Nature Immunology
5, 64 - 73 (2003)
Published online: 21 December 2003; | doi:10.1038/ni1022
An essential function for the nuclear receptor ROR t in the generation of fetal lymphoid tissue inducer cellsGérard Eberl1, 5, Shana Marmon1, Mary-Jean Sunshine1, 2, Paul D Rennert3, Yongwon Choi4
& Dan R Littman1, 21 Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016, USA. 2 Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016, USA. 3 Department of Immunology, Biogen, Cambridge, Massachusetts 02142, USA. 4 Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. 5 Present address: Aaron Diamond AIDS Research Center, New York, New York 10016, USA.
Correspondence should be addressed to Gérard Eberl geberl@adarc.org or Dan R Littman littman@saturn.med.nyu.eduLymphoid tissue inducer (LTi) cells are associated with early development of lymph nodes and Peyer's patches. We show here that during fetal life the nuclear hormone receptor ROR t is expressed exclusively in and is required for the generation of LTi cells. RORt+ LTi cells provide essential factors, among which lymphotoxin- 1 2 is necessary but not sufficient for activation of the mesenchyma in lymph node and Peyer's patch anlagen. This early LTi cell−mediated activation of lymph node and Peyer's patch mesenchyma forms the necessary platform for the subsequent development of mature lymphoid tissues.Development of the murine lymphatic system begins at embryonic day 10.5 (E10.5; 10.5 days after conception) with the invagination of endothelial cells from veins and the formation of lymphoid sacs1. It is not known which events initiate the localized development of lymph nodes or Peyer's patches. However, LTi cells, characterized as CD4+CD3- and interleukin 7 receptor- (IL-7R)-positive cells, are among the first hematopoietic cells to be detected in lymph node and Peyer's patch anlagen2,
3,
4,
5,
6. These cells are found in spleen, blood and lymph node anlagen by E12.5 and in Peyer's patch anlagen by E16, but are present in only very low numbers after birth. The absence of IL-7R+ cells and Peyer's patches in mice deficient in IL-7R or Jak3 kinase, required for IL-7R-mediated signaling, has led to the suggestion that IL-7R+ cells are 'Peyer's patch inducers'4,
5,
7. In addition, purified CD4+CD3- cells induce the development of Peyer's patches or nasal-associated lymphoid tissue when transferred into mice otherwise deficient in Peyer's patches or nasal- associated lymphoid tissue8,
9. These data show that LTi cells are involved in the development of lymph nodes, Peyer's patches and nasal-associated lymphoid tissue.
The appearance of LTi cells in lymph node and Peyer's patch anlagen correlates with a lymphotoxin- (LT-)-dependent increase in local expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1)5,
10. LTi cells express the membrane-bound LT1 2 (refs. 3,5), whereas mesenchymal cells purified from Peyer's patch anlagen express VCAM-1, ICAM-1 and LTR11, the receptor for LT12. This indicates that LTi cells and mesenchymal cells interact in lymph node and Peyer's patch anlagen through LT12 and LTR to induce further development of lymph nodes and Peyer's patches. In accordance with a central function for this interaction12, lymph nodes and Peyer's patches are absent in mice deficient in LT13, LTR14 or factors required for signaling downstream of LTR15,
16,
17.
LTi cells, as well as lymph nodes and Peyer's patches, are absent from mice deficient in the inhibitory transcription factor Id2 (ref. 18) or the retinoic acid−related orphan receptors (RORs) ROR and RORt19,
20. ROR and RORt are two isoforms that differ in their N-terminal sequence, encoded by alternative 5' exons within the Rorc locus. Whereas ROR mRNA is detected in many adult tissues, including liver, lung, muscle, heart and brain, RORt mRNA and protein has been found only in immature double-positive CD4+CD8+ thymocytes21,
22,
23. The isoform expressed in LTi cells and required for lymph node and Peyer's patch development has not been described. To study the function of the individual ROR isoforms in lymph node and Peyer's patch development, we generated mice in which the gene encoding enhanced green fluorescent protein (EGFP) was targeted to the first exon of the gene encoding RORt (called Rorc(t) here). We found that during fetal life, RORt was expressed exclusively in CD4+ and CD4- LTi cells. This restricted fetal expression of RORt in LTi cells allowed us to visualize early lymph node and Peyer's patch development and to assess the function of different factors in the generation and recruitment of LTi cells into lymph node and Peyer's patch anlagen. The absence of LTi cells in RORt-deficient mice allowed us to evaluate directly the function of LTi cells in lymph node and Peyer's patch development.
Results
Generation of Rorc(t)-EGFP 'knock-in' mice
The mouse Rorc locus on chromosome 3 encodes both the ROR and RORt isoforms (Supplementary Fig. 1 online)22,
24. An alternative first exon (exon 1t), used in expression of RORt, is located approximately 2 kb downstream of exon 2, and is spliced to the common exon 3. Using homologous recombination in embryonic stem cells, we inserted the coding sequence of EGFP into exon 1t directly downstream of the ATG translational start codon (Supplementary Fig. 1 online). The translational stop codon of the coding sequence for EGFP was retained, but no additional splice or polyadenylation sites were introduced into exon 1t. The loxP-flanked neomycin-resistance (neor) cassette, used to select targeted embryonic stem cells, was excised in vivo by crossing heterozygous Rorc(t)+/GFPneo founder mice with 'Cre-deleter' mice (Supplementary Fig. 1 online). For subsequent experiments, we used only heterozygous Rorc(t)+/GFP mice to analyze expression of the Rorc(t) gene and homozygous Rorc(t)GFP/GFP mice to study the phenotype of RORt-deficient mice.
A segment encompassing exons 1−5 of the ROR mRNA was amplified by RT-PCR and detected in the liver of wild-type Rorc(t)+/+, mutant Rorc(t)+/GFP and Rorc(t)GFP/GFP mice, showing that insertion of the sequence encoding EGFP into exon 1t did not affect the transcription of Rorc() mRNA. In contrast, a segment encompassing exons 1t−5 of Rorc(t) mRNA was amplified from the thymi of Rorc(t)+/+ and Rorc(t)+/GFP mice, but not of Rorc(t)GFP/GFP mice, as expected (Supplementary Fig. 1 online). In accordance with these data, RORt protein was detected in the thymus of Rorc(t)+/+ and Rorc(t)+/GFP mice, but not of Rorc(t)GFP/GFP mice. However, no ROR protein could be detected in total liver samples of the different mouse lines, indicating very low expression of ROR or that only a small fraction of liver cells express ROR (Supplementary Fig. 1 online).
In the fetus, only LTi cells express RORt
We first analyzed EGFP expression, which reports Rorc(t) transcription, in the thymi of adult Rorc(t)+/GFP mice. Consistent with previous results22,
23, EGFP was expressed in double-positive thymocytes but not double-negative thymocytes (Fig. 1a). We also found small amounts of EGFP in CD4+ and CD8+ single-positive thymocytes, although Rorc(t) mRNA was not detected in these populations22,
23. This result may be because of the long half-life of EGFP protein (>24 h), which would be detected in single-positive thymocytes even after cessation of transcription of the sequence encoding EGFP. In addition, expression of EGFP was confined to the thymic cortex, the resident site for double-positive and immature single-positive heat-stable antigen−high thymocytes (Fig. 1a, right). We obtained similar results with a monoclonal antibody (mAb) recognizing both ROR isoforms19 (data not shown).
 | | Figure 1. In the fetus, RORt is exclusively expressed in LTi cells. | | |  | (a) Left: CD4 and CD8 expression in thymocytes from 6- to 8-week-old Rorc(t)+/GFP mice. Boxed areas indicate cell populations analyzed for EGFP expression. Center: blue histograms, thymocytes from Rorc(t)+/GFP mice; green lines, thymocytes from Rorc(t)+/+ mice. SP, single-positive; DP, double-positive; DN, double-negative. Right: staining of Rorc(t)+/GFP thymus with anti-EGFP. Cortex and medulla were defined by nuclear DAPI staining (data not shown). Data are representative of at least five independent experiments. (b−f) Sections from whole Rorc(t)+/GFP fetuses at age E16.5 (b−d,f) or E12.5 (e) were stained as indicated by color of lettering above sections. Thymus, spleen, intestine and lymph node anlagen in designated regions are shown here. Green autofluorescence was used to visualize tissue structure. Arrowheads in e point to EGFP+ cells. Pink cells in f are stained for both red (CD11b or MHC class II) and blue (CD45). Original magnifications: b,  100 (top) and 200 (bottom); c−f, 400. Sections are representative of at least ten individual sections and three to ten independent experiments.
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We assessed expression of EGFP during fetal life by immunofluorescence histology on serial sections of whole Rorc(t)+/GFP fetuses. We first analyzed E16.5 fetuses. At that stage, no mature B cells are yet produced25 and T cells can only be found in the thymus. We identified lymph node anlagen by the presence of large clusters of hematopoietic (CD45+) cells in the immediate vicinity of veins10, whereas we could not identify Peyer's patch anlagen on that basis because of the presence of CD45+ cells throughout the lamina propria and the submucosal region of the intestine4 (Fig. 1b). EGFP was expressed exclusively in tight clusters of cells found in lymph node anlagen, in the submucosal region of the intestine and around central vessels in the spleen (Fig. 1b). However, EGFP was not expressed in the thymi of E16.5 fetuses, in contrast to earlier findings based on the detection of Rorc(t) mRNA26. This discrepancy could be explained by the presence of parathymic lymph node anlagen that may have been isolated together with the thymus and thus account for the detection of Rorc(t) mRNA in thymus samples (data not shown). Most but not all RORt+ cells expressed CD4, whereas only rare cells expressed CD4 but not RORt (Fig. 1c). In contrast, all RORt+ cells expressed IL-7R (Fig. 1d). These results are in accordance with previous work showing that both CD4+IL-7RA+ cells and CD4-IL-7RA+ cells are present in similar numbers in Peyer's patch anlagen and express similar sets of chemokine receptors5,
27. Both the localization and the phenotype of CD4+ and CD4- RORt+ cells identify these as LTi cells. We detected RORt+ cells as early as E12.5 in the immediate vicinity of vessels among small clusters of CD45+ cells (Fig. 1e). From E12.5, RORt+ cell numbers increased up to birth (E20) and decreased thereafter within 2 d to barely detectable amounts (data not shown). We obtained similar results when we stained fetus sections with a mAb to both RORt and ROR (data not shown).
When we attempted to identify the CD45+ hematopoietic cells surrounding the clusters of RORt+ cells, we found that a fraction of these cells (10−20%) expressed CD11b (Fig. 1f), a 2 integrin confined to cells of the natural killer, polymorphonuclear and myeloid lineages28. However, CD45+ cells present in lymph node and Peyer's patch anlagen did not express additional markers of the myeloid lineage, such as Gr-1 and CD11c (data not shown). A larger fraction (approximately 50%) of CD45+ cells, as well as 25−50% of RORt+ cells, expressed major histocompatibility complex (MHC) class II proteins3 (Fig. 1f), usually expressed on antigen-presenting cells. The relevance of MHC class II expression in lymph node and Peyer's patch anlagen of E16.5 fetuses is not apparent, as at this stage CD4+ T cells are found only in the thymus.
RORt is required for the generation of LTi cells
We first examined whether lack of the RORt isoform accounts for the previously described increased cell cycle progression and susceptibility to apoptosis of thymocytes deficient in both the ROR and RORt isoforms19. Consistent with an essential function for RORt in thymocyte development, thymocytes from Rorc(t)GFP/GFP mice underwent apoptosis in culture within 2−3 h, whereas most thymocytes from heterozygous Rorc(t)+/GFP or wild-type mice survived in culture for more than 5 h (Fig. 2a). Moreover, 42% of the freshly isolated RORt-deficient thymocytes were in the S − and G2/M phases of the cell cycle, compared with 14% of wild-type thymocytes (Fig. 2b).
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Whereas heterozygous Rorc(t)+/GFP mice developed lymph nodes and Peyer's patches normally, RORt-deficient mice lacked all lymph nodes and Peyer's patches, as well as LTi cells. We detected no EGFP+ cells in sections of E16.5 Rorc(t)GFP/GFP fetuses (Fig. 2c), although EGFP+ double-positive thymocytes were present in adult mice. Rorc(t)GFP/GFP mice had fewer double-positive cells (30−50% that of wild-type mice) and expressed twice as much EGFP as did thymocytes from heterozygous Rorc(t)+/GFP mice (data not shown). These data demonstrate that RORt is necessary for the generation of LTi cells. As RORt was exclusively expressed in LTi cells in the fetus, these results also demonstrate that LTi cells are necessary for the development of lymph nodes and Peyer's patches. In contrast, clusters of hematopoietic CD45+ cells were still present in E16.5 Rorc(t)GFP/GFP fetuses, as were CD11b+ and MHC class II+ cells (Fig. 2c), showing that RORt is not involved in their generation.
Mice deficient in Id2, an inhibitor of basic helix-loop-helix transcription factors, also lack all lymph nodes, Peyer's patches and LTi cells18. We were unable to find RORt+ cells in sections of E16.5 Id2-deficient Rorc(t)+/GFP fetuses (Fig. 2d). However, in addition to LTi cells, Id2-deficient mice also lacked CD11b+ cells, whereas clusters of hematopoietic CD45+ cells and MHC class II+ cells were still present (Fig. 2d). These data show that Id2 is required for the generation not only of LTi cells but also of other fetal cells, including CD11b+ cells. The absence of fetal CD11b+ cells may account for the greatly reduced number of natural killer cells and splenic CD8+ dendritic cells, as well as the lack of Langerhans cells, in adult Id2-deficient mice18,
29.
LT12 is necessary but not sufficient for LTi cell function
Earlier reports have shown that LTi cells colocalize in lymph node and Peyer's patch anlagen at sites where ICAM-1 and VCAM-1 are expressed by mesenchymal cells5,
10,
11. Both adhesion molecules had low expression throughout the Rorc(t)+/GFP E16.5 fetuses on most endothelia (Fig. 3). However, ICAM-1 and VCAM-1 were most abundantly expressed in the thymus (data not shown) and in lymph node and Peyer's patch anlagen (Fig. 3a−c). High expression of ICAM-1 and VCAM-1 was not induced in emerging lymph node and Peyer's patch anlagen with low numbers of LTi cells (Fig. 3c). In the absence of LTi cells in Rorc(t)GFP/GFP fetuses, we found no expression of ICAM-1 and VCAM-1 in putative lymph node and Peyer's patch anlagen (Fig. 3d). This result shows that LTi cells are required for the activation of mesenchyma in lymph node and Peyer's patch anlagen.
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LT and its receptor, LTR, are essential in lymph node and Peyer's patch development13,
14. Little or no expression of VCAM-1 is detected in the intestine and lymph node anlagen of LT-deficient mice, even though IL-7R+ cells are still present5,
10. Consistent with these observations, the number of LTi cells present in lymph node anlagen of LT-deficient Rorc(t)+/GFP E16.5 fetuses was normal, but large amounts of ICAM-1 and VCAM-1 failed to be induced (Fig. 4a). In the fetus, LTi cells are thought to be the main if not the only producers of LT12, the ligand of LTR5,
11,
30. Activation of the LTR with an agonist mAb was previously shown to rescue lymph node and Peyer's patch development in LT-deficient mice12. Consistent with that earlier report, the agonist mAb induced ICAM-1 and VCAM-1 expression in lymph node anlagen and restored lymph node development in LT-deficient mice. In contrast, this mAb failed to do so in RORt-deficient mice (Fig. 4b,c). These results demonstrate that LTi cells induce lymph node and Peyer's patch development by activating the LTR signaling pathway in mesenchymal cells, and that additional LTi cell-derived factors are required and remain to be identified.
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Factors involved in recruitment of LTi cells
Accumulation of LTi cells in lymph node and Peyer's patch anlagen results from local expansion or from active recruitment. In sections of Rorc(t)+/GFP E16.5 fetuses stained for the proliferation marker Ki67, we found no proliferating LTi cells, and only very rare CD45+ hematopoietic cells were Ki67+ (Fig. 5a). In contrast, substantial numbers of CD45-RORt- cells stromal cells did proliferate. This indicates that LTi cells are recruited to lymph node and Peyer's patch anlagen.
| | Figure 5. LTi cells do not proliferate, but are recruited to lymph node and Peyer's patch anlagen. | | |  | (a) LTi cells do not proliferate in lymph node or Peyer's patch anlagen. Sections from whole Rorc(t)+/GFP E16.5 fetuses were costained for nuclei (DAPI), EGFP, CD45 and Ki67. For clarity, only stainings for two markers are shown together on the same panel. A parathymic lymph node anlage is shown. Original magnification, 400. Similar results were obtained in other lymph node anlagen, as well as in Peyer's patch anlagen. Sections are representative of at least ten individual sections and three independent experiments. (b) Blockade of IL-7R early in fetal development inhibits the accumulation of LTi cells. Sections from whole Rorc(t)+/GFP E16.5 fetuses, obtained from pregnant mothers treated at E12 and E15, or at E15 only, with a blocking mAb to IL-7R, were stained as indicated by color of lettering above sections. Only rare lymph node anlagen, such as the small cervical lymph node anlage at top left, were present in fetuses treated at E12 and E15. Original magnifications, 250 (top and bottom right) and 100 (remaining bottom).
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Many factors known to be required for lymph node and Peyer's patch development may function in the recruitment of LTi cells. In Rorc(t)+/GFP fetuses, it is possible to visually assess the function of these factors early in lymph node and Peyer's patch development. Mice deficient for BLC (CXCL13) or for its receptor, CXCR5, lack most lymph nodes and Peyer's patches, with the exception of mesenteric and deep cervical lymph nodes31,
32. Consistent with these data, LTi cells in BLC-deficient mice were present in normal numbers only in mesenteric and cervical lymph node anlagen (data not shown). Mice deficient in IL-7R or Jak3, or treated with a blocking mAb to IL-7R, lack LTi cells in Peyer's patch anlagen and fail to develop Peyer's patches5,
7 but develop most lymph node anlagen10,
27. In E16.5 Rorc(t)+/GFP fetuses treated on E12 and E15 with a blocking mAb to IL-7R, clusters of LTi cells were absent from the intestine and lymph node anlagen, or present only in reduced numbers (Fig. 5b). In fetuses treated with mAb to IL-7R only on E15, clusters of LTi cells were present in Peyer's patch anlagen and the number of LTi cells in lymph node anlagen seemed normal (Fig. 5b). These data indicate that IL-7R is required early in the recruitment of LTi cells to lymph node and Peyer's patch anlagen, and that other factors may provide recruiting functions later during lymph node development. Mice deficient in the tumor necrosis factor (TNF) family cytokine TRANCE33 or the intracellular adaptor TRAF6 (ref. 10), involved in the signaling downstream of the TRANCE receptor, have severely reduced numbers of LTi cells in mesenteric lymph node anlagen at birth. These mice fail to develop lymph nodes, whereas Peyer's patch development is not affected. In E16.5 fetuses treated on E12 and E15 with TRANCE receptor−immunoglobulin fusion protein, the number of LTi cells found in lymph node anlagen was generally reduced (Supplementary Fig. 2 online). Finally, mice deficient in TNF or TNF-R-I (a receptor for both TNF and LT3) have reduced numbers of Peyer's patches34,
35. Consistent with these data, we found only rare LTi cells in the intestine of E16.5 fetuses treated with TNF-R-I−immunoglobulin fusion protein (Supplementary Fig. 2 online).
Discussion
Using a knock-in mouse model, we have shown here that during fetal life RORt is expressed exclusively in LTi cells and that in its absence, LTi cells are not generated and lymph nodes and Peyer's patches do not develop. In lymph node and Peyer's patch anlagen, LTi cells induce activation of the local mesenchyma and subsequent lymph node and Peyer's patch development, by way of the LT12-LTR interaction and additional factors that remain to be defined. Moreover, accumulation of LTi cells into lymph node and Peyer's patch anlagen is the result of active recruitment by chemokines and cytokines, rather than local proliferation of LTi cells. Our data demonstrate that RORt is required for lymph node and Peyer's patch development as a consequence of its specific and essential function in the generation of LTi cells.
The results demonstrate that LTi cells, previously characterized as CD3-CD4+CD45+IL-7R+ cells2,
3,
4,
5,
6, are defined by the expression of RORt. The absence of LTi cells and of lymph nodes and Peyer's patches in RORt-deficient mice formally demonstrates that LTi cells are necessary for the development of lymph nodes and Peyer's patches. Like RORt, the inhibitor of basic helix-loop-helix transcription factors Id2 is also required for the generation of LTi cells18. However, unlike RORt, Id2 was also required for the appearance of fetal CD11b+ cells. Moreover, Id2 is involved in various cell differentiation programs during embryonic development36. RORt is thus the only molecule to our knowledge known to be expressed exclusively in fetal LTi cells.
The results also demonstrate that LTi cells interact with and activate mesenchymal cells present in lymph node and Peyer's patch anlagen, as suggested by earlier studies37. We assessed activation of mesenchyma by the expression of the adhesion molecules ICAM-1 and VCAM-1. In E16.5 fetuses, these molecules were highly expressed only in lymph node and Peyer's patch anlagen, as well as in thymus and spleen, consistent with a central function for these adhesion molecules in lymph node and Peyer's patch development. A threshold number of LTi cells was required in this process, reflecting a possible 'community effect' of clustered LTi cells33.
Mesenchymal cells express LTR11, whereas LTi cells express its ligand, membrane LT12 (refs. 3,5). The essential function of the LT12-LTR interaction in lymph node and Peyer's patch development is shown by the absence of lymph nodes and Peyer's patches in LT-deficient mice13. Lymph node and Peyer's patch development can be rescued in LT-deficient mice by treatment of embryos with an agonist mAb to LTR12. However, treatment of RORt-deficient mice with this mAb did not rescue lymph node and Peyer's patch development. Similarly, the agonist mAb does not rescue lymph node development in TRANCE-deficient mice, and transgenic TRANCE expression (in cells distinct from LTi cells) does not rescue lymph node development in LT-deficient mice6. These data show that the function of LTi cells in inducing lymph node and Peyer's patch development is not confined to providing LT12 for the activation of the mesenchyma. Other factors expressed by LTi cells, such as TRANCE33, may be part of the necessary arsenal of factors produced by LTi cells during lymph node development. As TRANCE is not involved in Peyer's patch development, additional factors provided by LTi cells remain to be defined.
The accumulation of LTi cells in lymph node and Peyer's patch anlagen may result from active recruitment of circulating cells, or from expansion of resident LTi cells or CD45+ precursor cells. In favor of the first possibility, we found that no LTi cells and only rare hematopoietic cells proliferated in lymph node and Peyer's patch anlagen. In addition, IL-7R, TRANCE, TNF-R-I and BLC (CXCL13) were all involved in the early recruitment of LTi cells to lymph node and Peyer's patch anlagen. The individual function of each of these factors is not apparent, and recent reports have shown that BCL (CXCL13) forms a redundant recruitment system with IL-7R and CCR7 for the development of particular lymph nodes27,
38.
Recruitment of LTi cells to lymph node and Peyer's patch anlagen may be favored by a positive feedback loop generated by activation of mesenchyma by LTi cells through the LT12-LTR interaction39. Ligation of the LTR induces expression of an array of chemokines in embryonic fibroblasts and in B cells, including BLC (CXCL13), ELC (CCL19) and SLC (CCL21)40,
41. BLC and ELC are also expressed by ICAM-1+VCAM-1+ mesenchymal cells in Peyer's patch anlagen11. In addition, the receptors for BLC (CXCR5) and ELC and SLC (CCR7) are expressed by LTi cells and transduce chemotactic signals in LTi cells in vitro11. LTi cells may therefore migrate to lymph node and Peyer's patch anlagen in response to basal levels of chemokine expression by mesenchymal cells and cytokines. There, LTi cells activate the mesenchyma through the LT12-LTR interaction, induce further chemokine expression and increase recruitment of LTi cells. However, even though the mesenchyma of lymph node and Peyer's patch anlagen failed to be activated in LT-deficient fetuses, we found that the number of LTi cells present was not affected. This indicates that a positive feedback loop may operate efficiently only at later stages of lymph node and Peyer's patch development; that is, after E16.5. At this stage, a threshold number of LTi cells may be present to exert a 'community effect' and induce activation of mesenchyma. The LT12-LTR interaction seems also to be required for prolonged recruitment or survival of LTi cells in lymph node anlagen, as few LTi cells are found at birth in lymph node anlagen of LT-deficient mice33.
In conclusion, the nuclear hormone receptor RORt defines by its expression a single population of fetal cells, the LTi cells. The function of RORt in the generation of LTi cells is not known. Previous findings on the requirement for RORt in double-positive thymocytes indicate that it could control survival of LTi cells19. However, forced expression of the anti-apoptotic protein Bcl-xL in place of RORt failed to rescue LTi cell generation (data not shown). It will therefore be useful to identify targets of RORt in LTi cells. Analysis of such genes is likely to provide insights into the mechanisms of lymph node and Peyer's patch development.
Methods
Generation of RORt-EGFP mice.
The mouse bacterial artificial chromosome (BAC) library ClTB (Research Genetics) was screened by PCR with primers amplifying a 280bp fragment containing Rorc() exon 1. We identified one clone, I15, that contained all the Rorc locus, as determined by restriction enzyme mapping, and used it as the source of genomic DNA for the Rorc locus. The targeting vector used for homologous recombination in embryonic stem cells consisted of a short homology arm followed by EGFP, a loxP-flanked neor cassette (from pL2neo; Supplementary Fig. 1 online), a long homology arm and the thymidine kinase gene, cloned into the pGEM-11Zf vector. The short arm consisted of a 1-kb fragment located immediately 5' of the ATG translational start site of Rorc(t) exon 1, and was amplified by PCR from BAC clone I15 with a NcoI site at its 3' end. The EGFP-coding sequence (BD Biosciences Clontech) was fused to the short arm through the NcoI site. The long arm consisted of a 7.8-kb fragment located immediately 3' of the ATG translational start site of Rorc(t) exon 1 and extending to Rorc() exon 5, and was amplified by PCR from BAC clone I15. Embryonic stem cells (E14.1; 129/Ola) were electroporated with 30  g of the linearized targeting vector, and 2 of 200 G418-gancyclovir double-resistant colonies had the correctly targeted Rorc(t) locus. Initial screening was done by PCR, amplifying a 1.1-kb fragment starting 100 bp 5' of the short arm in the Rorc() locus and ending at the 5' end of EGFP. Correct insertion of the sequence encoding EGFP was also confirmed by Southern blot (Supplementary Fig. 1 online). The neor-containing embryonic stem cells were injected into blastocyts and two chimeric males were obtained. Germline transmission of the mutant allele was obtained with one chimera and yielded heterozygos Rorc(t)+/GFPneo mice, which were then crossed with a mouse line expressing Cre under the control of a human cytomegalovirus minimal promoter (The Jackson Laboratory) to excise the neor cassette. The genotype of the Rorc(t)+/GFPneo and derived mice was established by PCR with primers surrounding Rorc(t) exon 1: the 5' primer was 5'-CCCCCTGCCCAGAAACACT-3' and the 3' primer was 5'-GGATGCCCCCATTCACTTACTTCT-3' (Supplementary Fig. 1 online).
Mice and treatments.
LT-deficient (Lta-/-) mice and Id2-deficient (Idb2-/-) mice18 were crossed with Rorc(t)+/GFP mice to obtain Lta-/-Rorc(t)+/GFP and Idb2-/-Rorc(t)+/GFP mice. BLC-deficient mice have been described31. In blocking experiments, pregnant mothers were injected intravenously at E12 and E15 with 100 g mAb or immunoglobulin fusion protein, unless indicated otherwise. All mice were bred and used in our specific pathogen-free animal facility according to the New York University School of Medicine Institutional Animal Care and Use Committee.
RT-PCR and immunoblotting.
Total RNA was extracted from livers and thymi of 6- to 8-week-old mice, and cDNA was synthesized with the Reverse Transcription System from Promega. The PCR primer pairs specific for the two ROR isoforms, as well as the PCR conditions and the blotting conditions of the PCR products, have been described22. For immunoblot, liver and thymus tissues were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet-P40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris-HCl, pH 8.0) supplemented with protease inhibitors and were sonicated. Equivalent amounts of protein were mixed with reducing loading buffer and separated by 12% SDS-PAGE. After transfer, membranes were incubated for 2 h at room temperature with 0.6 g/ml of a mAb to ROR19 and washed and were further incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated goat polyclonal antibody to armenian hamster (anti-armenian hamster; Jackson ImmunoResearch) at a dilution of 1:10,000.
Antibodies and immunoglobulin fusion proteins.
The following mAbs were purchased from PharMingen: phycoerythrin-conjugated anti-I-E/A (M5/114.15.2), anti-CD4 (RM4-5), anti-ICAM-1 (3E2), anti-Ki67 (B56), CyChrome-conjugated anti-CD4 (H129.19), anti-CD11b (M1/70), allophycocyanin-conjugated anti-CD8 (53-6.7), anti-CD45.2 (104), anti-CD11c (HL3), biotin-conjugated anti-VCAM-1 (429) and purified anti-CD16/32 (2.4G2). Rabbit anti-GFP, fluorescein isothiocyanate−conjugated goat anti-rabbit, indocarbocyanine-conjugated goat anti-armenian hamster and Alexa Fluor 488−, Alexa Fluor 546− and Alexa Fluor 647−conjugated streptavidin were purchased from Molecular Probes. Biotin-conjugated and purified endotoxin-free monoclonal anti-IL-7R were purchased from eBioscience. The hamster mAb to ROR and RORt19, the agonist mAb to LTR, AF.H6 (ref. 12), and the immunoglobulin (Ig) fusion proteins LTR-Ig, TNF-R-I-Ig42 and TRANCE-R-Ig43 have been described.
Flow cytometry.
Single-cell suspensions were prepared from thymus, spleen, lymph nodes, bone marrow and liver. Total liver cells were resuspended in a 40% isotonic Percoll solution (Pharmacia) and underlaid with an 80% isotonic Percoll solution. Centrifugation for 20 min at 2,000g yielded the mononuclear cells at the 40−80% interface. Cells were washed twice with PBS-F (PBS containing 2% FCS), preincubated with mAb 2.4G2 to block Fc receptors, then washed and incubated with mAb conjugates for 40 min in a total volume of 100 l PBS-F. Cells were washed, resuspended in PBS-F and analyzed on a FACScalibur flow cytometer (Becton-Dickinson). For cell cycle analysis of thymocytes, cells were fixed in 70% ethanol 30 min at 4 °C and washed with PBS-F, and 5 105 cells were incubated for 5 min at 37 °C with 12.5 g/ml of propidium iodide (Sigma) and 50 g/ml of RNAse A in 100 l STE buffer (100 mM Tris base, 100 mM NaCl and 5 mM EDTA at pH 7.5). Cells were then washed, resuspended in PBS-F and analyzed.
Thymocyte survival assay.
Thymocytes were isolated and cultured in DMEM supplemented with DMEM containing 10% FCS, 10 mM HEPES, 50 M -mercaptoethanol and 1% glutamine. After various periods of time, cells were stained with Annexin V (Pharmingen) and 1 g/ml of propidium iodide to exclude dead cells and were analyzed by flow cytometry.
Immunofluorescence histology.
Adult tissues or whole embryos were washed for several hours in PBS before being fixed overnight at 4 °C in a fresh solution of 4% paraformaldehyde (Sigma) in PBS. The samples were then washed 1 d in PBS, incubated in a solution of 30% sucrose (Sigma) in PBS until the samples sank, embedded in optimal cutting temperature compound 4583 (Sakura Finetek), frozen in a bath of hexane cooled with liquid nitrogen and stocked at -80 °C. Blocs were cut with a Microm HM500 OM cryostat (Microm) at a thickness of 8 m (tissues) or 12 m (embryos), and sections were collected onto Superfrost/Plus slides (Fisher Scientific). Slides were dried 1 h and processed for staining or were stocked at -80 °C. For staining, slides were first hydrated in PBS-XG (PBS containing 0.1% Triton X-100 and 1% normal goat serum; Sigma) for 5 min and were blocked for 1 h at room temperature with 10% goat serum and a 1:100 dilution of monoclonal anti-Fc receptor 2.4G2 in PBS-XG. Endogenous biotin was blocked with a biotin blocking kit (Vector Laboratories). Slides were then incubated overnight at 4 °C with primary polyclonal antibody or conjugated mAb (in general, at a 1:100 dilution) in PBS-XG, washed three times for 5 min each with PBS-XG, incubated for 1 h at room temperature with secondary conjugated polyclonal Ab or streptavidin, washed once, incubated for 5 min at room temperature with 4',6-diamidino-2-phenylindole-2HCl (DAPI; Sigma), washed three times for 5 min each wash and mounted with Fluoromount-G (Southern Biotechnology Associates). Slides were examined with a Zeiss Axioplan 2 fluorescence microscope equipped with a charge-coupled device camera and were processed with Slidebook v3.0.9.0 software (Intelligent Imaging).
Note: Supplementary information is available on the Nature Immunology website.
Received 9 September 2003; Accepted 12 November 2003; Published online: 21 December 2003.
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