Development of rat CD45+ 13-day-old fetal liver cells in SCID mouse fetal thymic organ cultures

Luis-Miguel Alonso-C, Angeles Vicente1, Alberto Varas1 and Agustín G. Zapata

Department of Cell Biology, Faculty of Biology, and
1 Department of Cell Biology, Faculty of Medicine, Complutense University of Madrid, 28040 Madrid, Spain

Correspondence to: : A. G. Zapata


    Abstract
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 
A phenotypic analysis of the lympho-hemopoietic cells which occur in the liver of 13-day-old fetal rats was achieved by flow cytometry in an attempt to further characterize the rat lymphoid progenitor cells. A small fraction of rat 13-day-old fetal liver (r13FL) cells, which weakly expressed the leukocyte common antigen CD45, constituted a homogeneous Thy-1hi, CD71, CD44+, MHC class I+, CD43+ cell subpopulation negative for CD45RC, CD3, TCR{alpha}ß, TCR{gamma}{delta}, CD2, CD5, CD4, CD8, CD25, CD28, NKR-P1a and sIg. On the contrary, the CD45 cells were a heterogeneous cell subset which expressed Thy-1, CD71 and CD44 at distinct levels. After MACS separation, the CD45+ r13FL cells, but not the CD45 cell subset, in vitro repopulated 14-day-old SCID mouse fetal thymic lobes providing rat T cells, both TCR{alpha}ß and TCR{gamma}{delta}, NK cells, and thymic dendritic cells but not B lymphocytes. Interestingly, NKR-P1alo TCR{alpha}ß+ or TCR{gamma}{delta}+ cells developed in the xenogeneic cultures, and a rare CD4+CD8+ double-positive subpopulation among the TCR{gamma}{delta}-expressing cells accumulated in the oldest cultures. These results are discussed from the double perspective of the nature of the precursor cells which colonize the fetal thymus and the relevance of the xenogeneic SCID mouse fetal thymic microenvironment for supporting rat lymphopoiesis.

Keywords: dendritic cells, NK cells, T cell development, xenogeneic culture


    Introduction.
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 
The phenotype of early cell progenitors which colonize the fetal thymus has been well studied in both humans and mice (16), but not in other species. In rats, the strong expression of Thy-1 on hematopoietic stem cells (HSC), a molecule also known to be expressed in human HSC (7), together with that of CD43 has been exploited to isolate enriched populations of lympho-hematopoietic progenitors either from fetal liver (8,9) or bone marrow (10). In addition, Crook and Hunt (11) recently reported that a CD71Thy-1hiOX82+ cell fraction contained all the HSC activity of 14-day-old rat fetal liver. On the other hand, both human (12) and mouse (13) lympho-hematopoietic progenitors express the leukocyte common antigen, CD45, as rat bone marrow cells with CFU-S capacity (10). In an attempt to further characterize the phenotype of early lympho-hematopoietic precursors occurring in the rat fetal liver, we have assayed the expression of this antigen together with that of Thy-1, CD71, CD44, and a large panel of T and non-T cell related antigens in 13-day-old rat fetal liver (r13FL) as well as the capacity of either CD45+ or CD45 r13FL cells to in vitro differentiate in fetal thymic lobes of SCID mice, a helpful xenogeneic in vitro system not previously assayed for rat T cell differentiation (1417).


    Methods
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 
Animals
Thirteen-day-old rat fetuses were obtained from timed pregnant, 2- to 3-month-old, Wistar Furth rats maintained at the animal facilities of the UCM (Madrid, Spain). Mating was carefully controlled for 5 h. C.B17/SCID mouse fetuses, kindly provided by the animal facilities of CBM (UAM, Madrid, Spain), were used at day 14 of gestation.

Preparation of r13FL cell suspensions
Livers from 13-day-old rat fetuses were mechanically disrupted by repeated pipetting in cold RPMI 1640 medium (Gibco, Eragny, France) supplemented with 25 mM HEPES, 5 mM EDTA plus 5% FCS, scraped on a stainless steel mesh of 230 µm pore size and finally filtered through a 25 µm pore size mesh. Viable cells, fetal erythrocytes being excluded, were counted in a hemocytometer by Trypan blue exclusion. Routinely, ~1x105 cells per r13FL were obtained by this method.

In order to separate the obtained r13FL cell suspension into two populations, i.e. CD45+ versus CD45 cells, the r13FL cell suspension was first incubated with 0.2 µg biotinylated OX1 mAb per 1x105 cells at a concentration of 1x107 cells/ml, for 1 h on ice, followed by incubation with a saturating concentration of biotinylated rabbit anti-mouse Ig (Sigma, St Louis, MO) for 30 min. After washing twice with PBS + 5 mM EDTA without FCS, the cell suspension was allowed to react for 10 min with 20 µl per 1x107 cells of a streptavidin–MicroBeads (SA-MB) stock solution (Miltenyi Biotech, Bergisch Gladbach, Germany). Positive selection of CD45+ cells was performed by passing the cell suspension through a mini-MACS separation column (Miltenyi Biotech). The non-retained fraction was incubated a second time with the SA-MB and newly passed through a second mini-MACS column in order to deplete it of residual non-retained CD45+ cells. Both cell fractions, i.e. CD45+ and CD45, were collected separately, counted and phenotypically analyzed by flow cytometry. The whole fetal erythrocytes were recovered in the CD45 fraction.

Immunophenotyping by flow cytometry
For flow cytometric immunophenotyping, 1–2x105 cells, suspended in 50 µl of PBS/0.1% BSA/0.1% sodium azide, were incubated with saturating concentrations of either fluorochrome or biotin conjugated mAb. Streptavidin–CyChrome (SA-CY) (PharMingen, San Diego, CA) was used as a second-step reagent when a biotinylated mAb was present. Either FITC- or phycoerythrin (PE)-conjugated F(ab')2 of rabbit anti-mouse Ig (Serotec, Oxford, UK) was also employed for indirect staining of cell suspensions pre-incubated with 50 µl of a mAbcontaining supernatant. In all cases, non-specific binding of mAb or antibody-binding free sites in the rabbit anti-mouse Ig molecules were blocked using purified mouse immunoglobulins (Sigma).

Cell acquisition, 10–30x103 events, was performed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed with PC-Lysys software (Becton Dickinson).

Fluorochrome-conjugated mAb, listed in Table 1Go, were purchased from PharMingen and Serotec. Biotinylated mAb were developed in our laboratory after Protein G purification from hybridoma cell supernatants (see Table 1Go). All the mAb employed were devoid of cross-reactivity with mouse cell surface antigens.


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Table 1. mAb employed in the present worka
 
Hybrid rat -> SCID mouse fetal thymic organ cultures (rSCID-FTOC)
rSCID-FTOC cultures were established as follows. An aliquot of 30 µl of a r13FL cell suspension was put in each well of a Terasaki plate containing one 14-day-old SCID mouse fetal thymic lobe. After 2 days of culture in hanging drops, lobes were placed on the surface of 0.8 µm pore size nitrocellulose filters (MilliPore Ibérica, Madrid, Spain), six per filter, which were layered over gelfoam rafts (Pharmacia-Upjohn, Madrid, Spain) in 35 mm sterile Petri dishes. The lobes, repopulated and control ones without rat cells, were cultured for different times (see later) and then dispersed into single-cell suspensions for flow cytometric or immunocytochemical analysis (at least 12 lobes per experiment). In all cases, viable cells were counted by Trypan blue exclusion and the phenotype of rat-derived cells analyzed using mAb specific for different rat cell markers (see Table 1Go) which did not show cross-reactivity with mouse cells. rSCID-FTOC cultures were first performed with different numbers of whole, non-enriched, r13FL cell suspensions in order to determine the suitability of the system for sustaining rat T cell development (results not shown). According to these preliminary results, a minimum of 5x103 total r13FL cells was necessary to in vitro colonize the SCID fetal thymic lobes. In addition, we determined to harvest the cells at every 2 days from day 8 to 12, then at day 16 and finally at day 24 in an attempt to identify both early stages of T cell differentiation as well as the presence of mature T cells. Cultures were maintained in a humidified incubator, at 37°C and 7.5% CO2, in culture medium consisting of RPMI 1640 supplemented with 10% FCS plus 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM 2-mercaptoethanol and antibiotics (all from Gibco/BRL, Eragny, France). Half of the medium was exchanged every 4 days with freshly prepared supplemented RPMI 1640 medium.

Immunocytochemical analysis
For immunocytochemical analysis, cytospin preparations were carried out with ~3x104 cells from rSCID-FTOC cell suspensions. They were fixed in acetone, incubated with supernatants from hybridoma cell line OX6, followed by rat anti-mouse Ig-peroxidase (Dako, Glostrup, Denmark). Peroxidase activity was routinely developed using DAB as chromogen and slides were counterstained with toluidine blue.


    Results
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 
Phenotype of r13FL cells
Rat 13FL cells expressed Thy-1 (44–70%), CD45 (11–17%), CD44 (80–98.5%), CD43 (95–100%), MHC class I (11–20%) and CD71 (60–80%) (Fig. 1AGo) but no reactivity was found for CD3–TCR, CD2, CD5, CD8, CD4, NKR-P1a, CD45RC, MHC class II, CD25, CD28 or surface Ig (Fig. 1BGo). When the phenotype of either CD45+ or CD45 cells was analyzed (Fig. 1CGo), the first ones represented a highly homogeneous Thy1hiCD44+CD71 cell subpopulation. In addition, they expressed MHC class I and CD43 molecules (data not shown). On the contrary, the CD45 cells were a highly heterogeneous subpopulation nearly showing the same phenotypic pattern as described above for the whole r13FL (Fig. 1CGo).



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Fig. 1. Expression of different cell surface markers in r13FL cell suspensions stained with the mAb listed in Table 1Go and examined by flow cytometry. The flow cytometric profiles of single-stained whole r13FL cell preparations are shown in (A) and (B). (C) Expression of Thy-1, CD44 and CD71 on CD45 versus CD45+ r13FL cells. These gated cell populations were phenotypically equivalent to the CD45+ and CD45 cell subsets separated by MACS.

 
CD45+ but not CD45 r13FL cells in vitro repopulated SCID mouse fetal thymic lobes
According to the above-described phenotype for r13FL cells, we reasoned that CD45+ r13FL cells, which highly expressed Thy-1 but not CD71, in agreement with the findings of other authors for fetal liver stem cells (8,11), could contain hematopoietic precursor cells. Therefore, we assayed the ability of CD45+ cells and, for comparison, of the CD45 cell population selectively separated by MACS, as described in Methods, to colonize in vitro fetal thymic lobes of 14-day-old SCID mice.

The rat cell production was determined by specific rat CD45 reactivity (rCD45+) (Fig. 2AGo). Thus, when 1x103 CD45+ r13FL cells were assayed, the first rCD45+ cells appeared after 10 days of culture, sharply increasing between days 10 and 16, and more gradually until day 24 (Fig. 2BGo). On the contrary, neither 1x103 nor 5–10x103 CD45 cells yielded rCD45+ cells at any time after co-culture in SCID mouse FTOC (Fig. 2BGo). Accordingly, CD45 seemed to be a suitable cell marker to determine the lympho-hematopoietic capacity of cell suspensions from early rat fetal liver.



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Fig. 2. rSCID-FTOC, established as described in Methods, were analyzed for the presence of rat-derived cells (rCD45+), as judged by specific rat CD45 staining (A), and their absolute number per cultured lobe determined at days 8, 10, 12, 16 and 24 of culture (B). rCD45+ cells, as gated in (A), were analyzed on the indicated days for their CD4 versus CD8 subset composition (C) and size (D). Numbers in (C) are percentages referring to the total rCD45+ cells recovered per lobe. All values are means from at least three independent experiments. Cell suspensions were first incubated with unconjugated mouse Ig and triple stained with biotinylated anti-CD45, PE-labeled anti-CD4 and FITC-labeled anti-CD8 mAb followed by SA–CY. A control staining for rCD45 performed with non-populated SCID-FTOC cells is shown in (A, patterned histogram).

 
Rat cell maturation in SCID mouse FTOC supplied with CD45+ r13FL cells
Phenotypic analysis of rat cell development in the rSCID-FTOC was achieved by using an extensive battery of mAb specific for different lymphoid and non-lymphoid cell markers. Rat T lymphocytes, NKR-P1a+ cells and dendritic cells (DC), but not B lymphocytes, were recovered from the rSCID-FTOC.

CD4/CD8 and TCR {alpha}ß expression by the rCD45+ cells recovered from rSCID-FTOC
After 10 days of culture, most cells (~74%) identifiable in a three-color staining for rat CD45, CD4 and CD8 were large-sized elements, which mainly corresponded to CD4CD8 double-negative (DN) cells (~30%) and immature CD8+ single-positive (SP) thymocytes (~34%) (Fig. 2CGo), and ~30% thymocytes had already matured to CD4+CD8+ double-positive (DP) cells which accounted for the small sized cells found at this time point (Fig. 2DGo). In addition, ~6% of cells corresponded to CD4+ SP cells which exhibited a low expression of the CD4 molecule (Fig. 2CGo). On the other hand, only a few rat thymocytes obtained after 10 days in culture weakly expressed the TCR{alpha}ß (Table 2Go).


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Table 2. TCR{alpha}ß expression in the rCD45+ cell fraction achieved from rSCID-FTOCa
 
As the absolute number of rCD45+ cells started to increase, higher numbers of both DP cells and CD8+ SP thymocytes appeared in correlation with a decreased frequency of the DN cell subset (Fig. 2CGo). This change in the subset composition of rCD45+ cells, which reflected a progression to more mature T cell developmental stages, was accompanied by increased numbers of both TCR{alpha}ß+ cells, most of them showing, however, a low expression of the receptor (Fig. 3AGo and Table 2Go) and proportions of small sized cells which were mainly DP thymocytes (Fig. 2DGo). At day 16, DP cells (~53%) and then CD8+ SP thymocytes (~35%) were the most prominent cell subpopulations (Fig. 2CGo), although all four cell subsets increased in absolute terms. At this stage, ~60% rat thymocytes expressed TCR{alpha}ß and ~24% of them corresponded to TCR{alpha}ßhi cells (Table 2Go). A more detailed analysis of these cells demonstrated that most TCR{alpha}ßhi cells corresponded to both CD8+ SP and DP thymocytes while, on the contrary, the CD4+ SP cells were minimally represented (Fig. 3BGo). Besides, at any time of our study, the representation of TCR{alpha}ßhi cells, in terms of both frequency and absolute numbers, was higher in the CD8+ SP subset than in the DP cell compartment (Table 3Go). This finding was specially evident when cultures were kept for 24 days (Table 3Go). At that time, the proportions of DN, DP and CD4+ SP cells decreased while those of the CD8+ SP subset markedly increased (Fig. 2CGo).



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Fig. 3. rSCID-FTOC colonized by CD45+ r13FL cells were analyzed for their rat TCR{alpha}ß, CD4, CD8 and TCR Vß expression. (A) TCR{alpha}ß expression on the cells recovered at days 12, 16 and 24. Their relative values refer to the whole rCD45+ cells as determined in Fig. 2Go. TCR{alpha}ßhi-expressing cells were gradually accumulating with time [TCR{alpha}ß: negative(–); high (H); low (L)]. (B) Expression of CD4 versus CD8 on TCR{alpha}ßhi cells gated as showed in (A). Expression of either CD4 (C) or CD8 (D) and cell size (E) were analyzed for comparison in DP cells gated either from TCR{alpha}ßhi or TCR{alpha}ß–/lo cells. (F) Relative contribution of TCR Vß8.2, Vß10 and Vß16 to the total TCR{alpha}ßhi population of SP cells at the indicated days. Triple stainings for flow cytometry were performed with a combination of biotinylated anti-TCR{alpha}ß, PE-labeled anti-CD4 and FITC-labeled anti-CD8 mAb (A–E) or biotinylated anti-CD8, PE-labeled anti-CD4 and FITC-labeled anti-Vß8.2, anti-Vß10 or anti-Vß16 mAb (F) followed by SA–CY.

 

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Table 3. TCR{alpha}ßhi expression of either rat DP or CD8 SP cellsa
 
On the other hand, when the expression of either the CD4 or CD8 co-receptors was analyzed in both the DP-TCR{alpha}ßhi cells and the DP-TCR{alpha}ß–/lo cells recovered from rSCID-FTOC, the former were enriched in CD8hiCD4lo cells, whereas the later mainly contained CD8intCD4hi cells (Fig. 3C and DGo). In addition, the two DP cell subpopulations defined by their TCR{alpha}ß expression also differed in their cell size; the DP-TCR{alpha}ßhi cells were mainly large sized cells while most DP-TCR{alpha}ß–/lo cells were small sized thymocytes (Fig. 3EGo).

Finally, a partial analysis of the TCR Vß repertoire expressed by the rat SP-TCR{alpha}ßhi thymocytes recovered from the rSCID-FTOC after either 16 or 24 days of culture was carried out using mAb specifically raised against the rat Vß8.2, Vß10 or Vß16 segments (Fig. 3FGo). No important variations were found in the expression of either Vß8.2 or Vß16 at both time points studied; however, the low proportion (<3%) of Vß10+ cells observed at day 16 of culture disappeared in the 24-day-old cultures (Fig. 3FGo).

TCR {gamma}{delta} T cells
At all days tested, rat {gamma}{delta} T cells were recovered from the rSCID-FTOC cultures. While they gradually increased from day 12 to 24 (Fig. 4AGo), their absolute number markedly increased between day 12 and 16 (Fig. 4DGo). Rat {gamma}{delta} T cells were divided into three cell subpopulations according to the expression of CD4 and/or CD8 cell markers. At any time, no CD4+ SP {gamma}{delta} T cells were found but both CD8+ SP and DP {gamma}{delta} T cells were recovered from the rSCID-FTOC (Fig. 4BGo). Moreover, among the CD8+ SP {gamma}{delta} T cells, it was possible to distinguish two cell subsets, CD8lo and CD8hi, which differentially progressed in the cultures (Fig. 4CGo). The proportion of {gamma}{delta} T cells which weakly expressed the CD8 molecule decreased over the culture period and, in parallel, that of CD8hi {gamma}{delta} T cells also increased (Fig. 4CGo). The absolute numbers of CD8lo {gamma}{delta} T cells remained, however, roughly constant over time, while both DP and CD8hi {gamma}{delta} T cells sharply increased between days 12 and 16 (Fig. 4DGo). At day 24, a decrease of CD8hi {gamma}{delta} T cells was accompanied by increasing values of the DP subpopulation (Fig. 4DGo).



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Fig. 4. Rat TCR{gamma}{delta} cells recovered from the rSCID-FTOC at 12, 16 and 24 days of culture (A) (indicated numbers correspond to the percentages of TCR{gamma}{delta} cells in the recovered rCD45+ fraction of cultured lobes). (B) CD8 versus CD4 dot-plots of TCR{gamma}{delta}-expressing cells as gated in (A) (upper- and lower-left quadrants contained <0.1% cells). (C) Percentages of CD8lo (L) and CD8hi (H) cells in the CD8 SP subpopulation of TCR{gamma}{delta}+ cells. (D) Absolute numbers of total TCR{gamma}{delta} cells and their subsets over the culture period. Cell suspensions were incubated with a combination of FITC-labeled anti-TCR{gamma}{delta}, PE-labeled anti-CD4 and biotinylated anti-CD8 followed by SA–CY.

 
NKR-P1a-expressing cells
Occurrence of rat NKR-P1a+ cells in rSCID-FTOC was demonstrated by reactivity with 3.2.3 mAb. On all the days studied, both NKR-P1alo cells and NKR-P1ahi cells were detected, the former being the most prominent (Fig. 5AGo). The expression of different T cell markers including TCR{alpha}ß, TCR{gamma}{delta}, CD4 and CD8 was additionally studied in both cell subsets (Fig. 5BGo). In general, both cell populations, i.e. NKR-P1alo and NKR-P1ahi, up-regulated these cell surface markers throughout the culture period although there were significant differences in the pattern of progression of each one. At day 12, as many as 70% of NKR-P1ahi cells were negative for both CD4 and CD8 cell markers, whereas ~40 and 37% of NKR-P1alo were CD8+ SP or DP respectively. In the following days, the proportion of DN cells decreased in both cell populations but, at day 24, ~56.5% NKR-P1alo cells expressed both CD4 and CD8 while only ~28.5% NKR-P1ahi were DP (Fig. 5BGo, upper panels). Likewise, the proportion of TCR{alpha}ß+ cells (Fig. 5BGo, middle panels) was very low at 12 and 16 days in the NKR-P1ahi subset but already reached ~40% in the NKR-P1alo cells. At day 24, there was a remarkable increase in the proportion of NKR-P1ahi TCR{alpha}ß+ cells, which reached ~64% of this population, and to a lesser extent in the NKR-P1alo subset (~60% expressed the TCR{alpha}ß), both of them expressing the CD8 co-receptor, as well. Accordingly, the proportion of CD8+ TCR{alpha}ß cells decreased in both NKR-P1a cell subsets (Fig. 5BGo, middle panels). On the other hand, the two studied cell subsets differentially up-regulated the TCR{gamma}{delta} expression throughout the culture period (Fig. 5BGo, lower panels). Thus, after 12 days in culture, nearly 33.5% of NKR-P1alo cells already expressed TCR{gamma}{delta} while the NKR-P1ahi cells were negative for this marker. In the following days, the proportion of {gamma}{delta} T cells slightly varied among the NKR-P1alo cells while only after 24 days of culture ~12.5% of NKR-P1ahi cells became TCR{gamma}{delta}+. As mentioned above for TCR{alpha}ß+NKR-P1a+ cells, most TCR{gamma}{delta} cells of both NKR-P1a cell subsets also co-expressed CD8 (Fig. 5BGo).



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Fig. 5. Presence of NKR-P1a-expressing cells in rSCID-FTOC colonized by CD45+ r13FL cells at culture days 12, 16 and 24 (A). High (H) and low (L) NKR-P1a-expressing populations were defined as marked by horizontal bars in the histogram profiles. Patterned histograms represent control staining with an isotype-matched mAb (mouse anti-human CD45, clone HI30). Numbers are percentages and refer to the whole rCD45+ population. (B) NKR-P1a antigen expression was confronted to CD8 versus CD4 (upper panels), CD8 versus TCR{alpha}ß (middle panels) and CD8 versus TCR{gamma}{delta} (lower panels) in triple-color stainings for flow cytometry (see below). As gated in (A), both NKR-P1ahi (H) and NKR-P1alo (L) populations were analyzed for their relative composition in the T cell-related antigens. All numbers are means of at least three independent experiments. In all cases, stainings were performed with a combination of FITC-labeled anti-NKR-P1a and biotinylated anti-CD8 along with either PE-labeled anti-CD4, PE-labeled anti-TCR{alpha}ß or a supernatant against TCR{gamma}{delta} plus PE-labeled rabbit anti-mouse Ig followed by SA–CY.

 
DC but not B cells develop in rSCID-FTOC
Absence of rat B cells in rSCID-FTOC was demonstrated by flow cytometry after staining with OX-12 mAb, which specifically binds to the {kappa} light chains of rat Ig (Fig. 6AGo).



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Fig. 6. (A) The presence of rat B cells was evaluated by the expression of {kappa} L chains recognized by mAb OX12 conjugated to PE. No reactivity was found among the recovered rCD45+ cells. (B) Cell suspensions from rSCID-FTOC were stained for rat MHC class II expression (FITC-labeled mAb OX6). Both MHC class IIhi cells (left histogram: H) and large size cells (right histogram: L) were gated as indicated by horizontal bars in order to determine the presence of rat DC. Percentages of such gated cells were determined at days 12, 16 and 24 of culture (C). (D) Percentages of rat DC were also evaluated by detection of MHC class II expression on cytospin preparations from cell suspensions of rSCID-FTOC cultured lobes.

 
On the other hand, DC of rat origin were determined by flow cytometry with OX6 mAb which recognizes the RT1.B product of rat MHC class II, a homologue of the mouse I-A molecule. Its identification was achieved by gating for large size and MHC class IIhi cells (Fig. 6BGo) in an attempt to exclude the MHC class IIlo-expressing thymocytes. Their relative values remained nearly the same from day 12 to 16 but they sharply increased from day 16 to 24 (Fig. 6CGo). To further corroborate the presence of rat DC in rSCID-FTOC, cytospin preparations stained for MHC class II were carried out from the cultures and the numbers of MHC class II+ DC estimated. Accordingly, DC were clearly distinguished from the rest of the MHC class II+ cells by their stronger expression of the antigen, which further correlated with cells with an eccentrically located nucleus, irregular shape and long cytoplasmic processes (Fig. 6DGo), characteristic of thymic DC.


    Discussion
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 
In the present work, we used flow cytometry to study the cell populations contained in the liver of 13-day-old fetal Wistar rats. In addition, both, highly enriched, CD45+ and CD45 r13FL cells were assayed for their capacity to in vitro repopulate thymic lobes from 14-day-old fetal SCID mice.

A small fraction of r13FL expresses the leukocyte common antigen CD45 as previously demonstrated for lymphoid committed cells from human (12) and mouse (13). In addition, Thy-1-expressing cells, and CD71-, CD43-, MHC class I- and CD44-reactive cells occur in the rat liver at this stage of development. In contrast, T cell related antigens including CD3, TCR{alpha}ß, TCR{gamma}{delta}, CD2, CD5, CD8, CD25 or CD28 as well as NKR-P1a, MHC class II, CD45RC or sIg are not detected in r13FL, confirming the lack of mature lymphoid cells. On the other hand, combined analysis of these cell markers shows that CD45+ r13FL cells constitute a homogeneous Thy-1hiCD71CD44+ cell population, whereas CD45 cells from r13FL heterogeneously express Thy-1, CD71 and CD44 antigens. Accordingly, this pattern of cell marker expression makes this CD45+ early fetal liver cell population phenotypically distinct from immature cells found in both fetal and adult rat thymus which express CD45RC, CD5 and CD2 (18) (unpublished observations). On the contrary, it resembles both the CD45RCThy-1hiCD43lo (10,19,20) and CD45R subpopulations (17) described in rat bone marrow to contain thymus repopulating cells.

In addition, CD45+ r13FL cells, but not the CD45 cell subpopulation, are capable of repopulating FTOC established from 14-day-old SCID mice, and differentiating into rat T lymphocytes (both TCR{alpha}ß+ cells and TCR{gamma}{delta}+ cells), NK cells and DC but not B lymphocytes. However, other attempts for further separation of the r13FL cells using expression of either MHC class II, CD4 or other cell lineage markers (see above), as reported in humans and mice (2124) were unsuccessful. In agreement with our results, all stem cell activity demonstrated in the liver of 14-day-old fetal rats resides in the CD71Thy-1hi fraction (11). Thus, our results indicate that CD45 is a suitable cell marker to separate rat fetal liver cells with distinct lympho-hemopoietic activities.

In the absence of clonal assays it is impossible, however, to assess whether CD45+ r13FL cells are really oligopotent cell progenitors or whether our results reflect, on the contrary, the existence of a mixture of unipotent cell precursors with similar cell surface phenotype. In fact, the nature of cell progenitors which colonize the thymus is really a matter of discussion. Several authors have proposed the existence of a common multipotent T/B/NK/thymic DC cell precursor which arrives at the thymus (2531). However, a single-cell assay which discards the arrival of oligopotent progenitors for each cell type rather than a unique common precursor is lacking. Early fetal thymic cells with a capacity to generate T cells as well as B lymphocytes and myeloid cells have also been described (32) but the occurrence of a single common precursor remains again to be demonstrated. Despite these results, which support that the thymus is colonized by multipotent cell precursors, Rodewald et al. (6) demonstrated the existence of a circulating T-restricted cell precursor in the blood of 15.5-day-old fetal euthymic and nude mice. In addition, Kawamoto et al. (33) recently reported the occurrence of such a restricted T cell precursor in the liver of 12-day-old fetal mice as well as of bipotent myeloid/T cell precursors and multipotent myeloid/T/B cell progenitors but not bipotent T/B cell precursors. Accordingly, the thymus could be colonized by multipotent cells but also by restricted T cell precursors.

The xenogeneic model of SCID mouse FTOC used in the current study represents, on the other hand, a better system to in vitro assay the lympho-hematopoietic capacities of partially purified rat cell populations than other ones previously reported. By employing SCID animals as a source of fetal thymic lobes, it overcomes the necessity of a prior treatment with deoxiguanosine (dGuo) or irradiation, both of which would result in severe damage of the thymic stroma. Thus, thymopoiesis was much less prominent in in vivo rat SCID mouse chimeras when they were prepared with 400 rad than in those established with 250 rad (15). Likewise, dGuo-treated mouse FTOC co-cultured with rat bone marrow cells provide a poor cell recovery and incomplete rat T cell development (14) (unpublished observations) although the different source of cell progenitors could also explain the differences.

Moreover, rat TCR{alpha}ß+ cell development is similar to that occurring during rat thymus ontogeny (34). Immature TCR{alpha}ß–/lo cells occur during the first few days of culture (days 10–12) to be later substituted by DP-TCR{alpha}ß+ cells (day 16) and then SP-TCR{alpha}ßhi cells, the number of which expands to day 24. Whole liver cell suspensions from 15- to 17-day-old fetal rats intravascularly injected in adult C.B17-SCID mice also provide a full spectrum of both immature and mature TCR{alpha}ß-expressing cells ({gamma}{delta} expression was not reported) but the process requires a long time period and seeding of donor progenitors from the chimeric bone marrow into the thymus cannot be discarded (15). In addition, in dGuo-treated mouse FTOC colonized by rat bone marrow cell precursors, DP TCR{alpha}ßlo cells are generated but only a few CD8+ SP TCR{alpha}ßhi cells mature (14,17).

Both, CD4+ SP and CD8+ SP, TCR{alpha}ßhi cells are generated in our rSCID-FTOC but the latter accumulated in the oldest cultures (day 24). In rat FTOC we have already observed a similar skewing of the SP cells to the CD8 cell lineage (35), and, as mentioned above, rat bone marrow cells repopulate dGuo-treated mouse FTOC providing both DP and a few CD8+ SP thymocytes but not CD4+ SP cells (17). In this regard, it is important to remark that the accumulation of CD8+ SP TCR{alpha}ßhi cells from day 16 to 24 is accompanied by a parallel increase of TCR{alpha}ßhi cells among the DP cells which are mainly CD4loCD8hi and large sized elements. This observation, which correlates with that reported previously for mouse DP thymocytes committed to the CD8+ SP cell lineage (36,37), seems to indicate that CD8+ SP cells, rather than CD4+ SP cells, are being preferentially generated. Furthermore, the lower number of CD4+ SP than CD8+ SP TCR{alpha}ßhi cells observed at day 24 was accompanied by a lack of TCR Vß10 usage, a TCR Vß segment largely expressed by rat T cells restricted to MHC class II recognition (38). Also, a higher proliferative activity of the CD8+ SP rather than the CD4+ SP cell subset could alternatively contribute to this skewing. On the other hand, it could be attributed to the fetal origin of both rat cell precursors and SCID mouse thymic stroma, as previously indicated (39). Remarkably, rat FL cells give rise to an adult CD4:CD8 cell ratio when in vivo inoculated in neonatal SCID mice either intravascularly (40) or intrathymically (unpublished observations). In the other hand, Mitnach et al. (41) observed that mouse and rat CD4+CD8+ DP thymocytes show opposite lineage decisions upon identical in vitro culture conditions, i.e. the former generate CD4 SP while rat DP cells exclusively produce CD8 SP cells. Thus, the CD8 SP skewed phenotype observed by us could be also partially explained by this in vitro preferential CD8 cell lineage by rat DP cells.

Not only rat TCR{alpha}ß+ thymocytes but also {gamma}{delta} cells are recovered from the rSCID-FTOC supplied with CD45+ r13FL cells. In this regard, the reconstitution with {gamma}{delta} T cells by rat lymphoid precursors has not been examined to date. In mice, total 12- to 14-day-old mouse FL cell suspensions almost completely recover the thymic and peripheral {gamma}{delta} cell population of neonatal SCID mice (42). Most recovered rat TCR{gamma}{delta} cells highly express the CD8 co-receptor, suggesting that they represent mature cells, as previously reported (43), as compared to CD8lo {gamma}{delta} T cells, which could be precursors of the former ones. Furthermore, DP {gamma}{delta} T cells accumulate in the long-term cultures (24 days). A similar phenotype has been reported to occur among {gamma}{delta} thymocytes obtained from mouse FTOC after long periods of culture and in the thymus of late embryonic and perinatal but not adult mice (44,45). It is tempting to speculate that DP TCR{gamma}{delta} cells could constitute a rare population of mature TCR{gamma}{delta} cells largely present during thymic development which expands in some experimental conditions as reported after IL-10 addition to mouse FTOC (46).

The presence of NK cells among the recovered rat CD45+ cells was monitored by detection of the NKR-P1a molecule which is expressed at high levels on rat NK cells (47). In our rSCID-FTOC cultures, a heterogeneous population of NKR-P1a+ cells already occurred at day 12, gradually increasing to reach 7% of the total rat recovered cells after 24 days of culture. The proportion of NKR-P1alo cells is higher than that of thymic NK cells highly expressing NKR-P1a and, throughout the culture period, both cell subpopulations, but especially the NKR-P1alo cells, up-regulate the expression of CD8, CD4, TCR{alpha}ß and TCR{gamma}{delta}. These NKR-P1alo cells could, therefore, be related to a recently defined NK T cell population in rats (48) as in mice and humans (49) which shares typical NK cell markers and TCR. In rats, NKR-P1aloTCR{alpha}ß+ are mainly CD8+ SP cells (48, 50) rather than CD4 SP or DN, as described for mouse NK T cells (49). Accordingly, NKR-P1a+ TCR+ cells recovered from rSCID-FTOC were mainly CD8 SP but, strikingly, DP as well. We have previously observed DP NKR-P1a+ cells in fetal rat thymus (unpublished observations). Thus, this rare cell population could be expanded in the current xenogeneic rSCID-FTOC conditions. In fact, the relevance of culture conditions was manifested by Knudsen et al. (51) who cloned rare NK T cell populations from rat spleens which were mainly CD4CD8 DN or CD4+ SP and restricted in their expression of TCRVß antigens but only after culture in MIP-1{alpha} plus IL-2 containing medium.

On the other hand, the origin of NK T cells is a matter of discussion. Because of both their abundance in the peripheral lymphoid organs and occurrence in athymic nude mice, it has been suggested that these cells develop extrathymically (5254) although it does not preclude their intrathymic generation. In fact, other results, in agreement with our current study, show that NK T cells could also develop within the thymus and are, at least in part, the precursors of peripheral NK T cells (55,56).

On the contrary, NKR-P1ahi cells which gradually increase the CD8 expression could be `conventional' rat NK cells which strongly express NKR-P1a but not TCR{alpha}ß (47,48). However, an important proportion of this cell population positively regulates the expression of both TCR{alpha}ß and TCR{gamma}{delta} molecules after 24 days in culture. The occurrence of NKR-P1ahi TCR{alpha}ß+ cells in our cultures could be a consequence of the special SCID thymic microenvironment in which rat FL cell precursors develop. In this regard, Shimamura et al. (52) recently demonstrated that 14-day-old fetal liver cultures established from either normal or athymic mice in the presence of supernatants of WEHI-3 cells and phorbol myristate acetate-stimulated EL-4 cells provide TCR{alpha}ß+ cells, virtually all co-expressing NK1.1, a mouse equivalent molecule to rat NKR-P1a. Alternatively, NKR-P1alo TCR{alpha}ß+ cells could up-regulate the NKR-P1a expression to become NKR-P1ahi TCR{alpha}ß+ cells. Accordingly, we might conclude that CD45+ r13FL cells contain NKR-P1a cell precursors which differentiate in the SCID thymic environment.

DC are professional antigen-presenting cells essential for initiating T cell immune responses in the periphery and involved in T cell selection inside the thymus. Occurrence of a common cell precursor for thymocytes and thymic DC was demonstrated in the mouse adult thymus (2527) and further confirmed by several studies (28,5759). Our results further support the existence of precursors for both thymocytes and thymic DC in the CD45+ r13FL cells which mature concomitantly in the rSCID-FTOC. Previously, immunohistochemical analysis of thymic cryosections from mild-irradiated adult SCID mice in vivo repopulated with rat 15- to 17-day-old FL cells showed rat MHC class II+ cells, which were identified as presumptive macrophages and/or DC (15). Moreover, the kinetics of development of both thymocytes and thymic DC appears to occur in parallel in our cultures, as previously described in mice (27,57). In this regard, it has been suggested that this mechanism assures that immature T cells are selected predominantly by intrathymic self antigens (57). Remarkably, DC yields sharply increase from day 16 to 24 of culture, when a prominent appearance of TCR{alpha}ßhi cells takes place.

Despite the above-mentioned occurrence of a presumptive multipotent cell progenitor for thymocytes, B lymphocytes, NK cells and DC, we were unable to detect B lymphocytes in our cultures. Experimental conditions could, at least in part, explain our results. Only in a small number and, sometimes, only transient B cells are generated when dGuo-treated fetal thymic lobes and FL cells are co-cultured (60). On the other hand, the capacity of mouse fetal thymic stroma for supporting B lymphopoiesis reported by Kawamoto et al. (33) only occurred after addition of a cytokine cocktail to the cultures. In addition, committed lymphoid precursors have been isolated from the mouse thymus which upon intrathymic injection give rise to only T cells and thymic DC but when i.v. injected they also generated B lymphocytes (2527). Alternatively, it could be possible that our cultures contain immature B cells which we cannot detect with the reagents used (i.e. a mAb against {kappa} chain of Ig) because, as known, the appearance of light chains of Ig is a late event during B cell maturation. A similar situation was reported after 15 days of recolonization of dGuo-treated mouse FTOC by 1–2x105 total rat bone marrow cells (14). Further analysis using rat B cell markers specifically expressed in early B lymphopoiesis is necessary to clarify these results.


    Acknowledgments
 
This work was supported by CAYCIT grant no. PB94-0332, from the Spanish Ministry of Education and Science. L. M. A.-C is supported in part by a fellowship from the Spanish Ministry of Education and Science. We wish to thank Dr M. A. Rodríguez-Marcos and Dr J. Palacín from the CBM (UAM, Cantoblanco, Madrid) for the generous supply of SCID mice, Dr T. Hünig for generous gift of antibodies and Carmen Hernández for help in preparing the manuscript. The FACScan flow cytometer was from the Servicio Común de Investigación (Faculty of Biology, UCM, Madrid).


    Abbreviations
 
CYCyChrome
DCdendritic cell
dGuodeoxiguanosine
DNdouble negative
DPdouble positive
HSChematopoietic stem cell
MBMicroBeads
PEphycoerythrin
rSCID-FTOChybrid rat -> SCID mouse fetal thymic organ culture
r13FLrat 13-day-old fetal liver
rCD45rat CD45
SAstreptavidin
SPsingle positive

    Notes
 
Transmitting editor: T. Hünig

Received 18 September 1998, accepted 25 March 1999.


    References
 Top
 Abstract
 Introduction.
 Methods
 Results
 Discussion
 References
 

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