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* Memorial Sloan-Kettering Cancer Center, New York, NY 10021; and
University of Miami School of Medicine, Miami, FL 33101
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Abstract |
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Introduction |
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Once inside the thymus, each newly recruited progenitor undergoes 
20 relatively symmetric cell divisions (reviewed in Ref.3) to generate a large pool of immature thymocytes for TCR-mediated selection. Approximately 12 of these cell divisions occur over a 14-day period that spans the CD4/CD8 lineage double-negative (DN)3 stages of development (3). The remaining proliferation occurs over a 2- to 4-day span that includes the double-positive (DP) stages of differentiation, in particular, the early DP phase (pre-DP or DN4; see Refs.3, 4, 5). Again, the conditions within the thymus that regulate control of proliferation at the DN or DP stages remain unclear.
In this study, we measure the ability of i.v.-transplanted wild-type marrow to proliferate and differentiate in the thymus of different types of immunodeficient or wild-type hosts. We show that thymus size is controlled independently at the DN and DP stages by dramatically different mechanisms. Remarkably, the total cellularity of the thymus does not appear to have any impact on regulation of progenitor recruitment or expansion processes, or to feed back at all in the regulation of thymus size. Instead, expansion at the DN stage is limited by the availability of stromal niches in the thymus, and competition for them by DN cells. At the DP stage, proliferation appears to be intrinsically limited by the DP cells themselves, and does not appear to correlate with the availability of niches, or to any measure of thymic cellularity. Together with finite importation of new progenitors from the blood (2) and their asymmetric commitment to replenish the DN and DP pools (6), these functions cooperate to maintain continuous T cell output while limiting progenitor growth and thymus size.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6J (CD45.2) and congenic B6/Ly5.2/Cr (CD45.1) mice were purchased from the National Cancer Institute (Frederick, MD). IL-7R
–/– mice (7) (C57BL/6J background) were purchased from The Jackson Laboratory (Bar Harbor, ME), and subsequently maintained by homozygous breeding at Memorial Sloan-Kettering Cancer Center (MSKCC, New York, NY). RAG-2–/– mice (8) were originally purchased from Taconic (Germantown, NY), and subsequently bred at the Animal Care Facility of MSKCC. The mice were rested for at least 1 wk after receipt or weaning before transplantation. All mice were housed under pathogen-free conditions in accordance with the procedures outlined in National Institutes of Health Publication No. 86-23. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of MSKCC.
Cells
Bone marrow was recovered by flushing cells from iliac crests, femurs, and tibias, followed by filtration through mesh. RBC were lysed using 0.15 M NH4Cl/1 mM KHCO3/0.1 mM EDTA. White cells from peripheral blood were prepared using 2% Dextran T500 (Pharmacia Biotech, Piscataway, NJ) and hypotonic red cell lysis as previously described (9). Single-cell suspensions of thymocytes were prepared by gentle dissociation through wire mesh. Hypocellular thymus preparations were prepared using microfuge tubes and micropestles (Brinkmann Instruments, Westbury, NY).
Nonirradiation chimeras
Recipient mice received 2–20 x 106 viable nucleated donor bone marrow cells isolated from congenic B6/Ly5.2/Cr (CD45.1) mice. Administration was via tail vein injection in 100 µl of PBS. Recipient mice received no prior conditioning. The majority of recipient mice were sacrificed for evaluation of thymic and marrow chimerism 5–8 wk later. Other cohorts were sacrificed up to 5 months after transplant for analysis of long term reconstitution (data not shown).
Flow cytometric analyses
All cell suspensions were stained and analyzed at 4°C in mouse tonicity HBSS containing 5% FBS and 0.5% DNase (buffer). Samples of cells prepared as described above were washed with buffer and incubated with optimal concentrations of mAbs. Except as indicated, all Abs were prepared, purified, and conjugated on site at MSKCC. Conjugation of Abs to Alexa dyes was performed as recommended by the manufacturer (Molecular Probes, Eugene, OR). Blocking of nonspecific binding was performed with purified rat IgG and anti-FcR (clone 2.4G2). Bone marrow and blood were stained with Alexa680-conjugated anti-CD45.1 (clone 100-4), Alexa633-conjugated anti-CD45.2 (clone A-20), PE-conjugated anti-Gr1 (clone RB6-8C5; Caltag Laboratories, Burlingame, CA), and biotin-conjugated anti-B220 biotin (Caltag Laboratories) followed by streptavidin-conjugated FITC (BD Pharmingen, San Diego, CA). Thymocytes were stained with anti-CD45.1-A680, anti-CD45.2-A633, Alexa488-conjugated anti-CD4 (clone GK-1.5), and PE-conjugated anti-CD8 (clone 53-6.7; Caltag Laboratories) for assessment of DP and single-positive (SP) stages. DN populations from chimeric mice were identified by staining with lineage mixture (anti-CD4, -CD8, -CD3, -Gr1, -ID3, -Mac1, -Ter119) followed by Red613-conjugated anti-rat IgG (Caltag Laboratories), then by Alexa488-conjugated anti-CD44, PE-conjugated anti-CD25 (clone PC-61; BD Pharmingen), anti-CD45.1-Alexa680, and anti-CD45.2-Alexa633. DN cells from nonchimeric mice were prepared by depletion of lineage+ cells using paramagnetic beads as described in Ref.4 , followed by staining with Alexa488-conjugated anti-c-kit (clone ACK-2), PE-conjugated anti-CD24 (clone M1/69; BD Pharmingen), Alexa633-conjugated anti-CD44, and Alexa660-conjugated anti-CD25. 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes) was used at 0.1 µg/ml for dead cell exclusion, and at 10 µg/ml for cell cycle analysis, as described in Ref.10 . Fixation of stained cells for cell cycle analysis was performed as previously described (10). Doublets were eliminated by forward light scatter pulse processing. Samples were acquired on an LSR cytometer (BD Biosciences, San Jose, CA) with modifications as described (10). Postacquisition analysis was performed using FlowJo software (Tree Star, San Carlos, CA). Cell cycle statistics were generated using the Dean-Jett-Fox algorithm in the FlowJo cell cycle platform.
Generation of enhanced GFP (EGFP) transgenic mice
A vector containing EGFP under the control of the chicken 
-actin promoter and CMV enhancer (pCX-EGFP) (11) was provided by Dr. A. Nagy (Lumenfeld Research Institute, Toronto, Canada) with the kind permission of Drs. M. Okabe and J.-I. Miyazaki (Osaka University, Osaka, Japan). The promoter/enhancer/coding sequence was injected into C57BL/6 blastocysts and founder mice were generated by the Transgenic Core Facility of MSKCC. Three founders demonstrating germline transmission were used to establish stable transgenic lines by crossing with wild-type C57BL/6 mice (The Jackson Laboratory). All lines expressed EGFP in all tissues tested, consistent with similar transgenic strains made by others (11).
Establishment of triple-chimeric mice
CD45.1+ RAG-2-deficient mice (B6.SJL-PtprcaBoCrTac-Rag2tm1 N10) were purchased from Taconic. Bone marrow from these mice (2 x 107 nucleated white cells) was transplanted into standard (CD45.2+) nonirradiated IL-7R–/– mice. Recipient mice were allowed to recover for 3 wk, following which they received 5 x 106 bone marrow cells derived from EGFP transgenic mice. After an additional 4 wk, recipient mice were euthanized and their thymuses analyzed after staining with Abs against CD45.1 (Alexa680 conjugate, recognizing the RAG-2–/– donor) and CD45.2 (Alexa633 conjugate, recognizing the wild-type EGFP transgenic donor, as well as the IL-7R–/– recipient). Recipient and wild-type donor cells were discriminated on the basis of EGFP expression. Samples were also stained with anti-CD4-PE (BD Pharmingen) and anti-CD8-biotin (clone 53-6.7.2) followed by Red613-streptavidin (Caltag Laboratories).
Histology and immunofluorescent microscopy
Tissues were removed after euthanasia and embedded in OCT (Fisher Scientific, Pittsburgh, PA). Transverse cryosections of 5-µm thickness were prepared and fixed in ice-cold acetone. Light microscopy was performed after staining with Harris’ modified hematoxylin (Fisher Scientific). Fluorescence microscopy was performed essentially as described (6), using FITC-conjugated anti-pancytokeratin (clone C11; Sigma-Aldrich, St. Louis, MO) and Alexa594-conjugated anti-CD25. Samples were mounted in Prolong (Molecular Probes) containing DAPI (0.25 µg/ml), and photographed on a fluorescent microscope using mercury illumination. Monochrome blue (DAPI), green (FITC), and red (Alexa594) images were merged and colorized using IPLab software (Scanalytics, Fairfax, VA).
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Results |
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To assess whether overall thymic cellularity or differences in progenitor number could affect thymic progenitor homing and expansion, we measured thymic responsiveness to reconstitution by wild-type marrow in various types of nonirradiated immunodeficient mice. Administration of even small doses of bone marrow (5 x 106 unfractionated cells) revealed a range of different responses in such mice. For instance, thymuses from mice lacking the -chain of the IL-7R (7), which forms part of the heterodimeric receptors for IL-7 and thymic stromal lymphopoietin (12) were stably reconstituted to normal size by such treatment (Fig. 1). The wild-type donor thymocytes in reconstituted mice had normal proportions of all developmental stages (DN1, DN2, DN3, DP, SP, data not shown), suggesting that proliferative expansion was normal. In contrast, other lymphopenic strains, such as mice lacking MLR-2 (RAG-2) (8), developed trace levels of thymic chimerism proportional to that found in marrow stem cells, but did not reconstitute to normal size (Fig. 1). This was not a consequence of a stromal defect in RAG-deficient thymuses, because they can be reconstituted to normal size and structure in radiation chimeras (13).
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-chain (c) (17, 18, 19) deficiencies, both of which exhibit impaired responses to IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (reviewed in Ref.20), reconstituted in a manner similar to mice lacking IL-7R, while mice lacking the TCR -chain (21) were as refractory as RAG-2-deficient mice. Mice lacking DNA protein kinase (more commonly known as SCID) (22) displayed an intermediate level of reconstitution, as discussed later in the study. In the remainder of experiments shown in this study, IL-7R–/– and RAG-2–/– mice were used as models for responsive or refractory strains. Reconstitution seen in responsive (e.g., IL-7R) vs refractory (e.g., RAG-2) strains was thymus specific, because marrow chimerism was present at similar low levels in both types of mice (Fig. 1). This level of marrow chimerism is consistent with what is expected after transplantation into nonirradiated recipients, based on the findings of others (23). However, thymuses from refractory strains could not be reconstituted even after large doses of bone marrow (i.e., 2 x 107 cells; Fig. 3, and data not shown). Such doses are sufficient to generate detectable chimerism in nonirradiated recipients (23), thus indicating that thymic reconstitution is controlled by factors other than the availability of marrow-derived progenitors. It is important to note that these doses of marrow are sufficient to induce reconstitution of immunodeficient strains after radiation (for example, see Ref.13), indicating that the levels of thymic reconstitution are influenced mainly by the radiation-sensitive component, i.e., the lymphoid cells.
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-deficient thymuses from a responsive to a refractory phenotype
The thymus is composed of cells of hemopoietic origin (mostly immature T lymphocytes) as well as nonhemopoietic stroma (24). Mutations that lead to a deficiency in lymphoid cells, including RAG-2 and IL-7R, can also indirectly affect the thymic stroma (25). To determine whether the difference between responsive (e.g., IL-7R–/–) and refractory (e.g., RAG-2–/–) strains was primarily a reflection of the hemopoietic components of the thymus, or of other differences in the thymuses of these mice, several types of experiments were performed. In the first of these (Fig. 4), nonirradiated IL-7R-deficient mice were transplanted with RAG-2-deficient bone marrow 3 wk before transplantation with wild-type marrow. The resulting triple-chimeric mice were then analyzed as described for the experiments shown in Fig. 1. Cells of the three different origins could be identified in chimeric mice because wild-type donor cells were EGFP+ as well as CD45.2+, while IL-7R–/– (recipient) cells were CD45.2+ and EGFP–, and RAG-2–/– cells were CD45.1+. Transplantation with RAG-2-deficient marrow transformed the thymus of IL-7R-deficient mice from a responsive phenotype (i.e., capable of reconstitution by wild-type cells) to a refractory one. Donor cell chimerism (both RAG-2–/– and wild type) in the marrow of IL-7R–/– mice remained low, as is expected for nonirradiated recipients (23), and consistent with the data shown in Fig. 1. Together, these data show that responsiveness of the IL-7R–/– thymus to reconstitution by wild-type progenitors can be modulated by the presence of RAG-2-deficient thymocytes, i.e., DN cells, and the effects that they confer on the thymic stromal microenvironment.
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The data in Fig. 4 indicate that RAG-2-deficient DN thymocytes are capable of rendering the IL-7R-deficient thymus refractory to reconstitution by wild-type cells. To evaluate the mechanism of this, we compared DN cell distributions in these two strains of immunodeficient mice. Consistent with their published phenotype (8), thymuses from RAG-2-deficient mice consisted solely of DN cells (Fig. 5). In contrast, thymocytes from IL-7R–/– mice displayed essentially normal CD4 vs CD8 distributions, as previously reviewed (26). Because thymuses from these two strains are virtually the same size (Fig. 1) and contain similar total cell numbers (Table I), this means that thymuses from IL-7R–/– mice have only 2–3% as many DN cells as those from RAG-2–/– mice. The phenotype of the thymus in mice with IL-7R signaling deficiencies is complicated, because despite having apparently normal proportions of DP and SP cells, they appear to mostly lack cells at the CD25+44low DN3 stage (Fig. 5, and Refs.27, 28, 29). Nonetheless, those cells that do fall in the DN3 region of a CD44 x CD25 plot appear to be bona fide DN3 cells, because they undergo a reduction in proliferative rate similar to that of wild-type cells (Fig. 6). Further, IL-7R–/– DN2 cells (CD25+44+) proliferate at levels indistinguishable from wild-type DN2 (Fig. 6), suggesting that the apparent paucity of DN3 cells is not due to the inability of thymocytes from IL-7R-deficient mice to proliferate, but rather to the absence of a survival signal.
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–/– mice, and the ability of RAG-2–/– thymocytes to render an IL-7R–/– thymus refractory to reconstitution by wild-type cells, suggests that homing of new progenitors to the thymus (and thus, thymic reconstitution) might be influenced by the absolute number of DN cells present. This is consistent with predictions made by previous studies (2), which showed that importation of new progenitors into the thymus is an actively regulated periodic event initiated by a decrease in thymocyte progenitor number. The data in Fig. 5 and published findings of others (27, 28, 29) suggest that the most obvious impact of IL-7R–/– signaling deficiency is at the DN2/DN3 transition. However, DN3 cells are physically removed from the site of blood progenitor entry into the thymus (6, 30), and thus, it is not intuitive that they would influence new progenitor recruitment. To more fully evaluate whether other DN subsets were also reduced, absolute DN1, DN2, and DN3 cell numbers in various immunodeficient strains were calculated (Table I). Note that proper identification of DN1 (CD25–44+) and DN2 stages requires markers in addition to CD44 and CD25. For instance, many nonlymphoid cells express CD44 (31), and because they are nonlymphoid, they are, likewise, predictably negative for markers such as CD3, CD4, CD8, and CD25. A number of surface markers can help to identify the lymphoid progenitor component within the CD44+25–lin– region (i.e., DN1), but CD117 (c-kit) is especially useful, because it allows clear discrimination between DN2 and DN3 cells (32).
In strains that were readily reconstituted by wild-type marrow (e.g., IL-7R–/–, common -chain (c)–/–; see Fig. 2), we found that total DN cell numbers were markedly reduced compared with refractory strains (RAG-2–/–, TCR–/–, and wild type). Interestingly, SCID mice, which had an intermediate number of DN cells (Table I), displayed an intermediate level of reconstitution by wild-type marrow (Fig. 2). Together, these data would appear to indicate that the overall number of DN cells might be the most important factor regulating expansion of intrathymic progenitors. However, analysis of individual DN subsets (DN1, DN2, and DN3) reveals that IL-7R–/–, c–/–, SCID, and RAG-2–/– mice all have greatly reduced numbers of DN1 cells, yet reconstitute quite differently, suggesting that DN1 number does correlate well with sensitivity to reconstitution. There is some correlation between DN2 cell number and reconstitution capacity (Table I). However, SCID and RAG-2–/– mice have very similar numbers of DN2 cells, yet differ in reconstitution capacity, while TCR–/– mice have far more DN2 cells than RAG-2–/–, yet both strains are similarly resistant to reconstitution. By far, the subset that correlates best with reconstitution capacity is DN3; all mice exhibiting DN3 numbers equal to or greater than wild-type mice are resistant to reconstitution by transplanted marrow, while all mice with less than wild-type numbers are proportionally sensitive (i.e., IL-7R–/– 
c–/– < SCID). Thus, the triple-chimeric experiments (Fig. 4) indicate that regulation of thymus cell number is clearly a DN cell-related function, while the data in Table I suggest that it probably involves counting the numbers of DN2 and/or DN3 thymocytes (see Discussion).
The IL-7R–/– mouse has a miniature, but normally structured, thymus
The presence of DP and SP cells in the IL-7R–/– thymus (Fig. 5, and Ref.33) results in the production of a normally compartmentalized thymus, despite the dramatic overall decrease in cell number (Fig. 7). For instance, in contrast to the RAG-2–/– thymus, defined regions of densely packed cortex and less dense medulla are clearly obvious. The cortex is densely permeated by a reticular matrix of cytokeratin+ stromal cells, while the medulla is defined by cytokeratin+ cells that are less frequent and stellate in morphology. In contrast, the RAG-2-deficient thymus lacks large regions defined by medullary stromal cells and low cell density, as previously described (34). CD25+ progenitors (i.e., DN2 and DN3 cells) are found scattered at low density throughout the cortex of IL-7R–/– thymuses, while CD25+ cells are virtually ubiquitous in the RAG-2-deficient thymus, consistent with the results of flow cytometric staining (Fig. 5). Thus, the IL-7R-deficient thymus recapitulates the structure and composition of a normal thymus, except that it is atrophic, deficient in early progenitors, and responsive to reconstitution by normal progenitors. These characteristics indicate that it may be a useful model for studying secondary immunodeficiencies associated with aging and/or viral or chemical agents, as well as for studies of normal blood progenitor homing and thymic reconstitution, as discussed in the next section.
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Discussion |
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-deficient mouse is restored to normal cellularity by the same treatment (Fig. 1). The thymus of an IL-7R–/– mouse is virtually the same in overall size and cellularity as a RAG-2-deficient thymus (Table I). However, RAG-2-deficient thymocytes consist solely of DN cells, while IL-7R–/– thymocytes display roughly normal proportions of DN, DP, and SP cells (Figs. 4 and 5, and reviewed in Ref.26). Thus, the IL-7R–/– thymus has only 2–3% as many DN cells as that of RAG-2-deficient or wild-type mice (Table I).
Our demonstration that administration of RAG-2–/– marrow to IL-7R–/– mice converts them to a RAG-2–/– phenotype (Fig. 4) confirms that the critical feedback parameter involves the number of DN cells, because these are essentially the sole thymic cell type produced by RAG-deficient marrow (Fig. 4). More specifically, these data, which are consistent with the findings of others (32, 35, 36), suggest that of all the DN stages, DN3 cells appear to most strongly factor into the equation, with a possible secondary contribution by DN2. DN1 cells appear unlikely to be involved, because IL-7R–/–, RAG-2–/–, SCID, and c–/– mice all have reduced numbers of DN1 cells (Table I, and Refs.32 , and 35, 36, 37), but differ substantially in their abilities to be reconstituted by wild-type marrow (Fig. 1). DN2 cell numbers correlate somewhat with reconstitution capacity, with IL-7R–/– and c–/– mice having the fewest DN2 cells and the greatest sensitivity to reconstitution. However, SCID mice actually have slightly more DN2 cells than RAG-2–/– mice, yet reconstitute substantially better. The subset that correlates best with sensitivity to reconstitution is DN3; all strains with normal (or supernormal) numbers of DN3 cells are refractory to reconstitution, while strains with subnormal DN3 numbers are reconstituted proportionally to their number (Table I and Fig. 2). Thus, our data indicate that thymus size is controlled by counting DN cell numbers, specifically at the DN3 and, potentially, the DN2 stages.
The ability of DN2/DN3 progenitors to regulate reconstitution in the thymus is consistent with feedback inhibition resulting from competition for occupancy of limited stromal niches. This is noteworthy because we have previously shown that DN2 and DN3 cells (but not DN1) remain constantly in contact with VCAM-1+ stromal cells in the cortex (38). Competition for stromal niches is a widely accepted concept in the hemopoietic system, and has also been shown to play a role in the recruitment of new progenitors to the thymus (2). However, regulation of new progenitor recruitment seems unlikely to contribute to the difference between responsive and refractory strains as defined in this study, because stem cell chimerism in bone marrow was present at virtually identical levels in both categories of mice (see Fig. 1). Further, DN1 cells are, phenotypically and anatomically, the most closely related to thymic homing progenitors from marrow (6, 30, 39), and would appear to be the most likely candidates to feed back to on the progenitor recruitment process. However, as mentioned above, both responsive and refractory strains exhibited a reduction in DN1 cell numbers (Table I, and Refs.32 , and 35, 36, 37). Thus, while new progenitor homing to the thymus is almost certainly controlled by competition for specific stromal niches (2), our data suggest that once inside the thymus, competition for intrathymic stromal niches acts independently to regulate the intrathymic expansion process. Thus, there appear to be two phases of postnatal thymic lymphopoiesis that are independently impacted by competition for stromal niches (i.e., new progenitor recruitment, and expansion at the DN stages of differentiation).
In contrast to DN cells, which compete for occupancy of a limited number of thymic stromal niches, our data indicate that DP cells, which represent the bulk of thymocytes, do not enter into the equation by which thymus size is constrained. For instance, wild-type cells that do enter the thymus in RAG-2–/–-deficient hosts generate normal proportions of DN, DP, and SP cells (Fig. 4, and additional data not shown), but fail to restore the RAG-2–/– thymus to normal size. This indicates that proliferative expansion at the DP stage must be intrinsically limited; otherwise, any wild-type cells that gained access to the RAG-2–/– thymus should fully reconstitute the DP and SP compartments, where there is no competition by RAG-2–/– cells. This conclusion is completely consistent with a previous report by Almeida et al. (40), who showed that the number of DP cells in the thymuses of competitive (radiation) chimeras was directly proportional to the number of DN cells present, and was not regulated by thymus size. Thus, it appears that thymic size is limited by two coordinate functions: 1) competition at the DN stage for limited availability of stromal niches, and 2) intrinsic limits on proliferative expansion of each DN progenitor transiting to the DP stage. The finding that DN cells compete for limited stromal niches while DP cells do not is also consistent with previous findings from our laboratory showing that while DN cells must remain in constant contact with the stromal matrix that permeates the cortex, DP cells do not exhibit this requirement (38).
The remarkable susceptibility of IL-7R–/– mice, and other mutant strains with DN deficiencies, to thymic reconstitution by wild-type marrow has a number of implications. An obvious one is in regard to the characterization of the elusive nature of intrathymic stromal niches, and the signals they deliver to developing lymphocytes. Another is in regard to the plasticity of thymic stroma and its response to engraftment by lymphoid progenitors, including the modulation of signals that occur in response to occupancy or vacancy of stromal niches. The current model also has the advantage of facilitating analysis in untreated recipients, because it does not require high level marrow chimerism, and consequently, the administration of treatments (drugs or radiation) that may irreversibly damage thymic stroma are not necessary. Irradiation is generally required to induce marrow "space", and our data using nonirradiated chimeras are consistent with this requirement to generate high-level chimerism (Fig. 1). However, despite generation of only trace levels of stem cell chimerism in marrow (Fig. 1), the thymuses of nonirradiated IL-7R–/– mice are readily reconstituted by those few wild-type progenitors that are present, due to their characteristic low numbers of DN cells (Figs. 3 and 5). This raises the potential for a second, less obvious application, which is the use of this model to evaluate various T cell immunodeficiencies, especially secondary immunodeficiency disorders resulting from chemical or radiation damage, or age. For instance, therapeutic conditioning before bone marrow transplantation results in prolonged T cell lymphopenia, particularly in adults (41, 42). The basis for this is unclear, but it appears to be an intrinsic characteristic of the thymic stroma (43). Because our data show that the IL-7R–/– mouse thymus is exquisitely responsive to engraftment by transplanted progenitors, it may make an excellent model for assessing the detrimental effects of various conditioning regimens in the adult thymus, and for the aging thymus in general.
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Acknowledgments |
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Footnotes |
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1 This work was supported by Public Health Service Grants AI33940 and AI53739 (to H.T.P.) and CA08748 (to Memorial Sloan-Kettering Cancer Center). 
2 Address correspondence and reprint requests to Dr. Howard T. Petrie, University of Miami School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101. E-mail address: hpetrie{at}med.miami.edu
3 Abbreviations used in this paper: DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; DN, double negative, DP, double positive; EGFP, enhanced GFP; c, common cytokine receptor -chain; SP, single positive.
Received for publication January 30, 2004. Accepted for publication May 21, 2004.
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K. M. Williams, P. J. Lucas, C. V. Bare, J. Wang, Y.-W. Chu, E. Tayler, V. Kapoor, and R. E. Gress CCL25 increases thymopoiesis after androgen withdrawal Blood, October 15, 2008; 112(8): 3255 - 3263. [Abstract] [Full Text] [PDF]
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 R. N. Trotter-Mayo and M. R. Roberts Leptin Acts in the Periphery to Protect Thymocytes from Glucocorticoid-Mediated Apoptosis in the Absence of Weight Loss Endocrinology, October 1, 2008; 149(10): 5209 - 5218. [Abstract] [Full Text] [PDF]
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R. M. Kelly, S. L. Highfill, A. Panoskaltsis-Mortari, P. A. Taylor, R. L. Boyd, G. A. Hollander, and B. R. Blazar Keratinocyte growth factor and androgen blockade work in concert to protect against conditioning regimen-induced thymic epithelial damage and enhance T-cell reconstitution after murine bone marrow transplantation Blood, June 15, 2008; 111(12): 5734 - 5744. [Abstract] [Full Text] [PDF]
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Y. Wang, A. Kissenpfennig, M. Mingueneau, S. Richelme, P. Perrin, S. Chevrier, C. Genton, B. Lucas, J. P. DiSanto, H. Acha-Orbea, et al. Th2 Lymphoproliferative Disorder of LatY136F Mutant Mice Unfolds Independently of TCR-MHC Engagement and Is Insensitive to the Action of Foxp3+ Regulatory T Cells J. Immunol., February 1, 2008; 180(3): 1565 - 1575. [Abstract] [Full Text] [PDF]
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E. Sitnicka, N. Buza-Vidas, H. Ahlenius, C. M. Cilio, C. Gekas, J. M. Nygren, R. Mansson, M. Cheng, C. T. Jensen, M. Svensson, et al. Critical role of FLT3 ligand in IL-7 receptor independent T lymphopoiesis and regulation of lymphoid-primed multipotent progenitors Blood, October 15, 2007; 110(8): 2955 - 2964. [Abstract] [Full Text] [PDF]
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L. Gautreau, M.-L. Arcangeli, V. Pasqualetto, A.-M. Joret, C. Garcia-Cordier, J. Megret, E. Schneider, and S. Ezine Identification of an IL-7-Dependent Pre-T Committed Population in the Spleen J. Immunol., September 1, 2007; 179(5): 2925 - 2935. [Abstract] [Full Text] [PDF]
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T. Serwold, K. Hochedlinger, M. A. Inlay, R. Jaenisch, and I. L. Weissman Early TCR Expression and Aberrant T Cell Development in Mice with Endogenous Prerearranged T Cell Receptor Genes J. Immunol., July 15, 2007; 179(2): 928 - 938. [Abstract] [Full Text] [PDF]
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 K. Mori, M. Itoi, N. Tsukamoto, H. Kubo, and T. Amagai The perivascular space as a path of hematopoietic progenitor cells and mature T cells between the blood circulation and the thymic parenchyma Int. Immunol., June 1, 2007; 19(6): 745 - 753. [Abstract] [Full Text] [PDF]
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M. Cavazzana-Calvo, F. Carlier, F. Le Deist, E. Morillon, P. Taupin, D. Gautier, I. Radford-Weiss, S. Caillat-Zucman, B. Neven, S. Blanche, et al. Long-term T-cell reconstitution after hematopoietic stem-cell transplantation in primary T-cell-immunodeficient patients is associated with myeloid chimerism and possibly the primary disease phenotype Blood, May 15, 2007; 109(10): 4575 - 4581. [Abstract] [Full Text] [PDF]
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Y.-W. Chu and F. T. Hakim KGF boosts thymic archiTECture Blood, May 1, 2007; 109(9): 3613 - 3614. [Full Text] [PDF]
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S. W. Rossi, L. T. Jeker, T. Ueno, S. Kuse, M. P. Keller, S. Zuklys, A. V. Gudkov, Y. Takahama, W. Krenger, B. R. Blazar, et al. Keratinocyte growth factor (KGF) enhances postnatal T-cell development via enhancements in proliferation and function of thymic epithelial cells Blood, May 1, 2007; 109(9): 3803 - 3811. [Abstract] [Full Text] [PDF]
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M. H. Dallas, B. Varnum-Finney, P. J. Martin, and I. D. Bernstein Enhanced T-cell reconstitution by hematopoietic progenitors expanded ex vivo using the Notch ligand Delta1 Blood, April 15, 2007; 109(8): 3579 - 3587. [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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S. S. Perry, R. S. Welner, T. Kouro, P. W. Kincade, and X.-H. Sun Primitive lymphoid progenitors in bone marrow with T lineage reconstituting potential. J. Immunol., September 1, 2006; 177(5): 2880 - 2887. [Abstract] [Full Text] [PDF]
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F. J. Schnell, A. L. Zoller, S. R. Patel, I. R. Williams, and G. J. Kersh Early growth response gene 1 provides negative feedback to inhibit entry of progenitor cells into the thymus. J. Immunol., April 15, 2006; 176(8): 4740 - 4747. [Abstract] [Full Text] [PDF]
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O. Alpdogan, V. M. Hubbard, O. M. Smith, N. Patel, S. Lu, G. L. Goldberg, D. H. Gray, J. Feinman, A. A. Kochman, J. M. Eng, et al. Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration Blood, March 15, 2006; 107(6): 2453 - 2460. [Abstract] [Full Text] [PDF]
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D. Bhattacharya, D. J. Rossi, D. Bryder, and I. L. Weissman Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning J. Exp. Med., January 23, 2006; 203(1): 73 - 85. [Abstract] [Full Text] [PDF]
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C. Lagresle-Peyrou, F. Yates, M. Malassis-Seris, C. Hue, E. Morillon, A. Garrigue, A. Liu, P. Hajdari, D. Stockholm, O. Danos, et al. Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity Blood, January 1, 2006; 107(1): 63 - 72. [Abstract] [Full Text] [PDF]
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O. Adjali, R. R. Vicente, C. Ferrand, C. Jacquet, C. Mongellaz, P. Tiberghien, K. Chebli, V. S. Zimmermann, and N. Taylor Intrathymic administration of hematopoietic progenitor cells enhances T cell reconstitution in ZAP-70 severe combined immunodeficiency PNAS, September 20, 2005; 102(38): 13586 - 13591. [Abstract] [Full Text] [PDF]
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T. S. P. Heng, G. L. Goldberg, D. H. D. Gray, J. S. Sutherland, A. P. Chidgey, and R. L. Boyd Effects of Castration on Thymocyte Development in Two Different Models of Thymic Involution J. Immunol., September 1, 2005; 175(5): 2982 - 2993. [Abstract] [Full Text] [PDF]
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I. Zubkova, H. Mostowski, and M. Zaitseva Up-Regulation of IL-7, Stromal-Derived Factor-1{alpha}, Thymus-Expressed Chemokine, and Secondary Lymphoid Tissue Chemokine Gene Expression in the Stromal Cells in Response to Thymocyte Depletion: Implication for Thymus Reconstitution J. Immunol., August 15, 2005; 175(4): 2321 - 2330. [Abstract] [Full Text] [PDF]
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J. D. Carter, G. M. Calabrese, M. Naganuma, and U. Lorenz Deficiency of the Src Homology Region 2 Domain-Containing Phosphatase 1 (SHP-1) Causes Enrichment of CD4+CD25+ Regulatory T Cells J. Immunol., June 1, 2005; 174(11): 6627 - 6638. [Abstract] [Full Text] [PDF]
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