Requirement for Notch1 signals at sequential early stages of ... - Nature

© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

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Requirement for Notch1 signals at sequential early stages of intrathymic T cell development Joanne B Tan, Ioana Visan, Julie S Yuan & Cynthia J Guidos Signaling through the transmembrane Notch1 receptor directs thymus-seeding progenitors (TSPs) to suppress their B cell potential and ‘choose’ the T cell fate. Present paradigms suggest that TSPs are contained in the multipotent early T lineage precursor (ETP) subset of thymocytes. However, we show here that the B cell potential of ETPs was not augmented in microenvironments that limited Notch1 activation. Furthermore, low-threshold Notch1 signals suppressed B cell production by TSPs before they reached the ETP stage. Notch1 signals of a higher threshold were needed to drive proliferation of ETPs and development into CD4+CD8+ double-positive thymocytes. Thus, TSPs can be differentiated from all previously identified early T cell progenitors by their robust B cell potential and exquisite sensitivity to Notch1 signals.

Signaling through the Notch1 receptor is required at the earliest stages of T cell development in the postnatal thymus. Binding of Notch receptors to Delta or Jagged ligands induces proteolytic release of the Notch intracellular domain from its membrane tether, allowing it to enter the nucleus. The Notch intracellular domain then interacts with the transcription factor RBP-Jk (also called ‘suppressor of hairless’) to regulate expression of genes encoding ‘hairy and enhancer of split’ (Hes) transcriptional repressors and other Notch target genes1,2. Bone marrow precursors lacking Notch1 (refs. 3,4) or RBP-Jk5 generate B cells rather than T cells in the thymus. Overexpression of Notch inhibitors in early lymphoid progenitors produces similar effects6,7. Moreover, many wild-type thymus-seeding progenitors (TSPs) incorrectly adopt the B cell fate when the molecule lunatic fringe (L-Fng) is misexpressed in cortical thymocytes8. L-Fng is a glycosyltransferase that modulates Notch receptor–ligand interactions9,10. Conversely, complimentary gain-of-function approaches, using overexpression of the Notch1 intracellular domain or culture of lymphoid progenitors with bone marrow stromal cells expressing the Notch ligands Delta-like 1 (DL1) or DL4 (refs. 11–14), have shown that Notch1 activation inhibits B cell development and promotes thymus-independent T cell development. Collectively these studies have demonstrated that bone marrow–derived TSPs must activate Notch1 soon after entering the postnatal thymus to suppress their B cell potential and undergo T lineage specification. However, neither TSPs nor the earliest targets of Notch1 signaling in the adult thymus have been identified. TSPs have proven difficult to study directly, as they seed the thymus at low frequencies in periodic waves, rather than continuously15,16. The earliest intrathymic T cell precursors, known as early T lineage precursors (ETPs), have high expression of CD117 (also called c-Kit) and are found in the CD44+CD25 subset of double-negative 1 (DN1)

thymocytes17,18. ETPs can also produce B cells, natural killer cells and dendritic cells when transferred intravenously into an irradiated host, but they have minimal myeloid potential19–23, suggesting that they are not hematopoietic stem cells (HSCs). Thus, the developmental potential of ETPs resembles that of bone marrow– derived common lymphoid progenitors (CLPs)24,25. This subset is lineage negative (Lin) CD117intAA4.1+ and also expresses the Flk2 growth factor receptor (also called Flt3), which ‘marks’ primitive multipotent bone marrow progenitors26,27. However, studies suggest that CLPs do not circulate or seed the postnatal thymus23,28. A ‘downstream’ lymphoid-restricted B220+CD117 subset (CLP-2) isolated from bone marrow can seed the thymus when high numbers are injected into the bloodstream29, but whether these are physiological TSPs is unknown. Because ETPs include B cell progenitors as well as robust T cell precursor activity, present models suggest that they include TSPs that adopt the T cell fate in response to Notch1 activation1,2. Thus, this model predicts that the production of ETPs should be Notch1 independent and that B cell production from ETPs should be augmented by limiting their ability to activate Notch1. However, this idea has not been tested experimentally. A CD117intCD24hi DN1 population has been identified and shown to contain both T cells and B cell progenitors when cultured on OP9 stromal cells versus OP9 cells expressing DL1, respectively30. Thus, it is not clear whether Notch1 signals direct ETPs to suppress their B cell potential and undergo T lineage specification or whether other DN1 subsets contain TSPs that are the earliest targets of intrathymic Notch1 signaling. Conditional deletion of Notch1 or RBP-Jk in DN3 thymocytes (after loss of B cell potential) impairs T cell receptor b (TCRb) selection and production of double-positive (DP) thymocytes31,32, identifying a second essential function for Notch1 signaling after

Program in Developmental Biology, Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada, and Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Correspondence should be addressed to C.J.G. ([email protected]). Published online 12 June 2005; doi:10.1038/ni1217

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T cell commitment has taken place. DN2 thymocytes proliferate and differentiate to the DP stage in vitro when cultured on OP9 cells expressing DL1 (refs. 33,34), and Notch1 and pre-TCR signals act cooperatively to induce survival and proliferation during the DN3-toDP transition in vitro33,35. However, whether committed T cell progenitors also require continuous Notch signaling in their physiological microenvironment is not clear. To gain insights into the earliest Notch1-dependent events in postnatal T cell development, we have used two partial loss-offunction experimental strategies to limit Notch1 activation in vivo. The first approach uses L-Fng-transgenic thymocytes to inhibit Notch1 activation in wild-type TSPs8, whereas the second exploits the considerable sensitivity of the Notch pathway to small changes in the amount of Notch receptor or ligand expressed36,37. Because each strategy limits rather than ablates Notch1 activation, T cell and B cell development occurs simultaneously in the thymus. TSPs that fail to activate Notch1 ‘choose’ the B cell fate, whereas TSPs that activate Notch1 ‘choose’ the T cell fate. In contrast, conditional Notch1 deletion or gain-of-function strategies allow only one developmental outcome to occur in a given situation. We show here that contrary to predictions of present models, ETP production was highly Notch1 dependent and B cell production by ETPs was not augmented in conditions that limited Notch1 activation. ETPs showed minimal B cell production even after direct intrafemoral injection into the bone marrow, suggesting that ETPs cannot account for the robust B cell production seen from TSPs when intrathymic Notch1 activation is impaired. Finally, we show that sensitivity to intrathymic Notch1 signals decreased from the TSP to the ETP stage. Collectively these data show that multipotent TSPs are exquisitely sensitive to Notch1 signals that direct them to develop into ETPs with limited B cell potential. Notch1 signals of a higher threshold then promote ETP proliferation, loss of residual B cell potential and development into DP thymocytes.

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Figure 1 Failure of ETPs to produce B cells in L-Fng-transgenic thymic lobes. ETPs (3 103) or Lin bone marrow cells (BM Lin; 2 105) purified from B6.CD45.1 mice were injected intrathymically into sublethally irradiated B6.CD45.2 wild-type (a) or L-Fng-transgenic (b) hosts. Then, 3 weeks later, thymocytes were stained with anti-CD45.1–phycoerythrin and anti-CD45.2–FITC together with anti-CD4–biotin (avidin-CyChrome) versus anti-CD8– allophycocyanin or a ‘cocktail’ of biotinylated anti-CD3, anti-CD4 and anti-CD8 (avidinCyChrome) versus anti-B220–allophycocyanin. Data are presented as two-parameter contour plots (2% probability, showing outliers). Plots of anti-CD45.1–phycoerythrin versus anti-CD45.2– FITC (left column) were gated to show the phenotype of donor-derived (CD45.1+) cells (middle and right columns). Numbers in quadrants and beside outlined areas indicate percent cells in those areas. Data are summarized in Supplementary Table 1 online. The experiment was repeated three times with similar results.

RESULTS ETPs lack robust B cell potential Although TSPs have not been identified directly, it has been suggested that they are contained in the CD117hi ETP subset of DN1 thymocytes, as this population is multipotent and can generate T cells, B cells, dendritic cells and natural killer cells in appropriate assay conditions17,18,38. Notch1 signaling suppresses the B cell potential of TSPs4,6,8, inducing them to adopt the T cell fate in the thymus. Therefore, if ETPs include TSPs, their B cell potential should be augmented in microenvironments that limit Notch1 activation. We tested this prediction using several independent experimental approaches. First we assessed the ability of purified ETPs to produce T cells and B cells after injection into wild-type versus L-Fngtransgenic thymic lobes. L-Fng overexpression in thymocytes enables the thymic microenvironment to support vigorous B cell development from multipotent precursors that fail to activate Notch1 efficiently8. Inhibition of Notch1 occurs because L-Fng-expressing thymocytes inappropriately bind Delta-like Notch ligands, excluding TSPs from a limiting microenvironmental niche in which T cell commitment occurs (I.V., J.B.T., J.S.Y., J. Harper, U. Koch and C.J.G., unpublished data). As expected, ETPs produced large numbers of DP thymocytes but no B cells by 3 weeks after injection into wild-type thymic lobes (Fig. 1a and Supplementary Table 1 online). Bone marrow Lin precursors, which are enriched for CLPs and HSCs, also produced a large burst of DP thymocytes in wild-type lobes. However, no B220+ or CD19+ B cells were produced 12 d or 3 weeks after ETPs were injected into L-Fng-transgenic thymic lobes (Fig. 1b, Supplementary Table 1 online and data not shown). In contrast, bone marrow Lin precursors made many more B cells than T cells after injection into L-Fng-transgenic thymic lobes, confirming previous findings8. The ability of L-Fng-transgenic thymocytes to augment the B cell potential of bone marrow Lin precursors but not ETPs may have been because of differences in their microenvironmental localization

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after injection. For example, ETPs may preferentially localize to a Notch ligand–rich microenvironmental niche after injection, whereas bone marrow Lin precursors may do so less efficiently. We therefore used two other experimental approaches to determine if limiting Notch1 activation could augment the B cell potential of ETPs. The first was to culture ETPs on the OP9 bone marrow stromal cell line, which supports vigorous B cell development from HSCs and committed lymphoid or B cell precursors but does not promote T cell development from HSCs13,39. Although we noted robust B cell development to the pro-B and pre-B stages when HSCs, CLPs or bone marrow Lin precursors were cultured on OP9 cells, ETPs survived poorly and did not generate B lineage cells (data not shown). The failure of ETPs to produce B cells on bone marrow stromal cell lines in vitro has been reported before30,38. ETPs can produce B cells after injection into the bloodstream, although the process is slow and does not yield high numbers of B cells18. To test whether the inefficient B cell production from intravenously injected ETPs might reflect poor homing to the bone marrow, we assessed B cell production from ETPs after direct intrafemoral injection into sublethally irradiated mice. This approach can identify previously unknown HSC classes that are not detected after intravenous injection40,41. As expected, bone marrow Lin precursors engrafted efficiently in the bone marrow and produced CD11b+GR-1+ (myeloid) and B220+ B cells (Fig. 2) 3 weeks later.

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Figure 3 High expression of Hes1 by all DN thymocyte subsets. The poly(A)+ mRNA from each purified subset was reversed-transcribed and the abundance of Hes1 and Cd45 cDNA was determined by quantitative RT-PCR. The number of Hes1 cDNA templates was divided by the number of Cd45 templates to yield a normalized Hes1 expression value for each thymic subset. Data represent the mean normalized values (7 s.d.) of triplicate measurements. The experiment was done three times (independent sorts) with similar results.

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Figure 2 Minimal B cell potential of ETPs Bone marrow Thymus Donor cells Bone marrow Thymus 104 in the bone marrow. ETPs (3 105) or Lin 1 3 0 100 bone marrow cells (2 105) purified from 103 ETP B6.CD45.1 mice were injected into one femur 4 87 95 102 of each sublethally irradiated wild-type host 101 (B6.CD45.1;CD45.2). Then, 3 weeks later, cells 0 from the spleen (data not shown) bone marrow 100 4 10 20 1 and thymus were stained with anti-CD45.1– 15 98 phycoerythrin and anti-CD45.2–FITC together 103 BM Lin– 80 with biotinylated anti-CD11b and anti-GR-1 99 102 73 (avidin-CyChrome) versus anti-B220– 101 2 allophycocyanin or biotinylated anti-CD3, anti100 0 CD4 and anti-CD8 (avidin-CyChrome) versus anti10 101 102 103 104 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 B220–allophycocyanin. Plots of anti-CD45.1– CD45.2 B220 phycoerythrin versus anti-CD45.2–FITC (left) were gated to show the phenotype of donorderived (CD45.1+) cells (right). Numbers beside outlined areas indicate percent cells in those areas. Plots are representative of four mice injected with ETPs and two mice injected with bone marrow Lin precursors. The experiment was done twice with similar results.

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Although there was lower engraftment of ETPs in the bone marrow, we detected small numbers of ETP-derived B220+ cells but no CD11b+ or GR-1+ myeloid cells in the bone marrow (Fig. 2) and spleen (data not shown). The frequencies of CD19+ (data not shown) and B220+ cells were similar, suggesting the latter were B lineage cells. Moreover, both bone marrow–engrafted Lin precursors and ETPs generated T cell progeny in the thymus. The extent of thymic colonization by ETP progeny was unexpectedly high, given their low frequency in the bone marrow, suggesting that ETPs retain the capacity for efficient thymic seeding from the bone marrow. These data confirm previous findings17–19,23 that ETPs can produce small numbers of B cells in nonthymic microenvironments. However, our data show that the inefficient B cell production from ETPs was not augmented in microenvironments that limited Notch1 activation, such as the L-Fng-transgenic thymus or the bone marrow. Collectively these results indicate that ETPs and TSPs are distinct thymic subsets. L-Fng-transgenic DN thymocyte subsets The ability of L-Fng-transgenic thymocytes to promote efficient B cell development from TSPs but not ETPs suggests that in wild-type mice, most ETPs have already responded to Notch1 signals and thus have minimal B cell potential. Consistent with this idea, we found that ETPs had high expression of the Notch1 target Hes1, similar to that of DN2 and DN3 thymocytes (Fig. 3). To further explore the idea that ETPs are generated from earlier TSPs in a Notch1-dependent way, we compared the abundance of ETPs in wild-type versus L-Fngtransgenic thymi. The numbers of both ETPs and DN2 thymocytes were significantly less in L-Fng-transgenic than in wild-type thymi (P ¼ 0.0004 and P o 0.0001, respectively; Fig. 4). Moreover, wildtype mice showed an increase of two- to fourfold from the ETP to the DN2 stage, whereas L-Fng-transgenic thymi showed a decrease across this transition (Fig. 4b). Thus, few ETPs were generated in the L-Fngtransgenic thymus and they produced very few DN2 thymocytes. Notably, DN3 thymocytes had lower expression of CD25 and were also considerably decreased in L-Fng-transgenic thymi (Fig. 4). The generation of DN3 thymocytes from the few DN2 cells present in L-Fng-transgenic thymi seems paradoxical, but we have found that the expression of transgenic L-Fng begins at the DN2-to-DN3 stage and transforms these subsets into ‘supercompetitors’ for limiting microenvironmental niches expressing Notch ligand (I.V., J.B.T., J.S.Y., J. Harper, U. Koch and C.J.G., unpublished observations). Thus, overexpression of L-Fng renders DN3 thymocytes highly

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efficient at generating downstream DN4 and DP thymocytes. It is also possible that inhibition of Notch1 activation by L-Fng-transgenic thymocytes is less efficient in the microenvironment in which DN3 and DN4 thymocytes are generated. CD117-versus-CD24 staining can be used to identify distinct DN1 subsets that have T or B cell progenitor activity in vitro30, so we used these markers to determine how the L-Fng transgene altered the abundance of DN1 subsets. DN1 thymocytes from L-Fng-transgenic mice contained almost no true ETPs that were CD117hiCD24hi (Fig. 5a,b). Moreover, they contained increased proportions of a CD117intCD24hi subset referred to as ‘population DN1c’30, as well as a high proportion of a CD117intCD24lo subset not described before. To determine whether these CD117int cells resembled bone marrow CLPs27, we looked for coexpression of Flk2 and CD117 among DN1 thymocytes from wild-type and L-Fng-transgenic mice. In both strains, all CD117int DN1 cells were Flk2 and thus were unlikely to be CLPs (Fig. 5c). However a small fraction (5–20%) of CD117hi DN1 cells coexpressed Flk2. The number of Flk2+CD117hi DN1 cells was similar in wild-type and L-Fng-transgenic mice. Because Flk2 ‘marks’

primitive bone marrow progenitors and signaling through this receptor is required for very early stages of T cell and B cell development, we suggest that Flk2+CD117hi cells represent a primitive DN1 subset whose abundance is not affected by L-Fng-mediated inhibition of Notch1. The substantial increase in the CD117intCD24lo subset in L-Fngtransgenic thymi relative to that in wild-type thymi suggests that they are probably early B cell progenitors. Consistent with this, some

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Figure 5 DN1 thymocyte subsets in L-Fng-transgenic mice. Wild-type and L-Fng-transgenic thymi were depleted of Lin+ cells as described for Figure 4. (a) Lin thymocytes were stained with anti-CD25–biotin (avidinCyChrome), anti-CD44–phycoerythrin, anti-CD117–allophycocyanin and anti-CD24–FITC. Data represent CD44 versus CD25 expression and the quadrants used to identify DN1, DN2, DN3 and DN4 thymocytes (clockwise from top left). Numbers in quadrants indicate percent cells in those areas. (b) CD117 versus CD24 expression gated on CD44hiCD25 DN1 thymocytes from the samples in a. Outlined areas indicate gates used to quantify the frequency of CD117hiCD24hi, CD117intCD24hi and CD117intCD24lo subsets. Numbers in and above outlined areas indicate percent cells in those areas. Representative of three experiments. (c) Lin thymocytes were stained with anti-CD25–FITC, anti-CD44–phycoerythrin, anti-CD117–allophycocyanin, and anti-Flk2–biotin (or biotinylated rat IgG2a isotype control antibody) followed by avidin-CyChrome. Data represent expression of CD117 versus Flk2 gated on CD44+CD25 DN1 thymocytes; numbers beside outlined rectangular areas indicate the proportions of CD117hiFlk2, CD117hiFlk2+ and CD117intFlk2 subsets. Less than 0.3% of DN1 cells stained with the biotinylated rat IgG2a isotype control antibody in all samples. Representative of three experiments.

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Figure 4 Paucity of ETPs and DN2 thymocytes in L-Fng-transgenic mice. (a) DN subsets in wild-type (WT) versus L-Fng-transgenic (Tg+) mice. Thymocyte samples were immunomagnetically depleted of cells expressing B220, CD11b, GR-1, CD4, CD8a, CD3e and Ter119 and were stained with anti-CD117–allophycocyanin versus anti-CD25–FITC. Quadrants indicate the gating used to define the ETP, DN2, DN3 and DN4 subsets (clockwise from top left). Numbers in quadrants indicate percent cells in those areas. (b) Numbers of ETPs, DN2 and DN3 thymocytes in individual thymi from 4- to 6-week-old wild-type (n ¼ 6) and L-Fng-transgenic (n ¼ 5) mice. Thymocytes were stained with a lineage ‘cocktail’ of biotinylated antibodies specific for B220, CD11b, GR-1, CD4, CD8a and CD3e (avidin-CyChrome), and anti-CD117–allophycocyanin and anti-CD25–FITC. The frequencies of Lin thymocytes that were CD117hiCD25 (ETPs), CD117hiCD25+ (DN2) and CD117loCD25+ (DN3) were measured and were multiplied by the total number of cells in each thymus. Data represent the number of each subset per thymus, with lines connecting values from the same mouse. Two L-Fng-transgenic mice had no detectable DN2 thymocytes. Mean values 7 s.e.m.: ETP, 34,340 7 4,240 (wild-type) and 8,676 7 2,390 (L-Fng-transgenic); DN2, 84,740 7 11,730 (wild-type) and 2,748 7 1,175 (L-Fng-transgenic); DN3, 2 106 7 1.6 105 (wild-type) and 6.9 105 7 1.9 105 (L-Fng-transgenic). The differences between genotypes were statistically significant at each developmental stage: P ¼ 0.0004, ETP comparison; P o 0.0001, DN2 comparison; P ¼ 0.0002, DN3 comparison.

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Figure 6 Cells with an early B cell progenitor phenotype in L-Fng-transgenic thymi. (a) Wild-type and L-Fng-transgenic thymocytes were stained with antiCD117–allophycocyanin, were positively selected with immunomagnetic beads and then were stained with biotinylated anti-B220, anti-CD25, anti-CD4, anti-CD8a, anti-CD3e, anti-CD11b, anti-GR-1, anti-NK1.1 and anti-CD11c (avidin-CyChrome) and anti-AA4.1–FITC. Plots show expression of CD117 versus AA4.1 gated on LinCD25 cells. Numbers beside outlined rectangular areas indicate frequencies of CD117hiAA4.1 and CD117loAA4.1+ cells. Although many CD117 cells remained after positive selection, the frequency of CD117hi cells was increased by 83- to 250-fold relative to that of unfractionated thymocytes. Representative of two experiments. (b) Wild-type and L-Fng-transgenic thymocyte samples from 4- to 6-week-old mice were depleted of Lin+ cells as described for Figure 1, except that anti-B220 was replaced with anti-CD19. The samples after depletion, which contained less than 0.2% Lin+ thymocytes, were stained with anti-CD117–allophycocyanin, anti-CD25–biotin (avidin-CyChrome), antiAA4.1–phycoerythrin and anti-B220–FITC. Data represent expression of markers (along margins) gated on CD25 cells. Numbers above outlined rectangular areas indicate frequencies of CD117+AA4.1, CD117AA4.1+ and B220+AA4.1+ thymocytes. Representative of two experiments.

CD117int thymocytes were B220 AA4.1+ in L-Fng-transgenic thymi (Fig. 6a) and these cells coexpressed the interleukin 7 receptor-a (IL7Ra) chain (data not shown). This phenotype corresponds to ‘fraction Ao’, which is thought to include the earliest B cell progenitors in the bone marrow42. Fraction Ao cells then become B220+ pre–pro–B cells, so we evaluated the abundance of AA4.1+B220+CD19 cells in wildtype versus L-Fng-transgenic thymi. A sizable fraction of the LinCD19CD25 population coexpressed B220 and AA4.1 in LFng-transgenic but not wild-type mice, suggesting they were pre–pro– B cells (Fig. 6b). Collectively these data strongly suggest that the impairment of Notch1 activation by L-Fng misexpression in thymocytes allows TSPs to produce early B cell progenitors similar to those found in the bone marrow.

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Figure 7 Notch1+/ progenitors are highly sensitive transgenic L-Fng. Bone marrow Lin progenitors from Notch1+/+ (B6.CD45.1) or Notch1+/ (B6.CD45.1;CD45.2) donors were injected intrathymically into sublethally irradiated wild-type or L-Fng-transgenic B6.CD45.2 hosts (5 105 cells/ lobe). Then, 3 weeks later, thymocytes from each lobe were stained with anti-CD45.1–phycoerythrin, anti-CD45.2–FITC, biotinylated anti-CD3e, anti-CD4 and anti-CD8a (avidin-CyChrome) and anti-B220–allophycocyanin. (a) Expression of CD3e, CD4 and CD8a versus B220, gated on donorderived cells as in Figure 2. (b) Numbers of T cells and B cells derived from Notch1+/+ versus Notch1+/ donors in individual wild-type and L-Fngtransgenic hosts. Bars represent the mean values of three to four lobes per group. Similar results were obtained in four independent experiments.

B cell production by Notch1+/– TSPs We sought confirmation that TSPs generate ETPs in a Notch1dependent way using an independent experimental approach. In flies and worms, Notch signaling is subject to ‘gene dosage’ effects such that Notch+/ precursors often show inefficient Notch activation when they are adjacent to Notch+/+ cells36,37. Thus, in these situations, competition for Notch signals critically regulates cell fate ‘choices’. For example, Notch1+/ HSCs generate very few DP thymocytes when competing with Notch1+/+ HSCs in mixed bone marrow chimeras43, a finding we have confirmed (Supplementary Fig. 1 online). Notch1+/ bone marrow Lin progenitors generated many more B cells than did wild-type progenitors after intrathymic injection into L-Fngtransgenic thymic lobes8 (Fig. 7). Nonetheless, T cell production was normal and B cell development was minimal at steady state in thymi from Notch1+/ mice (Supplementary Fig. 2 online). Thus, Notch1+/ progenitors can make T cells efficiently in the wild-type thymic microenvironment but show enhanced B cell generation in

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L-Fng-transgenic thymic lobes and have difficulty making T cells in competitive conditions. Collectively these data demonstrate that Notch1+/ lymphoid progenitors are less able to activate Notch1 than are their wild-type counterparts. To determine the developmental stages at which Notch1+/ T cell progenitors become disadvantaged in mixed bone marrow chimeras, we assessed their contribution to bone marrow HSCs and thymic DN, DP and B cell subsets after 6–9 weeks (Supplementary Fig. 3 online). On average, the contribution of each donor to the bone marrow HSC subset (Fig. 8a) as well as B and myeloid bone marrow subsets (data not shown) was relatively equal, reflecting the input HSC frequency. Thus, Notch1+/ and Notch1+/+ HSCs showed comparable abilities to home to the bone marrow and establish multilineage hematopoiesis, in keeping with studies of conditionally Notch1-deficient mice2,44. Notably, however, 80% of ETPs were derived from Notch1+/+ HSCs when data from all chimeras were averaged (Fig. 8a; n ¼ 12). In most individual chimeras, the proportion of Notch1+/+-derived cells increased by 20–40% from the HSC to the ETP stage (Fig. 8b). The proportion of Notch1+/+-derived cells increased again at each subsequent stage, such that the average contribution of Notch1+/+ cells to the DP subset was over 90%. In some chimeras, Notch1+/+ HSCs engrafted preferentially and their contribution to the ETP subset did

DISCUSSION We have shown here that in contrast to B cell production from TSPs, B cell production from ETPs is inefficient and is not augmented by limiting intrathymic Notch1 activation. The Flk2+ ETP subset is probably more primitive and thus may be responsible for the inefficient B cell production from ETPs after intrafemoral or intravenous injection. We have also shown that TSPs and ETPs can be distinguished by differential sensitivity to low-threshold Notch1 signals. In the wild-type thymic microenvironment, Notch1+/ TSPs were no more likely to generate thymic B cells than were Notch1+/+ TSPs, suggesting that suppression of B cell potential by TSPs is exquisitely sensitive to low-threshold Notch1 signals that Notch1+/ and Notch1+/+ TSPs are equally able to transduce. However, Notch1+/ TSPs showed impaired generation of ETPs in mixed chimeras, suggesting that Notch1 signals of a higher threshold are required for this developmental transition. Notably, the decreased signaling competency of Notch1+/ progenitors was apparent only in competitive conditions, suggesting that access to Delta-like Notch ligands may be limiting in the normal thymus. This limitation probably explains why even Notch1+/+ TSPs do not efficiently suppress their B cell potential in the L-Fng-transgenic thymic microenvironment. Collectively these data show that Notch1 signals of low threshold act to suppress the B cell potential of TSPs before their development into

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Figure 8 Production of ETPs and thymic B cells from HSCs is differentially sensitive to decreased Notch1 ‘dosage’. Lethally irradiated Notch1+/+ B6.CD45.2 mice were injected intravenously with a 50:50 mixture of bone marrow Lin cells from Notch1+/+ (B6.CD45.1) and Notch1+/ (B6.CD45.1; CD45.2) donors (total of 4 105 cells/mouse). The contribution of each donor subset to bone marrow B cells and thymocyte DP subsets was determined 6–9 weeks later as described for Figures 1 and 2. (a) An aliquot of bone marrow cells and thymocytes from each host was depleted of Lin+ cells as described for Figure 4 and the contribution of each donor to bone marrow HSCs versus thymic DN subsets was analyzed as in Supplementary Figure 3 online. Data from four different experiments are averaged and data represent mean contribution (7 s.d.) of each donor to bone marrow HSCs versus the DN and DP thymocyte subsets (n ¼ 12). (b) Contribution of Notch-1+/+ (diamonds) versus Notch1+/ (circles) to bone marrow HSCs versus the DN and DP thymocyte subsets in an individual chimera. (c) Proportion of Notch1+/+-derived thymic B cells in individual chimeras from each of the four experiments in a.

not increase substantially, but the proportion of Notch1+/+ cells increased from the ETP to the DN2 stage (Fig. 8b, chimera 6). These data confirm a salient Notch1 dependence for the generation of ETPs from TSPs and document that T cell progenitors require continuous Notch1 signals from the ETP to the DN3 stage in vivo. Unexpectedly, however, the proportion of Notch1+/+ thymic B cells was more than 50% on average and was typically equal to or greater than the Notch1+/+ HSC frequency in each mixed chimera (Fig. 8c). The frequency of bone marrow B cells derived from each donor was also very similar to the donor HSC frequency in each individual chimera (data not shown). These experiments demonstrate that Notch1+/ TSPs are no more likely to generate thymic B cells than Notch1+/+ TSPs and thus show that suppression of B cell potential by TSPs is exquisitely sensitive to Notch1 signals. When intrathymic Notch1 activation is impaired, B cell production is favored at the expense of T cell production in the thymus4–8. These data have been interpreted to suggest that TSPs are minimally ‘bipotent’ and that intrathymic Notch1 activation determines whether they will ‘choose’ a T cell or B cell fate. However, direct evidence in support of this model is lacking, as TSPs have not been identified directly. It remains possible that the thymus is seeded by progenitors already committed to the T or B cell lineage. Impairment of intrathymic Notch1 activation would then allow the proliferation and maturation of committed B cell progenitors at the expense of committed T cell progenitors. To test this model, we injected purified pro–B cells into wild-type versus L-Fng-transgenic thymic lobes and found that pro–B cell population expansion and maturation into CD22hi pre–B cells occurred similarly in both environments (Supplementary Fig. 4 online). In contrast, bone marrow Lin progenitors produced many more B cells in L-Fng-transgenic than in wild-type thymic lobes and most were CD22lo at this time, reflecting their origin from an earlier progenitor. Thus, the L-Fng transgene favored intrathymic B cell development from multipotent progenitors (HSCs and bone marrow Lin cells)8 but had no effect on committed B cell progenitors. Collectively these data strongly support the idea that TSPs are not yet T cell committed and can adopt the B cell fate when intrathymic Notch1 activation is suppressed.

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ARTICLES ETPs, whereas Notch1 signals of a higher threshold are required for the generation of ETPs by TSPs. Thus, contrary to present models1,2, these results indicate that TSPs and ETPs are functionally distinct subsets. That conclusion is supported by other data suggesting that TSPs and ETPs are also phenotypically distinct. Cells that seed the thymus after very large numbers of heterogeneous bone marrow cells are injected into the bloodstream include progenitors for the B cell, T cell, dendritic cell and natural killer cell lineages but not HSCs29,30,45. However, none of those studies detected a prominent CD117hi subset among the earliest thymic immigrants, and in two of the studies29,45, cells with an ETP phenotype were not detectable until several days after injection. Nonetheless, the most efficient circulating T cell progenitors are contained in a rare CD117hi subset of blood cells that also contains HSCs28. Although CD117int DN1c thymocytes have T cell and B cell potential in vitro30, their population expansion potential is very limited. In agreement with those findings, we detected only weak T cell and B cell production from this subset after intrathymic or intrafemoral injection. Because DN1c thymocytes do not have robust T cell and B cell potential in vitro or in vivo, we believe it is unlikely that this subset contains TSPs. Our data as well as several published reports have demonstrated that intrathymic Notch1 activation is required for suppression of the B cell potential of multipotent progenitors in postnatal mice. Although these data do not demonstrate that the cells that normally seed the thymus are ‘bi-potential’, we believe that this is likely, as suppression of intrathymic Notch1 activation by transgenic L-Fng had no effect on committed B cell progenitors. In addition, L-Fng-transgenic thymi had decreased numbers of the earliest T cell progenitors and increased numbers of cells closely resembling very early bone marrow–derived B cell progenitors. The most parsimonious interpretation of these findings is that TSPs have robust T cell and B cell potential and that intrathymic Notch1 activation suppresses the B cell potential. However, in fetal mice, both circulating progenitors and the first thymuscolonizing cells seem to be T cell restricted46–48. Although the function of Notch in promoting fetal T cell commitment remains to be demonstrated, these data emphasize another potential mechanistic difference between fetal and adult lymphopoiesis49. Our data strongly indicate that there are different downstream effects for Notch1 signals of low threshold versus higher threshold, as they have different biological outcomes. In flies and lower vertebrates, Notch signaling induces expression of Hes genes to suppress particular cell fates1,2,10. Hes1 is required at the earliest stage of T cell development50, and overexpression of Hes1 or the related Hes5 is sufficient to block B cell generation by bone marrow lymphoid precursors51. Thus, the high expression of Hes1 in ETPs (and DN2 and DN3 thymocytes) is consistent with their low B cell potential. We suggest that lowthreshold Notch1 signals may be sufficient to induce Hes gene expression to suppress the B cell potential of TSPs. However, as Hes1 or Hes5 overexpression is not sufficient to promote ectopic T cell development from bone marrow lymphoid progenitors51, Notch1 signals of higher threshold may be necessary to induce expression of the additional target genes required for T lineage specification. Notably, in vitro experiments suggest that induction of Hes1 transcripts can be temporally uncoupled from expression of several transcription factors thought to be important for T lineage specification52. Both of our experimental approaches demonstrated a considerable Notch1 dependence for the ETP-to-DN2 and DN2-to-DN3 transitions. The small numbers of ETPs generated in L-Fng-transgenic thymi produced very few DN2 cells, in contrast to the population expansion

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seen in wild-type mice during this transition. In mixed bone marrow chimeras, the contribution of Notch1+/ cells decreased between the ETP and the DN2 stage and further decreased between the DN2 and the DN3 stage, suggesting that Notch1 activation is needed continuously to promote survival or proliferation throughout the early stages of intrathymic T cell development. This conclusion is supported by studies showing that overexpression of the Notch inhibitor Nrarp blocks progression through the DN phases of thymocyte development53 and that DN2 and DN3 thymocytes depend on interactions with the Notch ligand DL1 to survive and proliferate on OP9 stromal cells in vitro33–35. Notch1 signaling may also promote TCRb rearrangement32, the ab-versus-gd lineage ‘decision’31,43 or cell migration54 during the ETP-to-DN3 stages. Finally, it may promote the loss of residual B cell, macrophage, natural killer or dendritic cell potential that has been detected in these subsets19,23,30,38,55,56. An important goal of future studies will be the identification of Notch1 targets that regulate these early stages of intrathymic T cell development. METHODS Mice and adoptive transfer experiments. B6.SJL-Ptprca (B6.CD45.1) and C57BL/6NTac-Ptprcb (B6.CD45.2) mice were purchased (Taconic) and were bred in our specific pathogen–free facility (Hospital for Sick Children, Toronto, Ontario, Canada). These mice were also intercrossed to produce B6.CD45.1;CD45.2 heterozygous donor or host mice for adoptive transfer experiments. L-Fng-transgenic mice have been described8; all mice used in the experiments described here were backcrossed ten generations or more to B6.CD45.2 mice. B6.129-Notch1tm1Con/J (Notch1+/) mice were originally obtained from the Jackson Laboratory and have been backcrossed at least ten generations to B6.CD45.2 mice. For adoptive transfer experiments, Notch1+/ B6.CD45.2 and Notch1+/+ B6.CD45.1 mice were intercrossed to generate Notch1+/ B6.CD45.1;CD45.2 heterozygous donor mice. Mouse genotypes were determined by PCR amplification of DNA obtained from tail tissue, as described8. Intrathymic8 and intrafemoral40 injections were made as described into anesthetized host mice (4–7 weeks old) that had been sublethally irradiated (650 cGy with a 137Cs g-irradiator) up to 4 h before injection. For mixed bone marrow chimeras, host mice between 6 and 8 weeks of age were lethally irradiated (1,000 cGy) up to 4 h before tail vein injections. All animal experiments followed protocols approved by the Hospital for Sick Children Animal Care Committee (Toronto, Canada). Flow cytometry and cell purification. Single-cell suspensions were stained with fluorochrome-conjugated antibodies and secondary reagents (Supplementary Methods online) and immunofluorescence was analyzed on a FACSCalibur (BD Biosciences) using standard techniques. Data files were uploaded into FlowJo (Tree Star) for analysis. Dead cells and debris were excluded by propidium iodide and forward-scatter gating. Statistical significance was calculated using the unpaired t-test (one-tailed; Fig. 4). For lineage depletions, cells were stained with a ‘cocktail’ of rat immunoglobulin G (IgG) antibodies specific for B220, CD11b, GR-1, CD4, CD8a, CD3e and Ter119 (each used at predetermined saturating concentrations) and then samples were depleted of Lin+ cells with immunomagnetic beads coated with antibody to rat IgG (anti–rat IgG) using the AutoMacs DEPLETE-S mode according to manufacturer’s instructions (Milteny Biotech). Fewer than 0.5% Lin+ cells were detected in samples after depletion. Other antibody ‘cocktails’ used are identified in the figure legends. After magnetic depletion or enrichment, samples were blocked with 20% normal rat serum before being stained with additional antibodies. CLPs and HSCs were sorted from bone marrow depleted of Lin+ cells as LinIL7Ra+CD117lo and LinSca-1hiCD117hi cells, respectively. For purification of ETPs, single-cell suspensions of B6.CD45.1 thymocytes (6- to 10-week-old mice) were stained with purified anti-CD117 or anti-CD117–allophycocyanin, were incubated with magnetic beads coated with anti–rat IgG and were positively selected using the POSSELD-S mode of an AutoMacs (Miltenyi Biotech). Positively selected cells were then stained with a ‘cocktail’ of biotinylated antibodies specific for CD3e, CD4, CD8a, CD11b, GR-1 and B220,

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together with anti-CD44–phycoerythrin and anti-CD25–phycoerythrin– indotricarbocyanine. Biotinylated markers were visualized by staining with avidin conjugated to phycoerythrin–Texas Red (BD Biosciences), and ETPs were sorted as LinCD25CD44hi CD117hi cells. Cells were sorted on a MoFlo (DakoCytomation) equipped with Argon I-90 and Krypton 302c lasers emitting excitation wavelengths of 488 and 647 nm, respectively, or on a BD FACStar Plus equipped with Argon I-70 and HeNe lasers emitting excitation wavelengths of 488 and 633 nm, respectively. Isolation of mRNA and quantitative real-time PCR. DP (TCRbintCD4+CD8+) thymocytes were sorted from total thymocytes stained with anti-CD4–phycoerythrin, anti-CD8a–allophycocyanin and anti-TCRb–fluorescein isothiocyanate (FITC). Before sorting of CD4 cells (TCRbhiCD4+CD8) or CD8 cells (TCRbhiCD4CD8+), thymocyte samples were immunomagnetically depleted of either CD8+ or CD4+ cells, respectively, were stained with goat anti–rat IgG– Alexa 633 (Molecular Probes), were blocked with 20% normal rat serum and then were stained with anti-TCRb–FITC and either anti-CD8–allophycocyanin or anti-CD4–phycoerythrin. DN subsets were sorted from thymocyte samples that had been depleted of Lin+ cells and were stained with goat anti–rat IgG–phycoerythrin (Cedarlane), followed by normal rat serum, antiCD117–allophycocyanin and anti-CD25-FITC. ETP (CD117hiCD25), DN2 (CD117hiCD25+), DN3 (CD117CD25+) and DN4 (CD117CD25) subsets were sorted on a MoFlo or on a BD FACStar Plus. Purity was more than 98% for all sorted populations used in this study. The poly(A)+ mRNA from each sorted thymic subset was isolated using the mMACS mRNA Isolation Kit (Miltenyi Biotech) and was reverse-transcribed. The abundance of Hes1 and Cd45 cDNA in each sample was determined using the Taqman qRT-PCR Assay Kit (Applied Biosystems) together with amplification primers and dual-labeled probes complementary to an internal sequence of the Hes1 or Cd45 amplicons (Supplementary Methods online). OP9 cultures. Sorted ETPs (1 103 to 1 104 cells/well), CLPs (5 102 to 1 103 cells/well), HSCs (4 103 to 1 104 cells/well) or bone marrow Lin cells (3 103 to 3 104 cells/well), were seeded onto 80% confluent monolayers of OP9 cells (24-well plates) in DMEM 10-013V medium (Wisent) supplemented with 10% fetal bovine serum, 25 mM HEPES, pH 7.0, 55 mM 2-mercaptoenthanol, 2 mM L-glutamine, a 0.1 mM solution of nonessential amino acids, 100 ng/ml of human Flt3 ligand (R & D System) and 10 ng/ml of mouse IL-7 (Stem Cell Technologies). Cells were incubated at 37 1C in a humidified atmosphere with 5% CO2. The presence of B lineage cells was evaluated between 8 and 14 d by gating on viable lymphoid cells that coexpressed B220 and CD19. The percentage of B220+CD19+ cells produced from HSCs, CLPs and bone marrow Lin cells ranged from 20% to 90% in these experiments, whereas few viable cells and no B220+CD19+ cells were recovered from cultures seeded with ETPs. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank C. Paige (Ontario Cancer Institute, Toronto, Canada) for OP9 cells; J. Dick and M. Doedens (University Health Network, Toronto, Canada) as well as L. Nutter for help with intrafemoral injections; S. Zhao for cell sorting; H. Petrie (University of Miami, Miami, Florida), W. Pear and A. Bhandoola (University of Pennsylvania, Philadelphia, Pennsylvania) for sharing data before publication; H. Petrie for Ter119; and J. Danska and M. Anderson (Sunnybrook & Women’s College Hospital, Toronto, Canada) for comments on the manuscript. Supported by the Canadian Institutes of Health Research (C.J.G.) and Hospital for Sick Children (Restracom Studentship to J.Y.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 24 February; accepted 23 May 2005 Published online at http://www.nature.com/natureimmunology/

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Erratum: Requirement for Notch1 signals at sequential early stages of intrathymic T cell development Joanne B Tan, Ioana Visan, Julie S Yuan & Cynthia J Guidos Nature Immunology 6, 671–679 (2005).


On page 673, the second sentence of the legend to Figure 2 should begin “ETPs (3 × 104) or Lin– bone marrow cells (2 × 104) purified from B6.CD45.1 mice….”

Erratum: Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway Wouter J de Jonge, Esmerij P van der Zanden, Frans O The, Maarten F Bijlsma, David J van Westerloo, Roelof J Bennink, Hans-Rudolf Berthoud, Satoshi Uematsu, Shizuo Akira, Rene M van den Wijngaard & Guy E Boeckxstaens Nature Immunology 6, 844–851 (2005). On page 845, the last sentence should begin “The selective non-α7 nAChR antagonist dihydro-β-erythroidine.…” On page 848, in the legend for Figure 7, lines 15–16, the scale bar should be described as “Scale bar, 20 µm (40 µm for far left image).”

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