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* Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; and
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Developmental relationships between multipotent hemopoietic stem cells (HSC)4 in the marrow and T-lineage committed cells in the thymus have long been investigated (7, 8, 9). HSC are known to be present in the circulation and could enter the thymus (10, 11). HSC start producing thymocytes 3 wk after either i.v. or intrathymic transplant (12, 13); however, cells with stem cell properties have not been found in the thymus (8, 11, 14, 15). This observation and the fact that other marrow fractions can more rapidly seed the thymus under experimental conditions suggest that HSC are not directly responsible for maintaining thymocytopoiesis (12, 16). Our focus was on testing marrow cells that rapidly generate large numbers of thymocytes, and on assessing the loss of other differentiation options.
Of the many categories of lymphoid progenitors that have been identified in bone marrow, the most robust thymus repopulating ones have been found among a primitive Lin–Sca1+c-Kithigh (LSK) multipotent progenitor fraction. Early lymphoid progenitors (ELP) were originally defined as a hormone-sensitive, Flt3/Flk-2+CD27+subset of the Lin–Sca1+c-Kithigh compartment (17). They represent the most primitive cells to express lymphoid-restricted genes such as EBF, TdT, RAG-1, or RAG-2 (18). Although ELP are heterogeneous with respect to the combined expression of these genes, RAG-1 represents a useful parameter for sorting viable cells from RAG-1/GFP mice (18). Although not firmly lymphoid committed, they are lymphoid specified and have greatly reduced nonlymphoid differentiation potential when compared with otherwise similar RAG-1– cells. In addition, ELP require much longer intervals to generate CD19+ lymphocytes in culture than Lin–c-Kitlow progenitors (18). Of particular importance, RAG-1+ ELP were more effective and persistent T cell precursors than were c-KitlowRAG-1+ prolymphocytes (18).
Consistent with these findings, Adolfsson et al. (19) found effective lymphoid progenitors among LSK with the highest density of Flt3/Flk-2+. When compared with HSC, this fraction had greatly reduced erythroid differentiation potential. Lai et al. (20) found that down-regulation of VCAM-1 on LSK coincided with progression of HSC through the multipotent progenitor stage to become ELP. Cloning and transplantation assays revealed that this transition was accompanied by substantial loss of nonlymphoid differentiation potential.
Long and short term repopulating stem cells in a particular congenic strain of mice express the CD90.1/Thy1.1 marker (12, 21, 22, 23). Comparison of progressively smaller bone marrow subsets showed that thymic repopulating capacity was enriched in the Sca1+c-Kit+Thy1.1– bone marrow compartment (12). Testing with a panel of Abs revealed that the L-selectin adhesion molecule further defined primitive subsets with lymphoid potential. Intravenous transfer of marrow subsets into sublethally irradiated mice showed that Lin–Sca1+c-KithighThy1.1–L-selectin+ (L-selectin+ progenitors; LSP) are effective thymus-repopulating cells. LSP defined in this way represent 
17% of the LSK fraction (Ref. 16) and our unpublished observations). Moreover, they are distinctly better T than B cell progenitors (16).
Common lymphoid progenitors (CLP) were originally isolated as Lin–Sca1+c-KitlowThy1.1–IL-7R
+ cells with potential for T, B, and NK cells (23). However, primitive cells are present in the thymus of Ikaros knockout mice that have no CLP, and several studies have shown that primitive thymocytes express little IL-7R (14, 24). CLP removed from bone marrow and transferred intrathymically matured faster than cells present in the thymus (23, 24).
Benz and Bleul (25, 26) recently used a chemokine receptor reporter strain to isolate bone marrow cells with T lineage potential. Although some GFP-marked marrow cells were among the LSK subset, most of them were c-Kitlow and expressed IL-7R; i.e., they had properties overlapping with those of CLP.
Von Boehmer and colleagues (27, 28) identified more differentiated c-Kit–CD45R/B220+CD19–IL-7R+ progenitors in pre-T reporter transgenics. Although not directly compared with LSK subsets, these CLP2 cells homed to the thymus on transplantation and had B lineage potential.
Clues to the nature of thymus-replenishing cells have also been sought by characterizing primitive progenitors resident within this organ. The most primitive thymocytes lack markers associated with all hemopoietic lineages (Lin–), including CD4 and CD8 (double negative; DN) (29, 30, 31, 32). Furthermore, a consensus has developed that these early thymic progenitors (ETP) express high levels of c-Kit (9, 24, 33, 34). The primitive c-Kithigh ETP are uniformly L-selectin+ (16, 35), and regenerating thymocytes in irradiated mice are initially Thy1– (13). As noted here, ETP express little IL-7R, but all are Sca1+, and ones that retain some B potential are Flt3/Flk-2+ (3, 14, 15, 24, 33, 36). Thymocytes with high levels of CCR9 reporter activity are not T lineage restricted and also produce B, dendritic, and myeloid cells (25, 26). The CD44+CD25– DN1 category can also be divided into five subsets on the basis of CD117 (c-Kit), and CD24 (HSA) expression (14, 24, 33). Two c-Kithigh subsets, designated DN1a and DN1b, were potent T progenitors, but had little if any B lineage potential (14). Therefore, thymus-colonizing cells would likely be Lin–CD24–/low CD25–Thy1–Sca1+c-KithighFlt3/Flk-2+CD44+ L-selectin+ unless they quickly change their characteristics on entry to the thymus. Once arriving, effective T precursors must be capable of rapid and robust expansion to accommodate the rate of lymphocyte production intrinsic to that organ (1, 13, 37, 38).
We have now crossed C57BL6 (B6)-RAG-1/GFP mice to B6-Thy1.1 animals so that ELP and LSP, as well as CLP, could be isolated and compared. An experimental model was used where transplanted cells home and compete with host progenitors for limited niches in thymuses of sublethally irradiated mice. Although a range of marrow cells have some ability to produce T cells, our findings support reports that most thymic engraftment potential is concentrated among the LSK fraction of bone marrow. Although progenitors previously designated LSP are T vs B lineage biased, they include RAG-1+ ELP that are effective at restoring both cell types. However, none of the primitive ETP category of thymocytes expresses RAG-1, and ELP may replenish the thymus only under circumstances of special need.
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Materials and Methods |
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B6 (CD45.2 alloantigen), B6-Thy1.1, BALB/c, and B6-SJL (CD45.1 alloantigen) mice (The Jackson Laboratory) were bred and maintained in the Laboratory Animal Resource Center of the Oklahoma Medical Research Foundation (Oklahoma City, OK). B6-Thy1.1 were crossed with B6-RAG-1/GFP knockin (39) mice to produce animals expressing Thy1.1, RAG-1/GFP, and the CD45.2 alloantigen.
Isolation of cell populations and flow cytometry
Cell manipulations were performed in HBSS with 5% FCS. Marrow cells were isolated from the long bones of donor mice, and erythrocytes were lysed by briefly resuspending in NH4Cl– hypotonic solution. Progenitors were further enriched by labeling marrow with RB6-8C5 (Ly6G+C), M1/70 (CD11b), TER-119, RM2-5 (CD2), 17A2 (CD3), 53-7.3 (CD5), 53-6.7 (CD8), 1D3 (CD19), RA3-6B2 (B220), and then immunomagnetically depleting cells (Dynatech Laboratories). Because of the possibility of early or passive expression of CD4, this was not used as a lineage depletion parameter. Lineage-negative cells were stained with IL-7R PE (PE; SB/14), Thy1.1 biotin (Bio; OX-7; secondary streptavidin PE-Texas Red; Caltag Laboratories), Sca1 PE-Cy5 (D7; eBioscience), c-Kit allophycocyanin (2B8), and L-selectin allophycocyanin-Cy7 (MEL-14; eBioscience), then separated using a FACSAria flow cytometer (BD Biosciences) into specific populations. Dead cells were excluded by propidium iodide staining (Molecular Probes). Cells harvested were stained with Abs to determine the phenotypes of engrafted cells. Abs included 145-2C11 PE (CD3), GK1.5 PE (CD4), 53-6.7 allophycocyanin, HL3 PE (CD11c), 1D3 allophycocyanin, 1A8 PE (Ly-6G), PK136 allophycocyanin (NK1.1), RA3-6B2 allophycocyanin, AL-21 Bio (Ly6C), and 104 FITC (CD45.2). Analysis was done on a FACSCalibur (BD Biosciences). For TdT expression, progenitor populations were fixed with 5% acetic acid in ethanol and then incubated with rabbit anti-TdT IgG (Supertechs). Cells were illuminated with PE-conjugated goat anti-rabbit IgG (Caltag Laboratories). All Abs came from BD Pharmingen, unless otherwise stated. Purification of each lymphocyte subset was done according to published parameters (16, 18, 23) and was confirmed in each experiment by postsorting analyses (see Fig. 1).
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Recipient mice received 6.5 Gy radiation from a 137Cs source (Mark I gamma irradiator; J. L. Shepard and Associates). Mice were anesthetized with isofluorane (Isosol, Vedco), and sorted populations were infused i.v. Host mice were 6- to 12-wk-old B6-CD45.1; donor cells were from 4- to 10-wk-old B6-RAG-1/GFP-Thy1.1 mice expressing the CD45.2 alloantigen. Thymic lobes, spleen, and marrow from one femur were collected separately from recipient mice, and donor-derived cells were assayed by flow cytometry. Limiting dilution analyses of thymic progenitor frequencies were determined by transplanting graded numbers of progenitors to sublethally irradiated mice. At least eight thymic lobes were analyzed at each dose and percentages of thymic lobes containing greater than 105 donor-derived thymocytes at day 17 were determined. Data were pooled from two independent sorting experiments and used to construct lines of best fit (40). Correlation coefficients (r) exceeded 0.9 for each plot.
Peripheral blood and DN thymus preparations
Leukocytes were collected from peripheral blood as previously described (41). Lineage-committed cells were identified through incubation with biotin-conjugated lineage Abs, and cell markers were stained as outlined for marrow. A similar strategy was used to label primitive thymocytes, except that CD2 and CD5 were removed from the lineage mixture, and TCR-
(H57-597) and TCR-
/
(GL3) were added. DN subsets were identified using CD44 (IM7) and CD25 (PC61).
Fetal thymic organ culture
Fetal thymic organ culture was done as described (42). BALB/c fetuses were harvested at E15, and thymic lobes were cultured in RPMI 1640, 10% FCS with 0.36 mg/ml 2'-deoxyguanosine for 7 days. Lobes were then transferred to V-bottom culture plates, one lobe per well with 100 progenitors and 200 µl of culture medium. On day 7, medium was replaced with X-VIVO15 (BioWhittaker, Cambrex) supplemented with 1% BSA (StemCell Technologies), 100 ng/ml stem cell factor, and 1 ng/ml IL-7 (R&D Systems). Plates were sealed in gas sample bags (MiDan) with 70% O2, 25% N2, and 5% CO2 gas mixture and cultured at 37°C with medium changed every 5 days. After 14 days, lobes were disrupted for cell suspensions, stained with Abs for surface markers, and analyzed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Several subsets of bone marrow contain T cell precursors, but it remains unclear which is likely to maintain thymocytopoiesis (16, 18, 23, 28). We constructed a model for comparison of CLP (Lin–Sca1lowc-KitlowThy1.1–IL7R+), ELP (Lin–Sca1+c-KithighRAG-1/GFP+), and LSP (Lin–Sca1+c-KithighThy1.1–L-selectin+). C57BL6 (B6)-PL mice (Thy1.1 allele of CD90) were crossed with B6-RAG-1GFP/GFP mice so that markers would be available to isolate all progenitor populations in parallel (Fig. 1). One thousand sorted progenitors were transplanted i.v. into sublethally irradiated (6.5 Gy) CD45 congenic recipients and evaluated for their reconstitution of lineages. Particular attention was paid to the kinetics of thymic colonization, degree of restriction to the T lineage, and numbers of thymocytes produced.
Fig. 2A shows representative CD4 vs CD8 profiles of donor thymocyte recovery over time. All progenitors contributed to thymocyte recovery in advance of the 21 days required by hemopoietic stem cells (12). However, unseparated bone marrow and ELP yielded thymocytes earlier than the other populations.
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These findings indicated that all three subsets were relatively restricted to lymphoid lineages but differed in the kinetics of thymic recovery. LSP were effective sources of thymocytes, whereas ELP were enriched for T, B, and NK progenitors. CLP peaked early for B lineage differentiation in the marrow and were effective NK producers.
ELP and LSP, but not CLP, produce robust thymic engraftment
The thymus contains primitive cells that repopulate the organ within 9 days of i.v. transplantation (1, 12). A marrow progenitor might have similar kinetics but be capable of more sustained expansion. Therefore, our analysis focused on the speed and durability of T lineage regeneration from marrow subsets (Fig. 3). The kinetics of engraftment was similar in LSP- and ELP-grafted animals, whereas CLP had limited expansion potential (Fig. 3A, left). An early peak was observed in animals transplanted with whole bone marrow, followed by robust replenishment (Fig. 3A, right). This pattern was matched in mice with ELP, whereas LSP-transplanted mice had no early wave, but expansion proceeded from day 15. T lineage cells were generated in CLP-grafted mice from day 16 (data not shown). To determine whether the marrow contained rare progenitors comparable with primitive thymocytes, large numbers (107) of unfractionated cells were transplanted, but no repopulation was observed before 13 days (Fig. 3A, right).
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70%), nearly 2-fold and 7-fold greater than with LSP or CLP, respectively (Fig. 3B). However, LSP engraftment increased with time until the percentages of positive lobes for LSP and ELP recipients were identical. CLP engraftment increased with time but was consistently less than LSP or ELP.
Inspection of these curves suggested that the three subsets contained different frequencies of engraftable thymic progenitors (Fig. 3B), a point addressed with limiting dilution experiments. We calculate that there was at least one progenitor capable of producing detectable engraftment within 17 days of transplant per 1000 cells in the ELP fraction, as compared with one per 2000 LSP (Fig. 3C). Low frequencies of engraftment with CLP precluded a similar analysis.
These observations demonstrate that lymphoid progenitors generate an early wave of T lymphopoiesis, followed by a second, more robust engraftment. Whereas the ELP fraction was more enriched with respect to thymus-repopulating cells than LSP, both could restore thymic lobes to normal size within 3 weeks posttransplant. The CLP fraction contributed to T lymphopoiesis but with less efficiency than the other subsets.
The LSP subset contains a majority of the RAG-1+ ELP
As shown above, LSP and ELP have similar thymocyte potential after engraftment. We conducted six-color flow cytometry to determine the correlation between these populations (Fig. 4). This revealed that the majority of RAG-1+ ELP (Fig. 4A, left) reside in the Thy1.1– subset (>80%, left of gray line). Furthermore, evaluation of Thy1.1 and L-selectin expression on ELPs showed that 60% are within the previously designated LSP category (Fig. 4A, right). Gating LSPs for RAG-1/GFP expression showed that only 8% of LSP express RAG-1 (Fig. 4A, right). Therefore, many RAG-1+ ELP overlap with but still represent a small fraction of LSP.
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500 RAG-1+). Thus, rare RAG-1-expressing cells in the LSP category could account for a very small early wave of T lymphopoiesis. These observations raised the question of what T lineage potential in the LSP fraction corresponds to RAG-1– progenitors. Therefore, we sorted and transplanted several doses of RAG-1/GFP-negative LSP (RAG-1– LSP) and evaluated thymocyte production (Fig. 4C). By day 17, 2,000 RAG-1– LSP produced thymic engraftment comparable with that of 1000 whole LSP; by day 27, 800 RAG-1– LSP were comparable in thymocyte potential with unseparated LSP and ELP. These results suggest that the early thymic potential of LSP may be a function of the RAG-1+ cells. Even so, the LSP subset includes engrafting thymic progenitors apart from the RAG-1+ fraction. We also found that RAG-1– LSP are more T lineage restricted than the LSP population as a whole (data not shown).
Cells with ELP and LSP characteristics in blood and thymus
Marrow cells that colonize the thymus must traverse the blood stream. To determine whether LSP or ELP were present in the circulation, we analyzed peripheral blood leukocytes for expression of surface Ags. Analysis for L-selectin and RAG-1/GFP in Lin–Thy1.1– peripheral blood showed a fraction of ELP and LSP in the circulation (Fig. 5A). The ratio of LSP to ELP was reduced relative to the marrow (3:1 in blood vs 12:1 in marrow). In contrast to a previous report (13), we found CLP in the circulation (Fig. 5A, far right panel) and 85% of them were RAG-1/GFP+ (data not shown).
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The DN1 fraction of the thymus includes cells with LSP characteristics (16). We next asked whether RAG-1+ ELP overlapped with these progenitors. Fig. 5D shows RAG-1/GFP expression in the DN subsets. All c-Kithigh DN1 thymocytes were RAG-1/GFP–, and only this fraction generated thymocytes in fetal thymic organ culture (Fig. 5E). Therefore, cells phenotypically similar to ELP and LSP were found in the circulation, but L-selectin+TdT+ primitive thymocytes did not include RAG-1+ cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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+ CLP were much less effective T lineage precursors when assessed by i.v. transplantation. Although a wealth of information has been obtained by many laboratories, the nature of marrow cells that replenish the adult thymus under normal circumstances remains unclear (3). This could be an infrequent event, and it almost certainly involves very few cells. The issue is further complicated by the fact that many marrow fractions have T lineage potential under experimental conditions. Rigorous comparisons of previously defined progenitors could be helpful, especially when information is available about their relative potency, abundance in the bone marrow, and presence in the circulation.
Spangrude and colleagues (16) and Wu et al. (35) described candidate T lineage progenitors among the Thy1.1– subset of the LSK fraction of bone marrow that expressed the CD62L/L-selectin adhesion molecule. These LSP comprise 17% of the LSK fraction, and we have confirmed that they are better T progenitors than B progenitors in transplanted mice. We estimate that LSP are 12-fold more numerous than RAG-1+ ELP and 4 times more abundant than CLP. As in a previous study (16), we found that LSP contributed to T lymphopoiesis within 13 days, 9 days faster than reported for stem cells (12). Thus, the abundance of LSP relative to ELP and the increased frequency of thymic seeding progenitors indicate that LSP represent a rich source of thymus-replenishing cells.
Early lymphoid progenitors were originally identified as a hormone-sensitive Flt3/Flk-2+CD27+ subset of LSK, and some of them can be sorted in viable form from RAG-1/GFP mice (17, 18). We have now determined that 60% of RAG-1+ ELP express L-selectin and lack Thy1.1; i.e., they would be sorted as LSP. However, RAG-1+ ELP comprise only 8% of the total LSP subset. Approximately 23% of LSP were TdT+, another characteristic used to distinguish ELP (17). Overlaps between LSP/ELP subsets are depicted in Fig. 6.
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2-fold more enriched in the RAG-1+ ELP subset than in the LSP fraction. Considering the 2-fold higher percentages of successfully engrafted lobes on day 17 (Fig. 3C), RAG-1+ ELP may have been superior with respect to thymic homing. Elegant single cell studies performed by Kondo et al. (23) revealed that CLP have high T potential when injected into the thymus. This was confirmed in another study where CD93/AA4.1 was used as a gating parameter for CLP (24). CD93+ CLP produced lymphocytes as early as Flt3/Flk-2+ LSK after intrathymic injection but lost activity more rapidly (24). Therefore, we were surprised that T lymphopoiesis was slow and gradual following i.v. transplantation of CLP (Figs. 2D and 3A). It may be that these cells are inefficient with respect to thymic homing and, as suggested by others (3, 23, 24, 44), would normally differentiate to B and NK lineages if left in the marrow.
Primitive hemopoietic cells express low levels of transcripts corresponding to multiple lineages (45, 46), and there is no evidence that lymphoid genes are activated in a synchronous manner. Whereas some transcription factors are essential for initiating the process, patterns of cell surface marker expression are variable and a precise sequence need not be followed to produce lymphocytes of a given type (7, 47, 48). Early lymphoid cells sorted according to selected markers are heterogeneous with respect to others, and we have documented many combinations of CD62L, TdT, RAG-1, and a human µ transgene in individual cells (17, 18). It remains unclear whether acquisition or loss of any marker corresponds to lymphoid lineage specification. Mice transplanted with RAG-1+ ELP or large numbers of LSP produced early (day 13) and later (day 20) peaks of thymocytes. Large numbers (up to 64 lobes) of recipient thymuses were evaluated, and the phenomenon was consistent. This could reflect heterogeneity in the populations and may also indicate that some of the cells colonize other hemopoietic organs in sublethally irradiated recipients before seeding the thymus (49).
The degree of lymphoid lineage restriction of progenitors has been controversial and may be assay dependent. For example, CLP produce no myeloid cells in clonal assays driven by recombinant cytokines but do so when placed in stromal cell cocultures (23, 43). This has also been our experience, and we observed small numbers of GR-1+ cells in marrow of CLP recipients soon after transplantation (<day 13; data not shown). It is possible that cells sorted in our laboratory were contaminated with myeloid progenitors, but we used all four of the sort criteria originally described by Kondo et al., and similar results were obtained when CLP were sorted twice. The time after engraftment (before or after 4 wk) and tissues examined (blood vs marrow) represent additional variables between studies. In any case, myeloid potential is lost in a progressive rather than abrupt manner, given that multipotent progenitors acquire LSP or ELP and then CLP characteristics (50, 51).
The marrow cells we studied were sources of B and NK cells, and our experiments do not reveal when T progenitors completely lose those options (Fig. 2). However, there was notable bias associated with the subsets. LSP were particularly effective for producing T cells, whereas recipients of RAG-1+ ELP made all lymphoid cell types. Our findings suggest that segregation of cell fates starts at an extremely early stage in bone marrow. There is some evidence for this in lymphoid progenitors present in the fetus (52, 53). As a possible indication of maturity, B lineage lymphocytes emerged in the marrow of CLP recipients faster (<12 days) than in mice transplanted with LSP or ELP (14 days). CLP2 were not included in our study, and it would be useful to assess their potency as T cell progenitors relative to the other subsets (27, 28).
As noted in the Introduction, Lin–Sca1+c-KithighCD62L+CD44high cells resident in the thymus are potent T progenitors and produce lymphocytes within 9 days of transplantation (14, 33, 54, 55). This pattern of surface marker expression corresponds to the characteristics of marrow LSP and ELP. Flt3/Flk-2 is expressed on 15% of ETP, whereas almost all ELP have this marker (Refs. 15 , 17 , and 18 and our unpublished observations). TdT uniformly marked ETP in the thymus, whereas 23% of LSP and 43% of ELP had this intracellular marker (Fig. 5C). Given their effectiveness for producing T cells after transplantation, it was surprising to find no RAG-1+ ELP in the thymus (Fig. 5, D and E). None of the RAG-1+ DN1 cells was c-Kithigh, and RAG-1+ DN1 cells did not produce T cells in fetal thymic organ culture. This suggests that RAG-1+ ELP may colonize the thymus only under conditions of stress, as would result from the sublethal irradiation treatment we used. However, RAG-1+ ELP were detected in the circulation of unconditioned mice, which suggests that they have access to the thymus. If RAG-1+ ELP colonize the organ under normal conditions, they must rapidly down-regulate RAG-1 or become c-KithighRAG-1+CD25+ DN2 thymocytes.
It has been previously demonstrated that stem cells and progenitors with LSK characteristics are in the circulation (56, 57). This includes cells that express a RAG-2 BAC transgene. We now report that LSP, RAG-1+ ELP, and CLP are detectable in the blood. Resolution of the CLP subset was done with the original Kondo gating parameters and may explain a small discrepancy with a previous study (57).
Our analysis was not designed to establish developmental relationships between HSC and lymphoid progenitors present in marrow, blood, and thymus. However, the kinetics of thymocyte production and patterns of marker expression would be consistent with the presence of LSP and ELP immediately downstream of HSC. Although it is difficult to define rare cells that infrequently replenish the thymus, we have characterized defined categories of marrow progenitors and compared them for T lineage potential. It seems probable that some specification for T lymphopoiesis begins in the marrow, but loss of other differentiation options is clearly a gradual process.
Since completion of these experiments, Hardy and colleagues (58) compared marrow progenitors separated according to different parameters. Although not as lymphoid restricted as RAG-1+ ELP, a CD24lowCD93+ subset of Lin–c-Kithigh bone marrow cells was superior to CLP and later fractions with respect to T lineage reconstitution.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants AI 33597 and AI 056129 (to X.-H.S.), AI 20069 and AI 58162 (to P.W.K.), and T32-AI 07633 (to S.S.P.). X.-H.S. holds the Eli Lilly Distinguished Chair in Biomedical Research, and P.W.K. holds the William H. and Rita Bell Chair in Biomedical Research. 
2 S.S.P. and R.S.W. contributed equally to this study.
3 Address correspondence to Dr. Paul W. Kincade, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, OK 73104. E-mail address: kincade{at}omrf.ouhsc.edu
4 Abbreviations used in this paper: HSC, hemopoietic stem cell; LSK, Lin–Sca1+c-Kithigh; ELP, early lymphoid progenitor (Lin–Sca1+c-KithighRAG-1/GFP+); LSP, L-selectin+ progenitor (Lin–Sca1+c-KithighThy1.1–L-selectin+); CLP, common lymphoid progenitor (Lin–Sca1lowc-KitlowThy1.1–IL-7R+); ETP, early thymic progenitor; DN, double negative.
Received for publication April 7, 2006. Accepted for publication June 10, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-controlled reporter gene. Nat. Immunol. 3: 489-496. [Medline] chains than fetal progenitors. J. Immunol. 175: 5848-5856. This article has been cited by other articles:
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R. S. Welner, R. Pelayo, K. P. Garrett, X. Chen, S. S. Perry, X.-H. Sun, B. L. Kee, and P. W. Kincade Interferon-producing killer dendritic cells (IKDCs) arise via a unique differentiation pathway from primitive c-kitHiCD62L+ lymphoid progenitors Blood, June 1, 2007; 109(11): 4825 - 4931. [Abstract] [Full Text] [PDF]
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 A. Y. Lai and M. Kondo Identification of a bone marrow precursor of the earliest thymocytes in adult mouse PNAS, April 10, 2007; 104(15): 6311 - 6316. [Abstract] [Full Text] [PDF]
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B. A. Schwarz, A. Sambandam, I. Maillard, B. C. Harman, P. E. Love, and A. Bhandoola Selective Thymus Settling Regulated by Cytokine and Chemokine Receptors J. Immunol., February 15, 2007; 178(4): 2008 - 2017. [Abstract] [Full Text] [PDF]
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) and LSP (
) are shown. Numbers in parentheses are estimates of the ELP included at each dose of LSP administered, as predicted from the overlap between these populations. A minimum of 10 thymic lobes was analyzed at each transplant dose. C, T progenitor activity associated with RAG-1– LSP. Percentages are given for thymic lobes with at least 105 donor thymocytes after transplant with LSP depleted of RAG-1+ cells (RAG-1– LSP). Transplantation results with unseparated LSP and ELP (open and gray bars, respectively) are given for reference. Data in C represent two independent experiments; the smaller number of thymic lobes studied could explain the different in frequency of ELP thymic engraftment seen in B and C.
