The ontogeny of B cells in the thymus of normal, CD3{varepsilon} knockout (KO), RAG-2 KO and IL-7 transgenic mice

Rhodri Ceredig

Laboratoire d'Immunochimie, U548 INSERM, Commissariat à l'Energie Atomique-Grenoble, Département de Biologie Moléculaire et Structurale, Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble, France

Correspondence to: R. Ceredig, E-mail: ceredig{at}dsvsud.cea.fr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The ontogeny of thymic B cells was determined by three-color flow cytometry and the presence or absence of B cell progenitors confirmed by cell culture experiments. In the thymus of young normal mice, CD117+, B220low pro- and pre-B cells are present but disappear with age. B220low, CD5+, B-1 B cells are present in the thymus of older animals following the appearance of similar cells in the peritoneal cavity and blood. In CD3{varepsilon} gene-deleted mice, the phenotypic progression and number of thymic B cells remains unaltered, showing that blocking T cell development does not automatically result in an increase of thymic B lymphopoiesis. Pro-B cells in RAG-2 knockout mice are found in the fetal and neonatal blood, spleen and thymus, but with increasing age are only found in the bone marrow. B lymphopoiesis in adult IL-7 transgenic mice is dramatically altered with CD117+ pro- and pre-B cells present in spleen, lymph node and blood. In the thymus of adult IL-7 transgenic mice, the fraction of CD117+ thymic B cells is significantly increased. These results show that in the steady state, the phenotype of thymic B cells is critically dependent on both mouse age and the phenotype of circulating B cells.

Keywords: B cell development, B-1 cells, IL-7, T cell development


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It has been known for some time that B lymphocytes are present in the thymus (1,2), often associated with thymic fibroblasts (3). Based on several criteria, most early reports described thymic B cells as activated cells (1). With the division of peripheral B cells into so-called Ly-1/CD5+ (B-1) and Ly-1/CD5-, `conventional' (B-2) subpopulations, thymic B cells were found to represent B-1 cells (4,5). Along with other non-T cell elements in the thymus, thymic B cells were thought to be involved in the maintenance of T cell tolerance to self antigens, including those expressed by B cells (6,7), such as Ig molecules (8,9) or viral superantigens (6). However, it would appear that B cells are not the only cell type responsible for tolerance induction to the latter (10).

Much is known about the phenotypic and molecular stages of B cell development in the bone marrow (BM) (11–13). Cells in the process of rearranging Ig heavy chain (IgH) genes are subdivided into pro- and pre-B-1 cells, both of which express CD117 (or c-kit) and CD127. At the molecular level, pre-B-1 but not pro-B cells have IgH rearrangements. Following successful IgH rearrangement and expression, pre-B-1 cells proliferate and transit into the CD25+, CD127+, CD117- pre-B-2 compartment and following IgL chain rearrangements, they express a mature BCR and become IgM+, CD25-, CD127- immature B cells. Following migration from the BM, immature B cells become so-called transitional cells (14), increasing IgM expression and becoming IgD+. Until this point, developing B cells express the C1q receptor, recognized by mAb 493 (15) or AA4.1 (16). Expression of 493 is lost following differentiation of transitional cells to mature, conventional, (B-2) B cells, which are CD5-, CD11b-, CD23+ (15). Much less is known about the early and intermediate stages of B-1 cell development (17), but mature CD5+ B-1 cells are CD11b+ and CD23- (13).

The developmental status of thymocytes can be characterized by both flow cytometry and TCR gene rearrangements (18). Following successful TCRß gene rearrangements, cells expressing neither CD4 nor CD8, so-called double-negative (DN) cells differentiate to CD4+CD8+ double-positive thymocytes and following TCR{alpha} rearrangement and selection to single-positive cells. Based on CD44 and CD25 expression, DN cells can be further subdivided into four subpopulations (19), i.e. CD44+CD25- (DN#1), CD44+CD25+ (DN#2), CD25+CD44- (DN#3) and finally CD44-CD25- (DN#4) cells. As a population, DN#1 cells can reconstitute T, B, NK and dendritic cell lineages (20,21). Phenotypically, they are heterogeneous, containing some CD117+ cells (22), thought to represent recent thymic immigrants from the BM. It is possible that DN#1 cells contain a mixture of lineage-committed progenitors (23–25).

Recently, several reports have described B lymphopoiesis in the thymus (26–30). The thymus of transgenic mice carrying multiple copies of a human CD3{varepsilon} transgene was small (1.7x106 cells) containing 1.2x106 B cells, 6 times that of a normal mouse (27,31). Mice in which the Notch gene was selectively and inducibly deleted in adult BM progenitors had a 200-fold increase in thymic B cells (28). In TCRß knockout (KO) mice, the thymus contained 1.2x106 B cells (30) and, based on cell labeling experiments, the thymus was shown to participate actively in B lymphopoiesis. These studies concluded that cells migrating from BM to the thymus were capable of becoming either a T or a B cell. Such B/T bipotent cells, also called common lymphocyte progenitors (CLP), are B220+/-, CD117+, CD127+, AA4.1+ and express the receptor Flt3. CLP were initially described in the BM (32–34) and more recently in the fetal liver (35). However, it is unclear whether CLP are normally present in the thymus.

In order to analyze in more detail the phenotype of thymic B cells, an ontogenic study was carried out in normal, CD3{varepsilon} KO (36), RAG-2 KO (37) and IL-7 transgenic (38) mice. Results show that the phenotype of thymic B cells depends critically on the age of the mouse and that blocking thymocyte development does not result in a compensatory increase in thymic B lymphopoiesis.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice and cell suspensions.
Normal C57Bl/6 mice were produced in the conventional animal facility of the Commissariat à l'Energie Atomique (CEA-G, Grenoble, France). The day of finding a vaginal plug was considered as day 0 of development. RAG-2-/- (RAG-2 KO) (37) and CD3{varepsilon}{Delta}5/{Delta}5 (CD3{varepsilon} KO) (36) mice on a C57Bl/6 background were maintained in the specific pathogen-free animal facility at CEA-G. IL-7 transgenic mice on a C57Bl/6 background (38) were maintained at the Basel Institute for Immunology (BII) and analyzed there.

Thymus, BM, spleen and peritoneal cells (PerC) were obtained by standard procedures from mice killed by CO2 inhalation, and cell suspensions prepared in PBS, 1% FCS and 0.1% NaN2 (FACSwash). For peripheral blood lymphocytes (PBL), mice were anesthetized with chloroform and blood removed by cardiac puncture into heparinized syringes. To exclude parathymic lymph nodes, thymuses from RAG-2 KO and CD3{varepsilon} KO mice were removed using a dissecting microscope. Fetal mice were washed free of maternal blood in a large volume of FACSwash. Fetal PBL in heparinized FACSwash were obtained from the cut umbilical cord. Fetal organs were isolated using a dissecting microscope and cell suspensions prepared by passing them through needles of decreasing diameter. Lymphocytes were purified from heparinized PBL by centrifugation at 20°C over a cushion of Hypaque-Ficoll (Pharmacia, Uppsala, Sweden). Thymus cell suspensions were depleted of CD8+ and CD4+ cells by antibody and rabbit complement as previously described (39) and the resulting CD4-CD8- DN cells purified by Hypaque-Ficoll separation.

Cell culture
To culture pro- and pre-B cells, use was made of the ST-2 + IL-7 culture method as previously described (40). Replicate cultures, containing dilutions of 105 to 3x102 thymus cells suspended in 100 µl DMEM, 10% FCS and 20% IL-7-containing supernatant (a gift of Dr Ton Rolink, BII), were added to wells of 96-well microtiter plates containing 104 2000 rad-irradiated ST-2 cells in 100 µl DMEM and plates incubated at 37°C in a humidified incubator containing 5% CO2 in air for 7–10 days. Wells containing between one and three B cell colonies were pooled, washed, stained and analyzed by FACS. The plating efficiency of the culture system was verified using day 14 fetal liver cells as control; positive controls were included in all experiments.

Fetal thymus organ cultures (FTOC) were set up as previously described (41) in DMEM and 10% heat-inactivated FCS supplemented with 10% IL-7 supernatant. In some experiments, organ cultures were of neonatal RAG-2 KO or CD3{varepsilon} KO thymuses; in these instances, thymic lobes were cut into four pieces and fragments cultured on filters as for FTOC.

Flow cytometry
Three-color flow cytometry was carried out with a FACSCalibur (Becton Dickinson, Mountain View, CA) flow cytometer calibrated with CaliBRITE beads and AutoCOMP software and using, wherever possible, the same instrument settings. Photomultiplier voltages were: FL1 637, FL2 651, FL3 468 and FL4 757 with compensations FL1–FL2 0.8, FL2–FL1 24.3 and FL3–FL2 2.4. In order to remove cellular debris, all cell suspensions were filtered through fine nylon mesh prior to staining. Cells in 100 µl FACSwash were stained in 96-well microtiter plates as previously described (42) in combinations of pre-titrated FITC-, phycoerythrin (PE)-, Cy5-, allophycocyanin (APC)- and biotin-labeled antibodies. Labeled antibodies were either purchased from PharMingen (San Diego, CA) or were kindly provided by Drs Rolink and Andersson (BII). For analysis of B cells, cells were routinely stained with CD19–FITC (ID3; PharMingen), B220–Cy5 (RA3/6B2; BII) and biotin-labeled third mAb. For DN thymocytes, cells were also stained with CD25–FITC (PharMingen), CD44–APC (PharMingen) and biotin-labeled third mAb. Biotinylated antibodies used included anti-CD5 (clone 53-7.3; PharMingen), anti-CD25 (7D4; PharMingen), anti-CD117 (2B8, anti-c-kit; BII), anti-CD127 (A7R34, anti-IL-7R{alpha}; BII), 493 (identical to AA4.1; BII) and anti-IgM (II/41; PharMingen). The anti-CD23 mAb (B3B4, PharMingen) was a direct conjugate with PE. After a 20-min incubation at 4°C, cells were washed with 150 µl FACSwash and biotin-labeled antibodies revealed with streptavidin–PE (Southern Biotechnology, Birmingham, AL). After 20 min at 4°C and a further wash, stained cells were resuspended in FACSwash containing 1 µg/ml propidium iodide (PI) (Sigma, St Louis, MO). Viable lymphocytes were identified by a combination of FSC, SSC and PI- (FL3) signals, and list mode data from up to 3x105 events analyzed with WinMDI 2.8 software (Joe Trotter, Salk Institute, CA). Data is presented as cytograms where horizontal quadrant bars are set according to streptavidin–PE-labeled negative controls and vertical bars arbitrarily set according to B220–Cy5 or CD44–APC fluorescence intensity.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Heterogeneity of CD44 and B220 expression by thymic B cells
Based on CD44 and CD25 expression, DN thymocytes may be subdivided into four subpopulations, DN#1–DN#4 (Fig. 1AGo) (19). The DN#1 subset contains different cell types including CD19+ B, NK1.1+ NK or NKT and CD11c+ dendritic cells (not shown). CD117 expression by DN cells varies widely with 19.4% being CD117++ (Fig. 1BGo). Gating on the 3.7% CD117++ total DN cells (boxed area in Fig.1CGo), two distinct subpopulations of DN#1 and DN#2 cells can be clearly identified (Fig. 1DGo) with CD44 expression lower on CD25+ DN#2 than on CD25- DN#1 cells. CD117++ cells represent 3.12 ± 0.68% of DN thymocytes and in this experiment 1.6x105 cells/thymus. Using the lower level of CD44 expression on DN#2 cells, the distinction can be made between DN#2 and the subsequent CD25+ DN#3 subpopulation where CD44 and CD117 expression decreases progressively (Fig. 1A and CGo). DN#4 cells are characterized by low-to-negative expression of CD44, CD25 and CD117. Three-color analysis of purified DN cells indicated that CD44 expression by CD19+ thymic B cells was heterogeneous (Fig. 1EGo), extending below that defined by CD117++ DN#1 and DN#2 cells to that on DN#3 cells.



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Fig. 1. Heterogeneity of CD44 and B220 expression by normal thymic B cells. Purified DN cells were stained with the indicated markers. Cytogram displays from the indicated cells are from files containing 100,000 events gated on a combination of FLS, FSC and PI- signals, and are typical of one of four such experiments. Horizontal quadrant bars in each display were set using negative controls, whereas vertical bars were arbitrarily set. Values in each panel represent the percentage of events.

 
To identify thymic B, most previous studies have relied on B220 expression, a marker not confined to B cells, but expressed on some T cells (43), a heterogeneous group of cells including NK progenitors in the BM (44) and thymus (45), and some CD25+ DN#3 cells (46). In addition, not all CD19+ B cells express B220 at similar levels; so-called B-1 cells are characteristically B220low (13). CD19+, B220low cells were clearly evident among DN cells (Fig. 1FGo). In this experiment, the number of B cells in the thymus was 9.2x104. Note that B220 expression on thymic B cells extends to what would be regarded as B220-.

B220lowcells in the adult thymus are B-1 cells
Initially, heterogeneity of B220 expression was thought to be a unique property of CD19+ adult thymic B cells. However, analysis of B cells in other lymphoid organs and the PerC of adult mice indicated a correlation between the presence of CD19+, B220low cells and the presence of so-called B-1 or CD5+, CD11b+ cells (see below). As shown in Fig. 2Go, CD19+, B220low B-1 cells appear at about day 14 in both PBL (Fig. 2AGo) and PerC (Fig. 2BGo) and slightly later in the thymus (Fig. 2CGo). In these experiments, PBL, PerC and thymus cells were analyzed from the same mice, indicating that thymic B cells were not simply PBL contaminants. The separation between the two subsets of CD19+ B220++ (B-2) and B220low (B-1) cells was sometimes distinct (Fig. 2AGo, PBL day 14). Weak B220 expression is a property shared by pro/pre-B and B-1 cells, but the former are CD117+, CD127+, 493+, IgM-, CD5-, CD11b- and MHC class II- whereas B-1 cells are CD117-, CD127-, 493-, IgM+, CD5+, CD11b+, CD23- and MHC class II+. To fully characterize B220low thymic B cells throughout ontogeny, mAb to the above markers were routinely used. Some antigens, including CD117, CD127, CD25 and CD5, can be passively acquired by thymic B cells from surrounding thymocytes (7). In this regard, expression of the C1q receptor, as recognized by mAb. 493, CD23 and IgM are useful in that thymocytes are not stained.



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Fig. 2. Ontogeny of CD19 versus B220 expression by normal B cells. Shown are CD19 versus B220 cytogram displays of (A) PBL, (B) PerC and (C) thymic B cells from normal mice at the indicated ages. In each quadrant, the percent events are shown.

 
Shown in Fig. 3Go(A–F) are the profiles of total adult BM (containing pro/pre-B cells), and the CD19-gated profiles of PerC (containing B-1 B cells), neonatal thymus, neonatal PBL, adult thymus and adult PBL. In the neonatal thymus, CD117+, CD127+, 493+, CD19+ pro/pre-B cells were indeed present, but in the adult thymus, they were not detected. The level of CD117 expression by thymic (Fig. 3CGo) and BM (Fig. 3AGo) pro/pre-B cells is similar and is lower than that on CD117++ DN (Fig. 1B and CGo) and B220- BM cells (Fig. 3AGo, left panel). In contrast, CD5+ B220low cells were only seen in the adult thymus; such cells in PerC and thymus were CD11b+, MHC class II++ and larger in cell size (not shown). Among DN cells, 0.3–2% (2–12x104 cells/thymus) were CD19+, B220+ B cells (Fig. 3GGo). The decreasing proportion of CD117+ cells among the relatively constant number of thymic B cells indicates a decrease in their absolute number during development.




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Fig. 3 A–D Ontogeny of thymic B cells in normal mice. Shown are cytogram displays of (A) total cells from adult BM or the CD19-gated profiles of (B) adult PerC, (C) day 10 neonatal thymus, (D) neonatal PBL, (E) day 42 adult normal thymus and (F) adult PBL. In each quadrant, the percent events are shown. (G) Summary of a series of experiments with DN cells from normal mice and each point represents the mean data from at least two experiments. (Parts E–G are shown overleaf.)

 
Previous functional studies had indicated that clonable pre-B cells appear transiently in the PBL and spleen of neonatal mice (40), and therefore the disappearance of CD117+ thymic B cells could be a consequence of a decrease in circulating B cell precursors. FACS analysis (Fig. 4A and BGo) confirmed that CD117+, B220+ pro/pre-B cells were present in the fetal and neonatal spleen and PBL, but disappeared with increasing mouse age. In addition, the fetal blood was found to contain 493+ IgM+ B cells, even in embryos from RAG-2 KO-deficient mothers mated with RAG-2 KO-proficient fathers (Fig. 4CGo), confirming that fetal IgM+ B cells are embryo derived.



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Fig. 4. Ontogeny of B cell development in spleen and PBL of normal mice. (A and B) Summary of a series of experiments, each point representing at least two determinations. (C) Cytogram displays of RAG-2 KOxWT day 17 fetal PBL stained for CD19–FITC and B220–Cy5 (total cells, left panel), and the CD19-gated profiles of B220–Cy5 versus the indicated markers in PE (CD19+, right three panels).

 
Phenotypic analysis of B cells in CD3{varepsilon} KO mice
Reports indicated that thymic B cell numbers increased when T cell development was impaired (27,28,30,31). Therefore, it was decided to investigate the thymic B cell compartment in CD3{varepsilon} KO mice (36) which, because of a failure to express CD3-containing functional pre-TCR complexes, have a severe block in T cell development, the thymus containing 2–3x106 cells. Phenotypic analysis of CD3{varepsilon} KO thymocytes confirmed the presence of DN#1 to DN#3 cells (Fig. 5AGo); thymic B cells were found amongst CD44+, CD25- cells (Fig. 5AGo, lower right). Interestingly, the proportion and absolute number of CD117++ DN#1 cells among CD3{varepsilon} KO thymocytes was drastically reduced, representing only 0.3% (0.44 ± 0.22%) of total thymocytes at 4 weeks of age (Fig. 5AGo, top right) and in absolute number (9x103/thymus), only 5.6% that in a normal mouse. Most DN#1 cells (55.4%) expressed low levels of CD117 (Fig. 5AGo, top middle) and were NK1.1+, CD127+ (data not shown). Changes in the B220/CD19 profile of thymic B cells were again noted and B220low, CD19+ B-1 cells present by day 42 (Fig. 5BGo). In day 14 neonatal mice, CD117+, B220low thymic B cells could be clearly identified (Fig. 5CGo) and their relative proportion decreased with age (Fig. 5DGo). The proportion of CD19+ cells varied from 0.5 to 2.5% with no consistent changes with age (Fig. 5DGo). Analysis of B220low cells in PerC and PBL of CD3{varepsilon} KO mice indicated that, like their thymus counterparts, they were B-1 cells (data not shown). Thus, B-1 cell development is normal in CD3{varepsilon} KO mice and changes in thymic B cell phenotype reflect this.




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Fig. 5. Thymic B cells in CD3{varepsilon} KO mice. (A) Cytogram displays of thymocytes from mice stained with the indicated markers. See Fig. 1Go legend for details. (B) CD19 versus B220 cytograms of thymic B cells from mice of the indicated ages. (C) The CD19-gated profiles of thymic B cells stained with the indicated markers. (D) Summary of a series of such experiments.

 
Thymic B cells in RAG-2 KO mice
To investigate the distribution of pro-B cells during development, use was made of RAG-2 KO mice. The RAG-2 KO thymus contained few (2x104) CD117++ DN#1 and DN#2 cells (Fig. 6AGo), a number representing 7.5% of that in a normal mouse and which remained constant throughout development (from day 18 fetal to day 360 post-natal). Again, most (63%) CD117low DN#1 cells were NK1.1+, CD127+ cells (not shown). FACS analysis of lymphocytes from RAG-2 KO mice of different ages (Fig. 6BGo) showed that pro-B cells were present in the PBL, spleen and thymus of young (day 10 postnatal) animals but, except for the BM, gradually disappeared with increasing age (Fig. 6CGo). Thus, in the neonatal period, pro-B cells are disseminated via the blood independently of the presence of mature T cells and maternal Ig.



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Fig. 6. Thymic B cells in RAG-2 KO mice. (A) Cytogram displays of RAG-2 KO mice stained with the indicated reagents. See Fig. 1Go legend for details. (B) CD19 versus B220 cytogram displays of cells from the indicated organs of day 10 neonatal mice. (C) Summary of a series of such experiments.

 
Cell culture experiments
The proportion of CD19+, B220+ B cells in the RAG-2 KO neonatal thymus was extremely low, and in normal and CD3{varepsilon} KO mice there was an age-related decrease in the proportion of CD117+ B cell progenitors. To verify that in vitro clonable B cell progenitors were present in the neonatal thymus, but apparently absent in older mice, use was made of the ST-2 + IL-7 culture system (40), where pro- and pre-B cells have a high plating efficiency and where pro- and pre-B cell clones from normal mice after 7–10 days of culture comprise ~2x104 cells which are CD19+, B220+ CD117+/-, IgM-.

In cultures of day one neonatal DN cells (Fig. 7AGo, left panel), CD19+, B220+ cells could be clearly identified, representing 59.3% of harvested cells. The remaining 9.9% CD19-, B220+, cells were a mixture of NK.1.1+, NK and CD11b+ myeloid cells (not shown). Further analysis showed that 89.0% of CD19+ cells were CD117+, 98.9% 493+ and 0.3% IgM+, phenotypic properties typical of pre-B cells (not shown). In cultures of day 10 neonatal DN cells, a smaller number (1.6%) of CD19+, B220+ pre-B cells were detected (Fig. 7AGo, middle panel), but DN cells from older (day 42) mice (Fig. 7AGo, right panel) failed to generate CD19+ B cells. This experiment was repeated five times with similar results.



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Fig. 7. Cell culture experiments. Shown are CD19 versus B220 cytogram displays of (A) normal DN cells from mice of the indicated ages. (B) Day 14 CD3{varepsilon} KO FTOC. (C) Fresh (upper panels) or ST-2 + IL-cultured cells from young and old RAG-2 KO (left panels) or CD3{varepsilon} KO (right panels) mice.

 
Culturing day 17 FTOC or day 1 neonatal thymus fragments as organ cultures yielded 0.19 and 0.25% CD19+, B220+ cells respectively, which for the latter were 19.8% CD117+, 66.4% 493+ and 15.9% IgM+ (not shown). Thus, FTOC seemed relatively inefficient at revealing the presence of pre-B cells. In FTOC of day 10 RAG-2 KO (not shown) and day 14 CD3{varepsilon} KO mice (Fig. 7BGo), 59 and 52% respectively of cells were CD19+, B220+, which for CD3{varepsilon} KO mice were 38.4% CD117+, 98.7% 493+ and ~6% IgM+ cells (not shown). Pro-B cells were also present in FTOC of the day 18 fetal RAG-2 KO thymus (not shown), thereby confirming the early in vivo seeding of the thymus by circulating pro-B cells. Thus, as shown previously, the thymus stroma contains elements capable of supporting B cell development (29,47,48), but with an unknown cloning efficiency. Therefore, all further experiments were only carried out with ST-2 + IL-7.

In Fig. 7Go(C) are shown the results of ST-2 + IL-7 cultures of one of three young or old thymuses from either RAG-2 KO (Fig. 7CGo, left panels) or CD3{varepsilon} KO (Fig. 7CGo, right panels) mice. Two important findings emerge from these experiments. Firstly, even though the original day 14 neonatal RAG-2 KO thymus contained very few (0.08%) CD19+ cells as detected by FACS, after culture, a large fraction (58.8%) of recovered cells were CD19+ pro-B cells. Thus, the culture system reveals the presence of rare cells not reliably detectable by FACS analysis. Secondly, for older CD3{varepsilon} KO mice, where the fresh thymus contained B220low/+, CD19+ B cells and older RAG-2 KO mice containing no detectable CD19+, pro- and pre-B could not be cloned. The oldest RAG-2 KO thymus from which pro-B cells could be grown was 62 days (1% of recovered cells CD19+) and 56 days for the CD3{varepsilon} KO thymus (1.1% CD19+).

Thymic B cells in IL-7 transgenic mice
The massively perturbed B lymphopoiesis in the BM, spleen, lymph nodes and PBL of adult IL-7 transgenic mice has already been described (49,50). As shown in Fig. 8AGo, upper left panel), in the thymus of 6-week-old IL-7 transgenic mice, 5.3% of DN cells were CD19+, a significant increase above values in normal age-matched controls (Fig. 3GGo), containing distinct subsets of CD117+, 493+, IgM- immature B cells (Fig. 8AGo, upper right panels). Culturing these DN cells on ST-2 + IL-7 (Fig. 8AGo, lower panels) generated CD19+ cells which were mostly (47%) CD117+, 96% 493+ and 96% IgM-. In a series of five individual 20-week-old mice, 6.3 ± 2.3% of the 1.5 ± 0.3x106 recovered DN cells were CD19+ B cells. Thus, the IL-7 transgenic thymus contained on average 9.4x104 B cells, a value not significantly different from controls. However, unlike normal mice, in 20-week-old transgenics (Fig. 8BGo), 73 ± 15% of PBL (Fig. 8BGo, left two panels) and 14.7 ± 8.2% of thymic CD19+ cells (Fig. 8BGo, right two panels) were CD117+. Some (10.4 ± 6.9%) thymic B cells were CD5+, B220low B-1 cells. Thus, when B lymphopoiesis is dramatically increased, the thymus contains an increased proportion of B progenitor cells.



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Fig. 8. Thymic B cells in IL-7 transgenic mice. (A) Cytogram displays of fresh (upper panels) or ST-2 + IL-7-cultured (lower panels) DN thymocytes from 6-week-old IL-7 transgenic mice. In each case, cytograms are of total (left panel) or CD19-gated cells (right four panels). (B) Cytogram displays of PBL (left two panels) or DN thymocytes (right two panels) from one of four 20-week-old IL-7 transgenic mice. For PBL and DN thymocytes, cytograms are of total (left) or CD19-gated (right) cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report, the ontogeny of thymic B cells in normal, CD3{varepsilon} KO and RAG-2 KO mice was determined by three-color flow cytometry. In young normal mice, B220+, CD19+, CD117+, CD127+, 493+, IgM- B cells, a phenotype shared with pro/pre-B cells in the BM, were found in the thymus. Such pro/pre-B cells could also be found in the peripheral blood and spleen of young normal mice, but with time they were only found in the bone marrow. Results with RAG-2 KO mice show that perinatal pro-B cell mobilization to the blood, spleen and thymus is independent of the presence of mature T cells and maternal Ig. With increasing age, the number of thymic B cells in normal and CD3{varepsilon} KO mice remained relatively constant, yet their content of pro/pre-B cells decreased. The presence of pro/pre-B cells in the young thymus and their disappearance with age was confirmed by experiments using the ST-2 + IL-7 cell culture system and organ cultures. Finally, in adult IL-7 transgenic mice where B lymphopoiesis outside the BM is maintained into adulthood, the proportion of pro/pre-B cells in the thymus was significantly increased. Taken together, these results show that the phenotype of thymic B cells depends critically on both the age of the mouse and the phenotype of circulating B cells. Blocking intrathymic T cell development does not necessarily result in a compensatory increase in thymic B lymphopoiesis.

With increasing mouse age, B cells of larger cell size, expressing a marker constellation typical of so-called B-1 B cells, accumulated in the thymus. In the peritoneal cavity and peripheral blood, B-1 cells appear after conventional B-2 B cells (51). Thus, the delay in appearance of thymic B-1 cells may either reflect the slower expansion of these cells in peripheral organs or, as has been suggested by others (17,52,53), that B-1 cells may be derived from B-2 cells. That B-1 cells appear in CD3{varepsilon} KO mice shows that their development is independent of T cells. It was rather surprising to find so many B-1 cells in the blood of adult mice.

Anatomically, B cells in the thymus are found mostly at the cortico-medullary boundary (30), often in association with fibroblasts (3). Functionally, thymic B cells are thought to be important for the maintenance of T cell tolerance to antigens expressed by B cells. However, it would appear that B cells are neither necessary for nor the only cell types in the thymus involved in tolerance induction to viral superantigens (10). Turnover of thymic B cells, as demonstrated by Akashi et al. (30), may be necessary for efficient T cell tolerance to B cell antigens. In this study, more B cells emigrated from the thymus than returned via the circulation which was interpreted as showing that thymic B lymphopoiesis contributes to the peripheral B cell pool (30).

By flow cytometry, expression of B220, but not CD19, by B-1 cells was found to vary considerably, reaching a level overlapping that of the negative control. One implication of this very low B220 expression is that some B-1 cells may be inadvertently omitted from analysis where only two-color flow cytometry is employed. Secondly, expression of B220 alone as a selection criterion for eliminating B cells from analysis and sorting would be insufficient to eliminate all CD19+ B cells.

Based on the combined expression of CD44 and CD25, DN thymocytes have been subdivided into four subpopulations (19). The earliest (CD44+CD25-) DN#1 cells are thought to include CD117+ BM-derived T cell progenitors, or pro-T cells. CD117 expression by normal DN#1 cells was found to vary considerably (Figs 1, 5A and 6AGoGoGo). The proportion and absolute number of CD117++ was very much dependent on mouse genotype and in both CD3 (Fig. 5AGo) and RAG-2 KO (Fig. 6AGo) mice, both of which have small thymuses, their number was <10% that of euthymic controls. At the present time, these differences are difficult to interpret, but could indicate either that the thymus of CD3 and RAG-2 KO mice contains fewer `niches' for progenitors or that an expansion phase of early progenitors does not take place in the thymus of these mice.

The thymus of mice expressing a transgene of the human CD3{varepsilon} antigen (27), where the TCRß gene had been deleted in the germline (30) or reconstituted with BM progenitors in which the Notch gene had been recently deleted (28) contained more B cells. Some of these B cells contained transcripts characteristic of pre-B cells or expressed pre-B cell markers. One interpretation of these results was that the cell colonizing the thymus might have T/B bi-potent potential. Such cells, called CLP, have been found in the BM (32,33), but under physiological conditions their presence elsewhere, including the thymus, has not been directly demonstrated. Indeed, single-cell analysis of the developmental potential of DN#1 cells from fetal mice has failed to reveal the presence of B/T bi-potent cells (24); whether such cells exist in the normal adult thymus is less clear (25). Cells with the CD19-, B220+/-, CD117+, CD127+, AA4.1+ (CLP) phenotype were not found in organ cultures of CD3{varepsilon} KO adult thymus fragments to which Flt3 ligand and IL-7 were added (not shown).

B lymphopoiesis in the BM is a very dynamic process that can be dramatically perturbed by physiological signals such as infection (54) or pregnancy (55) and by experimental procedures such as irradiation. Experiments with inducible Notch deletion involved treating mice with IFN-{alpha} in order to activate deletion of the `floxed' Notch gene. IFN-{alpha} is known to perturb both B lymphopoiesis in the BM and T lymphopoiesis in the thymus (56). Altering the expression of adhesion molecules on BM stromal cells can release immature B cells into the circulation and prevent mature B cells returning there (57). In CD3{varepsilon} KO (herein) or PT{alpha} KO mice (not shown), mutants in which T cell development is severely or partly blocked respectively, the total number of thymic B cells did not increase during ontogeny and the proportion of cells expressing pro-or pre-B cell markers decreased with time. Thus, blocking intrathymic T cell development does not automatically result in an increase in the number of total or progenitor B cells in the thymus.

The nature and phenotype of cells that enter the adult thymus under constant physiological conditions are poorly defined, but there is general agreement that they are few in number (58). It would appear that colonization of the thymus by circulating B lineage cells occurs in utero (2,48,59), and pro-B cells are in the circulation of neonatal normal and RAG KO mice. Experiments designed to investigate thymus colonization following irradiation (60–62) are often criticized as being non-physiological and recent results would suggest that in such circumstances, not all cells entering the thymus have progenitor activity (63). Pre-B cell clones from Pax-5 KO mice could recolonize the RAG KO thymus (64), suggesting pre-B cells are capable of entering the irradiated thymus. Results presented herein with IL-7 transgenic mice indicate that if pro- and pre-B cells constitute the majority of PBL B cells, they can be found in the thymus. In the steady state, progenitor T cell entry into the thymus is not a continuous process in time; the thymus `gate' permitting intermittent entry of prothymocytes (58). However, it is not known whether in the steady state the thymus gate is selective for the cell type that is allowed to enter. Taken together, one interpretation of the results presented herein is that the thymus gate is not selective, the phenotype of thymic B cells depending on what is available in the circulation at that time.


    Acknowledgments
 
This work was supported by Institutional grants from INSERM and the CEA. I thank Dr Serge Candéias and Eve Borel for the provision of CD3 and RAG-2 KO mice maintained at CEA-G, Drs Patrice Marche and Evelyne Jouvin-Marche (U548 INSERM) for their support, Véronique Collin-Faure for running the FACS facility, and Dr Ton Rolink, Dr Jan Andersson and Ernst Wagner for maintaining the colony of IL-7 transgenic mice at BII and for the provision of numerous reagents.


    Abbreviations
 
APC allophycocyanin
BM bone marrow
CLP common lymphoid progenitor
DN double negative
FTOC fetal thymus organ culture
KO knockout
PE phycoerythrin
PerC peritoneal cells
PI propidium iodide

    Notes
 
Transmitting editor: E. A. Kincade

Received 8 August 2001, accepted 10 October 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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