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T Cell Development from Kit-Negative Progenitors in the Foxn1^/ Mutant Thymus¹

Department of Genetics, Coverdell Center, University of Georgia, Athens, GA 30602

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Foxn1 is a hypomorphic allele of the nude gene that causes arrested thymic epithelial cell differentiation and abnormal thymic architecture lacking cortical and medullary domains. T cells develop in the Foxn1^/ adult thymus to the double- and single-positive stages, but in the apparent absence of double-negative 3 (DN3) cells; however, DN3 cells are present in the fetal thymus. To investigate the origin of this seemingly contradictory phenotype, we performed an analysis of fetal and adult DN cells in these mutants. Neither adult bone marrow-derived cells nor fetal liver cells from wild-type or Rag1^–/– mice were able to differentiate to the DN2 or DN3 stage in the Foxn1^/ thymus. Our data suggest that thymopoiesis in the Foxn1^/ adult thymus proceeds from CD117^– atypical progenitors, while CD117⁺ DN1a cells are absent or blocked in their ability to differentiate to the T lineage. Wild-type cells generated by this pathway in the postnatal thymus were exported to the periphery, demonstrating that these atypical cells contributed to the peripheral T cell pool. The Foxn1^/ adult (but not fetal) thymus also preferentially supports B cell development, specifically of the B-1 type, and this phenotype correlated with reduced Notch ligand expression in the adult stroma.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The adult mouse thymus receives bone marrow (BM)⁴-derived hemopoietic progenitors, supports thymocyte proliferation, differentiation, and repertoire selection, and then releases the mature T cells to the thymic periphery (1). Most, if not all, stages of thymocyte differentiation are mediated by the thymic stroma, which is mainly composed of thymic epithelial cells (TECs) (2). Although initial TEC differentiation in the fetal thymus is TEC intrinsic, continued TEC differentiation, and maintenance of adult thymic architecture is dependent on interactions between TECs and thymocytes (2). This interdependence of thymocyte and TEC differentiation is referred to as thymic cross-talk (3, 4). Although the stages of thymocyte differentiation, from the immature double-negative (DN; CD4^–CD8^– DN), to double-positive (DP; CD4⁺CD8⁺ DP), then to functional single-positive (SP; CD4⁺ or CD8⁺ SP) T cells are well-known (5), the stages of TEC differentiation are just beginning to be described (6). Additionally, the molecular mechanisms through which these stepwise interactions mediate thymocyte and TEC differentiation are largely unknown.

The Foxn1 gene is known to be required for most, if not all, TEC differentiation. TEC differentiation in the Foxn1^null mutant mouse, nude (), is arrested at a very immature stage (7). We recently generated a Foxn1 hypomorphic allele, Foxn1, in which TEC differentiation initiates, but subsequently appears to arrest at an intermediate progenitor phenotype (8) corresponding to a thymocyte-dependent stage of TEC differentiation (9). The resulting adult thymus does not have identifiable cortical or medullary regions, and most of the thymic epithelial cells retain a fetal progenitor-like phenotype. This TEC phenotype results in blocks at both the DN1 (CD44⁺CD25^–) and DP thymocyte stages, with a striking loss of CD25⁺ DN2 and DN3 thymocytes. Interestingly, this thymocyte development phenotype was seen in adult, but not fetal, stages; in the Foxn1^/ fetal thymus, TEC differentiation was blocked, but thymocyte differentiation was only slightly delayed. These results suggested that Foxn1 is required for both initial and later stages of TEC differentiation, and further showed that the fetal and adult phenotypes were strikingly different. The difference in the fetal and adult thymocyte phenotypes and the extremely low number of SP T cells in the adult thymus further raises the possibility that the cells generated via this abnormal pathway in the adult thymus might not be exported to the periphery at all. This would suggest that the majority of cells in the adult mouse may originate from persisting cells that had developed in the initial postnatal period from fetal-derived progenitors.

DN1 cells, as defined by the cell surface markers CD44⁺CD25^–, are considered heterogenous prothymocytes. Since our initial report of the Foxn1^/ mutant phenotype, five subsets (DN1a, b, c, d, and e) of DN1 cells have been identified in the adult thymus, based on the expression of CD24 (HSA) and CD117 (c-kit) markers (10). These subsets retain the distinct capacity to differentiate into several different types of cells, including T cells, B cells, NK cells, and dendritic cells (10, 11, 12). Although all five subsets have the capacity to generate T cells when cultured on the OP9-DL1 coculture system, only DN1a (CD117^highCD24^–) and DN1b (CD117^highCD24⁺) do so efficiently and with high proliferative capacity. The data support the conclusion that DN1a are apparently the "canonical" hemopoietic progenitor cells that migrate in from BM, whereas DN1b cells are likely derived from DN1a, and then take the normal T cell development pathway to DN2. DN1a and b are also the only DN1 subsets to demonstrate NK cell potential. The other subsets are able to take a T cell developmental pathway in vitro, albeit with low proliferation capacity and an abnormal development profile; DN1c and d also have B lineage potential. Thus, consistent with other reports, CD117⁺ cells define the primary progenitors that produce most, if not all, of the T cells in a wild-type adult thymus, but are a small minority of the total DN1 population. As the abnormal T cell differentiation profiles shown by some of these CD117^low/– populations in vitro resemble the DN1 profile in the Foxn1^/ adult thymus, these results raise the possibility that differences in the differentiation of the various DN1 subsets could underlie the Foxn1^/ thymocyte differentiation phenotype. However, previous studies were unable to show that these other DN1 cell populations can generate T cells in vivo (10).

In the current study, we investigated the origins of the DN differentiation defects in the Foxn1^/ hypomorphic postnatal thymus. Neither adult nor fetal wild-type or Rag1^–/– BM-derived progenitors were able to differentiate to the DN2 or DN3 stages in the Foxn1^/ thymus, supporting the conclusion that the Foxn1^/ thymic microenvironment cannot support the normal thymocyte development pathway via the DN3 stage. Wild-type T cells differentiating by this pathway were exported to the periphery. CD177^high DN1a/b "canonical" T cell progenitors did not contribute to the T lineage. The thymocytes that develop into DP cells appear to arise primarily from atypical progenitors (DN1d and/or DN1e), which do not normally contribute significantly to thymopoiesis in vivo. Other recent data from our laboratory shows that the peripheral T cell phenotype of cells developing in this pathway is also atypical (13). B and NK cell development was much more normal, although B cells preferentially developed along the B-1 phenotype. Taken together, these data suggest that the microenvironment in the Foxn1^/ adult thymus generates T cells via an atypical differentiation pathway that may represent a minor pathway in the normal thymus.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

Foxn1^/ mice were generated and genotyped by PCR as described (8). Rag1^–/– Ly5.1⁺ mice were provided by E. V. Rothenberg (California Institute of Technology, Pasadena, CA). All experiments were performed using mice on a mixed 129SvJ, C57BL6/J genetic background. For timed matings to generate embryos, the day of the vaginal plug was designated E0.5. All experiments using animals were performed with the approval of the University of Georgia Institutional Animal Care and Use Committee.

Chimera generation

Donor adult BM cells and fetal liver (FL) cells were isolated by lysing RBC in ACK-lysing buffer (Cambrex Bio Science). CD45⁺ BM cells (and FL cells at age fetal day 15.5–18.5) from Rag1^–/– Ly5.1⁺ mice were purified by anti-CD45 microbeads and passing through a column (Miltenyi Biotec). Sorted BM cell (5 x 10⁶/recipient) and FL cells (12 x 10⁶/recipient) were injected i.v. into sublethal irradiated (600 rad) Ly5.2⁺ Foxn1^/ or Foxn1^+/ littermate control recipients (see Fig. 1). In Fig. 2, Adult BM cells from Bl6-Ly5.1 mice were first deleted for mature T cells by incubating with rat-anti CD3, CD4, and CD8 Abs (BD Pharmingen), followed by anti-rat IgG Dynal bead subtraction (Invitrogen Life Technologies). The T cell-deleted cells were further purified by anti-CD45 microbeads and passed through a column for CD45⁺ cells. The T cell-deleted CD45⁺ BM cells were transferred into sublethally irradiated mice. Three weeks later, profiles of thymocytes and spleen cells from donor and host cells were analyzed as above.

View larger version (49K): [in this window] [in a new window]   FIGURE 1. The thymic microenvironment in adult Foxn1/ mutant mice failed to support progenitor cell development to DN2 and DN3 stages. Sorted CD45+ progenitor cells derived from BM and FL of Rag–/– Ly5.1 mice were i.v. transferred into sublethal irradiated adult Foxn1/ mutant or Foxn1+/ control mice. After 3 wk, donor (Ly5.1+) and host (Ly5.1–) cells were analyzed. a, Expression of CD25 and CD44 on gated CD4–CD8– BM-derived cells. b, Similar analysis of CD45+ FL-derived cells.

View larger version (54K): [in this window] [in a new window]   FIGURE 2. The thymic microenvironment in adult Foxn1/ mutant mice produces DP and SP thymocytes that contribute to the peripheral T cell pool. Sorted T cell-depleted CD45+ progenitors derived from BL6 Ly5.1 BM cells were i.v. transferred into sublethally irradiated Foxn1/ and Foxn1+/ adult mice. After 3 wk, gated donor Ly5.1+ cells (right column) and host (Ly5.1–) cells (left column) were analyzed. a, Expression of CD25 and CD44 (top panels), and CD4 and CD8 (bottom panels) on thymocytes. b, Expression of CD4 and CD8 on peripheral T cells.

A total of 0.5–1 x 10⁶ cells in suspension were used for each sample. Cells were blocked by anti-CD16/32 Ab (BioLegend) plus rat serum before staining. For transfer experiments, PE anti-Ly5.1, biotin anti-mouse CD25 following streptavidin-PerCP, and allophycocyanin anti-mouse CD44 (BD Pharmingen), FITC anti-CD4 and CD8, or PE anti-CD4 (BioLegend) were used. Donor cells were analyzed by gating live Ly5.1-positive cells; Ly5.2⁺ host cells were gated on Ly5.1-negative cells. To characterize the profile of DN1 cells, thymocytes from adult Foxn1^/ and Foxn1^+/ littermate controls were deleted for most DP cells by gradient density centrifugation in 13.6% Opti-Prep (Greiner Bio-One) solution at 2000 rpm x 20 min. All lineage markers (anti CD3, CD4, CD8, CD11b, CD19, B220, Gr-1, TER-119, NK1.1) and anti-CD25 were PE conjugated (BD Pharmingen or BioLegend). Biotin anti-CD44 following streptavidin-PerCP, FITC-HSA, and allophycocyanin c-Kit Abs (BD Pharmingen) was used.

Four-color staining was used to analyze DN1 cells. DN1 cells were gated on all lineages and CD25-negative and CD44-positive subpopulations, and analyzed with HSA and c-Kit staining. For analysis of thymic B cells, single-cell suspensions were prepared from adult Foxn1^/ and Foxn1^+/ thymi and stained with PE-CD19, allophycocyanin-B220, and FITC-IgM or FITC-IgD or FITC-CD5 (BioLegend). All cells were acquired by dual-laser FACSCalibur system and analyzed by CellQuest software (BD Biosciences).

Reconstitution of DN1 cells in 2dGuo-treated fetal thymic organ culture (FTOC)

DN1 cells were enriched and sorted by MoFlo cytomation using PE-conjugated lineage markers as above (anti CD3, CD4, CD8, CD11b, CD19, B220, Gr-1, TER-119, NK1.1), CD25-FITC and CD44 allophycocyanin. E15.5 day thymi were isolated and treated with 10% FBS plus RPMI 1640 medium containing 1.35 mM 2dGuo (Sigma-Aldrich) in a high oxygen culture system at 37°C for 5 days to deplete hemopoietic cells. Each dGuo-treated fetal thymic lobe was put in a "hanging drop" coculture with 1000 sorted DN1 cells in total volume 25 µl of 10% FBS plus RPMI 1640 medium at 37°C, 5% CO₂ for 48 h. Fetal thymi were then washed and transferred to a membrane floating on 10% FBS plus RPMI 1640 medium and incubated at 37°C, 5% CO₂ for 7 or 10 days. Cultured thymi were ground through a mesh screen and single-cell suspensions were analyzed as above.

Immunofluorescence staining

Freshly isolated Foxn1^+/ and B cell-deleted Foxn1^/ thymocytes were seeded on the coverslips coated with 150 µg/ml poly-L-lysine (Sigma-Aldrich). After washing, attached cells were fixed with methanol at –20°C for 20 min. Cells were washed with PBS for three times, blocked by 5% donkey serum plus 1% BSA in PBS at room temperature for 1 h. Cells were incubated with rabbit anti--H2AX Ab (1/1,000; Sigma-Aldrich) for 1 h. Rabbit serum at the same dilution was used as negative control. Cells were washed and then incubated with donkey anti-rabbit Cy3 (1:300; Jackson ImmunoResearch Laboratories) for 1 h following nuclear staining with 4',6'-diamidino-2-phenylindole (1/10,000; Sigma-Aldrich) for 2 min. The coverslips were mounted with Aqua Poly/mount (Polysciences). Images were acquired with a Zeiss ApoTome microscope, at a x40 objective, using Axio Vision Rel.6 software.

Semiquantitative RT-PCR for Notch ligands and TCR

TRIzol (Invitrogen Life Technologies) was used to isolate total RNA from the E15.5 thymi by FTOC with 1.35 mM dGUO (depleting hemopoietic cells), or from sorted adult DN thymocytes. Reverse transcription and PCR amplification were done by using the SuperScript II (Invitrogen Life Technologies) and Taq (Qiagen) or SuperScript III One-step RT-PCR with Platinum Taq (Invitrogen Life Technologies). Each reaction contained 0.5 µg of RNA for Notch ligand genes or 0.025 µg for control CD45 or GAPDH genes. Reactions were repeated at least twice using RNA from different fetal litters and different adult animals. Primers (14, 15) for Notch ligands were Delta-like-1: (forward) 5'-GTC ACA GAG CTC TGC AGG AG-3'; (reverse) 5'-TGT GGG CAG TGC GTG CTT CC-3'; Jagged-1: (forward) 5'-CAT TAC GTG TTG CCT GTA AGC C-3'; (reverse) 5'-GTG GTT CAG CAT TAC ATA CG-3'; Delta-like-4: (forward) 5'-CAG AGA CTT CGC CAG GAA AC-3'; (reverse) 5'-ATC CAT TC TGC ACG GAG AG-3' and Jagged-2: (forward) 5'-GTC CTT CCC ACA TGG GAG TT-3'; (reverse) 5'-GTT TCC ACC TTG ACC TCG GT-3'. Primers for pre-TCR were pT-F: 5'-CAG AGC CTC CTC CCC CAA CAG-5'; pT-R: 5'-GCT CAG AGG GGT GGG TAA GAT-3' (16).

Southern blot analysis of TCRβ rearrangement

TCRβ gene rearrangement was assayed at the genomic level. Sorted DP and DN adult thymocytes (5 x 10⁴ per PCR), and FL cells (for germline control) were lysed in 5,000 cells/µl PCR lysis buffer (17) with 0.1 mg/ml proteinase K at 55°C for 2 h, then 100°C for 10 min. PCR was performed with primers of: (3' primer) Jβ2: 5'-TGA GAG CTG TCT CCT ACT ATC CAT T-3' (18); (5' primer) Dβ2: 5'-GTA GGC ACC TGT GGG GAA GAA ACT-3' (19); (5' primer) Vβ5.1: 3'-CCC AGC AGA TTC TCA GTC CAA CAG-3' (19). PCR products were run on a 1.8% agarose gel, denatured, neutralized, and transferred to Hybond N⁺ nylon membrane, then hybridized with a [-³²P]ATP 5'-end labeled Jβ2 probe 5'-TTT CCC TCC CGG AGA TTC CCT AA-3' (18).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The adult Foxn1^/ thymic microenvironment fails to support progenitor differentiation to DN2 and DN3

Our previously published data (8) showed that thymocyte development in adult Foxn1^/ thymi is blocked at the DN1 (CD44⁺CD25^–) stage. Despite a near total absence of DN2 and DN3 cells, DN4, DP, and SP cells were present, although in reduced numbers. There are several possible explanations for this steady-state profile: 1) DN2/3 cells could be present, but in too low numbers to reliably detect due to low efficiency of generation; 2) thymocytes could be transiting the DN2/3 stages with unusually rapid kinetics, then accumulating at DN4; or 3) thymocytes could progress directly from the DN1 to DN4 stages via an atypical differentiation pathway.

To distinguish between these possibilities, we compared the ability of Rag1^–/– and wild-type progenitors to differentiate in the Foxn1^/ thymus. If DN cells could develop to the DN2/3 stages, even at low efficiency, Rag1^–/– thymocytes should accumulate at the DN3 stage, revealing their presence in the Foxn1^/ thymus. Sorted CD45⁺ progenitors derived from Ly5.1⁺ BM were i.v. injected into sublethally irradiated Foxn1^+/ controls or Foxn1^/ mutants (Ly5.2⁺ congenic recipients). Differentiation of the donor and host cells in the thymus was analyzed at 3 wk postinjection by gating on the Ly5.1- and Ly5.2-positive populations and staining for CD44 and CD25. By 3 wk postinjection, >90% of Ly5.1⁺ thymocytes in the control thymus had progressed to the DN2 or DN3 stages (Fig. 1a). In contrast, all of the progenitors from Rag1^–/– BM (two experiments, total six mice) failed to generate DN3 cells, and were blocked at the DN1 stage in the Foxn1^/ thymus (Fig. 1a). This result clearly demonstrated that the Foxn1^/ thymus is unable to support the normal thymocyte differentiation pathway to the DN3 stage (possibility no. 3 above). This result also confirms that DN1 cells from these mutants require TCR rearrangement to progress to the DN4 stage.

Our previous analysis of Foxn1^/ mutants had also shown that DN2 and DN3 cells were present in relatively normal numbers in the fetal thymus, even though TEC differentiation was blocked. To test the capacity of fetal thymocytes to differentiate in the adult Foxn1^/ microenvironment, we performed a similar experiment using FL-derived CD45⁺Ly5.1⁺ Rag1^–/– progenitors. All of these cells were also blocked at the DN1 stage (Fig. 1b) (three experiments, total of eight mice injected with E15.5 or E18.5 cells, or with mixed E14.5, 15.5, and 17.5 cells). These results showed that the Foxn1^/ thymic microenvironment is completely nonpermissive for the DN2/3 stages of differentiation.

SP thymocytes develop in the Foxn1^/ thymus and are exported to the periphery

This atypical differentiation pathway was associated with a reduction in SP cell production by at least 500-fold in the postnatal Foxn1^/ thymus (Table I; Refs. 8 and 13). Other data from our laboratory showed that peripheral T cells in the adult have a phenotype similar to peripheral T cells in early postnatal stages (13). Furthermore, the Foxn1^/ postnatal thymus is highly disorganized with no discernible cortical or medullary compartments (8). These results raised question of whether SP cells were maturing in the absence of an organized medulla, and whether they could actually exit the postnatal thymus.

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Absence of CD117^high cells in Foxn1^/ adult thymus

The failure of transferred Rag1^–/– and wild-type or endogenous progenitors to differentiate to the DN2/3 stages could be due to either failure of progenitor immigration or an inability of these progenitors to differentiate in the mutant thymic microenvironment. To determine which DN1subpopulation is affected in Foxn1^/ mutants, we characterized the profile of freshly isolated, enriched DN thymocytes from adult Foxn1^/ and their littermate controls. The DN1 population of control mice displayed five subpopulations based on the expression of CD117 (c-kit) and CD24 (HSA) as previously described (Fig. 3a) (10). In contrast, very few or no CD117^high cells were present in the Foxn1^/ adult thymus, although some HSA^lowckit^low cells were present in the DN1a quadrant defined in the control mice (Fig. 3a, right panels, R3 gate). The c-kit^low"DN1a" cells in the mutants could reflect an inability of the / microenvironment to up-regulate or maintain high levels of c-kit on these cells, or reflect an absence of true DN1a cells and presence of separate class of atypical cells in these mutants. As expected, the percentage of CD117^high cells in BM was not significantly different between Foxn1^/ and their Foxn1^+/ littermate controls (data not shown). Although the very low numbers of DN1a cells made it difficult to determine conclusively whether they were present by this approach, DN1b cells were clearly absent (Fig. 3a). These results suggested that progenitors for the "canonical" DN1a-DN1b-DN2 pathway were either not present or were blocked in their differentiation along the usual pathway, via the DN2 and DN3 stages. DN1c cells were also significantly decreased, resulting in the majority of DN1 cells being DN1d and DN1e subsets, which were previously shown to differentiate in the T lineage with atypical kinetics and with marker profiles similar to thymocyte differentiation in the Foxn1^/ adult thymus (10).

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Absence of CD117high cells in Foxn1/ adult thymus. a, Characteristic plot of DN1a–e subsets. DN-enriched thymocytes were stained with nine PE-conjugated lineage markers and PE-CD25, CD44-biotin plus avidin PerCP, CD117-allophycocyanin, HSA-FITC. CD44+, lin–, CD25– cells were gated as DN1. b, Reconstitution of sorted DN1 cells in FTOC. Cocultured thy1.2-positive cells were collected and analyzed by flow cytometry after 7 or 10 days of culture. Top panel, Expression of CD4, CD8; bottom panel, expression of CD44, CD25. c, Expression of thy1.2 and B220 at FTOC day 10.

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Foxn1^/ DN4 cells contain DP precursors

The data above suggest that DP cells in the Foxn1^/ thymus are generated from atypical DN1d or DN1e progenitor cells, differentiating from DN1 to DN4, then through to DP and SP stages. As DN4 cells are primarily defined by the absence of markers, we determined whether the DN4 cells in the Foxn1^/ thymus generated via this atypical pathway represented authentic pre-DP cells by testing whether they could differentiate directly to the DP stage in suspension culture (20). We cultured 5 x 10³ sorted DN4 cells/well (lin^–, CD44^–25^–, Thy1.2⁺). To obtain sufficient numbers of DN4 cells from Foxn1^/ mutants, cells from 10 adult mutant thymi were pooled. Controls contained the same number of sorted DN4 cells from individual mice. The pooled cells from the Foxn1^/ mutants did differentiate to the DP stage with a similar profile and at a frequency similar to that of control cultures (Fig. 4a). The low level of DP cells produced in this assay was due to the low density of seeding required by the poor recovery of cells from the mutant thymi, because increasing the density of seeding for control DN4 cells progressively increased DP cell production (Fig. 4a).

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Characteristics of DN cells in Foxn1/ mutant mice. a, Sorted DN4 cells (Lin–, CD25–, CD44– cells) derived from Foxn1/ mutant and Foxn1+/ mice were cultured in medium only. The expression of CD4 and CD8 were analyzed after 18 h. Top row, Results before and after culture of control and mutant-derived cells seeded at 5 x 103 cells/well. Second row shows that the number of DP cells recovered from control cultures depends on initial seeding density. b, TCRβ gene rearrangement in sorted thymocytes. Southern blot analysis of TCRβ chain Dβ2-Jβ2 (left panel) and Vβ5.1-Jβ2 (right panel) rearrangement in FL cells (lanes 1 and 6), sorted DP cells from Foxn1+/ (lanes 2 and 7) and Foxn1/ (lanes 4 and 9) thymi, and sorted DN cells from Foxn1+/ (lanes 3 and 8) and Foxn1/ (lanes 5 and 10) thymi. The 1.8-kb Dβ2-Jβ2 germline band for Dβ2-Jβ2 is indicated with an arrow. c, Presence of punctate -H2AX staining in control and mutant thymocytes from 1-mo-old adult mice. Examples of cells with one or two foci of H2AX staining indicative of active TCR rearrangement are indicated with arrowheads. d, Expression of pT mRNA in sorted DN thymocytes.

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B and NK cells in the Foxn1^/ adult thymus

Because the results presented above showed that the Foxn1^/ mutant adult thymic microenvironment is not favorable to canonical T cell differentiation, we investigated whether B and NK cell development in the thymus was affected as well. Overall, the percentage of B cells was greatly increased in Foxn1^/ mice, reflecting the reduction in thymocyte number, although variation in the percentage of thymic B cells in Foxn1^/ mice was large (CD19⁺ cells: 490%). The total number of B cells was reduced, although not as severely as for thymocytes (Table II).

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View larger version (54K): [in this window] [in a new window]   FIGURE 5. B cells in adult and E17.5 embryonic thymi. Freshly isolated adult (a–c) or E17.5 fetal (d) thymic lymphocytes and splenocytes were analyzed for surface expression of B cell markers (CD19, B220 and CD5, IgM, IgD) as indicated. Each data point on the graphs represents one thymus or spleen. Bar is the mean. *, p < 0.001. a, Expression of CD19 and B220 on thymocyte and splenocytes. b, Cell size on forward scatter (FSC) linear scale, and expression of CD5, IgM, and IgD on gated thymic CD19med, B220high (R3, thin line) and CD19high, B220–/low (R2, bold line) subpopulations only from Foxn1/ mice. c, Ratio of B-1 cells to B-2 cells in thymus and periphery. d, Analysis of total B cell number in E17.5 fetal thymus.

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As we have previously reported that fetal thymocyte differentiation is less severely affected than adult stages in Foxn1^/ mutants, we analyzed thymic B cell development at fetal stages. The percentage of B220⁺ cells was increased in the fetal mutant thymus, but cell numbers were decreased by 50% (Fig. 5d). This is very similar to the results for B-1 cells in the adult thymus, and suggest that this phenotype remains constant throughout the fetal and adult stages.

As the only DN1 populations reported to have NK lineage potential are the DN1a and b subsets (10), absence of these cells might result in a decrease in thymic NK cells. NK1.1⁺ cells were present in variable but similar numbers in the control and mutant thymi, although the relative percentage was increased in the mutants due to the general hypocellularity (data not shown). Given that both B and T cells were reduced in the Foxn1^/ adult thymus, this result is consistent with normal generation of thymic NK cells, possibly from the DN1a/b lineage (10).

Reduced Notch ligand expression in the Foxn1^/ mutant thymus

Notch signals have been strongly implicated in the choice between B or T lineage commitment in the thymus (23, 24, 25, 26). Reduced Notch signaling has been suggested to favor B-lineage commitment early in differentiation (27), and the presence of DL-1 on OP9 stromal cells is required to support T cell development in coculture (28, 29, 30, 31). There are four Notch ligands expressed on mouse TECs: Delta-like 1, Jagged-1, Delta-like 4, and Jagged-2 (14, 15, 32). We assayed the expression of these four Notch ligands in fetal and adult Foxn1^/ mutant thymic epithelial cells by semiquantitative RT-PCR. Expression of all four ligands was strongly decreased in adult Foxn1^/ thymus RNA enriched for stromal cells, with DL-4 being most markedly reduced (Fig. 6a). There were no differences in any of the ligands in the fetal thymus compared with controls (Fig. 6b, and data not shown), again consistent with the milder thymocyte phenotype at this stage (Fig. 5d). Thus, Notch ligands are either down-regulated or fail to be up-regulated in the postnatal Foxn1^/ thymus, and may contribute to the observed phenotypes.

View larger version (62K): [in this window] [in a new window]   FIGURE 6. Expression of Notch ligands in fetal and adult TECs and cleaved Notch1+ cells in DN subsets. a, mRNA expression of Notch ligands Delta-like 1, Delta-like 4, Jagged-1, and Jagged-4 in collagenase treated TEC-enriched adult thymi. b, Expression of Notch ligands Delta-like 1 and Jagged-1 in dGUO-treated E15.5 fetal thymi by RT-PCR.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

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The extremely low number of DN1a cells even in wild-type thymus makes it difficult to confirm whether they are physically present, especially in the very hypocellular Foxn1^/ thymus. One possibility is that the DN1a progenitors never enter the postnatal thymus, although clearly the DN1c, d and e cells can. Previously published data (8) and our more recent analysis of SP cells in Foxn1^/ mice (13) have also shown that peripheral T cells can easily recirculate into the postnatal thymus, so there is no physical barrier to their entry. However, we cannot exclude the possibility that DN1a progenitor cells never enter the postnatal Foxn1^/ thymus. If they do enter the thymus, what is the fate of these DN1a cells? The normal number of NK cells raises the possibility that these cells may still be present and able to differentiate along this lineage in the Foxn1^/ thymus. Alternatively, they could fail to proliferate, survive, migrate, or differentiate properly, due to reduced availability of kitl. The low levels of c-kit on the putative DN1a subset could represent an inability of the Foxn1^/ microenvironment to up-regulate or maintain high levels of c-kit on these cells, or reflect an absence of true DN1a cells and presence or expansion of a novel class of progenitor cells in these mutants. Signaling through the Kit receptor is dosage sensitive, and kitl expression in Foxn1^/ adult thymic stromal cells is reduced by 50% (our unpublished data). This reduction, and/or changes in the location and/or presentation of kit signals in the highly disorganized and abnormal Foxn1^/ thymus combined with low levels of c-kit expression on thymocytes could result in much lower effective availability of kitl to DN1a cells, resulting in loss of or differentiation defects in these cells.

The conclusion that the T cells generated in the Foxn1^/ adult thymus are derived from atypical or noncanonical progenitors raises the question of whether the T cells produced in these mice contribute to the peripheral T cell pool, and have normal function. These atypical DN1 populations do not proliferate or differentiate in the context of a normal thymic microenvironment (10), and therefore it is unknown what type of T cells, if any, these progenitor populations can generate in a normal thymus. The Foxn1^/ mice, therefore, provide an in vivo situation in which these populations can differentiate, albeit inefficiently, to produce mature T cells which are exported to the periphery. Other data from our laboratory has shown that the peripheral T cells in Foxn1^/ mice have an atypical phenotype (13). Taken together, these data raise the possibility that under some conditions, these cells may give rise to a minor subpopulation of T cells which may have different functional characteristics from those arising from canonical precursors, even in a wild-type mouse.

As DN1c and d cells were previously shown to have significant B cell potential, it is perhaps not surprising that B cell development is much less affected than T cell development in these mice. Although total B cell numbers are reduced, the number of B-1 cells is least affected, and the ratio of B-1 to B-2 cells is increased 5-fold. Our data suggest that these B-1 cells are mostly produced inside the thymus, and not recirculated from the periphery. Interestingly, the ratio of B-1 to B-2 cells was also increased in the spleen, suggesting that the thymus may be a significant source of B-1 cells in the periphery as well. In thymocytes, Notch signaling has been linked to both cell lineage commitment and proliferation (32, 33). Notch1 is present in murine thymocytes mostly at the DN2 and DN3 stages, with the highest levels of expression at DN2 stage. Thus, even if a low number of DN2 cells is produced, decreased Notch ligand availability in the Foxn1^/ adult thymus could contribute to the loss of these cells. In addition, this altered microenvironment is likely to selectively promote thymic B cell development. Reduced availability of Notch ligands might also be a contributing factor in the lower proliferation of Foxn1^/ T lineage thymocytes. Taken together, all of the above could contribute to the thymic B and T cell phenotypes in Foxn1^/ adult thymus.

The differences in thymocyte development profiles between fetal and adult Foxn1^/ thymus remain an intriguing aspect of this phenotype. However, as the DN1a–e subsets defined in adult stages are not clearly present in the fetal thymus, the difference in fetal and adult T cell phenotypes may not be as simple as presence or absence of DN1a/b cells. One clear difference is in Notch ligand availability in the fetal vs adult mutant thymus. This difference in Notch ligand levels may be compounded by a difference in the response of fetal vs adult progenitor cells to the Foxn1^/ microenvironment. Although the ratio of B to T cells changes from the fetal to adult stages, the relative numbers of B cells produced in the thymus at fetal and adult stages are affected to a similar degree (both are reduced by 50%). Alternatively, it is entirely possible that changes in Notch ligand availability play a role in the phenotypic differences in T cell differentiation at fetal and adult stages.

	Acknowledgments

 
We thank Drs. Howard Petrie (Scripps/Florida Research Institute, Jupiter, FL) and Juan-Carlos Zuniga Pflucker (Sunnybrook and Women’s College Health Sciences Centre, Toronto, Ontario, Canada) for helpful discussions. We also thank Dr. E. V. Rothenberg (California Institute of Technology, Pasadena, CA) for Rag^–/– LY5.1 mice, and Julie Nelson in the Center for Tropical and Emerging Global Diseases Flow Cytometry Facility (University of Georgia, Atlanta, GA) for help in cell sorting.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health, National Institute of Allergy and Infectious Disease Grant AI055001 (to N.R.M.).

² Current address: Department of Biomedical Research, University of Texas Health Center, Tyler, TX 75708.

³ Address correspondence and reprint requests to Dr. Nancy R. Manley, Department of Genetics, S270 Coverdell Center for Biomedical and Health Sciences, University of Georgia, Athens, GA 30602. E-mail address: nmanley{at}uga.edu

⁴ Abbreviations used in this paper: BM, bone marrow; TEC, thymic epithelial cell; DN, double negative; SP, single positive; DP, double positive; FL, fetal liver; FTOC, fetal thymic organ culture.

Received for publication February 21, 2007. Accepted for publication November 11, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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