Impaired Development of T Lymphoid Precursors from Pluripotent Hematopoietic Stem Cells in New Zealand Black Mice

Yoshiko Hashimoto, Encarnacion Montecino-Rodriguez, M. Eric Gershwin and Kenneth Dorshkind
J Immunol January 1, 2002, 168 (1) 81-86; DOI: https://doi.org/10.4049/jimmunol.168.1.81

Abstract

Bone marrow cells from autoimmune-prone New Zealand Black (NZB) mice are less efficient at colonizing fetal thymic lobes than cells from normal strains. This study demonstrates that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Rather, the T lymphopoietic defect of NZB mice is due to an impaired ability of pluripotent hematopoietic stem cells (PHSCs) to generate more committed lymphoid progeny, which could include common lymphoid precursors and/or other T cell-committed progenitors. Although PHSCs from NZB mice were not as efficient at thymic repopulation as comparable numbers of PHSCs from control strains, the ability of common lymphoid precursors from NZB mice to repopulate the thymus was not defective. Similarly, more differentiated NZB T cell precursors included in the intrathymic pool of CD4−CD8− cells also exhibited normal T lymphopoietic potential. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that generation of lymphoid progeny from the PHSCs is a regulated event.

The New Zealand Black (NZB)3 mouse is a well-described model for autoimmune disease, and reports of immune system abnormalities in this strain are extensive (1). Included are defects in primary lymphocyte development, and those in the B cell lineage are well documented (2, 3, 4, 5, 6). There are indications that primary T lymphopoiesis is also abnormal in NZB mice (7, 8). In addition to abnormalities in the thymic microenvironment (9, 10, 11), a recent report from this laboratory demonstrated that defects in the ability of bone marrow cells from NZB mice to generate T lymphocytes also contributes to reduced T lymphopoiesis in that strain (12).

This conclusion is based on the finding that NZB bone marrow cells are less efficient at differentiating into mature T cells in fetal thymic organ cultures (FTOCs) compared with cells from control mice. However, the mechanism(s) responsible for this deficiency was not defined. Doing so is critically important, because such information would provide insights into how T lineage differentiation is regulated and could potentially provide a basis for understanding how defects in that process contribute to development of the autoimmune phenotype.

One possibility is that T cell precursors in the bone marrow of NZB mice are deficient in their ability to migrate to the thymus in response to chemotactic signals produced by that organ (13, 14). Upon reaching the thymus, cells have to enter the thymic parenchyma (15) and this process, which involves the interaction between cell surface receptor expressed on the precursors with ligands presented by the vascular endothelium and thymic stroma, might also be impaired in NZB mice. In either case, fewer T cell precursors would enter the thymus, and this in turn could be manifested as a reduction in the number of T lineage cells that are produced.

Additional defects in NZB T cell precursors must also be considered. For example, previous results from this laboratory demonstrated that the diminished thymus repopulating potential of NZB bone marrow cells was an intrinsic defect that could be localized to the pluripotent hematopoietic stem cell (PHSC) compartment (12). However, PHSCs are difficult to detect in the thymus and they may not be the cell population that normally migrates to that organ and sustains thymopoiesis. Instead, more differentiated PHSC progeny, such as the common lymphoid precursor (CLP) (16) or a T cell-committed prothymocyte (17, 18, 19) may be the bone marrow emigrant population. If development of these progeny from the PHSCs is defective, NZB bone marrow would contain fewer T cell precursors, thus explaining their deficiency in thymus repopulation. Alternatively, CLPs and prothymocytes may develop in normal numbers, but their ability to respond to developmental signals upon entry into the thymic microenvironment could be impaired.

The aim of this study is to distinguish between these possibilities to define a basis for the decreased thymus repopulating potential of NZB bone marrow. The data indicate that the reduced capacity of NZB bone marrow cells to repopulate the thymus does not result from their inability to migrate to or enter the thymus. Instead, the results suggest that their T lymphopoietic defect results from an impaired ability of PHSCs to generate CLPs and/or T cell-committed progenitors. Taken together, the results identify an unappreciated defect in NZB mice and provide further evidence that the generation of lymphoid progeny from the PHSCs is a regulated event.

Materials and Methods

Mice

C57BL/6J (H-2b; Thy 1.2), NZB/BNJI (H-2d; Thy-1.2), and BALB/c By (H-2d;Thy-1.2) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and were housed in the Division of Laboratory Animal Medicine vivarium at the University of California (Los Angeles, CA). Timed pregnant Swiss/Webster (S/W; H-2s/q;Thy 1.1) mice were purchased from Taconic Farms (Germantown, NY).

Preparation of cell suspensions

Bone marrow cell suspensions were prepared by flushing femurs and tibiae with 3 ml of RPMI 1640. Thymocyte suspensions were prepared by gently pressing thymuses through a fine mesh screen into RPMI 1640. Cells were counted with a hematocytometer, and cell viability, determined by eosin dye exclusion, was always >95%.

FTOCs

FTOCs were initiated and maintained based on the protocol established by Jenkinson et al. (20), as described previously (12). Briefly, embryonic thymuses were aseptically harvested from timed pregnant S/W mice at day 14 of gestation. The thymus lobes were dissected free from extraneous tissue and cultured for 5 days in the presence of 1.35 mM deoxyguanosine (dGuo; Sigma-Aldrich, St. Louis, MO) to deplete endogenous lymphohematopoietic cells. The lobes were then rinsed and incubated in hanging drop cultures in Terasaki plates (Nunc; Fisher, Pittsburgh, PA) for 48 h with bone marrow cells enriched in lineage-negative population of bone marrow (Lin−) or CLPs. Subsequently, lobes were transferred to FTOCs on filter/gelfoam rafts in RPMI 1640 supplemented with 10% FCS, 5 × 10−5 2-ME, 100 U/ml streptomycin, and 100 U/ml penicillin and placed in a humidified incubator (37°C, 5% CO2, and air). After 14–21 days of culture, thymocytes were harvested by gently pressing the lobes through fine mesh sieves with the plunger of a sterile 1-ml syringe. Donor cell origin was always confirmed on the basis of Thy-1-allotypic differences.

Thymus reaggregate cultures

Thymus reaggregate organ cultures were established based on the protocol described by Anderson et al. (21). Deoxyguanosine-treated S/W fetal lobes were digested in collagenase dispase (Sigma-Aldrich) in Ca2+, Mg2+-free PBS for 30 min at 37°C. The reaction was stopped by the addition of Ca2+, Mg2+-free PBS, and the suspension was vigorously pipetted and dispersed with a syringe fitted with a 21-gauge needle. To make reaggregates, sorted Lin− bone marrow cells were mixed with dispersed stromal cells at a ratio of 1:10. After centrifugation, the cell slurry was placed on the surface of a millipore filter in organ culture. After 3 wk of culture, the reaggregates were processed as described above for FTOCs.

Migration assay

Deoxyguanosine-treated fetal thymic lobes were placed in wells of six-well plates in RPMI 1640 supplemented with 10% FCS, 5 × 10−5 2-ME, 100 U/ml streptomycin, and 100 U/ml penicillin. Transwells with 0.4-μm pore membranes, into which smaller transwells with 5-μm pore membranes were inserted, were placed over each well. A total of 106 bone marrow cells were then deposited into the 5-μm wells. Following a 24-h incubation at 37°C, the number of cells trapped in the 0.4-μm chamber was determined on a FACScan (BD Biosciences, San Jose, CA) calibrated for automatic cell counting. Incubation of 106 cells in the absence of fetal thymic lobes in this experimental system was used to determine random migration through the 5-μm membranes. A chemotactic index was then calculated by dividing the number of cells that migrated toward wells that included thymic lobes by the number of cells that migrated randomly.

Hematopoietic cell isolation

Bone marrow cell suspensions were depleted of mature lineage cells (Lin−) (22, 23) by magnetic depletion according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Briefly, cell suspensions were incubated with the appropriate concentrations of rat anti-lineage Ags CD45R/B220 (clone RA3-6B2), TER-119, Gr-1 (clone RB6-8C5), CD8 (clone 53-6.7), CD2 (clone RM2-5), and CD11b (clone M1/70) (all from BD PharMingen, San Diego, CA) at 4°C in Ca2+, Mg2+-free PBS. Cells were then washed and incubated with goat anti-rat IgG Abs conjugated to MACS microbeads (Miltenyi Biotec) before application to a preconditioned MACS separation column. The Lin−-enriched fraction was then incubated with an anti-rat Ig Ab conjugated with Tricolor to exclude remaining mature cells during the sorting procedure.

After blocking of residual anti-rat Ig activity by incubation with unlabeled rat IgG, Lin−-enriched cells were labeled with appropriate Abs to enrich for PHSCs (Lin−Sca-1+ (clone E13-161.7), CD117+ (clone 2B8)) and CLPs (CD127+Sca-1lowCD117low). All Abs were obtained from BD PharMingen. Cells with these phenotypes were isolated by flow cytometry on a dual-laser BD Biosciences FACSVantage flow cytometer located in the Jonsson Comprehensive Cancer Center (Los Angeles, CA).

CD4−CD8− thymocytes were isolated by magnetic depletion after incubation with anti-CD4 (clone GK1.5)- and anti-CD8 (clone 53-6.7)-conjugated MACS microbeads, according to the manufacturer’s instructions. FACS analysis demonstrated that an enrichment of >95% could be routinely achieved by this procedure.

Immunofluorescence

Cells were analyzed for the expression of the following cell surface determinants: Thy-1.2, CD44, CD25, CD4, and CD8, using Abs conjugated to fluorescein, PE, or biotin (all from BD PharMingen). Biotinylated Abs were revealed with PerCP-streptavidin (BD Biosciences). The optimal working dilution was determined for each reagent before use. Before staining, FcγII/IIIR on cells were blocked by preincubation of the samples with a rat anti-CDw32/16 Ab (BD PharMingen). Cells were then incubated with the first-step Ab for 30 min at 4°C, washed with Ca2+, Mg2+-free PBS, and incubated an additional 30 min at 4°C with the second-step reagent. After the last wash, the cells were resuspended in Ca2+, Mg2+-free PBS, and 103 viable cells from each sample were analyzed on a FACScan (BD Biosciences).

Statistics

The Student t test was used to determine the significance of FTOC data.

Results

Bone marrow cells from NZB mice do not effectively differentiate in fetal thymic lobes

Previous observations demonstrated that bone marrow cells from NZB mice are deficient in their ability to repopulate fetal thymic lobes (12). As shown in Fig. 1⇓A, although 96% of cells recovered from FTOCs seeded with Lin− NZB bone marrow cells were donor derived, a consistent finding was that total cellularity was lower than in lobes seeded with cells from control mice. Further analysis demonstrated that the frequency of cells that had matured to the CD4+CD8+ stage of development was also lower in lobes colonized with NZB compared with control bone marrow cells (Fig. 1⇓B). In the experiment shown, the frequency of CD8+ cells is higher in lobes repopulated with NZB donor cells, but this is not a consistent finding (12). The lower cell number in thymic lobes repopulated with NZB bone marrow cells is in agreement with histological analysis showing that thymuses seeded with NZB donor cells are relatively hypocellular (Fig. 1⇓C).

FIGURE 1.
FIGURE 1.

Reduced thymus-repopulating capacity of NZB bone marrow cells. A total of 104 Lin− bone marrow cells from NZB mice were used to repopulate FTOC. A, Total number of cells recovered is lower in lobes repopulated with NZB bone marrow. B, CD4 and CD8 expression on cells harvested from lobes 2 wk after seeding. C, Lobes were sectioned and stained with H&E. Histologic analysis reveals fewer cells in the medulla of lobes repopulated with NZB bone marrow cells (×400). Data are representative of five experiments.

Previous results from this laboratory demonstrated that the diminished thymus repopulating potential of NZB bone marrow cells was due in part to intrinsic defects in bone marrow T cell precursors (12). One explanation for this defect is that bone marrow cells from NZB mice are defective in their ability to migrate to and enter the thymus. Additional possibilities are that the development of lymphoid progeny from PHSCs is defective or that CLPs and prothymocytes that do develop fail to respond normally to developmental signals upon entry into the thymic microenvironment. Subsequent experiments were designed to distinguish between these alternatives.

NZB bone marrow cells can migrate to the thymus

The possibility that NZB bone marrow cells were defective in their ability to migrate to the thymus was examined using a diffusion chamber culture system depicted in Fig. 2⇓A. Bone marrow cells from NZB and BALB/c mice were placed in a diffusion chamber that separated them from deoxyguanosine-treated thymic lobes present in a lower chamber. Twenty-four hours later, cells that accumulated in an intermediate chamber were harvested and counted. As shown in Fig. 2⇓B, no significant difference in the ability of unseparated NZB and BALB/c bone marrow cells to migrate toward the thymic lobes was detected. Whole bone marrow is heterogeneous, and multiple cell types could have migrated toward the thymus, regardless of whether or not they had T lymphoid potential. Therefore, an additional experiment was conducted to measure the ability of Lin−, CD127 (IL-7Rα)-expressing cells to migrate toward the thymus. Lin−CD127+ cells were isolated from NZB or control bone marrow, and equivalent numbers were assayed as shown in Fig. 2⇓A. The data in Fig. 2⇓B indicate that lymphoid-enriched precursors from NZB mice migrate normally to the thymus.

FIGURE 2.
FIGURE 2.

Unseparated and Lin−CD127+ bone marrow cells from NZB mice can migrate to the thymus. A, Experimental design. Bone marrow cells or Lin−CD127+ cells were placed in a diffusion chamber with 5-μm pores. This was then placed into another diffusion chamber with 0.4-μm pores. These two chambers were then inserted into wells that contained medium only or deoxyguanosine-treated thymic lobes. Cells that migrated toward thymic lobes in the lower chamber were trapped in the intermediate one due to its small pore size. B, Number of cells present in the intermediate chamber was used to determine the chemotactic index (cells migrating in response to thymus/cells migrating in response to medium only). Data shown indicate indices for unseparated bone marrow (BM) or Lin−CD127+ cells. Differences were not statistically significant.

Upon reaching the thymus, precursors have to migrate into its parenchyma (15). To assess this function, bone marrow cells from NZB or BALB/c mice were labeled with CFSE, a dye that integrates into the cell membrane and whose intensity of staining diminishes by one-half with each cell division, and were incubated with thymic lobes in hanging drop culture. Upon disruption of lobes 24 h later, the frequency and absolute number of donor-derived cells in the lobes was comparable, and little cell division was observed with cells from either strain (data not shown).

Taken together, these results suggest that the thymopoietic defects of NZB bone marrow cells should be apparent, even when the need to migrate to and enter the thymus is obviated. This was confirmed in a thymic reaggregate system that allows direct interactions between T cell precursors and thymic stroma (21). Thymic reaggregate cultures were initiated with NZB or BALB/c Lin− bone marrow cells, and thymocyte development was analyzed 3 wk later. As shown in Fig. 3⇓, >90% of the thymocytes that developed in the reaggregated lobes were donor-derived cells as determined by expression of the donor Thy-1.2 allele. The data further demonstrate that thymocyte numbers and the frequency of CD4+CD8+, CD4+, and CD8+ cells were lower in lobes repopulated with Lin− bone marrow cells from NZB mice.

FIGURE 3.
FIGURE 3.

T cell development in thymic reaggregate cultures. Lin− cells from NZB or BALB/c mice were incubated with thymic stromal cells, and the mixture was allowed to reaggregate as described in Materials and Methods. Three weeks later, the frequency of donor-derived Thy-1.2 cells and of CD4- and CD8-expressing thymocyte subpopulations was determined. The data are representative of one of three experiments with identical results.

Thymopoietic potential of NZB-derived CLPs

In view of the above findings, subsequent experiments focused on the development of lymphoid progeny from PHSCs and their immediate progeny, such as the CLP. The CLP is one of the earliest PHSC-derived populations with T lymhopoietic potential (16). Fig. 4⇓A presents the sorting strategy used to identify and isolate PHSCs and CLPs. The PHSC purification strategy was described previously (12), and enrichment of CLPs was based on the Lin−CD127+CD117+Sca-1+ phenotype described by Kondo et al. (16). An aliquot of sorted CLP was used to confirm that cells so identified lacked myelopoietic potential (data not shown). Fig. 4⇓B presents data showing the isolation of CLPs from NZB and control strains, which were used to initiate FTOC.

FIGURE 4.
FIGURE 4.

Identification and isolation of PHSCs and CLPs in NZB mice. A, Strategy for isolation of Lin−c-kithighSca-1high PHSCs and Lin−CD127+CD117+Sca-1+ CLP-enriched bone marrow cells from control mice. B, Isolation of CLPs from NZB or C57BL/6 mice. For CLP isolation, Lin− bone marrow cells were isolated and subsequently stained with Abs to the IL-7R (CD127), Sca-1, and c-kit (CD117). After gating on the Lin−CD127+ cells, that population was further analyzed based on expression of CD117 and Sca-1.

The data in Fig. 5⇓A demonstrate that the total numbers of cells recovered from thymic lobes seeded with CLPs from NZB or C57BL/6 mice were comparable. More than 97% of these cells were donor derived, as assessed by expression of the donor Thy-1.2 determinant (Fig. 5⇓B). This contrasts with the observation that the number of cells recovered from FTOCs seeded with NZB PHSCs is lower than from C57BL/6 or BALB/c mice (Ref. 12 and data not shown). Furthermore, although PHSCs from NZB mice do not generate CD4- and/or CD8-expressing thymocytes as efficiently as PHSCs from the control strain, these populations are efficiently produced from NZB-derived CLPs (Fig. 5⇓C).

FIGURE 5.
FIGURE 5.

Repopulation of thymic lobes by CLPs from NZB mice. A, Total number of cells recovered from lobes 2.5 wk after seeding with CLP-enriched bone marrow cells. n, Number of lobes analyzed. B, More than 97% of cells in CLP-repopulated lobes expressed the donor-derived Thy-1.2 allele. C, CD4 and CD8 expression on cells harvested from FTOC after seeding with PHSCs or CLP-enriched bone marrow cells from NZB or C57BL/6 mice. A single pool of thymic lobes was prepared and subdivided into aliquots seeded with PHSCs or CLPs. The latter cells were isolated from the same bone marrow pool.

CD4−CD8− cells from NZB thymus have normal developmental potential

Taken together, the above data suggest that defects in the ability of NZB CLP to generate thymocytes are minimal to nonexistent. A prediction based on this conclusion is that differentiated, downstream progeny of NZB CLPs would also exhibit normal T lymphopoietic potential. To test this hypothesis, CD4−CD8− thymocytes, from NZB and BALB/c mice, which include the earliest intrathymic T cell progenitor population, were isolated and used to seed FTOCs. As shown in Table I⇓, no significant difference in cellularity between lobes seeded with the two donor populations was observed. These results are consistent with data indicating that CD4−CD8− cells from NZB and BALB/c mice proliferate to similar levels in response to IL-7 (data not shown). The data in Table I⇓ also show no differences in the generation of CD4+CD8+-expressing cells from NZB and BALB/c mice.

View this table:
Table I.

Colonization of fetal thymic lobes with limiting numbers of CD4−CD8− thymocytesa

Discussion

The aim of the present investigation is to determine why bone marrow cells from NZB mice are less effective at repopulating the thymus compared with cells from control strains. The results confirm our previous observation (12) that NZB mice exhibit a T lymphopoietic defect. This does not result from an impaired ability of bone marrow cells from NZB mice to migrate to or enter the thymus. Instead, the data support the existence of an intrinsic defect in hematopoietic cells that seems to be operative at the PHSC level. When PHSCs from NZB mice are used to colonize FTOCs, defects in thymus repopulating potential are observed. Such abnormalities are minimal to nonexistent when populations enriched in CLPs or intrathymic progenitors are used to reconstitute FTOCs.

One possibility that was considered to explain these data is that NZB PHSCs are defective in their ability to generate normal numbers of CLPs. In this case, fewer progenitors capable of rapid T cell generation in FTOC would be present in the bone marrow. A reanalysis of our previous data in which FTOCs were repopulated with PHSCs from NZB and control strains (12) supports this premise. For example, the number of thymocytes in FTOCs repopulated with 50 PHSCs from NZB mice was ∼3-fold lower than in lobes seeded with 50 PHSCs from BALB/c mice. If the PHSC defect is simply due to their inability to generate normal numbers of CLPs and/or prothymocytes, increasing numbers of PHSCs should compensate for this defect and mediate thymus repopulation comparable to that in control mice. In this regard, thymic cellularity in FTOCs repopulated with 500 NZB PHSCs was comparable to that in lobes seeded with 50 PHSCs from BALB/c or C57BL/6 mice (12).

Attempts were made to determine whether the frequency of CLPs was reduced in the bone marrow of NZB vs control strains of mice. However, performing accurate quantitative analysis on such populations between different mice is extremely difficult. One reason for this is that there are subtle strain differences in the expression of cell surface determinants used in the identification of rare hematopoietic precursor populations. This likely explains why, although the CLP frequency in NZB mice was lower than in the C57BL/6 strain (Fig. 4⇑), no difference was observed upon comparison to BALB/c mice (data not shown). Compounding these problems is the fact that there is no ideal control strain to which NZB mice can be compared (12).

As observed previously (12), PHSCs from NZB mice exhibited a defect in their capacity to generate CD4+CD8+ thymocytes in FTOC. However, when CLP-enriched cells from NZB mice were used, the defect in the generation of CD4+ and CD8+ thymocytes was not as severe. This finding supports the hypothesis that the primary developmental block in NZB mice occurs at the PHSC to CLP stage. However, further abnormalities in the ability of NZB CLPs to differentiate cannot be formally excluded because the frequency of CD4-expressing cells remained below that observed in lobes repopulated with CLPs from C57BL/6 mice (Fig. 5⇑B). Once again, these differences may be due to strain effects and not developmental defects in NZB CLPs. The fact that the frequencies of thymocyte subpopulations expressing CD44 and/or CD25 were comparable in lobes seeded with NZB and C57BL/6 CLPs (data not shown) and that the thymus-repopulating potentials of intrathymic progenitors from NZB and BALB/c mice were similar would support this latter conclusion.

Although the T lineage defects described herein were observed in NZB mice 3 mo of age and older, deficiencies in the total number and frequency of T cell subpopulations in the thymus of NZB mice in vivo are not observed. One reason for this is that the FTOC system may reveal defects that are not apparent in vivo. In vitro, only one wave of T cell development can occur from a finite number of T cell precursors present in the seed inoculum. However, in vivo there is a continuous input of bone marrow T cell precursors to the thymus (13, 14). Thus, over time enough progenitors might migrate to the thymus and fill all available microenvironmental niches (15). However, a prediction based on the findings herein is that the NZB T lineage generative defect might be exhibited in instances when rapid T cell generation is required. For example, after bone marrow transplantation, a detailed kinetic analysis with limiting numbers of NZB bone marrow cells might reveal a slower rate of thymic repopulation.

Proposing the existence of abnormalities in NZB PHSCs is not unprecedented (1, 26, 27), although the definition of their defect in generating lymphoid committed progeny heretofore has not been appreciated. Interestingly, Sudo et al. (28) recently reported that the ability of PHSCs from aging mice to generate lymphoid progeny is impaired. Those authors noted that mice that received a transplant of bone marrow cells from aged donors exhibited myeloid, but not lymphoid, reconstitution. Thus, the T lineage defect that occurs in NZB mice may be similar to what occurs during aging. Nevertheless, a clear definition of why lymphoid developmental defects occur in NZB mice remains elusive, because the events that regulate the commitment of PHSCs to the CLP and the subsequent decision of the latter cells to generate B- or T-committed progeny are incompletely understood. Based on the present findings, the NZB mouse may provide an appropriate model system by which such regulatory pathways can be studied.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants HL60658 (to K.D.) and AR44173 (to M.E.G.). The Flow Cytometry Core at the Jonsson Cancer Center is supported in part by National Institutes of Health Grant CA16042.

  • ↵2 Address correspondence and reprint requests to Dr. Kenneth Dorshkind, Department of Pathology and Laboratory Medicine, University of California School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1732. E-mail address: kDorshki{at}mednet.ucla.edu

  • ↵3 Abbreviations used in this paper: NZB, New Zealand Black; FTOC, fetal thymic organ culture; PHSC, pluripotent hematopoietic stem cell; CLP, common lymphoid precursor; S/W, Swiss/Webster; Lin−, lineage-negative population of bone marrow.

  • Received August 28, 2001.
  • Accepted October 22, 2001.

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