Abstract

Peyer's patch (PP) organogenesis proceeds through three histologically distinct steps: formation of organizing centers expressing VCAM-1 and ICAM-1 in segregated regions of the intestine at 15.5 days post-coitus (d.p.c.) (step I), accumulation of blood cells expressing different sets of surface markers to this region at 16.5–17.0 d.p.c. (step II), and entry of CD3+ and B220+ lymphocytes just before birth (step III). PP formation of both Il7ra–/– and Lta–/– mice is impaired from step I, suggesting involvement of the two molecules at the same timing in PP organogenesis. Expression of lymphotoxin (LT) α and LTβ in IL-7 receptor (IL-7R) α+ cells in the intestine indicates that defects of Il7ra–/– and Lta–/– mice are due to functional inability of IL-7Rα+ cells in the induction of PP anlage. Blocking of IL-7Rα function by a single injection of the antagonistic mAb in 15.5 d.p.c. embryos suppressed appearance of VCAM-1+ spots and expression of LTα and LTβ in the intestine, which eventually resulted in mice without PP but are otherwise normal. Intestinal IL-7Rα+ cells are lymphoid in morphology but CD3 and functional in both nu/nu and Rag2–/ mice. These results implicate IL-7Rα+ CD3 cells as the direct inducer of the organizing center of PP.

Introduction

Peyer's patch (PP) is a peripheral lymphatic tissue distributed along the intestinal tract. In adult, 6–12 and 180–240 PP are present in the intestine of mouse and human respectively (1). Histological organization of PP is similar to other peripheral lymphatic organs elsewhere, as T and B lymphocytes are major components of this tissue, but other hematopoietic cells such as macrophages and dendritic cells are also present (2). Moreover, specialized structures like the high endothelial venule (HEV) and germinal center are found in PP as well as the lymph node (LN) and spleen. It was also suggested that antigenic stimulation in PP induces preferentially Ig class switch to IgA, an Ig class which can be exported through the epithelial sheet (35). Based upon these observations and its anatomical location, PP is thought to form the first front in the mucosal immunity of the intestine.

Recently, similarity and differences in PP and LN organogenesis were demonstrated in mice which bear null mutation in the genes encoding lymphotoxins (LT) and their receptors (LTR). In Lta–/– mice, formation of all PP and LN is inhibited (6,7). Moreover, it was reported that Ltbr–/– mice display no PP nor LN (8). As LTα and LTβ form a heterotrimer which binds LTβR (9), these results support the idea that this heterotrimer plays an essential role in the induction of LN and PP. However, LTβ null mutant mouse lacks peripheral LN and PP but mesenteric LN remain intact (8,10). These results suggest a diversity in the role of LT in the formation of these peripheral lymphoid organs.

Despite clear evidence for involvement of LT in the formation of PP, little is known about temporal expression of LT in embryogenesis, which cells in the intestine produce them, or which cells are affected by LT. In an attempt to elucidate the cellular process by which PP is constructed, we have demonstrated that PP organogenesis in mouse proceeds with at least three histologically distinct steps (11). The first step can be defined by expression of VCAM-1 in the mesenchymal cell component of segregated regions of the intestine at 15–16 day post-coitus (d.p.c.). Round cells including IL-7Rα+, CD4+ and Ia+ cells accumulate in this VCAM-1+ region during the next step at ~16–17 d.p.c. CD3+ and B220+ mature lymphocytes, which are the major components of PP in later life, are found in this region just before birth. These observations indicate a chronological sequence of PP formation and molecules potentially involved in this process. Moreover, absence of VCAM-1+ spots in Il7ra–/– or Jak3–/ embryos provides direct evidence for the involvement of IL-7Rα signaling in the early process of PP organogenesis (12). Since the PP development of nu/nu, scid/scid or Rag2–/ develops normally to the second step (11,12), this IL-7Rα signaling step is independent of thymus-derived or mature lymphocyte development.

In this report, we investigated how IL-7Rα+ cells are involved in PP anlage formation. The results suggest that the IL-7Rα+ CD3 subset in the embryonic intestine expresses both LTα and LTβ upon IL-7 stimulation, and acts on surrounding mesenchymal components to form organization centers of PP.

Methods

Mice

Pregnant C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan). Rag2–/ mutant mice were purchased from Taconic Farms (New York, NY) and Lta–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME). 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 d.p.c. Aliquots of 3 mg of antibodies dissolved in PBS were injected i.v. into pregnant mice at 6:00 p.m. of each gestational day.

Antibodies

Names of clones and sources of mAb used in this study are listed below. Anti-TCR-δ (3A10) was a kind gift from Dr Itohara. Anti-IL-7Rα antibody (A7R34), anti-Flk-1 antibody (AVAS12), anti-c-fms antibody (AFS98), anti-Flk-2 antibody (A2F10) and anti-c-Kit antibody (ACK2) were established in our laboratory and prepared as previously described (1317). Anti-B220 antibody (RA3-6B2), anti-CD11b antibody (Mac-1), anti-CD8a (53-6.7) and anti-F4/80 (F4/80) were purified from hybridoma culture supernatant as described (11). Anti-CD4 (GK1.5; PharMingen, San Diego, CA), anti-Thy-1.2 (Caltag, San Francisco, CA), anti-CD3 (Y65.135; Seikagakukogyo, Tokyo, Japan; 2C11; PharMingen), anti-CD11c (HL3; PharMingen), anti-VCAM-1 (429 MVCAM.A; PharMingen), anti-ICAM-1 (3E2; PharMingen), anti-TCR-αβ (H57-597; PharMingen), anti-TCR-γδ (GL3; PharMingen), anti-CD45 (30F11.1; PharMingen), anti-NK1.1 (NKR-P1C; PharMingen), anti-CD44 (IM7; PharMingen) and anti-CD25 (7D4; PharMingen) were purchased.

Immunohistochemistry

Whole-mount immunohistostaining was performed as described previously (11,12,18). In order to immunostain the mAb-injected embryos, we used biotin-labeled mAb followed by horseradish peroxidase-conjugated streptavidin (Wako, Osaka, Japan). Briefly, the specimens were dissected and pre-fixed in 2% paraformaldehyde (pH 7.4) with microwave irradiation (30 s at 600 W) and fixed for 30–90 min in the same fixative on ice. After washing with PBS and dehydration by methanol, intrinsic peroxidase activities in the specimens was blocked by 0.3% of H2O2. Non-specific binding was blocked by incubation with PBSMT (1% skim milk/0.3% Triton X-100 in PBS) twice for 1 h each and then incubating with biotinylated antibodies for overnight. After washing the specimen in PBSMT 3 times for 1 h each and once in PBST (0.3% Triton X in PBS) for 30 min, the tissue was incubated with horseradish peroxidase–streptavidin for 1 h in PBST. After washing the specimen in PBST twice for 30 min each, the antigens were detected by the color reaction with diaminobenzidine and nickel chloride.

Preparation of single-cell suspension for flow cytometry and cell sorting.

All organs of embryos were dissected under a binocular stereomicroscope with fine forceps and then dissociated by incubating with dispase (Boehringer, Mannheim, Germany) for 10–25 min at 37°C. The whole gut of adult mice was chopped to small pieces with dissecting scissors and digested by type IV collagenase (Sigma, St Louis, MA) for 1 h at 37°C. After pipetting with a 21 gauge needle and syringe, dissociated cells were washed with HBSS containing 20% FCS and DNase (Sigma). Cells were filtered through nylon mesh to remove large clumps, washed and surface stained as described (13). Stained cells were analyzed or sorted by a FACS Vantage (Becton Dickinson).

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; Gibco, LOCATION??). In each PCR reaction for detecting the expression of LTα, LTβ and hprt genes, cDNA corresponding to an amount from 1000 cells was incubated with 100 pg of each primer set, 0.4 mM of each dNTP and 2.5 U of ExTaq polymerase (Takara, Kyoto, Japan). PCR conditions were determined for each primer set and control reactions were carried out in tubes without cDNA. The following oligonucleotides were used for PCR: LT-α: TCACCTTGTTGGGTACCCCAGCAA, ATACACAGACTTCTGCGCAC; LT-β: TTGTTGGCAGTGCCTATCACTGTCC, CTCGTGTACCATAACGACCCGTAC; hprt: GAGCTACTGTAATGATCAGTCAACGG, GATTCAACTTGCGCTCATCTTAGGC.

For semiquantative RT-PCR analysis, the amount of the template cDNA from the each specimens was equalized by comparing the concentration of RT-PCR products with hprt primer sets at various amplifying cycles. The minimum number of amplifying cycles those detect the PCR products was determined for each specimen by each primer sets.

Cell culture

The cells sorted by FACS were co-cultured with stromal cell line OP-9, which support hematopoiesis from variable sources (19), in the presence of IL-7 (20 U/ml) and stem cell factor (SCF; 100 ng/ml). To block the function of IL-7Rα, A7R34 was added at the concentration of 20 μg/ml. All cells were harvested 24 h later and the mRNA expression of each sample were examined by RT-PCR.

Results

Process of PP formation in the mouse embryo

PP organogenesis develops in three distinctive steps designated as steps I, II and III distinguished by several lineage-specific marker-expressing cells distributions (11,12). In addition, we detected ICAM-1 expression in step I, and CD11c-, F4/80- or c-fms-expressing cells accumulation in step II in this study (Figs 1 and 3B). The distribution pattern of ICAM-1-expressing cells in developing PP was similar to VCAM-1-expressing cells. In contrast, the distribution patterns of various lineage marker-expressing blood cells at step II was different from each others (cf. the distribution patterns of IL-7Rα+, CD11c+, F4/80+ and c-fms+ cells in Figs 1 and 3B). This result indicates the various lineages of blood cells accumulate independently into developing PP at step II.

We have previously shown that the Il7ra–/– mouse does not have PP and all three steps were undetectable (12). As accumulation of blood cells in developing intestine was not detected in the Il7ra–/– mouse by using anti-CD45 mAb recognizing all leukocyte cell lineages (Fig. 1), we confirmed that step II is completely absent in the Il7ra–/– mouse. Since IL-7Rα+ cells were already found in embryonic gut as early as 13.5 d.p.c. and distributed mainly to the ante mesenteric region of the whole intestine by 15.0 d.p.c. (data not shown), the PP defect of the Il7ra–/– mouse must be an outcome of a failure in step I or more early step rather than involvement of IL-7Rα signaling when IL-7Rα+ cells accumulate to PP as a part of step II event.

We also investigated the Lta–/– mouse to specify the stage in which LTα plays a role in PP organogenesis. It displayed no VCAM-1+ (Fig. 1E) nor ICAM-1+ spots (data not shown) in the intestine, indicating the involvement of LTα in step I. Likewise, no cluster formation of CD45+ cells was observed (data not shown). Interestingly, IL-7Rα+ cells remained widespread, but not in cluster, neither in the intestine of 17 d.p.c. Lta–/– mice (Fig. 1F and G) nor in neonatal Lta–/– mice (data not shown), just as seen in 15 d.p.c. wild-type embryos (Fig. 1H).

Involvement of IL-7Rα signal in a short time-window during the initial step of PP formation

In order to specify the timing of IL-7Rα+ cell function, we carried out an experiment to block the IL-7Rα signal at various stages of embryogenesis. A single i.v. injection of an antagonistic mAb to IL-7Rα (13) was given to pregnant mice on one of the days from 14.5 to 18.5 d.p.c. and the presence of PP in the offspring was examined at 2 months of age. As a class-matched control mAb, we used non-antagonistic anti-Flk1 mAb (14).

PP could not be detected in any of the offspring from mice treated with mAb to IL-7Rα (A7R34) on 14.5–15.5 d.p.c. (Table 1). To confirm that the effect of A7R34 injection was restricted to PP, we collected each lymphoid tissue separately, and examined cell recovery and surface phenotype from 2-month-old animals treated with A7R34 at 14.5–15.5 d.p.c. Comparable numbers of cells were recovered from bone marrow, thymus, spleen, inguinal LN and mesenteric LN of control and A7R34-injected groups. Moreover, percentages of IL-7Rα+, CD4+, CD8+ and B220+ cells in each organ were nearly the same between two groups (data not shown).

Interestingly, mice that were treated later than 16.5 d.p.c. generated PP, though only in the upper intestine (Table 1 and Fig. 2). It is intriguing that all offspring of the 16.5 d.p.c. injected group generated only one PP in the upper intestine. In the 17.5 d.p.c. injected group, the number of organized PP was variable, but their localization was also restricted to the upper intestine. The injection on 18.5 d.p.c. had no effect on the PP development. It is of note that the lymphoid follicles in the cecum, which is absent in the Lta–/– or aly/aly but present in the Il7ra–/– mouse (Yoshida, unpublished observation), were unaffected by this treatment (Fig. 2, asterisk), suggesting the specificity of A7R34 treatment and involvement of other signals in the formation of the cecal follicle.

Phenotypes of IL-7Rα+ cells in the intestine

To determine the lineage of IL-7Rα+ cells in the intestine, we investigated the surface phenotypes of IL-7Rα+ cells in the intestine. Figure 3(A) displays an example of FACS analysis of the IL-7Rα+ cells present in the gut of C57BL/6 embryos at 16.5 d.p.c. when the IL-7Rα signaling system is necessary for PP development. They were further subdivided to CD4+ and CD4 populations, whereas all are CD3 at this embryonic stage. None of other lymphoid cell markers except Thy1 were detectable in this population (Fig. 3A). Thy1 expression was detectable both in CD4+ and CD4 populations.

In addition to the markers presented in Fig. 3(A), we analyzed the expression of a panel of surface markers for various cell lineages. The IL-7Rα+ cells expressed CD44, CD45, c-Kit, integrin α4 (100% of IL-7Rα+ cells) and Mac-1 at low level (<10% of IL-7Rα+ cells). IL-7Rα+ cells in the gut at this embryonic stage did not express the surface markers for T cells: CD25, CD3, CD8, TCRδ, TCRαβ, TCRγδ; B cells, B220, CD19; NK cells, NK1.1; IEL, integrin αE; macrophages, c-fms, F4/80; dendritic cells, CD11c, Flk2/Flt3; erythrocytes, ter119; endothelial cells, Flk1; mesenchymal cells, PDGFRα, VCAM-1; epithelial cells, EC CD2 (Fig. 3A and Table 2). The mRNA expression analysis by means of RT-PCR of FACS-sorted cells detected lck and Ikaros mRNA, in both IL-7Rα+ and IL-7Rα CD45+ populations (data not shown). Interestingly, chemokine receptor BLR1 mRNA expression was found only in IL-7Rα+ cells but not in IL-7Rα populations (data not shown). Whole-mount immunostaining of 17.5 d.p.c. embryos showed that c-fms+ cells had a dendritic shape and were clustering in developing PP, F4/80+ cells were also dendritic, rather bigger than c-fms+ cells and distributed in the PP rather sparsely than IL-7Rα+ cells, and CD11c+ cells were also dendritic but distributed only in the periphery of developing PP (Fig. 3B). In contrast, IL-7Rα+ cells accumulating in the developing PP showed a round shape and diffuse distribution in the whole PP (11) (Fig. 1), indicating they are different from macrophages or dendritic cell lineages at this embryonic stage. Sorted CD4IL-7Rα+ and CD4+ IL-7Rα+ cells were small in size and showed a spherical shape, dark nucleus and scanty cytoplasm, which is consistent with the phenotyope of cells in the lymphoid lineage (Fig. 3C). Taken collectively, the surface marker expression, mRNA expression, morphology of in situ or sorted cells and the distribution patterns in the developing intestine indicated that IL-7Rα+ cells present in the embryonic intestine are different from mature T cells, B cells, NK cells, macrophages, dendritic cells and non-blood cells.

Since all the gut IL-7Rα+ cells express c-Kit, we examined the PP development in c-Kit the defective mutant KitW/KitW mice (20) to investigate the importance of this receptor tyrosine kinase in PP formation. Although this strain lacks the function of c-Kit, all three steps were detected during embryogenesis (data not shown).

Intestinal IL-7Rα+ cells represent a subset specific to the abdominal cavity of embryos

In order to gain an insight into the origin of the IL-7Rα+ cells in the embryonic intestine, we compared the surface phenotype of IL-7Rα+ cells in various organs from embryos. Although, the number of IL-7Rα+ cells in fetal liver is quite few, <1%, they are present in fetal liver, thymus, intestine, mesentery and spleen of the embryo at 14.5 d.p.c. Interestingly, however, CD4+ IL-7Rα+ double-positive cells are only present in the intestine, mesentery and spleen (Fig. 4). This result suggests that the co-existence of CD4+ IL-7Rα+ and CD4 IL-7Rα+ cells is a common feature of tissues in the abdominal cavity of early embryos. IL-7Rα+ cells disappeared rapidly from the spleen, while they were maintained in the intestine and mesentery before birth. When compared to mesenteric cells which have only a few CD3+ cells at 18.5 d.p.c., those in the intestine of the same embryos already contained many CD3+ cells.

Production of LT by IL-7Rα+ cells

To test whether LT are involved in PP anlage formation as effector molecules expressed in IL-7Rα+ cells or not, we sorted CD4 IL-7Rα+, CD4+ IL-7Rα+ and CD45+ IL-7Rα cells from 15.5 and 16.5 d.p.c. intestine, and analyzed for expression of LT by RT-PCR. LTα and LTβ mRNA were detected both in CD4+ and CD4 IL-7Rα+ cells but not in CD45+ IL-7Rα cells in the same intestine (Fig. 5A). Similarly, IL-7Rα+ cells in spleen or mesentery also expressed LTα or LTβ mRNA but CD45+ IL-7Rα cells in these organs did not. In contrast, LTα and LTβ mRNA were not detected in the IL-7Rα+ cells of fetal liver (Fig. 5B). These results are representative of three independent examination.

IL-7Rα+ cells are activated within the intestine to induce PP anlage

Next, we investigated an immediate effect of A7R34 injection on the formation of PP anlage and also on the expression of LT in the intestine. Mice were given a single injection of A7R34 on 15.5 d.p.c., and the appearance of VCAM-1+ and ICAM-1+ segregated spots in the intestine was examined 24 h later. AVAS12 mAb was injected as the class-matched control mAb. Consistent with the result that the same treatment blocked PP organogenesis, generation of VCAM-1+ and ICAM-1+ spots was inhibited completely by A7R34 injection, whereas AVAS12 injection had no effect on this process (Fig. 6). We next prepared mRNA from intestines from A7R34- or AVAS12-injected mice and analyzed expression of LT semiquantitatively by RT-PCR. As compared with the control embryos, A7R34 injection suppressed LTα and LTβ expression of the intestine.

To further confirm that the IL-7Rα signal is involved in LT production of IL-7Rα+ cells, we investigated in vitro whether or not the IL-7Rα signal is required for production of LT. IL-7Rα+ CD45+ and IL-7Rα CD45+ cells were sorted from 15.5–16.5 d.p.c. intestine, and cultured with OP9 feeder layer for 24 h in the presence of IL-7 and SCF. As shown in Fig. 7, IL-7Rα+ cells but not IL-7Rα cells expressed LTα and LTβ under these culture conditions. Consistent with our in vivo result, addition of A7R34 blocked the production of LT, although the recovery of blood cell numbers was not different between the two cultures (data not shown). These results indicate that IL-7Rα is required for the production of LT in IL-7Rα+ cells.

Discussion

A molecular and cellular scenario for PP anlage formation

Molecular genetics have identified a number of molecules that play an essential role in PP organogenesis (68,10,2131). However, as yet little is understood about how these molecules are involved in PP formation. One impediment to our understanding has been lack of knowledge about the histological events during PP organogenesis; clarification of the PP bauplan allows the consequence of disparate mutations to be defined.

Previously, we presented a scenario in which three distinct steps were histologically defined in the embryonic process of PP organogenesis (11). Additionally, we showed that PP development of the Il7ra–/– mouse is impaired from step I (12). This finding implied that IL-7Rα+ cells are crucial to the induction of the VCAM-1/ICAM-1-expressing organizing center for PP organogenesis. In the study presented here, we demonstrated that (i) the IL-7Rα-mediated events for PP organogenesis are restricted to a short time-window in step I; (ii) PP organogenesis of the Lta–/– mouse is defective from step I; (iii) only IL-7Rα+ cells are the producer of LTα and LTβ in the intestine; and (iv) LT expression in the intestine is IL-7Rα dependent.

Although we detected the expression of IL-7 mRNA in developing gut by the RT-PCR method (data not shown), we could not identify cellular localization of the IL-7 in the intestine, despite extensive attempts using immunohistochemistry or in situ hybridization. Interestingly, by careful inspection of the intestinal surface as well as histological examination, we found the Il7–/ mouse has normal numbers of PP in its intestine (Honda, unpublished observation), although previously the mutant was reported to have no PP (22). This discordance in PP organogenesis of Il7–/ and Il7ra–/– mice implicates the role for another ligand for IL-7Rα, thymic stromal-derived lymphopoietin (32), stimulating IL-7Rα+ cells in the developing intestine.

That A7R34 injection on 14.5–15.5 d.p.c. completely blocks expression of VCAM-1 and ICAM-1 in the PP anlage strongly suggests an order of events: ligand-induced stimulation of IL-7Rα+ cells, expression of LT, and induction of VCAM-1 and ICAM-1. Alternatively, IL-7 may support survival of IL-7Rα+ cells, which respond to other unidentified signals to induce expression of LT. However, we could not detect organized PP in the Il7ra–/– mice harboring the bcl-2 gene under the control of the H-2 promoter (Yoshida et al., unpublished observation), in which IL-7Rα+ cells may survive in the absence of IL-7 (33). Hence, persistence of IL-7Rα+ cells in the intestine is not sufficient to induce PP organogenesis. We demonstrated here that LTα expression in the intestine is suppressed by A7R34 injection and also that in vitro production of LT by IL-7Rα+ cells is dependent upon IL-7Rα signal. Taken together, it is likely that IL-7Rα stimulation by specific ligands is directly involved in the production of LT for PP development. Whether or not LT are sufficient effectors for the induction of PP anlage is not clear, but it is implied by a recent study showing that ectopic expression of LTα induces formation of lymphoid follicles (34).

Once the organizing center of PP is formed, other molecules such as chemokines may also be expressed in the PP anlagen, thereby attracting various blood cell lineages to this center in step II. In fact, many cell types including IL-7Rα+ cells, c-fms+ or F4/80+ macrophages and CD11c+ dendritic cells accumulate to this region at the same time, although the staining pattern for each antibody was different. Thus, although we initially defined step II as the formation of segregated clusters of IL-7Rα+ and CD4+ cells, this should be a part of the secondary events induced after the formation of the organizing center of PP.

These findings indicate a molecular and cellular basis not only for IL-7Rα-dependent but also LT-mediated processes of PP organogenesis. Based upon this, we propose a model for PP organogenesis in which IL-7Rα+ CD3 cells play a pivotal role in the induction of PP anlage (Fig. 8).

Transient requirement of IL-7Rα stimulation for PP development

IL-7Rα+ cells are widespread in the intestine of the 14–16 d.p.c. embryo, whereas the organizing center of PP expressing VCAM-1 and ICAM-1 are segregated (11). Thus, the position of the PP anlage should be determined either by segregated expression of IL-7Rα ligands or receptors for LT. In either case, the positional signals for PP should be placed prior to the stimulation of IL-7Rα+ cells, while its molecular or cellular nature is totally obscure at present.

Histological analysis of VCAM-1+ cells showed that they are either fibroblastic or dendritic in shape (11). These findings agree with previous findings that constitutive expression of LTα in a non-lymphoid organ induces VCAM-1 and ICAM-1 in mesenchymal cells (35). At present it is not clear whether VCAM-1/ICAM-1-expressing cells in developing PP are the only cells that respond to the LT signal and are different from other mesenchymal fibroblastic cells or not. However, if so, presumptive VCAM-1/ICAM-1-expressing cells themselves may regulate this positional signal, because they should be accumulated in the presumptive PP region before being stimulated by IL-7Rα+ cells. Hence, isolation and characterization of these VCAM-1/ICAM-1-expressing mesenchymal cells in 14.5–15.5 d.p.c. embryo intestine is very important, and may clarify the cellular and molecular nature of the positional signals for PP organization.

The present results indicate that IL-7Rα is required in a very short time-window of PP organogenesis from 14.5 to 17.5 d.p.c. In contrast, Rennert et al. (36) reported that a single shot of LTβR–Fc chimera as late as 18 d.p.c. completely blocked PP organogenesis. This discrepancy may indicate that LTαβ is required even after the induction of PP anlage, while IL-7Rα functions transiently during the placement of the anlage, and the molecule which directs the transient positional signal of PP may be IL-7Rα ligands but not LTβR. We found IL-7Rα+ cells are the only source for LT production even in spleen or mesentery. Spleen or LN development was disrupted in Lta–/– mice (6,7) but not in Il7ra–/– or A7R-treated mice (data not shown). These results imply involvement of multiple signals in the initiation phase of the organogenesis of peripheral lymphoid tissues, though LTαβ may be functioning in all processes as the effector molecule.

A unique lymphocyte subset in the abdominal cavity.

As IL-7Rα+ cells in the PP are present in nu/nu, scid/scid and Rag2–/ mice (11,12), it is not thymus dependent nor does it express antigen receptor genes. Indeed, IL-7Rα+ cells in the intestine are negative for various lymphoid markers and widespread in the intestine as early as 14 d.p.c. Although these surface phenotypes or distributions cast doubt on their lymphoid lineage, they appeared lymphocytic in morphology and are negative in expression of surface markers for other blood cell lineage markers.

Recent studies of Mebius et al. (37,38) identified in the developing LN a similar subset CD3CD4+ IL-7Rα+, and demonstrated their potential to differentiate to NK cells and dendritic cells in response to various cytokines. It is likely that IL-7Rα+ CD3 cells described in this study represent the same population of them. However, all IL-7Rα+ cells in the embryonic intestine were c-Kit+ and CD25 (IL-2Rα), whereas Mebius reported that CD4+CD3 IL-7Rα+ cells in the developing LN were c-Kit–/dull and CD25+ (75%). Thus, they may represent distinct subsets or alternatively they are derived from the same precursor but those colonized LN lose c-Kit and gain CD25 expression. Moreover, Kanamori et al. (39) described a new intestinal lymphoid tissue, cryptic patch, that consists of IL-7Rα+ CD3 cells that have a potential to give rise to γδT cells. We also found that some IL-7Rα+ in the intestine of 18 d.p.c. embryos express CD3, although their numbers were very low in the mesentery. This may suggest that IL-7Rα+ CD3 cells differentiate to T cells in the intestine, although T cell entry through a non-mesenteric route can not be ruled out. Nevertheless, the results of ours and others suggest that the presence of IL-7Rα+ CD3CD4+/– cells is a characteristic feature of the developing peripheral lymphoid tissues. It is intriguing that this subset is found in the intestine, mesentery and spleen of embryos. Moreover, it is found also in the stomach and colon, though absent in the liver. This suggests that these organs in the abdominal cavity can provide a micro environment that can support survival, proliferation and/or differentiation of this subset.

In conclusion, this line of evidence suggests that the CD3 IL-7Rα+ subset in the embryonic intestine, mesentery and spleen represents a novel lymphoid subset. Altough they may have potential to undergo further differentiation, we speculate the major function of this subset is to control organogenesis of the PP anlagen. This is the first comprehensive model for the molecular and cellular basis for secondary lymphoid tissue organogenesis; however, it can only be proven by an experiment in which PP of the Lta–/– or Il7ra–/– mouse are reconstructed by transferring the wild-type CD3 IL-7Rα+ population. As shown in this study, the cell transfer should be performed at an appropriate timing during the initial stage of PP formation. To address this question, methods to transfer the cells to embryos and deliver them to the site of organogenesis of PP need to be developed.

Table 1.

Effect of single shot injection of anti-IL-7Rα mAb on PP organogenesis

Day of injection No. of litters Total no. of offspring No. of PP/mouse 
Pregnant C57BL/6 mice were given either 2 mg A7R34 or AVAS12 i.v. at the indicated date. Offspring were sacrificed at 2 months of age and intestines were inspected by stereo microscopy to examine the number of PP. No PP were formed in the offspring from 14.5 to 15.5 d.p.c. A7R34-treated mice. All offspring from 16.5 d.p.c. A7R34-treated mice bear only one PP in the upper intestine. A considerable variation in the number of PP was observed in the offspring from 17.5 d.p.c. A7R34-treated mice. An example of this group is displayed in Fig. 2
A7R34    
14.5 22 
15.5 44 
16.5 16 
17.5 40 2–4 
18.5 15 8–11 
AVAS12    
15.5 30 8–12 
17.5 16 8–12 
No treatment  40 8–12 
Day of injection No. of litters Total no. of offspring No. of PP/mouse 
Pregnant C57BL/6 mice were given either 2 mg A7R34 or AVAS12 i.v. at the indicated date. Offspring were sacrificed at 2 months of age and intestines were inspected by stereo microscopy to examine the number of PP. No PP were formed in the offspring from 14.5 to 15.5 d.p.c. A7R34-treated mice. All offspring from 16.5 d.p.c. A7R34-treated mice bear only one PP in the upper intestine. A considerable variation in the number of PP was observed in the offspring from 17.5 d.p.c. A7R34-treated mice. An example of this group is displayed in Fig. 2
A7R34    
14.5 22 
15.5 44 
16.5 16 
17.5 40 2–4 
18.5 15 8–11 
AVAS12    
15.5 30 8–12 
17.5 16 8–12 
No treatment  40 8–12 
Table 2.

Surface markers expression of IL-7Rα+ CD3 cells at 16.5 d.p.c.

Markers Expression 
Marker expression of IL-7Rα+ CD3 cells surface obtained from 16.5 d.p.c. gut was examined by flow cytometry. To clarify the cell lineage, the following surface markers were used, CD44, integrin α4, CD25; immature leukocyte marker, B220, CD19; B cell markers, TCRαβ, TCRγδ, integrin αE; T cell markers, F4/80, c-fms, Flk2/Flt3, CD11c; macrophage and dendritic cell markers, ter119; erythroid marker, Flk-1, VCAM-1, PDGFRα; endothelial or mesenchymal cell markers, E-cadherin; epithelial cell marker. 
CD44 
CD45 
Integrin α4 
c-Kit 
CD25 – 
B220 – 
CD19 – 
TCRαβ – 
TCRγδ – 
Integrin αE – 
F4/80 – 
c-fms – 
Flk2/Flt3 – 
CD11c – 
ter119 – 
Flk1 – 
VCAM-1 – 
PDGFRα – 
E-cadherin – 
Markers Expression 
Marker expression of IL-7Rα+ CD3 cells surface obtained from 16.5 d.p.c. gut was examined by flow cytometry. To clarify the cell lineage, the following surface markers were used, CD44, integrin α4, CD25; immature leukocyte marker, B220, CD19; B cell markers, TCRαβ, TCRγδ, integrin αE; T cell markers, F4/80, c-fms, Flk2/Flt3, CD11c; macrophage and dendritic cell markers, ter119; erythroid marker, Flk-1, VCAM-1, PDGFRα; endothelial or mesenchymal cell markers, E-cadherin; epithelial cell marker. 
CD44 
CD45 
Integrin α4 
c-Kit 
CD25 – 
B220 – 
CD19 – 
TCRαβ – 
TCRγδ – 
Integrin αE – 
F4/80 – 
c-fms – 
Flk2/Flt3 – 
CD11c – 
ter119 – 
Flk1 – 
VCAM-1 – 
PDGFRα – 
E-cadherin – 
Fig. 1.

Histological events during PP organogenesis of various strains of mouse. Expression of VCAM-1, ICAM-1, IL-7Rα, and CD45 in intestines of wild-type (WT) (A, B, C and H), Il7ra–/– (D) and Lta–/– (E, F and G) mice. Strains of mouse, stages of embryos and antibodies used for staining are indicated in the figure. VCAM-1+ or IL-7Rα+ spots have never been detected in the intestine of Lta–/– embryos (E, F and G) or neonates (data not shown). Although cluster formation of IL-7Rα+ cells could not be detected, they are widespread over the entire intestine of the 17.5 d.p.c. Lta–/– mouse (G) like those of the 15 d.p.c. wild-type embryo (H).

Fig. 1.

Histological events during PP organogenesis of various strains of mouse. Expression of VCAM-1, ICAM-1, IL-7Rα, and CD45 in intestines of wild-type (WT) (A, B, C and H), Il7ra–/– (D) and Lta–/– (E, F and G) mice. Strains of mouse, stages of embryos and antibodies used for staining are indicated in the figure. VCAM-1+ or IL-7Rα+ spots have never been detected in the intestine of Lta–/– embryos (E, F and G) or neonates (data not shown). Although cluster formation of IL-7Rα+ cells could not be detected, they are widespread over the entire intestine of the 17.5 d.p.c. Lta–/– mouse (G) like those of the 15 d.p.c. wild-type embryo (H).

Fig. 2.

Discordance of the effect of anti-IL-7Rα mAb injection at 17.5 d.p.c. on the upper and lower region of intestine. Pregnant mice were given an i.v. injection of either AVAS12 or A7R34 at 17.5 d.p.c. Intestines of representative offspring are displayed in the pictures. Arrows indicate the position where organized PP were detected. In the control intestine, 12 PP plus one cecal nodule were detected (A). On the other hand, in this particular intestine from the A7R34-treated mouse, four PP and one cecal nodule were present (B). A cecal nodule is present also in the Il7ra–/– mouse, indicating that its organogenesis is independent from IL-7.

Fig. 2.

Discordance of the effect of anti-IL-7Rα mAb injection at 17.5 d.p.c. on the upper and lower region of intestine. Pregnant mice were given an i.v. injection of either AVAS12 or A7R34 at 17.5 d.p.c. Intestines of representative offspring are displayed in the pictures. Arrows indicate the position where organized PP were detected. In the control intestine, 12 PP plus one cecal nodule were detected (A). On the other hand, in this particular intestine from the A7R34-treated mouse, four PP and one cecal nodule were present (B). A cecal nodule is present also in the Il7ra–/– mouse, indicating that its organogenesis is independent from IL-7.

Fig. 3.

Surface phenotypes of intestinal IL-7Rα+ cells in 16.5 d.p.c. embryos. (A) Single-cell suspension was prepared from intestines of 16.5 d.p.c. C57BL/6 embryos by collagenase treatment. Cells were three-color stained by phycoerythrin–anti-CD4, allophycocyanin–A7R34 and FITC-labeled mAb against various surface molecules indicated. Expression of CD8, CD3, δ-chain, Mac1, NK1.1 and Thy1.2 of IL-7Rα+ CD4+ and IL-7Rα+ CD4 cells were presented. The negative threshold was determined by using FITC-labeled anti-rat IgG. (B) Whole-mount immunostaining patterns of CD11c-, F4/80- and c-fms-expressing cells in developing intestine. Wild-type mouse embryonic intestine was whole-mount immunostained by anti-CD11c, anti-F4/80 and anti-c-fms antibody. Note the distribution patterns of immunoreactive cells are different from each other. (C) Morphology of sorted IL-7Rα+ CD4 and IL-7Rα+ CD4+ cells stained with May–Grünwald–Giemsa solution.

Fig. 3.

Surface phenotypes of intestinal IL-7Rα+ cells in 16.5 d.p.c. embryos. (A) Single-cell suspension was prepared from intestines of 16.5 d.p.c. C57BL/6 embryos by collagenase treatment. Cells were three-color stained by phycoerythrin–anti-CD4, allophycocyanin–A7R34 and FITC-labeled mAb against various surface molecules indicated. Expression of CD8, CD3, δ-chain, Mac1, NK1.1 and Thy1.2 of IL-7Rα+ CD4+ and IL-7Rα+ CD4 cells were presented. The negative threshold was determined by using FITC-labeled anti-rat IgG. (B) Whole-mount immunostaining patterns of CD11c-, F4/80- and c-fms-expressing cells in developing intestine. Wild-type mouse embryonic intestine was whole-mount immunostained by anti-CD11c, anti-F4/80 and anti-c-fms antibody. Note the distribution patterns of immunoreactive cells are different from each other. (C) Morphology of sorted IL-7Rα+ CD4 and IL-7Rα+ CD4+ cells stained with May–Grünwald–Giemsa solution.

Fig. 4.

IL-7Rα+ CD3 cells in the abdominal cavity of embryos. Single-cell suspensions of liver, thymus, intestine, mesentery and spleen of 14.5, 16.5 and 18.5 d.p.c. embryos were prepared, and three-color stained by FITC–anti-CD3, phycoerythrin–anti-CD4 and allophycocyanin–anti-IL-7Rα. Presence of both IL-7Rα+ CD4+ CD3 and IL-7Rα+ CD4CD3 cells is a common feature of intestine, mesentery and spleen of 14.5 d.p.c. embryo. The presence of the CD3+ population in these tissues became detectable from 18.5 d.p.c.

Fig. 4.

IL-7Rα+ CD3 cells in the abdominal cavity of embryos. Single-cell suspensions of liver, thymus, intestine, mesentery and spleen of 14.5, 16.5 and 18.5 d.p.c. embryos were prepared, and three-color stained by FITC–anti-CD3, phycoerythrin–anti-CD4 and allophycocyanin–anti-IL-7Rα. Presence of both IL-7Rα+ CD4+ CD3 and IL-7Rα+ CD4CD3 cells is a common feature of intestine, mesentery and spleen of 14.5 d.p.c. embryo. The presence of the CD3+ population in these tissues became detectable from 18.5 d.p.c.

Fig. 5.

Expression of LTα and LTβ in intestinal IL-7Rα+ cells. Single-cell suspensions prepared from either 15.5 or 16.5 d.p.c. embryos were three-color stained by FITC–anti-CD45, phycoerythrin–anti-CD4 and allophycocyanin–anti-IL-7Rα, and IL-7Rα+ CD4+, IL-7Rα+ CD4 and IL-7Rα CD45+ fractions were sorted. (A) Total RNA was prepared from each fraction collected from intestine, and expression of LTα, LT and hprt was analyzed qualitatively by RT-PCR. Spleen cells from normal mice were used as a control. Lanes from the left are: 1, size markers; 2, CD4+ IL-7Rα+ CD45+ (15.5 d.p.c.); 3, CD4 IL-7Rα+ CD45+ (15.5 d.p.c.); 4, IL-7Rα CD45+ (15.5 d.p.c.); 5, CD4+ IL-7Rα+ CD45+ (16.5 d.p.c.); 6, CD4 IL-7Rα+ CD45+ (16.5 d.p.c.); 7, IL-7Rα CD45+ (16.5 d.p.c.); 8, adult spleen cells. (B) Total RNA was prepared from each fraction collected from spleen, mesentery or liver. Lanes from the left are: 1, CD4+ IL-7Rα+ CD45+ (spleen); 2, CD4 IL-7Rα+ CD45+ (spleen); 3, IL-7Rα CD45+ (spleen); 4, CD4+ IL-7Rα+ CD45+ (mesentery); 5, CD4 IL-7Rα+ CD45+ (mesentery); 6, IL-7Rα CD45+ (mesentery); 7, IL-7Rα+ CD45+ (liver); 8, IL-7Rα CD45+ (liver). Arrows indicated the size of target bands.

Fig. 5.

Expression of LTα and LTβ in intestinal IL-7Rα+ cells. Single-cell suspensions prepared from either 15.5 or 16.5 d.p.c. embryos were three-color stained by FITC–anti-CD45, phycoerythrin–anti-CD4 and allophycocyanin–anti-IL-7Rα, and IL-7Rα+ CD4+, IL-7Rα+ CD4 and IL-7Rα CD45+ fractions were sorted. (A) Total RNA was prepared from each fraction collected from intestine, and expression of LTα, LT and hprt was analyzed qualitatively by RT-PCR. Spleen cells from normal mice were used as a control. Lanes from the left are: 1, size markers; 2, CD4+ IL-7Rα+ CD45+ (15.5 d.p.c.); 3, CD4 IL-7Rα+ CD45+ (15.5 d.p.c.); 4, IL-7Rα CD45+ (15.5 d.p.c.); 5, CD4+ IL-7Rα+ CD45+ (16.5 d.p.c.); 6, CD4 IL-7Rα+ CD45+ (16.5 d.p.c.); 7, IL-7Rα CD45+ (16.5 d.p.c.); 8, adult spleen cells. (B) Total RNA was prepared from each fraction collected from spleen, mesentery or liver. Lanes from the left are: 1, CD4+ IL-7Rα+ CD45+ (spleen); 2, CD4 IL-7Rα+ CD45+ (spleen); 3, IL-7Rα CD45+ (spleen); 4, CD4+ IL-7Rα+ CD45+ (mesentery); 5, CD4 IL-7Rα+ CD45+ (mesentery); 6, IL-7Rα CD45+ (mesentery); 7, IL-7Rα+ CD45+ (liver); 8, IL-7Rα CD45+ (liver). Arrows indicated the size of target bands.

Fig. 6.

Immediate effect of anti-IL-7Rα mAb injection on induction of the organizing center of PP and LT expression in the intestine. Pregnant mice were given an i.v. injection of either AVAS12 or A7R34 on 15.5 d.p.c. and sacrificed 24 h later. Intestines were dissected and whole-mount stained by biotinylated anti-VCAM-1 or -ICAM-1, followed by horseradish peroxidase–avidin. Appearance of segregated spots expressing (A) ICAM-1 and (C) VCAM-1 in the intestine of an AVAS12-treated mouse was absent (B and D) in the intestine of an A7R34-treated mouse. (E) Expression of LTα and LTβ of intestines treated either with A7R34 or control AVAS12 mAb was semiquantitatively analyzed by RT-PCR. Lanes from the left are: 1, normal spleen; 2, AVAS12-treated intestine; 3, A7R34-treated intestine.

Fig. 6.

Immediate effect of anti-IL-7Rα mAb injection on induction of the organizing center of PP and LT expression in the intestine. Pregnant mice were given an i.v. injection of either AVAS12 or A7R34 on 15.5 d.p.c. and sacrificed 24 h later. Intestines were dissected and whole-mount stained by biotinylated anti-VCAM-1 or -ICAM-1, followed by horseradish peroxidase–avidin. Appearance of segregated spots expressing (A) ICAM-1 and (C) VCAM-1 in the intestine of an AVAS12-treated mouse was absent (B and D) in the intestine of an A7R34-treated mouse. (E) Expression of LTα and LTβ of intestines treated either with A7R34 or control AVAS12 mAb was semiquantitatively analyzed by RT-PCR. Lanes from the left are: 1, normal spleen; 2, AVAS12-treated intestine; 3, A7R34-treated intestine.

Fig. 7.

Induction of LT expression by sorted IL-7Rα+ cells with IL-7. Expression of LTα and LTβ in FACS-sorted IL-7Rα+ cells cultured on stromal cell OP-9 (19) with IL-7 and SCF for 24 h with A7R34 (an antagonistic anti-IL-7Rα) or AVAS12 (a class-matched control antibody against Flk1). Lanes from the left are the products from: 1, normal thymus; 2, IL-7Rα+ cells with AVAS12; 3, IL-7Rα+ cells with A7R34; 4, IL-7Rα CD45+cells with AVAS12; 5, IL-7Rα CD45+ cells with A7R34; 6, the feeder layer alone with AVAS12.

Fig. 7.

Induction of LT expression by sorted IL-7Rα+ cells with IL-7. Expression of LTα and LTβ in FACS-sorted IL-7Rα+ cells cultured on stromal cell OP-9 (19) with IL-7 and SCF for 24 h with A7R34 (an antagonistic anti-IL-7Rα) or AVAS12 (a class-matched control antibody against Flk1). Lanes from the left are the products from: 1, normal thymus; 2, IL-7Rα+ cells with AVAS12; 3, IL-7Rα+ cells with A7R34; 4, IL-7Rα CD45+cells with AVAS12; 5, IL-7Rα CD45+ cells with A7R34; 6, the feeder layer alone with AVAS12.

Fig. 8.

A model for PP organogenesis. In this model, the significance of three steps defined histologically previously (11,12) was considered as steps for (i) induction of the organizing center for PP organogenesis, (ii) architecture formation of PP and (iii) homing of mature lymphocytes. As shown in the text, step I is essential for triggering subsequent steps. We presented evidence for involvement of two signal pathways in the induction of PP anlage. One is the IL-7Rα ligand/IL-7Rα/Jak3 pathway and the other is LTα. We also confirmed that no organized PP are formed in the γc gene null mutation (Yoshida et al., unpublished observation). As IL-7Rα+ CD3 cells are the producers of LTα and LTβ, IL-7Rα+ cells in the intestine are the direct inducers of the organizing center of PP. Indirect evidence supports a sequence of events: (i) IL-7Rα ligand stimulation of IL-7Rα+ CD3 inducer of the organizing center, (ii) production of LT by the inducer, (iii) LT-mediated activation of the organizing center of PP, and (iv) expression of VCAM-1 and ICAM-1 in the organizing center. A recent study by Alimzhanov et al. (8) indicated the involvement of both LTβ and LTβR in the final step. In this model, an IL-7Rα ligand-producing cell is the initiator of this cascade, but has not yet been identified.

Fig. 8.

A model for PP organogenesis. In this model, the significance of three steps defined histologically previously (11,12) was considered as steps for (i) induction of the organizing center for PP organogenesis, (ii) architecture formation of PP and (iii) homing of mature lymphocytes. As shown in the text, step I is essential for triggering subsequent steps. We presented evidence for involvement of two signal pathways in the induction of PP anlage. One is the IL-7Rα ligand/IL-7Rα/Jak3 pathway and the other is LTα. We also confirmed that no organized PP are formed in the γc gene null mutation (Yoshida et al., unpublished observation). As IL-7Rα+ CD3 cells are the producers of LTα and LTβ, IL-7Rα+ cells in the intestine are the direct inducers of the organizing center of PP. Indirect evidence supports a sequence of events: (i) IL-7Rα ligand stimulation of IL-7Rα+ CD3 inducer of the organizing center, (ii) production of LT by the inducer, (iii) LT-mediated activation of the organizing center of PP, and (iv) expression of VCAM-1 and ICAM-1 in the organizing center. A recent study by Alimzhanov et al. (8) indicated the involvement of both LTβ and LTβR in the final step. In this model, an IL-7Rα ligand-producing cell is the initiator of this cascade, but has not yet been identified.

Transmitting editor: D. Kitamura

We are grateful to Dr Itohara for providing anti TCRδ mAb, Dr Akashi for providing il7ra–/–×H2-Bcl-2 transgenic mice, and Dr Yokota for providing PCR primers for LTα and LTβ. We also thank to Drs R. Mebius, S. Fraser and D. Opstelten for reading our manuscript and many valuable comments. This work was supported by grants from Japanese Ministry of Education, Science and Culture (nos 07CE2005, 07457085 and 06277102), and a grant for Pediatric Research (9C-5) from the Ministry of Health and Welfare of Japan.

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