HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, H. Articles by Nishikawa, S.-I. Search for Related Content PubMed PubMed Citation Articles by Yoshida, H. Articles by Nishikawa, S.-I.

Expression of ₄₇ Integrin Defines a Distinct Pathway of Lymphoid Progenitors Committed to T Cells, Fetal Intestinal Lymphotoxin Producer, NK, and Dendritic Cells¹

Hisahiro Yoshida^2,*, Hiroshi Kawamoto, Sybil M. Santee, Hiroyuki Hashi^*, Kenya Honda^*, Satomi Nishikawa^*, Carl F. Ware, Yoshimoto Katsura and Shin-Ichi Nishikawa^*

^* Department of Molecular Genetics, Graduate School of Medicine, and Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo, Kyoto, Japan; and Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
During embryogenesis, the Peyer’s patch anlagen are induced by a cell population that produces lymphotoxin (LT) ₁₂ following stimulation of IL-7R. In this study, we show that the LT-producing cell is localized within the IL-7R⁺ and integrin ₄₇ (₄₇)⁺ population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of ₄₇. Before expression of ₄₇, the potential of IL-7R⁺ population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7R⁺₄₇⁺ cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7R⁺₄₇⁺ population migrates to the intestine, where it induces the Peyer’s patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7R⁺₄₇⁺ population can differentiate into fully competent NK1.1⁺ NK cells or CD11c⁺ APCs. Expression of ₄₇ is lost during differentiation of both lineages; IL-7R expression is lost during NK1.1⁺ cells differentiation. A newly discovered lineage^-IL-7R⁺c-Kit⁺₄₇⁺ population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peyer’s patch (PP)³ is an organized peripheral lymphoid tissue distributed along the antemesenteric site of the intestine. Our previous study demonstrated that induction of PP anlagen takes place during 14.5–15.5 days of gestation (days post coitus (dpc)), leading to formation of segregated regions expressing VCAM-1 and ICAM-1 (1). Two signal transduction pathways have been implicated in the initial phase of PP organogenesis by previous analyses of mutant mice that failed to induce VCAM-1/ICAM-1⁺ PP anlagen. The first pathway involves IL-7R, c, and Janus kinase (Jak) 3 (2), although the ligand triggering this signal pathway has not yet been identified. The second pathway involves lymphotoxin (LT) ₁₂ and its receptor LTR (3, 4, 5, 6, 7, 8). Mice carrying a mutation in the NF-B-inducing kinase, which is downstream of LTR, also lack PP and lymph nodes (LN) (9).

Although these two signal transduction pathways are independent of each other per se, we recently identified a cell population that serves as the convergence point of the two signals (10). This population bears IL-7R, CD45, c-Kit, CD44, but none of the lineage markers such as CD3, B220, CD19, NK1.1, CD11b, CD11c, Gr-1, or ter119 (Lin). This surface phenotype is reminiscent of lymphoid progenitors (11), indicating a close relationship of this population to lymphocytes. A fraction of this population expresses LT₁₂ and is suppressed in vivo by treatment with an antagonistic anti-IL-7R mAb (10). This strongly suggests that activation of the IL-7R/c/Jak3 signal pathway of these cells leads to the induction of LT₁₂, which subsequently activates surrounding LTR⁺ cells to form VCAM-1⁺ICAM-1⁺ PP anlagen (10). We refer to this Lin^-IL-7R⁺ population as fetal intestinal LT producer (FILyP) cells, as it is the only cell type in the fetal intestine to express LT₁₂ (10). Differentiation of IL-7R⁺CD3^-CD4⁺ cells is inhibited in mice with the null mutation of the Id2 gene (12). PP anlagen are not induced in these mice, indicating unequivocally that generation of IL-7R⁺CD3^-CD4⁺ cells in the embryonic intestine is indeed essential for the induction. Based upon these observations, we have proposed that Lin^-IL-7R⁺ subpopulation in the embryonic intestine includes the inducer cells of PP anlagen (10).

As this surface phenotype is common to several lymphoid progenitors, such as common lymphoid progenitors, T/NK/dendritic cell (DC)-committed progenitors, and B-committed progenitors (11, 13, 14, 15, 16), we investigated the relationship between FILyP cells and other lymphoid cell lineages. Our results show that commitment to FILyP cells is characterized by the expression of ₄₇ integrin (₄₇), an adhesion molecule that plays important roles in the migration of CD4⁺CD3^-LT⁺ cells to the neonatal LN (17). Upon commitment, the ₄₇⁺ populations can give rise to T and FILyP cells, but not B cells. This population subsequently loses the ability to produce T cells (T-potential) and then migrates to the intestine. Our results further demonstrate that the IL-7R⁺₄₇⁺ population in which FILyP cells are included represents an intermediate progenitor of NK and DC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Pregnant C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan). Ly-5.1⁺ C57BL/6J mice were maintained in our animal facility, as described (18). Female and male mice were mated overnight, and those with a vaginal plug were judged pregnant. Noon of the day when the vaginal plug was identified was calculated as 0.5 dpc.

Abs and rLTR human IgG1 chimerical protein

Names of clones and sources of mAbs used in this study are listed below: Anti-IL-7R Ab (A7R34) and anti-c-Kit Ab (ACK2) were established in our laboratory and prepared, as previously described (15, 19). Anti-B220 Ab (RA3-6B2), anti-CD11b Ab (Mac-1), anti-CD8 (53-6.7), and anti-F4/80 (F4/80) were purified from hybridoma culture supernatant, as described (14). Anti-Thy-1.2 was purchased from Caltag Laboratories (San Francisco, CA); anti-CD3 (Y65.135) was purchased from Seikagaku-kogyo (Tokyo, Japan); anti-integrin ₄₇ heterodimer (DATK32), anti-CD3 (2C11), anti-CD4 (GK1.5), anti-CD11c (HL3), anti-TCR- (H57-597), anti-TCR- (GL3), anti-CD45 (30F11.1), anti-NK1.1 (NKR-P1C), anti-Gr-1 (RB6-8C5), anti-CD44 (IM7), anti-CD25 (7D4), anti-CD80 (1G10), anti-CD86 (GL1), anti-Ly-49D (4E5), anti-2B4 (2B4), and anti-DX5 (DX5) were purchased from BD PharMingen (San Diego, CA).

The extracellular domain of the LTR was fused to the human IgG1 Fc portion (20) and expressed in baculovirus (Invitrogen, Groningen, The Netherlands), and the chimeric protein was purified as described (21). This chimeric protein was used in flow cytometric analysis to detect membrane-anchored LT₁₂ with biotinylated goat anti-human IgG (Southern Biotechnology Associates, Birmingham, AL) as the secondary reagent, followed by streptavidin conjugated with allophycocyanin (Molecular Probes, Eugene, OR). For a negative control, we used a fusion protein composed of the extracellular domain of platelet-derived growth factor receptor and human IgG1 Fc domain (22).

Preparation of single cell suspension for flow cytometry and cell sorting

All organs of embryos were dissected with fine forceps under the stereomicroscope, and then dissociated with dispase (Life Technologies, Grand Island, NY) for 10–25 min at 37°C. After gentle pipetting, dissociated cells were washed with HBSS containing 20% FCS and DNase (Sigma, St. Louis, MO). Cells were filtered through nylon mesh to remove large clumps, washed, and stained with mAbs, as described (16). These cells were analyzed or sorted by FACSVantage (BD Biosciences, Lincoln Park, NJ).

RNA isolation and RT-PCR analysis

Total RNA was isolated from 20,000 cells that were directly sorted to the vial containing ISOGEN LS (Nippon Gene, Tokyo, Japan). cDNA was prepared from the total RNAs by reverse transcriptase using oligo(dT) primers (Superscript; Life Technologies). In each PCR for detecting the expression of LT, LT, ₇ integrin, CD4, hypoxanthine phosphoribosyltransferase (HPRT), and GAPDH genes, cDNA corresponding to an amount from 1000 cells was incubated with 100 pg of following primer sets: LT-, sense, 5'-TCACCTTGTTGGGTACCCCAGCAA-3' and antisense, 5'-ATACACAGACTTCTGCGCAC-3'; LT-, sense, 5'-TTGTTGGCAGTGCCTATCACTGTCC-3' and antisense, 5'-CTCGTGTACCATAACGACCCGTAC-3'; HPRT, sense, 5'-GAGCTACTGTAATGATCAGTCAACGG-3' and antisense, 5'-GATTCAACTTGCGCTCATCTTAGGC-3'; ₇ integrin, sense, 5'-AACTTGGATGATGGCTGGTG-3' and antisense, 5'-TCCTAAGTCAGTCTGCTTCC-3'; CD4, sense, 5'-TAGTTCCAGGCCCTCGGTATACAC-3' and antisense, 5'-TGCCACAATGCTCTCTCCATAAAGG-3'; GAPDH, sense, 5'-TGCTGAGTATGTCGTGGAGTCTAC-3' and antisense, 5'-ATCACGCCACAGCTTTCCAGAG-3'.

Semiquantitative RT-PCR analysis was performed as follows. Comparing the concentration of RT-PCR products with HPRT primer sets at various amplifying cycles equalized the amount of the template cDNA from each specimen. The minimum number of amplifying cycles that detected the PCR products was determined for each specimen by each primer set.

Cell culture

In some experiments, sorted cells were cultured on stromal cell layers. Preparation of TsT4 and Op9 stromal cell layers was as described previously (10, 23). Sorted cells were suspended in RPMI 1640 (Life Technologies) containing 10% FCS and 5 x 10^-5 M 2-ME, and placed on the stromal cell layer with or without additional cytokines. These stromal cell lines have been shown to support differentiation of most blood cell lineages, including B lymphocytes from multipotent stem cells, although it has been difficult to induce mature T cells (23).

Unless indicated, U-bottom-shaped 96-well cluster dishes (Falcon) were used for all culture experiments without stromal cells. A total of 3000–5000 cells was cultured in each well. The concentrations of recombinant cytokines used in this study were murine stem cell factor (SCF; 100 ng/ml), murine IL-7 (20 U/ml), human IL-6 (20 U/ml) (Life Technologies), simian IL-15 (20 ng/ml) (Genzyme, Cambridge, MA), murine IL-3 (20 U/ml), murine GM-CSF (20 ng/ml), and murine TNF- (20 ng/ml) (PeproTech, Rocky Hill, NJ).

In vitro assays

Colony-forming cell assay in the semisolid medium containing methylcellulose was performed as described previously (24). In this study, we used GM-CSF (200 ng/ml; R&D Systems, Minneapolis, MN) or murine M-CSF (100 ng/ml; R&D Systems).

The T-potential was assessed by using a high oxygen thymus organ culture system, as described previously (25). A total of 3–100 cells was placed in wells containing a deoxyguanosine (Sigma)-treated thymic lobe. The cultures were maintained in the presence of 100 ng/ml SCF, 10 U/ml IL-7, and 30 U/ml IL-3. To facilitate the colonization of the inoculated cells, thymic lobes were cut into four pieces, which sealed spontaneously to form a lobe during incubation (26). Five cultures were prepared for each assay. After incubating for 14 days, each lobe was dissociated into single cell suspensions, split to aliquots, and three-color stained with mAb mixtures. Only cells with the light scatter profile of lymphocyte were gated and analyzed.

The NK activity was assessed by using YAC-1 target cells. In this experiment, we used the fluorescent dye release assay (27, 28). DC activity was assessed by [³H]thymidine incorporation of CD3⁺CD4⁺ T cells from BALB/c mice, according to the method described by Bjorck et al. (29).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Maintenance and stimulation of the FILyP cell in culture

We have previously demonstrated that CD45⁺CD44⁺IL-7R⁺c-Kit⁺₄₇⁺ Thy-1^+/-CD4^+/- cells in the embryonic intestine, which are also negative for the lineage markers B220, CD19, CD3, Ter119, Mac1, Gr1, CD11c, and NK1.1 (Lin), express LT and LT mRNA (10). For convenience, we will call this population simply Lin^-IL-7R⁺₄₇⁺ cell or FILyP cell. As induction of PP anlagen is dependent on LT₁₂, PP inducers should be a component of this population.

First, we wanted to determine whether LT₁₂ production in this population is a direct consequence of the stimulation of IL-7R. To identify appropriate culture conditions for this population, we cultured Lin^-IL-7R⁺₄₇⁺ cells of 15.5-dpc embryonic intestines with various cytokine combinations. A combination of SCF and IL-7 induced more than a 10-fold increase of Lin^-IL-7R⁺₄₇⁺ cells in 5 days of culture (data not shown). As expected, virtually all the cells, both CD4⁺ and CD4^-, in this culture expressed LT₁₂ (Fig. 1, A and B). A combination of IL-6 and SCF induced only a 2-fold increase in cell number (data not shown), and most cells in this culture did not express surface LT₁₂ (Fig. 1B). RT-PCR analysis of the same population demonstrated that LT gene expression was almost absent, although low level LT gene expression could be detected (Fig. 1D, lane 0). Although mlTBR:Fc, which is used to detect surface LT₁₂, will also detect LIGHT (the cytokine that is homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), mHVEM:Fc (mouse herpes virus entry mediator soluble receptor), which binds LIGHT (30), did not bind FILyP cells ex vivo from 15.5-dpc embryonic gut nor those maintained in vitro, indicating the presence of LT₁₂ only (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Rapid induction of LT12 after stimulation by IL-7. A, Lin-IL-7R+47+ cells were sorted from intestines of 15.5-dpc embryos. Three thousand cells were cultured in U-bottom cluster dishes for 3 days with IL-7 and SCF. A total of 100,000 of harvested cells from 10 dishes were analyzed for LT12 and CD4 expression in A. Note that most cultured cells showed LT12 expression, irrespective of CD4 expression. B, Expression of surface LT12 on FILyP cells after culture for 3 days with either IL-7 and SCF or IL-6 and SCF is illustrated. Note the FILyP cells maintained by IL-6 and SCF expressed LT12 in nearly background levels in contrast to that of the cells maintained with SCF and IL-7. C, Induction of surface LT12 expression by IL-7 stimulation. IL-7R+47+Lin- cells were cultured in the presence of IL-6 and SCF. After 3 days, cells were harvested and stimulated with IL-7 (10 U/ml). One and three hours after stimulation, cells were harvested and analyzed for LT12 expression. The number of cells harvested from each dish maintained with IL-6 + SCF for 5 days was 5000; therefore, the flow cytometry analysis shown in B–D indicates 3000 cells for each population. The results presented are representative of the seven independent experiments. D, An aliquot of cells was also used in RT-PCR analysis for LT or LT mRNA. The lane numbers indicate the hours following addition of IL-7. HPRT mRNA expression level was used as a control. E and F, Expression of LT12 on IL-7R+47+ cell surface freshly isolated from intestine. The IL-7R+47+CD4+ or IL-7R+47+CD4- cell population of 15.5-dpc intestines was gated and analyzed for expression of LT12. In CD4+ or CD4- population, 10–20% of cells expressed LT12. All experiments were conducted more than five times, and the representative results are shown.

To confirm that LT₁₂ expression of Lin^-IL-7R⁺₄₇⁺ cells is not the artifact induced in these in vitro culture conditions, we examined LT₁₂ expression of freshly isolated Lin^-IL-7R⁺₄₇⁺ cells from 16.5-dpc mouse intestine. Similar to our previous analyses of mRNA expression (10), we found both CD4⁺ and CD4^-Lin^-IL-7R⁺₄₇⁺ cell population expressed LT₁₂ on their surface (Fig. 1, E and F). In contrast to the in vitro culture, however, only a subset of the Lin^-IL-7R⁺₄₇⁺ cells produced LT₁₂ in vivo.

Expression of ₄₇ in the Lin^-IL-7R⁺ population is an early step during divergence of the FILyP cell population from the conventional lymphocyte

Lin^-IL-7R⁺₄₇⁺ cells in the mesenteric regions are lymphoid in morphology (10, 17). However, this population in the intestine could not give rise to T nor B cells under culture conditions that support the differentiation of lymphocytes (data not shown), indicating that it has already diverged from conventional lymphocytes. We speculated that the divergence of this lineage from the conventional lymphocyte occurs in the hemopoietic organs, and therefore investigated the differentiation potential of fetal liver cells.

Lin^-IL-7R^-c-Kit⁺CD45⁺, Lin^-IL-7R⁺₄₇^-, and Lin^-IL-7R⁺₄₇⁺ cells were purified from fetal livers of 12.5-dpc embryos and subjected to various progenitor assays (Fig. 2). When 100 cells from each population were cultured on the TsT4 stromal cell line in the presence of IL-7 and SCF, B cells were generated (B-potential) from Lin^-IL-7R^-c-Kit⁺CD45⁺ and Lin^-IL-7R⁺₄₇^- populations, but not from the Lin^-IL-7R⁺₄₇⁺ population (Fig. 3, A and C). Even using 5000 of Lin^-IL-7R⁺₄₇⁺ cells for cocultivation with stromal cells, we found no B-potential in that population (data not shown). In contrast, CD4⁺CD3^- cells that have been implicated in induction of PP and LN (10, 17) were generated from both Lin^-IL-7R⁺₄₇⁺ and Lin^-IL-7R⁺₄₇^- populations (Fig. 3, B and D). The CD4⁺CD3^- cells were more frequently generated from Lin^-IL-7R⁺₄₇⁺ population than from Lin^-IL-7R⁺₄₇^- population. Because myeloid cell differentiation occurred in the culture of Lin^-IL-7R⁺₄₇^- population at very low frequency (0.3% in Fig. 3A), we assessed the frequency of colony-forming cells reactive to either GM-CSF or M-CSF. The frequency of the granulocyte-macrophage CFU or macrophage CFU was 100-fold lower in Lin^-IL-7R⁺₄₇^- population than Lin^-IL-7R^-c-Kit⁺CD45⁺ population, and nearly absent in Lin^-IL-7R⁺₄₇⁺ population (Table I).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Experimental procedures for progenitor assays. In fetal liver of 11.5- to 15.5-dpc embryos, Lin-IL-7R+47+ cells are detectable at frequencies of 0.1–0.3% (top panel). Both Lin-IL-7R+47- and Lin-IL-7R+47+ cells were sorted (middle panels). In some experiments, Lin-c-Kit+IL-7R- cells were also sorted. These fractions were analyzed by stromal cell-dependent culture, thymus organ culture, or colony assay in the methylcellulose medium. The purity of sorted cells was >95%. Each assay condition allows generation of cell lineages indicated in the lower panels. The flow cytometric profile presented is representative of five independent experiments.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Lack of B cell potential in IL-7R+47+ cells from 12.5-dpc fetal liver. IL-7R+Lin- cells in the 12.5-dpc fetal liver were separated into 47+ and 47- populations. One hundred cells in each well were cultured on TsT-4 stromal cells in the presence of IL-7 + SCF. Ten days after incubation, the cells in two independent wells were harvested and pooled, and the small round cells, which are easily distinguished from large stromal cells, were counted. The cells were analyzed by FACS for expression of Mac1, B220, CD3, or CD4, and a representative result was shown in A and B, the expression of Mac1 and B220 of Lin-IL-7R+47- cells (A) and the expression of CD3 and CD4 of Lin-IL-7R+47+ cells (B). In the cultures of Lin-IL-7R+47+ cells, we could not detect Mac-1+ cells, whereas few Mac1+ cells (in A, 0.31%) could be detected in the culture of Lin-IL-7R+47- cells. The number of B220+ cells recovered in each culture was calculated and presented (C). CD3-CD4+ cells, as displayed (B), were generated in both cultures, and the cell number is calculated and presented (D). The figures are representative of three independent experiments.

View this table: [in this window] [in a new window]   Table I. Potential of Lin-IL-7R+47- and Lin-IL-7R+47+ cells to form M-CSF or GM-CSF induced colonies1

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Cells generated in thymus organ culture. Figures are representative examples of analyses summarized in Table II. Three criteria were used for judging the cell types generated in each thymus organ culture: 1) Ly-5.2 donor cell growth, 2) generation of and T cells as well as CD3+ cells, and 3) presence of CD3-CD4+ cells. In the first row, analyses on a thymus lobe that received three 47- cells are displayed. In this lobe, T cells are generated. Cells from these lobes were also three-color stained with CD4, CD3, and CD8 to exclude the presence of CD3-CD4+CD8+ immature T cells. Approximately 50% of CD3-CD4+ cells are CD8-, indicating the presence of FILyP cells. In the second row, analyses on a lobe receiving 10 47+ cells are presented. This lobe generated both T cells and FILyP cells. The third row represents analyses on a lobe receiving three 47+ cells. This lobe is positive for FILyP cells, but not T cells. In the lobe presented in the fourth row, we could detect the Ly-5.2+ donor cells with lymphocyte light-scattering pattern. However, because of the absence of CD3-CD4+ cells, we judged this lobe negative for FILyP cells. As LT12 expression is found also in CD4- cells, it is likely that such lobes that are negative in CD3-CD4+ growth, but positive in donor cell growth may contain FILyP cells. The figures are representative of results obtained from 10 independent lobes from two independent experiments for each condition. Lobes received three cells each, and results are similar if three or one hundred cells are placed in a thymic lobe.

View this table: [in this window] [in a new window]   Table II. Potential of Lin-IL-7R+47- and Lin-IL-7R+47+ cells to give rise to T and FLP cells1

To confirm differentiation from IL-7R⁺₄₇^- to IL-7R⁺₄₇⁺ populations, we sorted Lin^-IL-7R⁺₄₇^- cells from the liver of 15.5-dpc embryos and cultured them on OP9 stromal cell layer for 40 h in the presence of IL-7 and SCF. During this short-term culture, the cell number increased 6-fold and >20% of the recovered Lin^-IL-7R⁺ cells expressed ₄₇⁺ (Fig. 5A). Moreover, 25% of the Lin^-IL-7R⁺₄₇⁺ fraction also expressed CD4 (Fig. 5B), whereas no CD4⁺ cells were present in the initial ₄₇^- population (data not shown). These in vitro generated Lin^-IL-7R⁺₄₇⁺ cells expressed LT₁₂ regardless of CD4 expression (Fig. 5, C and D). This result indicates that Lin^-IL-7R⁺₄₇^- cells are indeed the progenitor of the Lin^-IL-7R⁺₄₇⁺ FILyP population. RT-PCR analyses revealed that fetal liver Lin^-IL-7R⁺₄₇^- cells expressed both ₇ integrin and CD4 mRNA (Fig. 5E).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Expression of 47 on the fetal liver-derived Lin-IL-7R+47- cells after 40-h coculture with stromal cells. Lin-IL-7R+47- cells were fractionated from fetal livers of 13.5-dpc embryos and cocultured with OP9 stromal cells in the presence of IL-7 and SCF for 40 h. A, Expression of 47 was induced during culture. B, Among these 47+ cells, 26.5% also expressed CD4. C and D, Surface expression of LT12 on these Lin-IL-7R+47+CD4+/- cells differentiated in vitro detected by flow cytometry. Note the expression level of LT12 is comparable between the CD4+ and CD4- population.

View larger version (41K): [in this window] [in a new window]   FIGURE 6. Effect of IL-7 on CD4 expression level. Combination of IL-7 + SCF or IL-6 + SCF could maintain the Lin-IL-7R+47+CD4+/- cells obtained from 15.5-dpc intestine. After 7 days of culture with each condition, CD4-positive and CD4-negative population was observed, and CD4 expression level seemed slightly higher with IL-7 + SCF than IL-6 + SCF (A). However, CD4 up-regulation was not observed 24 h after addition of IL-7 into the cells maintained with IL-6 + SCF for 6 days. Lin-IL-7R+47+ cells of 15.5-dpc intestine were sorted into CD4+ or CD4- population and maintained with IL-7 + SCF. In the former population, approximately one-half of the cells lost CD4 expression after 7 days of cultivation (C); in contrast, most of the cells did not express CD4 in the latter population (D). All of these cells expressed comparable level of LT12, regardless of being CD4+ or not (E, F).

Migration of IL-7R⁺ cells to the antemesenteric site of intestines

Our data suggested that Lin^-IL-7R⁺₄₇⁺ cells were generated in the fetal liver and then migrate to the intestine to be involved in induction of PP anlagen. Consistent with this expectation, the proportion of the Lin^-IL-7R⁺₄₇⁺ population in the mesenteric region increased before generation of PP anlagen (Fig. 7, upper and middle panels). Histological analyses demonstrated that at 12.5–13.5 dpc, IL-7R⁺ cells first appeared in the mesentery and then subsequently within the intestine from 13.5 dpc (Fig. 7, lower left panel). Interestingly, most IL-7R⁺ cells in the intestine of the 15.5-dpc embryo were distributed preferentially in the antemesenteric half of the intestine, where PP anlagen are formed (Fig. 7, lower right panel). Estimated numbers of each organ per embryo are listed in Table III.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Increase of IL-7R+47+ cells in the abdominal region during embryogenesis. The proportion of IL-7R+47+ cells gradually increased from 12.5 dpc in fetal mesentery (upper panels) and in the fetal intestine (middle panels). Note that this increase was observed 1 day earlier in the mesentery than in the intestine. Total cell numbers of each panel were 300,000 for 11.5-dpc specimen, and a million for 12.5- to 15.5-dpc specimen. The results are representative of more than five independent experiments for each gestational stage and each organ. Lower panels, Show immunohistochemical identification of IL-7R+ cells in the intestinal tissues. Left panel, 13.5 dpc. Many IL-7R+ cells are found in the mesenteric vessels, but rarely in intestinal vessels. Right panel, 15.5 dpc. IL-7R+ cells distributed in the antemesenteric region of developing intestine. From the total cell numbers of each organ at each embryonic day, Lin-IL-7R+47+ cell number was calculated and listed in Table III.

We have addressed the derivation and function of Lin^-IL-7R⁺₄₇⁺ populations that contain FILyP cells. Next, we investigated the fate of Lin^-IL-7R⁺₄₇⁺ populations in the embryonic intestine. It has been established that both NK and some DC are derived from lymphoid progenitors (13, 15). In addition, Mebius et al. (17) showed that CD3^-CD4⁺IL-7R⁺₄₇⁺ cells in mesenteric LN can differentiate into cytotoxic cells and APCs. Thus, we investigated the potential of the intestinal IL-7R⁺₄₇⁺ population to give rise to NK1.1⁺ and/or CD11c⁺ cells.

Lin^-IL-7R⁺₄₇⁺ cells were sorted and cultured under various conditions. In the presence of IL-7 and SCF, only a few NK1.1⁺ cells that coexpress IL-7R and ₄₇ were induced in the culture (Fig. 8A, upper panels). However, addition of IL-15, a cytokine implicated in the differentiation of NK cells (31, 32), induced a marked increase in NK1.1⁺ cells (Fig. 8A, lower panels). Interestingly, NK1.1⁺ cells induced by IL-15 stimulation down-regulated IL-7R and ₄₇ expression. These results are consistent with previous studies showing that IL-7 induces NK1.1⁺ progenitors, but their further differentiation requires IL-15 (33). Although IL-15 and SCF did not induce markers for more mature NK cells, such as Ly-49D and DX5 (34, 35), these NK1.1⁺ cells are competent, as evidenced by cytotoxicity against YAC-1 tumor cells (Fig. 8B).

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Differentiation of functionally competent NK1.1+ cells from intestinal IL-7R+47+ cells. A, Lin-IL-7R+47+ cells were sorted from cell suspensions prepared from 15.5-dpc intestine. Two thousand cells were cultured in the presence of either IL-7 and SCF or IL-15 and SCF in a 96-well U-bottom culture dish. Three days after culture, cells were harvested, and the presence of NK1.1+ cells was evaluated by FACS analysis. Expression of IL-7R and 47 integrin in NK1.1+ cells was also analyzed. B, The harvested cells were also analyzed for NK activity with YAC-1 tumor cells as the target at varying E:T ratios.

View larger version (21K): [in this window] [in a new window]   FIGURE 9. Differentiation of functionally competent CD11c+ DC from intestinal IL-7R+47+ cells. A, Lin-IL-7R+47+ cells were sorted from 15.5-dpc intestine. Two thousand cells were cultured in the presence of IL-3, TNF, and SCF. Three days later, the cells were harvested and analyzed for expression of various surface molecules. Although no CD11c+ cells were present in the initial population, 17% of cells expressed CD11c after culture. The CD11c+ population was gated and analyzed for other DC markers. B, DC activity of the harvested cells was analyzed by cell proliferation assay of CD4+CD3+ T cells from BALB/c mice (•). CD19+ B cells sorted from the same mice were used as a control (). Cells were incubated for 3 days, and proliferation was measured by [3H]thymidine incorporation during the last 16 h of the culture. Background level with T cells alone was 60 + 12 cpm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Induction of LT₁₂ by IL-7R signaling

The FILyP cell is part of the Lin^-IL-7R⁺CD4^+/- population in neonatal intestine. Lin^-IL-7R⁺CD4^+/- cells, which are freshly isolated from the intestine of 15.5-dpc embryos, express LT and LT mRNA (10). We examined whether IL-7 stimulation can induce LT₁₂ in this population. We found that a combination of IL-6 and SCF promoted the survival of sorted IL-7R⁺ cells without inducing the expression of LT₁₂. Most IL-7R⁺₄₇⁺ cells maintained under this condition rapidly expressed surface LT₁₂ as well as LT and LT mRNA upon stimulation with IL-7. This induction is completely blocked by an antagonistic anti-IL-7R mAb (data not shown). Taken together, Lin^-IL-7R⁺₄₇⁺ cells, be it CD4⁺ or CD4^-, are eligible to be termed FILyP cells, although only a portion of this population may be stimulated by the ligand for IL-7R in vivo to express LT₁₂. Consistent with this notion, less than 20% of the Lin^-IL-7R⁺₄₇⁺CD4^+/- cells in the intestine of 16.5-dpc embryo expressed LT₁₂.

Lineage derivation of FILyP cells

Surface expression of IL-7R in the absence of lineage markers is in common with lymphoid progenitors (12, 13). Although this observation suggests a close linkage between FILyP cells and lymphocytes, our previous study also demonstrated that the FILyP cell does not express RAG1 nor RAG2 (10), suggesting that this population in the intestine has already diverged from conventional lymphocytes. Consistent with this notion, Lin^-IL-7R⁺₄₇⁺ cells sorted from embryonic intestine could not generate B nor T cells (data not shown). Thus, we addressed the following question: At what stage do FILyP cells diverge from the differentiation pathway of T and B lymphocytes?

Lin^-c-Kit⁺IL-7R⁺CD44⁺ progenitors can give rise to B, T, NK, and DC (12, 13, 14, 15, 39). Although this population may contain colony-forming progenitors of myelomonocytic lineage at a low frequency, most are committed progenitors of lymphoid lineages. In this study, we further dissected this population into ₄₇⁺ and ₄₇^- populations and found that B-potential is lost in the ₄₇⁺ population, whereas T-potential remains to some extent. Hence, T cell progenitor and FILyP cell diverge later than the progenitors with B-potential. This is consistent with our findings that there are T-committed or B-committed lymphoid progenitor cells in fetal liver (40). As demonstrated in Table II, ₄₇⁺ cells showed a greater tendency toward giving rise to FILyP cells whereas ₄₇^- cells generated both FILyP and T cells equally. Moreover, our results demonstrated that fetal liver Lin^-IL-7R⁺₄₇^- cells quickly differentiated into Lin^-IL-7R⁺₄₇⁺ cells, indicating an order of differentiation from Lin^-IL-7R⁺₄₇^- to Lin^-IL-7R⁺₄₇⁺ populations. In addition, by means of RT-PCR analysis, we found that fetal liver Lin^-IL-7R⁺₄₇^- cells are already expressing ₇ integrin or CD4 mRNA. This result indicated at least a part of the Lin^-IL-7R⁺₄₇^- cells in fetal liver is committed to the Lin^-IL-7R⁺₄₇⁺CD4⁺ cells. As Lin^-IL-7R⁺₄₇⁺ cells could differentiate to NK and DC in vitro, our result is consistent with those of Zhang et al., who showed that NK or DC cells were induced from fetal liver cells using a stromal cell-dependent culture system (41). In addition, we have confirmed cells expressing NK or DC markers differentiating from fetal liver Lin^-IL-7R⁺₄₇⁺ or Lin^-IL-7R⁺₄₇^- cells (data not shown). Based upon these lines of evidence, we propose a model summarized in Fig. 9. In this model, the commitment to the FILyP cell pathway occurs in the fetal liver, and is thought to progress in the following order: loss of B-potential, expression of ₄₇ integrin, loss of T-potential, and migration to the intestine.

Relationship between T/FILyP cells and T/NK progenitors

The T/NK progenitor in fetal thymus (14), which was shown to be c-Kit^lowIL-7R⁺CD44⁺Thy-1⁺NK1.1⁺, appears similar to the liver-derived population that gives rise to T and FILyP cells. Because ₄₇⁺ cells from fetal liver or intestine contained both Thy-1-positive and Thy-1-negative cells and were NK1.1^- (10) (data not shown), liver-derived ₄₇⁺ cells may represent a more immature population. Nonetheless, when IL-7R⁺₄₇⁺ cells were stimulated with IL-15 in the thymus organ culture system, all lobes containing FILyP cells also contained NK1.1⁺ cells (Kawamoto, unpublished observation). Moreover, Lin^-IL-7R⁺₄₇⁺ cells in the intestine can give rise to NK cells in the culture. Thus, it is likely that T/FILyP and T/NK progenitors represent the same population. Kumar and colleagues (13) identified NK progenitors in the c-Kit⁺IL-7R⁺NK1.1^- population of the bone marrow. Due to the difficulty in distinguishing multipotent progenitors from committed progenitors, the presence of T/NK progenitors in hemopoietic tissues has not been documented, although their presence in the thymus is well established (13, 14). In this respect, our results raise the possibility that extrathymic hemopoietic tissues are also the sites in which T/NK progenitors are generated. Obviously, this notion does not exclude thymic development of T/NK progenitors.

Fate of FILyP cells in ontogeny

The Lin^-IL-7R⁺₄₇⁺ population containing FILyP cell then migrates to the intestine through the mesentery. Our observation that the proportion of ₄₇⁺ cells increases in the intestine suggests continuous recruitment of FILyP cells from fetal liver, although it is also possible that ₄₇⁺ cells may preferentially proliferate in the intestine. We also showed that they migrate preferentially to the antemesenteric site of the developing intestine, which is the site of PP induction. This observation implies the presence of an active process regulating the directed migration of the Lin^-IL-7R⁺₄₇⁺ population. Mebius et al. (42) have previously shown that the adhesion between mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and ₄₇ integrin plays an important role in the migration and/or proliferation of CD4⁺CD3^-₄₇⁺IL-7R⁺ cells to the developing LN, using injection of anti-MAdCAM-1 mAb. Because the cellular origin seems to be basically the same between our FILyP cells and their CD4⁺CD3^-₄₇⁺IL-7R⁺ cells, ₄₇ integrin may play some roles in FILyP cell migration to the PP anlagen. However, we found no difference in the PP anlagen formation with the blockade of MAdCAM-1 and ₄₇ integrin adhesion by injection of anti-MAdCAM-1 mAb MECA367 (43). Recently, we found that PP-specific mesenchymal cells expressing VCAM-1/ICAM-1 produce the chemokines B lymphocyte chemoattractant and EBI-ligand chemokine during development (37). It was reported that B lymphocyte chemoattractant-defective or CXCR5-defective mutant have a partial defect in PP organogenesis (44, 45), and that CXCR5 is expressed in FILyP cell population (10, 17). Combination of MAdCAM-1/integrin ₄₇ interaction and the function of those chemokines may play a pivotal role in FILyP cell migration to the periphery. The molecular nature of FILyP cell migration to the intestine should be studied in the future. Because the intestinal Lin^-IL-7R⁺₄₇⁺ population lacks the ability to differentiate to T cells, this population undergoes irreversible differentiation before migration to the intestine. After being seeded in the antemesenteric site of intestine, a portion of Lin^-IL-7R⁺₄₇⁺ cells comes into contact with cells expressing the ligand for IL-7R, thereby inducing LT₁₂ expression, resulting in the induction of the PP anlagen (10).

Induction of PP anlagen may not be the sole function of FILyP cells, as they can further differentiate into cytotoxic NK1.1⁺ and Ag-presenting CD11c⁺ populations under appropriate conditions. Consistent with the previous report that NK progenitors could be induced in bone marrow cultures with IL-7 and SCF (33), this cytokine combination induced a small proportion of NK1.1⁺ cells in the IL-7R⁺ population. This NK1.1⁺ population maintained expression of IL-7R, which was then down-regulated upon addition of IL-15. Thus, IL-7 can support differentiation of FILyP cells to c-Kit⁺IL-7R⁺NK1.1⁺ stage, but IL-15 is required for further differentiation. Although IL-15-induced cytotoxic NK1.1⁺ cells did not express mature NK markers such as Ly-49D nor DX5 (34, 35), they were functionally competent (Fig. 9). This is consistent with recent reports demonstrating a difference between adult and fetal NK cells in the ability to express Ly-49 family molecules (14, 46, 47).

From the present results, FILyP cells are shown to give rise to functional APCs expressing CD11c, I-A, CD80, CD86, ICAM-1, Mac-1, and CD8 upon IL-3 and TNF- stimulation. However, over a 4-day period in culture, the expression of IL-7R was not down-regulated. DC has been classified into Mac-1^-CD8⁺DEC205⁺ lymphoid DC and Mac-1⁺CD8^-DEC205^- myeloid DC in situ (15, 48). The surface phenotype of DC described in this study does not fit either phenotype, suggesting that they may represent a novel population or an immature stage of differentiation. Recent identification of CD8 and Mac-1 double-positive DC in the spleen (49) supports the former possibility that the FILyP cell-derived DC constitute a subset of DC in peripheral lymphoid organs.

The differentiation process, which occurs after acquisition of the FILyP cell phenotype, is depicted in Fig. 10. In this model, FILyP cells are the progenitors of NK and DC lineages, the potential to the former decreasing during differentiation from CD4^- to CD4⁺ cells. This scheme includes our unpublished observation that the potential to give rise to NK1.1⁺ cells decreased during differentiation of Lin^-IL-7R⁺₄₇⁺CD4^- to Lin^-IL-7R⁺₄₇⁺CD4⁺ cells. Our model conflicts with those of other groups (12, 14, 39). This could be due to the presence of multiple pathways of NK and DC development. In fact, our previous study on Id2^-/- mice indicates Id2-dependent and Id2-independent pathways of NK cell differentiation (11). DC can differentiate even from a pro-B cell population (29). It is also likely that these discrepancies reflect differences in stromal cell activity among organs. NK cell differentiation has been thought to occur in the central lymphoid organs such as bone marrow and thymus (13, 14). Recently, Iizuka et al. (50) reported that NK lineage differentiation is impaired in LT^-/- mice with defects in both the microenvironment and hemopoietic lineage. This suggests that establishment of the architecture of lymphoid tissue and generation of NK cells in the same tissue are concurrent processes in which LT₁₂-positive cells play important roles. If so, this notion could be extended to peripheral lymphoid tissues, in which LT₁₂-positive cells are required for organogenesis. There is a strong correlation between defects in PP/LN organogenesis and absence of functional NK cells (11, 51, 52), which is consistent with an idea of a common progenitor for FILyP and NK cells. However, there also appears to be a dichotomy in LN and PP formation, as PP can form in the absence of LN as in Osteoprotegerin ligand^-/- (53), receptor activator of NF-B^-/- (54), or TNFR-associated factor 6^-/- (55, 56) mice, and only PP is absent in the IL-7R^-/-, Jak3^-/-, IL-2R^-/- (2), and cpdm/cpdm mutant mice (57), implying presence of organ-specific processes (58). Nonetheless, IL-7R and ₄₇ integrin will serve as a useful marker for dissecting both common and organ-specific processes.

View larger version (27K): [in this window] [in a new window]   FIGURE 10. A model for the differentiation pathway of the FILyP cell. In this model, FILyP cells are derived from the IL-7R+c-Kit+47- population, which also gives rise to T and B cells, but not myeloid cells. As it was suggested that common lymphoid progenitors are not found in the fetal liver (17 34 ), this population may be a mixture of T- and B-committed progenitors. The FILyP lineage diverges from the B cell lineage before expression of 47 integrin, while T-potential remains in the 47+ population. In the liver-derived Lin-IL-7R+c-Kit+47+ population, more cells restricted to FILyP cells are present than those with T-potential. T-potential is lost completely from this population during their migration from fetal liver to the mesenteric tissues. After migration to the intestine, a portion of FILyP cells is activated by the ligand for IL-7R and involved in induction of PP anlagen by expressing LT12. During this process, expression of IL-7R and 47 integrin is maintained. In this model, CD4 expression is thought to associate with the divergence from NK lineage. If the appropriate signals are available in the microenvironment, this population can further differentiate to NK or DC lineages.

	Acknowledgments

 
We are grateful to Mizuho Satoh for technical assistance, to Dr. Ikawa and Dr. Ohmura for their advice on cytotoxic and cell proliferation assays, and to Dr. S. Frazer for critical reading of the manuscript.

	Footnotes

 
¹ This study was supported by Special Coordination Funds of the Science and Technology Agency of the Japanese Government (to H.Y.); by grants from the Ministry of Education, Science, Sports, and Culture of Japan (07CE2005, 09275102, and 11148213 to S.-I.N.); by U.S. Public Health Service, National Institutes of Health Grants AI33068 and PO1CA69381 (to C.F.W.); and by the State of California University-wide AIDS Research Program F98-LJIAI-111 (to S.M.S.).

² Address correspondence and reprint requests to Dr. Hisahiro Yoshida, Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Shogoin-Kawaharacho 53, Sakyo, Kyoto, Japan 606-8507. E-mail address: hyoshida{at}virus.kyoto-u.ac.jp

³ Abbreviations used in this paper: PP, Peyer’s patch; DC, dendritic cell; dpc, days post coitus; LT, lymphotoxin; FILyP, fetal intestinal LT producer; HPRT, hypoxanthine phosphoribosyltransferase; Jak, Janus kinase; Lin, lineage marker; LN, lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; SCF, stem cell factor.

Received for publication August 31, 2000. Accepted for publication June 20, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Adachi, S., H. Yoshida, H. Kataoka, S. I. Nishikawa. 1997. Three distinctive steps in Peyer’s patch formation of murine embryo. Int. Immunol. 9:507.[Abstract/Free Full Text]
2. Adachi, S., H. Yoshida, K. Honda, K. Maki, K. Saijo, K. Ikuta, T. Saito, K. Ikuta, S. Nishikawa, S. I. Nishikawa. 1998. Essential role of IL-7 receptor in the formation of Peyer’s patch anlage. Int. Immunol. 10:1.[Abstract/Free Full Text]
3. De Togni, P., J. Goellner, N. H. Ruddle, P. R. Streeter, A. Fick, S. Mariathasan, S. C. Smith, R. Carlson, L. P. Shornick, S. Schoenberger. 1994. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science 264:703.[Abstract/Free Full Text]
4. Banks, T. A., B. T. Rouse, M. K. Kerley, P. J. Blair, V. L. Godfrey, N. A. Kuklin, D. M. Bouley, J. Thomas, S. Kanangat, M. L. Mucenski. 1995. Lymphotoxin--deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
5. Alimzhanov, M. B., D. V. Kuprashi, M. H. Kosco-Vilbois, A. Luz, R. L. Turetskaya, A. Tarakhovsky, K. Rajewsky, S. A. Nedospasov, K. Pfeffer. 1997. Abnormal development of secondary lymphoid tissues in lymphotoxin -deficient mice. Proc. Natl. Acad. Sci. USA 94:9302.[Abstract/Free Full Text]
6. Koni, P. A., R. Sacca, P. Lawton, J. L. Browning, N. H. Ruddle, R. A. Flavell. 1997. Distinct roles in lymphoid organogenesis for lymphotoxins and revealed in lymphotoxin -deficient mice. Immunity 6:491.[Medline]
7. Futterer, A., K. Mink, A. Luz, M. H. Kosco-Vilbois, K. Pfeffer. 1998. The lymphotoxin receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 9:59.[Medline]
8. Nishikawa, S.-I., S. Nishikawa, K. Honda, H. Hashi, H. Yoshida. 1998. Peyer’s patch organogenesis as a programmed inflammation: a hypothetical model. Cytokine Growth Factor Rev. 9:213.[Medline]
9. Shinkura, R., K. Kitada, F. Matsuda, K. Tashiro, K. Ikuta, M. Suzuki, K. Kogishi, T. Serikawa, T. Honjo. 1999. Alymphoplasia is caused by a point mutation in the mouse gene encoding NF-B-inducing kinase. Nat. Genet. 22:74.[Medline]
10. Yoshida, H., K. Honda, R. Shinkura, S. Adachi, S. Nishikawa, K. Maki, K. Ikuta, S.-I. Nishikawa. 1999. Interleukin-7 receptor ⁺ CD3^- cells in embryonic intestine induces organizing center of Peyer’s patch. Int. Immunol. 11:643.[Abstract/Free Full Text]
11. Kondo, M., I. L. Weissman, K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91:661.[Medline]
12. Yokota, Y., A. Mansouri, S. Mori, S. Sugawara, S. Adachi, S.-I. Nishikawa, P. Gruss. 1999. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397:702.[Medline]
13. Williams, N. S., J. I. Klem, J. Puzanov, P. V. Sivakumar, J. D. Schatzle, M. Bennett, V. Kumar. 1998. Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems. Immunol. Rev. 165:47.[Medline]
14. Carlyle, J. R., J. C. Zuniga-Pflucker. 1998. Lineage commitment and differentiation of T and natural killer lymphocytes in the fetal mouse. Immunol. Rev. 165:63.[Medline]
15. Shortman, K., D. Vremec, L. M. Corcoran, K. Georgopoulos, K. Lucas, L. Wu. 1998. The linkage between T-cell and dendritic cell development in the mouse thymus. Immunol. Rev. 165:39.[Medline]
16. Sudo, T., S. Nishikawa, N. Ohno, N. Akiyama, M. Tamakoshi, H. Yoshida, S.-I. Nishikawa. 1993. Expression and function of the interleukin 7 receptor in murine lymphocytes. Proc. Natl. Acad. Sci. USA 90:9125.[Abstract/Free Full Text]
17. Mebius, R. E., P. Rennert, I. L. Weissman. 1997. Developing lymph nodes collect CD4⁺CD3^-LT⁺ cells that can differentiate to APC, NK, and follicular cells, but not T- or B-cells. Immunity 7:493.[Medline]
18. Kawamoto, H., K. Ohmura, S. Fujimoto, Y. Katsura. 1999. Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver. J. Immunol. 162:2725.[Abstract/Free Full Text]
19. Nishikawa, S., M. Kusakabe, K. Yoshinaga, M. Ogawa, S. Hayashi, T. Kunisada, T. Era, T. Sakakura, S.-I. Nishikawa. 1991. In utero manipulation of coat color formation by a monoclonal anti-c-Kit antibody: two distinct waves of c-Kit-dependency during melanocyte development. EMBO J. 10:2111.[Medline]
20. Force, W. R., B. N. Walter, C. Hession, R. Tizard, C. A. Kozak, J. L. Browning, C. F. Ware. 1996. Mouse lymphotoxin- receptor: molecular genetics, ligand binding, and expression. J. Immunol. 155:5280.[Abstract]
21. Crowe, P. D., T. L. VanArsdale, B. N. Walker, K. M. Dahms, C. F. Ware. 1994. Production of lymphotoxin (LT) and a soluble dimeric form of its receptor using the baculovirus expression system. J. Immunol. Methods 168:79.[Medline]
22. Takakura, N., H. Yoshida, T. Kunisada, S.-I. Nishikawa. 1996. A novel function of platelet derived growth factor receptor : its transient expression in the epidermis is essential for hair canal formation. J. Invest. Dermatol. 107:770.[Medline]
23. Watanabe, Y., O. Mazda, Y. Aiba, K. Iwai, J. Gyotoku, S. Ideyama, J. Miyazaki, Y. Katsura. 1992. A murine thymic stromal cell line which may support the differentiation of CD4^-8^- thymocytes into CD4⁺8^- T cell receptor positive T cells. Cell. Immunol. 142:385.[Medline]
24. Yoshida, H., S.-I. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, L. D. Shultz, S.-I. Nishikawa. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345:442.[Medline]
25. Dou, Y. M., Y. Watanabe, Y. Aiba, K. Wada, Y. Katsura. 1995. A novel culture system for induction of T cell development: modification of fetal thymus organ culture. Thymus 23:195.
26. Kawamoto, H., K. Ohmura, Y. Katsura. 1998. Presence of progenitors restricted to T, B, or myeloid lineage, but absence of multipotent stem cells, in the murine fetal thymus. J. Immunol. 161:3799.[Abstract/Free Full Text]
27. Slezak, S. E., P. K. Horan. 1989. Cell-mediated cytotoxicity. J. Immunol. Methods 117:205.[Medline]
28. Ikawa, T., H. Kawamoto, S. Fujimoto, Y. Katsura. 1999. Commitment of common T/natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190:1617.[Abstract/Free Full Text]
29. Bjorck, P., P. W. Kincade. 1998. CD19⁺ pro-B cells can give rise to dendritic cells in vitro. J. Immunol. 161:5795.[Abstract/Free Full Text]
30. Mauri, D. N., R. Ebner, R. I. Montgomery, K. D. Kochel, T. C. Cheung, G.-L. Yu, S. Ruben, M. Murpy, R. J. Eisenberg, G. H. Cohen, et al 1998. LIGHT, a new member of the TNF superfamily, and lymphotoxin are ligands for Herpesvirus entry mediator. Immunity 8:21.[Medline]
31. Carson, W. E., J. G. Giri, M. J. Lindemann, M. L. Linett, M. Ahdieh, R. Paxton, D. Anderson, J. Eisenmann, K. Grabstein, M. A. Caligiuri. 1994. Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J. Exp. Med. 180:1395.[Abstract/Free Full Text]
32. Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669.[Medline]
33. Williams, N. S., T. A. Moore, J. D. Schatzle, I. J. Puzanov, P. V. Sivakumar, A. Zlotnik, M. Bennett, V. Kumar. 1997. Generation of lytic natural killer 1.1⁺, Ly-49^- cells from multipotential murine bone marrow progenitors in a stroma-free culture: definition of cytokine requirements and developmental intermediates. J. Exp. Med. 186:1609.[Abstract/Free Full Text]
34. Takei, F., J. Brennan, D. L. Mager. 1997. The Ly-49 family: genes, proteins and recognition of class I MHC. Immunol. Rev. 155:67.[Medline]
35. Lanier, L. L.. 1998. NK cell receptors. Annu. Rev. Immunol. 16:359.[Medline]
36. Saunders, D., K. Lucas, J. Ismaili, L. Wu, E. Maraskovsky, A. Dunn, K. Shortman. 1996. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 184:2185.[Abstract/Free Full Text]
37. Honda, K., H. Nakano, H. Yoshida, S. Nishikawa, P. Rennert, K. Ikuta, M. Tamachika, K. Yamaguchi, T. Fukumoto, T. Chiba, S. Nishikawa. 2001. Molecular basis for hematopoietic/mesenchymal interaction during initiation of Peyer’s patch organogenesis. J. Exp. Med. 193:621.[Abstract/Free Full Text]
38. Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J.-I. Miyazaki, K. Ikuta. 1996. Interleukin 7 receptor-deficient mice lack T cells. Proc. Natl. Acad. Sci. USA 93:7172.[Abstract/Free Full Text]
39. Akashi, K., M. Kondo, I. L. Weissman. 1998. Role of interleukin-7 in T-cell development from hematopoietic stem cells. Immunol. Rev. 165:13.[Medline]
40. Kawamoto, H., T. Ikawa, K. Ohmura, S. Fujimoto, Y. Katsura. 2000. T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver. Immunity 12:441.[Medline]
41. Zhang, Y., Y. Zhang, Y. Wang, M. Ogata, S. Hashimoto, N. Onai, K. Matsushima. 2000. Development of dendritic cells in vitro from murine fetal liver-derived lineage phenotype-negative c-kit⁺ hematopoietic progenitor cells. Blood 95:138.[Abstract/Free Full Text]
42. Mebius, R. E., P. R. Streeter, S. Michie, E. C. Butcher, I. L. Weissman. 1996. A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4⁺CD3^- cells to colonize lymph nodes. Proc. Natl. Acad. Sci. USA 93:11019.[Abstract/Free Full Text]
43. Hashi, H., H. Yoshida, K. Honda, S. Fraser, H. Kubo, M. Awane, A. Takabayashi, H. Nakano, Y. Yamaoka, S. Nishikawa. 2001. Compartmentalization of Peyer’s patch anlagen before lymphocyte entry. J. Immunol. 166:3702.[Abstract/Free Full Text]
44. Forster, R., A. E. Mattis, E. Kremmer, E. Wolf, G. Brem, M. Lipp. 1996. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell 87:1037.[Medline]
45. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Forster, J. D. Sedgwick, J. L. Browning, M. Lipp, J. G. Cyster. 2000. A chemokine-driven positive feedback loop organizes lymphoid follicle. Nature 406:309.[Medline]
46. Van Beneden, K., A. De Creus, V. Debacker, J. De Boever, J. Plum, G. Leclercr. 1999. Murine fetal natural killer cells are functionally and structurally distinct from adult natural killer cells. J. Leukocyte Biol. 66:625.[Abstract]
47. Dorfman, J. R., D. H. Raulet. 1998. Acquisition of Ly49 receptor expression by developing natural killer cells. J. Exp. Med. 187:609.[Abstract/Free Full Text]
48. Wu, L., D. Vremic, C. Ardavin, K. Shortman. 1995. Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation. Eur. J. Immunol. 25:418.[Medline]
49. Leenen, P. J., K. Radosevic, J. S. Voerman, B. Salomon, N. van Rooijen, D. Klatzmann, W. van Ewijk. 1998. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J. Immunol. 160:2166.[Abstract/Free Full Text]
50. Iizuka, K., D. D. Chaplin, Y. Wang, Q. Wu, L. E. Pegg, W. Yokoyama, Y. X. Fu. 1999. Requirement for membrane lymphotoxin in natural killer cell development. Proc. Natl. Acad. Sci. USA 96:6336.[Abstract/Free Full Text]
51. Akashi, K., D. Traver, M. Kondo, I. L. Weissman. 1999. Lymphoid development from hematopoietic stem cells. Int. J. Hematol. 69:217.[Medline]
52. Georgopoulos, K., M. Bigby, J.-H. Wang, A. Molnar, P. Wu, S. Winandy, A. Sharpe. 1994. The Ikaros gene is required for the development of all lymphoid lineages. Cell 79:143.[Medline]
53. Kong, Y. Y., H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morony, A. J. Oliveira-dos-Santos, G. Van, A. Itie, et al 1999. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397:315.[Medline]
54. Dougall, W. C., M. Glaccum, K. Charrier, K. Rohrbach, K. Brasel, T. De Smedt, E. Daro, J. Smith, M. E. Tometsco, C. R. Maliszewski. 1999. RANK is essential for osteoclast and lymph node development. Genes Dev. 13:2412.[Abstract/Free Full Text]
55. Lomaga, M. A., W. C. Yeh, I. Sarosi, G. S. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al 1999. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13:1015.[Abstract/Free Full Text]
56. Naito, A., S. Azuma, S. Tanaka, T. Miyazaki, S. Takaki, K. Takatsu, K. Nakao, K. Nakamura, M. Katsuki, T. Yamamoto, J. Inoue. 1999. Severe osteopetrosis, defective interleukin-1 signaling and lymph node organogenesis in TRAF6-deficient mice. Genes Cells 4:353.[Abstract]
57. HogenEsch, H., S. Janke, D. Boggess, J. P. Sundberg. 1999. Absence of Peyer’s patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice. J. Immunol. 162:3890.[Abstract/Free Full Text]
58. Kim, D., R. E. Mebius, J. D. MacMicking, S. Jung, T. Cupedo, Y. Castellanos, J. Rho, B. R. Wong, R. Josien, N. Kim. 2000. Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE. J. Exp. Med. 192:1467.[Abstract/Free Full Text]

This article has been cited by other articles:

				D H Adams, B Eksteen, and S M Curbishley Immunology of the gut and liver: a love/hate relationship Gut, June 1, 2008; 57(6): 838 - 848. [Full Text] [PDF]

				M. Okuda, A. Togawa, H. Wada, and S.-i. Nishikawa Distinct Activities of Stromal Cells Involved in the Organogenesis of Lymph Nodes and Peyer's Patches J. Immunol., July 15, 2007; 179(2): 804 - 811. [Abstract] [Full Text] [PDF]

				F. Stevenaert, K. Van Beneden, V. De Colvenaer, A. S. Franki, V. Debacker, T. Boterberg, D. Deforce, K. Pfeffer, J. Plum, D. Elewaut, et al. Ly49 and CD94/NKG2 receptor acquisition by NK cells does not require lymphotoxin-{beta} receptor expression Blood, August 1, 2005; 106(3): 956 - 962. [Abstract] [Full Text] [PDF]

				T. Cupedo, M. F. R. Vondenhoff, E. J. Heeregrave, A. E. de Weerd, W. Jansen, D. G. Jackson, G. Kraal, and R. E. Mebius Presumptive Lymph Node Organizers are Differentially Represented in Developing Mesenteric and Peripheral Nodes J. Immunol., September 1, 2004; 173(5): 2968 - 2975. [Abstract] [Full Text] [PDF]

				G. Eberl and D. R. Littman Thymic Origin of Intestinal {alpha}{beta} T Cells Revealed by Fate Mapping of ROR{gamma}t+ Cells Science, July 9, 2004; 305(5681): 248 - 251. [Abstract] [Full Text] [PDF]

				H.-J. Kim, T. Kammertoens, M. Janke, O. Schmetzer, Z. Qin, C. Berek, and T. Blankenstein Establishment of Early Lymphoid Organ Infrastructure in Transplanted Tumors Mediated by Local Production of Lymphotoxin {alpha} and in the Combined Absence of Functional B and T Cells J. Immunol., April 1, 2004; 172(7): 4037 - 4047. [Abstract] [Full Text] [PDF]

				T W Spahn and T Kucharzik Modulating the intestinal immune system: the role of lymphotoxin and GALT organs Gut, March 1, 2004; 53(3): 456 - 465. [Full Text] [PDF]

				R. G. Lorenz, D. D. Chaplin, K. G. McDonald, J. S. McDonough, and R. D. Newberry Isolated Lymphoid Follicle Formation Is Inducible and Dependent Upon Lymphotoxin-Sufficient B Lymphocytes, Lymphotoxin {beta} Receptor, and TNF Receptor I Function J. Immunol., June 1, 2003; 170(11): 5475 - 5482. [Abstract] [Full Text] [PDF]

				D. V. Kuprash, M. B. Alimzhanov, A. V. Tumanov, S. I. Grivennikov, A. N. Shakhov, L. N. Drutskaya, M. W. Marino, R. L. Turetskaya, A. O. Anderson, K. Rajewsky, et al. Redundancy in Tumor Necrosis Factor (TNF) and Lymphotoxin (LT) Signaling In Vivo: Mice with Inactivation of the Entire TNF/LT Locus versus Single-Knockout Mice Mol. Cell. Biol., December 15, 2002; 22(24): 8626 - 8634. [Abstract] [Full Text] [PDF]

				J. L. Browning and L. E. French Visualization of Lymphotoxin-{beta} and Lymphotoxin-{beta} Receptor Expression in Mouse Embryos J. Immunol., May 15, 2002; 168(10): 5079 - 5087. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshida, H. Articles by Nishikawa, S.-I. Search for Related Content PubMed PubMed Citation Articles by Yoshida, H. Articles by Nishikawa, S.-I.