow

DN double negative (CD4-CD8-)

ECM extracellular matrix

FCM flow immunocytometric

i.t. intrathymic

LN lymph node

PCV post-capillary venule

	Notes

 
Transmitting editor: A. Singer

Received 4 September 2001, accepted 14 December 2001.

	References

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