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*Stem Cells
The Journal of Immunology, 2003, 170: 3514-3521.
Copyright © 2003 by The American Association of Immunologists

Two Developmentally Distinct Populations of Dendritic Cells Inhabit the Adult Mouse Thymus: Demonstration by Differential Importation of Hematogenous Precursors Under Steady State Conditions1

Elina Donskoy and Irving Goldschneider2

Department of Pathology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although a variety of lymphoid and myeloid precursors can generate thymic dendritic cells (DCs) under defined experimental conditions, the developmental origin(s) of DCs in the steady state thymus is unknown. Having previously used selective combinations of normal, parabiotic, and radioablated mice to demonstrate that blood-borne prothymocytes are imported in a gated and competitive manner, we used a similar approach in this study to investigate the importation of the hematogenous precursors of thymic DCs. The results indicate that two developmentally distinct populations of DC precursors normally enter the adult mouse thymus. The first population is indistinguishable from prothymocytes according to the following criteria: 1) inefficient (<20%) exchange between parabiotic partners; 2) gated importation by the thymus; 3) competitive antagonism for intrathymic niches; 4) temporally linked generation of thymocytes and CD8{alpha}high DCs; and 5) absence from prothymocyte-poor blood samples. The second population differs diametrically from prothymocytes in each of these properties, and appears to enter the thymus in at least a partially differentiated state. The resulting population of DCs has a CD8{alpha}-/low phenotype, and constitutes ~50% of total thymic DCs. The presence of two discrete populations of DCs in the steady state thymus implies functional heterogeneity consistent with evidence implicating lymphoid DCs in the negative selection of effector thymocytes and myeloid DCs in the positive selection of regulatory thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Despite intensive study over the past decade, the developmental origin(s) of thymic dendritic cells (DCs)3 still is unclear (reviewed in Refs. 1 and 2). This question is of considerable importance, as thymic DCs appear to be the primary mediators of negative selection among developing thymocytes (3), and as they may also function to induce the formation of immunoregulatory thymocytes (4, 5, 6). Initial evidence in mice suggested that the thymus contained a lymphoid-derived subset of DCs only (7, 8). Unlike presumptive myeloid-derived DCs in secondary lymphoid tissues and skin, CD11c+ MHCII+ thymic DCs had a CD8{alpha}+ Mac-1- DEC-205+ phenotype and appeared to be generated intrathymically (i.t.) from a common T/DC precursor. Similar evidence subsequently was educed for human thymic DCs (9, 10).

Recently, key aspects of this hypothesis have been challenged. For example, it has been reported that: 1) thymic DC cell development is independent of Notch 1 function, which is required for early T cell development (11); 2) the development of thymocytes and thymic DCs is dissociated in c-kit- {gamma}c-, RAG-2-/-, and RelB mutant mice (12, 13); and 3) the ectopic expression of Id2 and Id3 inhibits the development of putative lymphoid, but not myeloid DCs by progenitor cells from human thymus (14). In addition, it has been reported that both myeloid- and lymphoid-restricted precursors can generate CD8{alpha}+ and CD8{alpha}- thymic DCs (15, 16, 17, 18, 19), and that CD8{alpha}- DCs can generate CD8{alpha}+ splenic DCs (20). Although none of these studies formally excludes the origin of thymic DCs from a common i.t. T/DC cell precursor, they do raise the possibility that myeloid- and/or other lymphoid-derived DCs also contribute to the pool of thymic DCs. This notion has been reinforced by the recent description of two phenotypically disparate subsets of presumptive lymphoid and myeloid DCs in the human thymus (21, 22), and by the detection of a biphasic bromodeoxyuridine-labeling pattern of DCs in the murine thymus (23). It is also supported by our recent demonstration (4, 5) (unpublished observations) that some hematogenous DCs from mice injected intraocularly with Ag migrate to the thymus of naive recipients where they appear to trigger the activation and exportation of immunoregulatory NKT cells.

Nonetheless, it must be cautioned that the ability of both myeloid- and lymphoid-restricted precursors to generate DCs in vitro or to generate thymic DCs in myeloablated recipients does not prove that they normally do so under steady state conditions. Neither does the presence or absence of subsets of DCs in the disorganized thymus of various mutant mice necessarily document their lymphoid or myeloid origins. A similar caution is warranted regarding the designation of separate lineage status to thymic DCs based on differences in antigenic phenotype or cytokine usage/production. Even our description of a subset of putative thymus-seeking myeloid DCs from an immunologically privileged site (4, 5) (unpublished observations) may represent the exception rather than the rule.

Although formal proof of the derivation of thymic DCs from a common T/DC cell precursor ultimately will require in vivo clonal analysis analogous to that described in fetal thymus organ cultures (24), an essential prerequisite is that the hematogenous precursors of these DCs be indistinguishable from prothymocytes by a number of discriminating criteria. For example, we have recently demonstrated that the importation of prothymocytes is a gated phenomenon in normal adult mice (25), that newly generated prothymocytes are exported from bone marrow (BM) intermittently rather than continuously (D. Foss and I. Goldschneider, manuscript in preparation), and that newly imported prothymocytes compete for a finite number of i.t. binding sites (putative microenvironmental niches) (26). It therefore would be expected that the precursors of at least a subset of thymic DCs shared these properties, and that the generative kinetics of their progeny was linked to that of thymocytes. Conversely, the precursors of nonprothymocytically derived thymic DCs would be expected to have discernibly different properties. Hence, study of the pattern(s) of importation of DC precursors by the steady state thymus should provide key insights into the developmental origin(s) of the resulting DC population(s).

We approached this objective by using a combination of unmanipulated, parabiotic, and irradiated mice similar to that which we previously used to define the kinetics of prothymocyte importation and thymocytopoiesis in normal adult mice (25, 26, 27). The results provided clear evidence for the existence of two developmentally distinct populations of thymic DCs whose precursors are differentially imported from the blood. The first population (~50% of total thymic DCs) appears to arise i.t. from precursors that are indistinguishable from prothymocytes. The second population appears to arise extrathymically from at least partially differentiated precursors, presumably of myeloid origin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Cohorts of 4- to 6-wk-old female Ly-5 congeneic C56BL/6 mice, obtained from the National Cancer Institute, were housed in the Center for Laboratory Animal Care, The University of Connecticut Health Center, until they reached the designated ages. Animals were maintained on commercial mouse chow and water ad libitum.

Preparation of cell suspensions

BM cell suspensions were prepared by flushing the marrow from tibia and femur of 4- to 5-wk-old donor mice with cold RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with sodium bicarbonate (2 mg/ml) and 1% HEPES (1.5 M). Suspensions of nucleated peripheral blood cells (PBCs) were prepared by mixing 0.5 ml of heart blood with 10 ml Alsever’s solution and washing with RPMI 1640. After lysing the RBCs with 0.165 M NH4Cl, the nucleated cells were washed in cold medium and centrifuged at 4°C for 5 min at 1500 rpm. The modified enzymatic digestion technique of Wu et al. (27), using type II collagenase B and grade II bovine pancreatic DNase I (Boehringer Mannheim, Mannheim, Germany), was used to optimize recovery of DCs from thymus lobes stripped of attached lymph nodes. Nucleated cells were counted on a Z1 Coulter Counter (Beckman Coulter, Hialeah, FL).

Intrathymic 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) 2–4 h before BM or PBC injection. After anesthesia (ketamine/acepromazine), the thymus was surgically exposed, and the indicated numbers of cells were 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 (28). The skin incision was closed with Nexaband Liquid (Veterinary Products, Phoenix, AZ).

Intravenous adoptive transfer assay for prothymocytes

The indicated number of BM cells suspended in 0.5 ml of RPMI 1640 was injected through a 28-gauge needle into the lateral tail veins of irradiated (6 Gy) unanesthetized recipient mice. Control mice were injected with RPMI 1640 alone.

Flow immunocytometric (FCM) analysis

Thymic cells were harvested at timed intervals after BM cell transfer. The percentages of donor and host origin cells were determined by FCM analysis (FACScan; BD Biosciences, Sunnyvale, CA) after development with anti-Ly-5.1 and anti-Ly-5.2 mAbs (The Jackson Laboratory, Bar Harbor, ME), and the expression of MHCII, CD11b, CD11c, CD8{alpha} chain (BD PharMingen, San Diego, CA), and/or NLDC-145 (Serotec, Raleigh, NC) was determined by multicolor analysis using combinations of FITC, PE, Red 670, and APC-labeled Abs. Between 1 x 106 and 2 x 106 viable cells were collected in each file. Specificity and sensitivity of staining were controlled by checkerboard analysis against normal 5.1 and Ly-5.2 thymocytes and purposeful mixtures thereof. The percentage of positive cells was calculated by using the intersection of the fluorescence histogram with its control profile to determine the cutoff points.

In preliminary experiments, >95% of the total MHCII+ CD11c+ CD11b- thymic DCs were found among the 1% of thymic cells having the highest forward and side angle light scatter profiles. The DC cells constituted ~0.1% of total thymic cells, were mostly (>=70%) NLDC-145+, and showed typical DC morphology after sorting. CD3+ thymocytes were restricted to and constituted almost all of the cells in the lower light scatter fractions. As reported (29), many of the CD11b+ macrophages in the thymus exhibited autofluorescence and therefore also appeared to be CD11c+. However, as neither the CD11b- thymic DCs nor the thymocytes were autofluorescent, we did not find it necessary to remove or exclude autofluorescent cells before analysis.

Parabiosis

Pairs of 4- to 5-wk-old, Ly-5 congenic mice were surgically joined by cutaneous vascular anastomosis, as described previously (30). Parabiotic mice were maintained for periods of 2–8 wk before sacrifice, or were surgically separated at semiweekly intervals and killed 28 days later. Thymi from four to six pairs of unseparated or separated parabiotic partners were harvested at the indicated time points, and the respective degrees of thymocyte and thymic DC chimerism were determined by FCM analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of thymic involution and bone marrow regeneration on thymocyte and thymic DC precursor activities

If, as postulated, a major subset of thymic DCs is generated i.t. by a common T/DC precursor, the number of DCs per thymus should decrease roughly in parallel with that of thymocytes after the onset of age-dependent thymic involution. Similarly, the generation of the hematogenous precursors of thymocytes and many thymic DCs also should be linked. Although neither prediction would establish a common origin for thymocytes and thymic DCs, failure to satisfy either one would seriously jeopardize this hypothesis.

To test the first prediction, the mean numbers of thymocytes and thymic DCs in a cohort of weanling mice were determined at weekly intervals between 5 and 17 wk of age. The results confirmed that the onset of thymic involution at ~12 wk of age was associated with a progressive decrease of both thymocytes and DCs (data not shown).

To test the second prediction, the kinetics of regeneration of thymocyte and thymic DC precursor activities in BM of sublethally irradiated (6 Gy) mice was determined over a period of 9 wk. As shown in Fig. 1A, the levels of thymocyte and thymic DC chimerism induced by the i.v. injection of regenerating BM cells into irradiated recipients were essentially superimposable at all time points. The results also showed that, at most time points, the numbers of donor and host origin DCs (Fig. 1B), like those of thymocytes (Fig. 1C), were inversely related. Based on our demonstration of competitive antagonism for binding sites between recently imported prothymocytes (26), these latter observations suggest that the precursors of many thymic DCs also compete for a finite number of i.t. niches.



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  		FIGURE 1. Kinetics of regeneration of the precursors of thymocytes and thymic DCs in BM of sublethally irradiated mice. A cohort of 5-wk-old Ly-5.1 C56BL/6 mice was sublethally irradiated (6 Gy) and divided into nine groups of six mice each. Regenerating BM cells were harvested from the femura of a different group of mice at weekly intervals after irradiation and injected i.v. into sublethally irradiated 5-wk-old Ly-5.2 recipients. Each recipient received the total cells obtained from a single femur. The levels of thymocyte and thymic DC chimerism (% donor origin cells) (A) were determined by FCM analysis 28 days after BM cell transfer, and the respective numbers of donor and host origin dendritic cells (B) and thymocytes (C) per thymus were calculated. Results are expressed as the means per group at each time point.

 
Differential importation of two populations of DC precursors by the thymus

In previous experiments (25), we used Ly-5 congenic pairs of parabiotic mice in two complementary protocols to demonstrate that the importation of circulating prothymocytes is a gated phenomenon in the adult thymus (periodicity, 3–5 wk; duration of gate opening, 7–10 days). The first protocol, in which conjoined mice were sacrificed at timed intervals after surgical union, provided insights into both the onset of importation of donor origin prothymocytes and the duration of the ensuing wave of thymocytopoiesis. The second protocol, in which the parabiotic partners were surgically separated at timed intervals before sacrifice, provided insights into the pattern of importation of prothymocytes (i.e., continuous or gated).

Based on these observations, we reasoned in this study that if a major subset of thymic DCs was derived i.t. from a common T/DC precursor, its hematogenous progenitors should be imported synchronously with prothymocytes. Conversely, the precursors of nonprothymocytically derived DCs would be expected to be imported independently of prothymocytes. As before (25), these predictions were tested in Ly-5 congenic pairs of both unseparated and separated parabiotic mice. In the first group, the kinetics of development of thymocyte and thymic DC chimerism was determined at biweekly intervals over an 8-wk period. In the second group, the parabiotic partners were surgically separated at semiweekly intervals over a 9-wk period, and the respective levels of chimerism induced by recently imported precursors were determined 28 days later.

The results in the cohort of separated parabionts (Fig. 2) showed almost complete concordance between the kinetics of importation of the precursors of donor origin thymocytes and DCs. This was true even though gate opening was not synchronized between the parabiotic partners (25, 30). Thus, peak levels of thymocyte and DC chimerism (modal points of gate opening) approximated 10% and occurred in the Ly-5.1 partners at days 24 and 59–63 of parabiosis, and in the Ly-5.2 partners at days 10, 37–39, and 56–59 of parabiosis. These results indicated that the precursors of thymocytes and a major population of thymic DCs: 1) are both inefficiently (~10%) exchanged between parabiotic partners; 2) enter the thymus in a synchronously gated manner; and 3) coordinately generate mature progeny (25, 30). It therefore is likely that the donor origin thymocyte and DC populations in each of the separated parabionts were derived from a common hematogenous precursor.



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  		FIGURE 2. Kinetics of importation of hematogenous precursors of thymocytes and thymic DCs in parabiotic mice. Ly-5.1 and Ly-5.2 congenic mice were parabiosed at 5 wk of age, and groups of three to six parabiotic pairs were surgically separated at semiweekly intervals thereafter over a 9-wk period. The mean levels of thymocyte and thymic DC chimerism (% donor origin cells) were determined in the respective Ly-5.1 (A) and Ly-5.2 (B) parabiotic partners 28 days after separation. Representative experiment (repeated for days 4–44).

 
In contrast, no concordance whatsoever was observed between the kinetics of establishment of peak levels of thymocyte and thymic DC chimerism in the cohort of unseparated parabionts. As shown in Fig. 3, maximal DC chimerism (50–60% donor origin cells) occurred within 2 wk of parabiotic union and plateaued thereafter. Maximal thymocyte chimerism (~20% donor origin cells (30)), in contrast, did not occur until at least 8 wk of parabiosis. As peak thymocyte chimerism occurs ~4 wk after the gated importation of prothymocytes (25), the present results indicate that, unlike prothymocytes, the precursors of this rapidly appearing population of thymic DCs: 1) are randomly exchanged between parabiotic partners; and 2) enter the adult thymus in an ungated fashion. In addition, as most immature lymphohemopoietic cell precursors fail to mix randomly in the blood of parabionts (30, 31, 32), the results further suggest that these DC precursors enter the thymus in at least a partially differentiated state. Finally, the fact that this rapidly appearing population of DCs was not readily apparent in the cohort of separated parabionts (Fig. 2) suggests that its i.t. t1/2 is less than 4 wk.



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  		FIGURE 3. Kinetics of the establishment of thymocyte and thymic DC chimerism in parabiotic mice. Ly-5.1 and Ly-5.2 congenic mice were parabiosed at 5 wk of age, and the thymi from groups of three parabiotic pairs were examined for the onset and progression of chimerism at biweekly intervals thereafter. Results indicate the mean levels of thymocyte and thymic DC chimerism (% donor origin cells) in the respective Ly-5.1 (A) and Ly-5.2 (B) parabiotic partners. Representative experiment (one of two).

 
It was important, in reaching these conclusions, to exclude the possibility that the rapid appearance of donor origin thymic DCs relative to thymocytes simply reflected differential generative kinetics from a common i.t. precursor. We therefore compared the kinetics of appearance of DCs and thymocytes in sublethally irradiated mice as a function of time after the i.t. injection of normal BM cells. The results in Fig. 4A show that donor origin DCs and thymocytes were initially detected on days 7 and 10, respectively, and that they reached peak levels on days 21 and 24. This is consistent with other reports of linked thymocyte and thymic DC development (7, 8). Given that the rate of establishment of thymocyte chimerism is similar in irradiated and nonirradiated recipients (25, 28), this 3-day differential in the generative kinetics of thymocytes and thymic DCs was insufficient to explain the 6-wk differential observed in the unseparated parabiotic mice (Fig. 3).



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  		FIGURE 4. Kinetics of the establishment of thymocyte and thymic DC chimerism in mice injected i.t. with normal BM cells. Each member of a cohort of 4-wk-old sublethally irradiated (6 Gy) Ly-5.1 mice was injected i.t. with 1 x 106 BM cells from 4-wk-old Ly-5.2 donors. Thymi from groups of three recipients were examined for the onset and progression of chimerism at semiweekly intervals thereafter. Results indicate the mean numbers of donor origin (A) and host origin (B) thymocytes and DCs per thymus at each time point.

 
We presume that the population of nonprothymocytically derived thymic DCs did not appear promptly in these recipients because of the immaturity of their precursors in the donor BM (as opposed to blood). Therefore, it is important to note that this population was detected when the regenerative kinetics of host origin thymic DCs was examined. As shown in Fig. 4B, two waves of host origin thymocyte and DC accumulation occurred in the month following sublethal irradiation. In the first wave, peak levels of DCs and thymocytes, presumably arising from a common radioresistant i.t. precursor (26, 28), were reached at days 7 and 10, respectively. However, in the second wave, peak levels of host origin DCs were reached at least 2 wk before the expected peak levels of thymocytes. As the precursors for the second wave originated in the regenerating host BM (26), these results suggest that the bulk of the early appearing thymic DCs was nonprothymocytically derived.

Differential generation of thymic DCs by prothymocyte-rich and prothymocyte-poor aliquots of blood

We have demonstrated in other experiments (E. Donskoy and I. Goldschneider, manuscript in preparation) that, under steady state conditions, the export of thymocyte progenitors from the BM is coordinated with the periodic opening of the i.t. gate for prothymocytes. Hence, at most time points, peripheral blood contains few prothymocytes (28). We therefore determined whether naturally occurring aliquots of prothymocyte-poor as well as prothymocyte-rich blood could generate thymic DCs in adoptive recipients. Toward this end, total leukocytes in 0.5 ml of heart blood from each of 60 normal Ly-5.1 mice, ages 7–12 wk, were injected i.t. into sublethally irradiated 5-wk-old Ly-5.2 recipients (one donor per recipient). The generation of donor origin thymocytes and thymic DCs in each recipient was determined by FACS analysis 24 days later to permit detection of both prothymocytically and nonprothymocytically derived DCs.

The results in Table I show that, although only 17 (28%) of the 60 recipients of normal PBCs developed significant thymocyte chimerism (>5% donor origin cells), all displayed substantial thymic DC chimerism. However, the mean level of DC chimerism was more than twice as great (59 vs 23%; p < 0.01) in the group of mice that exhibited thymocyte chimerism (designated THY+) than in that which did not (designated THY-); and the total number of donor origin DCs was ~3 times as great (34 x 103 vs 12 x 103; p < 0.01). Extrapolations of prior log-dose titrations of i.t. injected BM cells (26, 28) indicated that the THY+ mice must have received at least 10-fold more prothymocytes on average than did the THY- mice to achieve the observed bimodal distribution of thymocyte chimerism (mean 55 vs 2% donor origin cells; p < 0.01). This is consistent with the observation that the number of host origin thymocytes was significantly lower in the THY+ than in the THY- group of mice (Table I), due to competition with donor origin prothymocytes for a finite number of i.t. niches (26). As the number of host origin DC also was lower in the THY+ than the THY- group, their precursors also appeared to compete for i.t. niches. Hence, it seems likely that the donor origin thymic DCs in the THY+ group arose predominantly from prothymocytic precursors, whereas those in the THY- group arose almost exclusively from nonprothymocytic precursors.


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  		Table I. Induction of thymocyte and thymic DC chimerism by PBCsa

 
This inference was further supported by the differential staining for the CD8{alpha} chain among the thymic DCs in the two groups of mice. As shown in Table II, 81% of the donor origin DCs in the THY+ group were CD8+, as compared with only 54% of those in the THY- group (p < 0.01). Furthermore, the mean fluorescence intensity of the CD8+ DCs in the THY+ group exceeded that in the THY- group by 2-fold (p < 0.01; data not shown). Hence, assuming that the mean numbers of CD8+ nonprothymocytically derived DCs per thymus were equivalent in both the THY+ and THY- groups of mice (~8 x 103), it can be calculated that virtually all of the prothymocytically derived DCs in the THY+ group were CD8+ and that most were CD8{alpha}high.


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  		Table II. Staining for CD8{alpha} on donor and host thymic DCs in chimeras generated by PBCs

 
Reciprocal results were obtained with host origin DCs. Thus, as shown in Table II, the proportions, total numbers, and mean fluorescence intensities (data not shown) of the CD8+ host origin DCs in the THY- group of mice were equivalent to those of their donor origin counterparts in the THY+ group of mice, and the mean fluorescence intensity of the CD8+ host origin DCs in the THY- group was twice as great as that in the THY+ group (p < 0.01). However, the numbers of CD8- donor and host origin DCs remained constant in the THY- and THY+ groups, and the number of CD8- host origin DCs in the THY+ group matched that of their CD8+ counterparts (p > 0.1). These results suggest that, unlike prothymocytes, the precursors of the nonprothymocytically derived population of thymic DCs (CD8{alpha}-/low) do not compete with each other (or prothymocytes) for i.t. niches.

To estimate the proportionate representation of prothymocytically and nonprothymocytically derived DCs in the steady state thymus, we determined the ratio of CD8{alpha}+ to CD8{alpha}- thymic DCs in a cohort of normal 7- to 12-wk-old mice. A mean of 74 ± 16% of the thymic DCs was found to be CD8+, and of these approximately two-thirds were CD8{alpha}high. Hence, assuming that 100% of prothymocytically derived (CD8{alpha}high) and 50% of the nonprothymocytically derived DCs (CD8{alpha}-/low) are CD8+, it can be calculated that both DC populations are equally represented in the young adult mouse thymus.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although a series of recent in vivo studies have indicated that more than one lineage of DCs may populate the mouse thymus under conditions in which thymocytopoiesis, myelopoiesis, and/or the thymic architecture are abnormal (11, 12, 13, 15, 16, 17, 18, 19), it was not possible to determine whether the observed results were applicable to the normal host. Using normal mice, Kamath et al. (23) recently observed two-phased 5-bromo-2'-deoxyuridine-labeling kinetics consistent with, but not conclusive for, the existence of two types of thymic DCs in normal adult mice. In this study, we document the differential importation of two developmentally discrete populations of DC precursors by the thymus of parabiotic mice. One population is indistinguishable from prothymocytes by the following criteria: 1) parallel kinetics of production in regenerating BM; 2) inefficient (<20%) exchange between parabiotic partners; 3) gated and simultaneous importation by the thymus; 4) competitive antagonism for a finite number of i.t. binding sites; 5) linked generation of thymocytes and thymic DCs; 6) absence from blood samples lacking detectable prothymocytic activity; and 7) parallel onset of age-dependent i.t. involution. Although it theoretically is possible that separate precursors for thymocytes and thymic DCs are simultaneously imported and processed by the thymus, this is highly unlikely in view of the evidence for a common i.t. T/DC precursor of prothymocytic origin (7, 8, 9, 10). Rather, the most parsimonious explanation for the striking similarities between prothymocytes and the hematogenous progenitors of a major population of thymic DCs is that they are identical. Furthermore, the slightly shorter lag period observed for the generation of thymic DCs as compared with thymocytes is consistent with the proposed origin of thymic lymphoid DCs from stage I and stage II triple-negative (CD3-, CD4-, CD8-) thymocytes (8).

The second major population of thymic DC precursors can be readily distinguished from prothymocytes by the following criteria: 1) efficient (random) exchange between parabiotic partners; 2) ungated importation by the thymus; 3) absence of competitive antagonism; 4) unlinked generation of thymocytes and thymic DCs; and 5) presence in blood samples lacking detectable prothymocytic activity. Their efficient exchange in the blood of parabionts, their rapid accumulation in the thymus within 1 wk after the establishment of vascular anastomosis (~2 wk after parabiotic union), and their relatively short i.t. t1/2 suggest that these nonprothymocytic precursors are imported as at least partially differentiated DCs. Furthermore, the absence of gating or competition for i.t. niches suggests that they enter the thymus continuously and in numbers reflective of the size of the circulating pool. The differences in the kinetics of importation/accumulation/generation/persistence of these two populations of thymic DCs are schematically illustrated in Fig. 5.



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  		FIGURE 5. Schematic representation of the differential importation of two developmentally distinct populations of DCs and/or their precursors by the thymus. A, Gated importation of the precursors of a prothymocytically derived, i.t. generated population of thymic DCs. As documented previously (25 26 ), blood-borne prothymocytes are imported by the thymus in a gated fashion (periodicity ~4 wk; duration ~1 wk), and, after a lag of 10–14 days, generate discrete waves of thymocytes (total elapsed time ~8 wk). The present results indicate that these imported prothymocytes also generate linked waves of CD8{alpha}high thymic DCs, presumably by the formation of common T/DC precursors. The lag period for these i.t. generated DCs is ~3 days shorter than that for thymocytes. The clusters of vertical black arrows and the horizontal black bars represent receptive periods (open gate) and refractory periods (closed gate), respectively, for the importation of prothymocytes. Black curves comprised of differing symbols represent sequentially generated waves of thymocytes (x10-6); and the associated gray curves represent sequentially generated waves of thymic DCs (x10-3). The duration of each set of linked waves exceeds the periodicity of gate opening by 2-fold, so as to maintain relatively constant levels and ratios of total thymocytes and i.t. generated DCs. The idealized time scale approximates, but is not necessarily identical with, chronological age. B, Ungated importation of the precursors of a nonprothymocytically derived, extrathymically generated population of thymic DCs. Closely spaced and progressively shaded vertical arrows designate continuous importation by the thymus of at least partially differentiated blood-borne DC precursors. Progressively shaded curves represent arbitrary semiweekly accumulations of these DCs (population t1/2 <4 wk), ~50% of which are CD8{alpha}- and 50% CD8{alpha}low. Some, but not necessarily all, are of myeloid origin. The number of nonprothymocytically derived DCs per thymus (B) is estimated to approximate that of prothymocytically derived DCs (A).

 
Two other laboratories have reported the rapid establishment of thymic chimerism from hematogenous precursors. Beschorner et al. (33) observed the accumulation of donor origin DCs in the corticomedullary regions of the thymi of cyclosporine-treated rats after the i.v. infusion of lymphohemopoietic cells. Inasmuch as: 1) splenocytes were more efficient than BM cells at reconstituting thymic DCs in these rats; 2) DC recruitment was more efficient when the spleen cells were transferred during than after cyclosporine treatment; and 3) skin grafts could also act as a source of thymic DCs, it is likely that most of the donor origin DCs were of nonprothymocytic origin. Kampinga et al. (34) also found that host origin DCs appeared in the medullary regions of rat vascular thymus grafts within 7 days of transplantation. However, as host origin thymocytes also began to appear in the cortical regions at 10 days, it is likely that many of these DCs were of prothymocytic origin.

Study of CD8{alpha} staining showed additional differences between the prothymocytic and nonprothymocytic populations of thymic DCs. All of the prothymocytically derived, but only one-half of the nonprothymocytically derived thymic DCs in recipients of PBCs stained for CD8, the former being mostly CD8{alpha}high and the latter mostly CD8{alpha}low. Hence, assuming that these phenotypic differences also apply to thymic DCs in the normal host, it can be calculated that each subset of DCs is equally represented in the steady state thymus. It is not surprising that the prothymocytic population of thymic DCs uniformly displayed high levels of CD8 (presumably the {alpha}-chain homodimer), as originally demonstrated by transfer of thymocyte precursors (8). However, the variable staining for CD8 by members of the nonprothymocytic population leaves open the possibility that they originate from a heterogeneous pool of extrathymic precursors, although differences in maturity or state of activation within a single lineage are alternative explanations. It is also possible that the CD8{alpha}low subset has passively acquired the CD8 {alpha}{beta} heterodimer (35), and is actually CD8{alpha}{alpha}-. At a minimum, it is likely that most of the CD8- thymic DCs are of myeloid origin, inasmuch as CD8- DCs predominate in the thymi of c-kit- {gamma}- mice (12). However, some could, at least theoretically, be generated by extrathymic lymphoid precursors (for examples, see Refs. 10 , 16 , 35 , and 36). It will be of interest therefore to determine whether either the CD8+ or CD8- subset of nonprothymocytically derived thymic DCs is analogous to the plasmacytoid population of DCs in human thymus (21), especially as similar cells have recently been identified in murine lymph nodes and spleen (37). Although our preliminary data indicate that ~10% of CD11b- CD11c+ thymic DCs have a phenotype consistent with that of plasmacytoid DCs (e.g., B220+, CD205-), we have not yet defined their developmental origins or lineage associations.

In addition to the two populations of CD11b- thymic DCs described in this work, it is possible that yet another subset of DCs exists among the population of CD11b+ murine thymic cells, as reported for human thymic DCs (21). Like Vremec et al. (29), we found that the vast majority of the CD11b+ CD11c+ cells resembled macrophages (unpublished observations). Nonetheless, because most CD11b+ cells in the mouse thymus are autofluorescent, it is difficult to determine their precise antigenic phenotypes.

The presence of at least two developmentally discrete populations of DCs in the thymus implies the existence of functional heterogeneity as well. One major function ascribed to i.t. generated lymphoid DCs involves negative selection of potentially autoreactive clones of developing thymocytes (3). The present results suggest that this function is optimized by the gated entry of a common T/DC precursor, which generates parallel waves of thymocytes and lymphoid DCs (see Fig. 5). Not only does this mechanism help to maintain the optimal thymocyte:DC ratio, but it presumably ensures that DCs of the appropriate lineage and stage of development/differentiation/activation are available to interact at the appropriate time and place with developing thymocytes. Such a mechanism theoretically could function under conditions of thymic growth, involution, or regeneration, as well as under steady state conditions. Furthermore, as proposed by Ardavin et al. (7), the synchronization of DC and thymocyte production may provide a mechanism whereby each wave of newly generated thymocytes is selected against a unique panel of i.t. expressed self peptides, thereby periodically updating the TCR repertoire.

The function(s) of the nonprothymocytic population of thymic DCs is more problematic, due not only to its possible heterogeneous origins, but also to possible functional differences between the CD8{alpha}+ and CD8{alpha}- subsets (38, 39, 40, 41, 42). However, several experimental systems suggest that at least some extrathymically generated thymic DCs can efficiently induce the formation, activation, and/or export of immunoregulatory thymocytes. For example: 1) i.t. injected donor MHC II-positive cells (presumably DCs) are responsible for the induction of specific unresponsiveness to cardiac allografts (43); 2) APC-depleted pancreatic islet allografts fail to induce unresponsiveness to donor strain islets when inoculated i.t. (44); and 3) Ag-pulsed thymic DCs protect against the induction of experimental autoimmune encephalomyelitis when transfused into euthymic, but not thymectomized, mice (45). The later study is particularly intriguing, as it suggests that thymic DCs can transfer Ag-specific dominant tolerance to a naive recipient, but only after returning to the thymus.

We have recently observed that i.v. infusion of F4/80+ CD1+ mononuclear cells from the blood of mice injected with Ag in the anterior chamber of the eye rapidly induces the appearance and release of immunoregulatory NKT cells from the thymus of naive recipients (4, 5). Importantly, some of these presumptive APCs, which acquire regulatory properties in the immunosuppressive (immunologically privileged) environment of the eye (46), accumulate as CD11c+ DCs in the thymus of the adoptive recipients (5) (our unpublished observations). It is possible that, under steady state conditions, these regulatory DCs are represented by the minor population of CD8{alpha}-/low F4/80low thymic DCs (29) or by B220+ plasmacytoid DCs, which have been reported to have tolerogenic potential (1, 47). Chiffoleau et al. (6) have provided yet another example of the ability of presumptive DCs to migrate to the thymus under tolerogenic conditions. In their experiments, donor origin MHCII+ APCs from a cardiac allograft were identified in the thymus of rats treated with the deoxyspergualine analog, LF15-0195, and were associated with the expansion of Ag-specific CD4+ CD25+ immunoregulatory thymocytes (48).

In summary, the present study supports the existence of at least two developmentally discrete populations of DCs in the steady state thymus. One population (~50%) appears to be generated i.t. from prothymocytic precursors, and the second extrathymically from nonprothymocytic precursors. When combined with evidence from the literature and our ongoing studies, these results support a novel paradigm whereby thymus-seeking DCs of nonprothymocytic origin supplement and reinforce the functions of i.t. generated DCs of prothymocytic origin by broadening the repertoire of extrathymic self Ags available for negative selection, and by inducing the i.t. formation, positive selection, activation, and/or export of regulatory T cells on demand.


   Acknowledgments
 
We thank Frances Tausche and Deborah Foss for expert technical assistance, and Cathy Mitchell for excellent secretarial assistance.


   Footnotes
 
1 This study was supported in part by National Institutes of Health Grants AI33741 and AI49882. Back

2 Address correspondence and reprint requests to Dr. Irving Goldschneider, Department of Pathology, School of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3105. E-mail address: igoldsch{at}neuron.uchc.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; BM, bone marrow; FCM, flow immunocytometric; i.t., intrathymic; PBC, peripheral blood cell. Back

Received for publication November 14, 2002. Accepted for publication January 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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