Surface expression of differentiation antigens on lymphocytes in the ileal and jejunal Peyer's patches of lambs

The surface phenotype of lymphocytes in the ileal (IPP) and jejunal (JPP) Peyer's patches (PP) of lambs was compared using flow cytometry and immunohistology with a panel of monoclonal antibodies (mAb). The B-cell markers p220, BAS9A and surface Ig molecules were detected on 70-95% of cells from the IPP. T-cell markers were detected on less than 1% of IPP lymphocytes, confirming that the IPP in lambs contains virtually only B lymphocytes. The JPP contained a lower proportion of B cells and 16% T cells, nearly all of which expressed the CD4 molecule. Interestingly, the reactivity of a fourth B-cell markers, BAQ44a, differed from this pattern; only 12% of IPP lymphocytes were positive whereas 70% of JPP lymphocytes expressed this marker. A majority of both IPP and JPP lymphocytes (80-95%) expressed the cell adhesion molecules CD11a (LFA-1) and LFA-3. Other adhesion molecules, such as CD2 and CD44, were expressed by fewer cells from the IPP than from the JPP. MHC class I antigens were detected on more than 95% of lymphocytes from both the IPP and JPP. In the case of MHC class II antigens, more positive cells occurred in the IPP (greater than 95%) than in the JPP (80%). The in situ localization of cell-surface antigens was assessed by immunohistology. CD4+ T cells occurred in the interfollicular T-cell regions and in JPP follicles, whereas CD8+ T cells localized only in the interfollicular regions and were absent from follicles. The pattern of expression of B-cell markers, adhesion molecules and MHC antigens indicated that a gradient of increasing maturity of B cells existed within follicles from the base towards the dome region. The data presented here lend support to the notion that the IPP in lambs represents a novel B-cell lymphoid tissue with a function different from that of the conventional Peyer's patches found in the jejunum.     

Fetal lambs are depleted of IgM+ cells following a single injection of an anti-IgM antibody early in gestation.

B-cell depleted fetal sheep were created following a single injection of an anti-IgM monoclonal antibody early in gestation. Six sheep fetuses were given a single intraperitoneal injection of a monoclonal antibody directed against IgM at 63 days of gestation (gestation in sheep = 150 days). The fetuses were killed at 138-142 days of gestation and lymphoid tissues were collected for subsequent light microscopy and immunohistochemical examination. The ileal and jejunal Peyer's patch (PP) follicles in four of the six injected fetuses were markedly reduced in size. Cells in the rudimentary follicles of the ileal PP of these animals showed no reactivity for IgM and most were negative for CD45. The dome regions contained many T cells, which were predominantly CD8+ cells and included gamma delta T cells. The interfollicular areas of the PP of the markedly affected fetuses contained large populations of T cells. The spleen and lymph nodes were also markedly depleted of IgM+ cells and these tissues contained only a small, scattered population of weakly IgM+ cells. Follicular accumulations of IgM+ cells were absent. Large populations of T cells were present in the white pulp of the spleen and cortex of the lymph nodes. The liver did not contain IgM+ cells and the medulla of the thymus was depleted of IgM+ cells. The results of this study suggest that a surface IgM+ B-cell population is present in the sheep fetus at 63 days of gestation, which is essential for the colonization of the ileal PP and subsequent B-cell development.     

Histological studies on the ontogeny of bovine gut-associated lymphoid tissue: appearance of T cells and development of IgG+ and IgA+ cells in lymphoid follicles

The development and distribution of lymphocyte subsets in bovine gut-associated lymphoid tissues (ileal and jejunal Peyer's patches (PP)) were examined. Before birth, the composition of lymphocyte subsets in both PP follicles did not differ except for the dimensions of the interfollicular area and the dome region. Many IgM+ cells were observed in these follicles, but very few CD3+, IgG+, and IgA+ cells could be found. At neonatal period, the IgG+ cells, which did not produce IgG mRNA, were dominant within both PP follicles. From 1 month after birth, many CD3+ cells, IgG mRNA expression, and IgA mRNA expression were detected within the jejunal PP follicles, but very few were in the ileal PP follicles. These data suggest that the characteristics of the jejunal PP follicles metamorphose into secondary lymphoid tissue such as germinal centers at around 1 month after birth, whereas the characteristics of ileal PP follicles were distinct from those of germinal centers.     

An analysis of the growth and differentiation of B cells isolated from follicles of the ileal Peyer's patch of sheep.

We developed a method to isolate and culture cells from the lymphoid follicles of the ileal Peyer's (PP) patch of young sheep (6-12 weeks). These cells were 98% sIgM+ B cells and 1% T cells. Cultured follicular cells were used to investigate B-cell proliferation and differentiation. Less than 50% of B cells were viable after 24 hr of culture and this decrease in B-cell viability also occurred following co-stimulation with pokeweed mitogen (PWM) and recombinant bovine interleukin-1 (rBoIL-1) or rBoIL-2. In contrast, co-stimulation with PWM and either rBoIL-1 or rBoIL-2 induced a marked proliferative response that was maximal on Day 4 of culture. Cytokine-induced proliferation of the B cells required PWM co-stimulation and proliferation induced by rBoIL-1 and rBoIL-2 was neither additive or synergistic. This suggests that PWM bound a molecule or molecules that signalled responsiveness to both rBoIL-1 and rBoIL-2. Culture of follicular cells with PWM and both rBoIL-1 and rBoIL-2 also resulted in B-cell differentiation. This differentiation was associated with decreased proliferation, an increased number of viable B cells, and increased expression of both surface IgM and non-Ig membrane molecules. Thus, co-stimulation of ileal PP follicular cells with PWM and rBoIL-1 and rBoIL-2 resulted in both B-cell proliferation and differentiation.     

A comparative study of gut-associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G-positive (IgG(+)) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG-producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4(+) cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4(+) T cells would be prerequisite for Ig class switching in these follicles, IgG(+) cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B-cell repertoire might be selected by gut-derived antigens in the ileal PP and the BF before seeding the periphery.     

Negative signaling by surface IgM on B cells isolated from ileal Peyer's patch follicles of sheep

Lymphoid follicles from the sheep ileal Peyer's patch (PP) were used to prepare a cell suspension consisting of 98% surface IgM-positive (sIgM+) B cells and 1% T cells. Co-stimulation of follicular cells with pokeweed mitogen and either recombinant bovine interleukin 1 (IL 1) or IL 2 resulted in a marked proliferative response. In contrast, the addition of soluble F(ab')2 rabbit anti-sheep Ig completely inhibited the proliferative response induced by pokeweed mitogen and IL 1 or IL 2 co-stimulation. Anti-Ig inhibition of B cell proliferation was specific for ileal PP follicular cells and was not observed with mesenteric lymph node cells or splenocytes. Furthermore, suppression of ileal PP follicular B cell proliferation required at most divalent cross-linking of sIg was independent of Fc receptors, but was dependent on the concentration of anti-Ig and required 48 h for maximal effect. Negative signaling by sIgM indicates that ileal PP follicular B cells are functionally distinct from B cells in other secondary lymphoid tissues. Also, the present observations are consistent with previous reports indicating that B cell proliferation in ileal PP follicles is antigen independent.     

Lymphoid follicles of different phenotype appear in ileum during involution of the sheep ileal Peyer's patch

The ileal Peyer's patch (IPP) of young sheep is a site of diversification of the primary antibody repertoire and where involution takes place at young age. Tissue samples from the ileum were collected in 134 animals aged from 1 month to 6 years, and IPP follicle phenotypes were characterised. We describe a new type of ileal lymphoid follicles that became relatively more frequent during involution, and had numerous intrafollicular T-cells and BAQ44A+ B-cells and large interfollicular T-cell areas. As opposed to classical IPP follicles in which the BAQ44A+ cells were confined to the narrow follicle-neck region, the novel atypical ileal lymphoid follicle had these cells distributed throughout the follicle. The relative distribution of cell types in the typical IPP follicle remained fairly constant during involution. Many animals older than 9 months (64/92) still had had typical IPP follicles and even sheep 4 years and older (5/9) had IPP-type follicles.     

A comparative study of gut‐associated lymphoid tissue in calf and chicken

The calf contains two types of Peyer's patches (PPs): jejunal and ileal. The ileal PP has been thought to be equivalent to the bursa of Fabricius (BF) as a central lymphoid organ. The morphologies of ileal and jejunal PPs in the calf were compared with those of the BF and the caecal tonsil (CT) in the chicken. Immunoglobulin G–positive (IgG⁺) cells appear in the follicles of them all and exhibited a dendritic appearance after birth. We investigated whether the IgG in these follicles was produced in situ. IgG‐producing cells were detected in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. CD4⁺ cells were distributed in the follicular medullas of the jejunal PP and the CT, but not in those of the ileal PP and the BF. The data suggest that Ig class switching occurs in both jejunal PP follicles and CT follicles, but does not occur in either the ileal PP follicles or the bursal follicles. Because CD4⁺ T cells would be prerequisite for Ig class switching in these follicles, IgG⁺ cells of the follicular medullas in the ileal PP and the BF would trap immune complexes from the gut lumen. The primary B‐cell repertoire might be selected by gut‐derived antigens in the ileal PP and the BF before seeding the periphery. Anat Rec 266:207–217, 2002. © 2002 Wiley‐Liss, Inc.     

Evidence for a stromal cell-dependent,self-renewing B cell population in lymphoid follicles of the ileal Peyer's patch of sheep

Lymphoid follicles of the ileal Peyer's patch (PP) of young sheep function as the major source of B cells and a site of immunoglobulin (Ig) receptor diversification. However, extensive cell death in culture has restricted investigations of ileal PP follicular (iPf)B cell biology. We investigated the possibility that sustained iPfB cell proliferation may require an interaction with mesenchymal stromal cells (SC). Four SC lines, cloned from lymphoid follicles of the ileal PP, and various sheep and xenogeneic mesenchymal cells were used to characterize the nature of iPfB cell-SC interactions. A sustained proliferative response was unique to iPfB cells, required iPfB cell-SC contact, and SC membranes functioned as intact SC to either enhance or inhibit iPfB cell proliferative responses. The iPfB cell proliferation in SC co-cultures was accompanied by extensive cell death and a slow decline in viable cell number. Flow cytometric analysis confirmed that viable lymphocytes, present in SC co-cultures, were immature B cells that expressed surface IgM, with either λ or χ Ig light chain, and that SC co-culture inhibited iPfB cell differentiation. Finally, addition of soluble anti-sheep Ig to iPfB cell-SC co-cultures did not inhibit SC-dependent iPfB cell proliferation or iPfB cell binding to SC. These data indicate that an interaction between specific SC membrane molecules and non-Ig molecules of iPfB cells either supported or inhibited a self-renewing proliferative response by immature (sIgM^Lo, BAQ44A^?) iPfB cells. Finally, SC-dependent iPfB cell proliferation was independent of T cells and extrinsic antigen which further suggests that a functionally distinct B cell population resides in lymphoid follicles of the ileal PP.     

Effect of early fetal splenectomy on prenatal B-cell development in sheep

The contribution of early splenic B-cell populations to the colonization of the ileal Peyer's patch was investigated following the surgical removal of the spleen in a series of 56-day-old fetal sheep. The fetuses were killed at 140 days of gestation and the ileal Peyer's patch, the distal jejunal lymph node which drains the Peyer's patch, and a peripheral lymph node, the superficial cervical lymph node, were examined. Enzyme and immunohistochemical evaluation concluded that the distribution of B cells, T cells and stromal cells in the ileal Peyer's patch was similar in splenectomized and normal fetal sheep. Thus, the presence of the fetal spleen was not essential for the colonization of the ileal Peyer's patch and other early sites of B-cell accumulation would appear capable of generating the necessary precursor populations. Investigation of B-cell populations in lymph nodes used a combination of terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate nick-end-labelling (TUNEL) histochemistry and immunofluorescence to determine the average number of apoptotic B cells in the primary follicles of the outer cortex of splenectomized and normal lambs. A significantly increased number of apoptotic B cells was present in the distal jejunal lymph node but not in the superficial cervical lymph node of splenectomized lambs. This finding suggests that splenectomy affected prenatal B-cell development in fetal sheep and raises questions as to the regulation of B-cell lymphopoiesis in a species using a post-rearrangement organ of diversification.     

CD40 signalling in ileal Peyer's patch B cells: implications for T cell-dependent antigen selection

The ileal Peyer's patch (PP) plays a central role in B celldevelopment in young sheep and it is hypothesized that thisB cell development occurs independent of extrinsic antigen andT cells. Therefore, it was of interest to examine ileal PP folllcular(iPf) B cell responses to CD40 ligand, a molecule integral toT cell-dependent B cell development. A variable level of CD40expression was detected on a subpopulation of iPfB cells andJ558L cells, expressing a membrane form of mouse CD40 ligand(mCD40L), interacted specifically with the CD40 molecule oniPfB cells. In response to mCD40L the non-S phase iPfB cellswere rescued from apoptotic cell death and there was a markedproliferative response but viable cell number remained relativelyconstant. The mCD40L also induced decreased cytoplasmic cAMPlevels, blocked anti-Ig-induced iPfB cell death and inducedfunctional IL-2 receptor expression on a subpopulation of iPfBcells. Many of the mCD40L-induced responses of iPfB cells weresimilar to those reported for germinal centre and immature Bcells, and indicated that a cognate T cell-B cell interactioncould influence iPfB cell proliferation and differentiation.Finally, that mCD40L induced iPfB cell activation and differentiationwas evident as increased expression of CD5, the BAQ44A molecule,the CACT65A molecule and the expansion of surface IgG1⁺ B cells.These mCD40L-induced phenotypic changes were also observed onsubpopulations of freshly isolated iPfB cells and jejunal PPfollicular B cells. However, few iPfB cells had a phenotypesimilar to that observed in co-culture with mCD40L and thissuggested that T cell-dependent B cell development may playa minor role in ileal PP B cell development. The possible significanceof CD40 signalling is discussed in terms of the selection ofiPfB cells during development.     

Ontogeny of leukocyte populations in the ileal Peyer's patch of sheep.

Enzyme- and immunohistochemical methods were used to characterize the leukocyte populations present in the ileal Peyer's patches of sheep foetuses between 68 and 135 d of gestation and particularly in the period around 100 d of gestation, when active lymphopoiesis begins. A wide variety of leukocytes including IgM+, CD5+, CD4+, CD8+ cells, and MgATPase+ dendritic cells were present at an early stage. Groups of IgM+ cells were seen immediately beneath the epithelium as early as 70 d of gestation. Conventional morphometric and computer-assisted morphometric techniques were used to confirm the significant expansion of these cell populations from 90 d of gestation. IgM+ and CD5+ cells were responsible for the vast majority of the increase in cell numbers. It was concluded that a diverse leukocyte population was present at the initiation of active lymphopoiesis in the ileal PP of the sheep foetus and that all members of this population were associated with the emergence of the dome/follicle primordia from which the B-cell follicle develops.     

Differential cytokine mRNA expression in single lymphatic follicles of the calf ileal and jejunal Peyer's patches

The ruminant gut-associated lymphoid tissues are broadly classified into ileal and jejunal Peyer's patches (PP). We isolated single lymphatic follicles from ileal and jejunal PP and examined mRNA expression of 13 cytokines using RT-PCR. Four patterns of differential expression were identified. In Pattern 1, the cytokines IL-7, IL-10, IL-12, and IL-18 were detected in all follicles of both ileal and jejunal PP. In Pattern 2, the cytokines IL-2, IL-4, and IL-13 were expressed in most jejunal PP follicles, but were undetectable in the ileal PP follicles. The cytokines characterizing Pattern 3 (IL-1beta, IFN-gamma, and IL-6) were detected in all follicles of the jejunal PP, but were differentially expressed in each follicle of ileal PP. In Pattern 4, the cytokines IL-8, TNF-alpha, and GM-CSF were variably expressed in follicles of both ileal and jejunal PP. More detailed knowledge about differential expression of cytokines in ileal and jejunal PP will facilitate a better understanding of the immune responses of primary and secondary lymphoid organs in the bovine small intestine.     

Ileal Peyer's patch emigrants are predominantly B cells and travel to all lymphoid tissues in sheep

The ileal Peyer's patch (PP) was selectively labeled with fluorescein isothiocyanate by extracorporeal perfusion in 7-12 week-old lambs and the lymphocyte lineage and fate of the emigrants was determined by fluorescence microscopy and flow cytometry. PP emigrants were found in all tissues examined, accounting for 10%-15% of ileal mesenteric lymph node (MLN). 1%-2% of jejunal MLN, jejunal PP, prescapular lymph node (PLN) and 3%-4% of spleen cells. All ileal PP emigrants enter the ileal MLN on their way to the circulation. Removal of the MLN prior to perfusion enabled emigrants to go directly to the circulation and extravasate in distant tissues faster than in intact animals. The ileal MLN might provide an additional level of regulation for ileal PP emigrants. The perfused ileal PP contained about 25 times more B cells than T cells. The emigrant cells found in different tissues included both T and B cells but came to reflect, although to a lesser degree, the B cell composition of the tissue from which they were derived. One day after perfusion the composition of PP emigrants was similar to that of the tissue within which they were found; the spleen was the exception with a bias towards B cells. By day 3 the ratio of B to T cells in the PP emigrants was 1 for jejunal MLN and PLN. 1.5 for ileal MLN and jejunal PP, and 4-5 for the spleen and blood. It was concluded that the PP-derived T cells were recirculating T cells that were in the ileal PP at the time of perfusion. These cells emigrated rapidly and equilibrated such that they accounted for about 1.5% of the T cell pool in various tissues. Most PP-derived B cells were probably produced in the PP. The greatest contribution (24.4%) that ileal PP emigrants made to the B cell pool of a tissue was with the ileal MLN through which they are obliged to pass. The contribution was lower but still very significant in blood (8.9%), spleen (6.8%), PLN (3.9%), jejunal MLN (3.5%) and jejunal PP (1.8%). There was no evidence that ileal PP emigrants made a greater relative contribution to either T or B cell populations in MLN or jejunal PP than to non-gut-associated sites. The B cells were distributed throughout the immune system, which is in accordance with the proposal that the ileal PP is a site of primary B cell genesis in sheep.     

Oligoclonal Peyer's patch follicles in the terminal small intestine of cattle

In small ruminants, the development of B cells differs from that in mice or in man. The anti-body repertoire is expanded in the Peyer's patches of the terminal ileum where each B-cell follicle is found by a few cells. To investigate the amount of founder clones in bovine ileal follicles, we have used sex mismatched cattle twins. These animals are chimeric due to placental anastomoses. Y-chromosome targeted in situ hybridization was used to trace donor-derived cells of the male genotype in a female recipient (called a freemartin). A strong clustering of lymphoid cells originating from either twin was seen in the ileal Peyer's patches (IPPs). Furthermore, the follicles displayed a low amount of immunoglobulin heavy chain gene configurations in comparison with the splenic or jejunal follicles. These findings strongly suggest that as in sheep, the B-cell follicles in cattle IPPs develop oligoclonally.     

Jejunal and ileal Peyer’s patches in pigs differ in their postnatal development

The postnatal development of the jejunal and ileal Peyer’s patches was studied before and after weaning in 1-, 1.5- and 2-month-old pigs. The follicles of the jejunal Peyer’s patches grew with age and were two times longer and wider in specified pathogen-free and conventional pigs than in germ-free animals, thus indicating an influence of the living microbial antigens from the gut lumen. In germ-free pigs the size of the ileal Peyer’s patch follicles increased between the 1st and 2nd month, whereas in the specified pathogen-free and conventional animals these follicles were comparable in size in all three age groups. In 1- to 1.5-month-old pigs the interfollicular area of jejunal Peyer’s patches was wider (0.1 ± 0.04 mm) than that of the ileal Peyer’s patch (0.04 ± 0.03 mm). Immunohistological studies showed that in germ-free pigs preferentially surface IgM⁺ but few IgA⁺ B cells were present in the follicles, domes and dome epithelia. In specified pathogen-free and conventional pigs the B cells expressed different levels of surface or cytoplasmic IgM or IgA. In all groups studied, more T cells were observed in the jejunal than in the ileal Peyer’s patch. Here, few T lymphocytes were found because of the small interfollicular areas. Small numbers of Null cells were distributed in the interfollicular regions of all animals. The results show that living microbial antigens have a major influence on the jejunal and ileal Peyer’s patches in pigs. The morphological differences between the two types of Peyer’s patches are an indication that they develop differently during postnatal life. So far it remains unclear whether these morphological differences reflect a specific function of the pig’s ileal Peyer’s patch, such as the expansion of the genetically determined B cell repertoire as has been reported for sheep.     

Ontogeny of leucocyte populations in the spleen of fetal lambs with emphasis on the early prominence of B cells.

The presence and distribution of B cells and other early leucocyte populations are described in the spleen of fetal lambs from 40 to 134 days of gestation (length of gestation 150 days). Computer-assisted morphometric analysis and flow cytometry were used to quantify the early predominance of B cells in mid-gestation. B cells appeared at about 48 days and increased in number to occupy over 20% of the spleen area at 77 days. All spleens were collected on their respective livers and at no stage did the livers contain more than a few IgM-positive (+) cells, which were usually close to blood vessels. Two-colour flow cytometry demonstrated that only 1-2% of IgM+ cells expressed CD5 at 81 days. Beyond 77 days, with the expanding presence of T cells, the percentage of area occupied by IgM+ cells declined to stabilize at about 7% during late gestation. The conventional organization of the splenic white pulp was observed from 90 days along with 5' nucleotidase-positive primary follicles. Double staining technique using immunohistochemical methods demonstrated that IgM+ cells were proliferating in the spleen from as early as 51 days and that clusters of proliferating IgM+ cells were prominent between 60 and 77 days. The results of the present study suggest that during the ontogeny of fetal lambs the spleen is a site of B-cell development or expansion before colonization of the ileal Peyer's patch and the subsequent generation of the preimmune antibody repertoire.     

Systematic characterization of porcine ileal Peyer's patch, I. apoptosis-sensitive immature B cells are the predominant cell type

It is now apparent that the Peyer's patches of some species exhibit structural, functional and developmental heterogeneity. In sheep, for example, the ileal Peyer's patch (IPP) is the primary, antigen-independent site for the generation of the primary immunoglobulin repertoire and consequent production of the systemic B-cell pool. The pig has three distinct Peyer's patches, including an IPP, but the functional status of this organ, as primary or secondary lymphoid tissue, is not clear. Here, we have systematically characterized pig IPP follicular lymphocytes and show that about 90% B cells that are positive for surface immunoglobulin G (sIgM+) and express an immature phenotype characterized by expression of myeloid marker sWC3 (74-22-15) and two molecules recognized by IPP B-cell-specific monoclonal antibodies (F10/4, F12/35). Extensive apoptosis in vivo and in vitro was demonstrated by electron microscopy, immunohistology with TdT-mediated dUTP nick end labelling, DNA analysis and fluorescence-activated cell sorter analysis. Thus, when isolated IPP follicular cells were incubated at 37 degrees in vitro, the majority of them became apoptotic. The few that survived, however, had lost their expression of sWC3, F10/4, F12/35, but showed an increased expression of sIgM and major histocompatibility complex class II indicating that such surviving cells were of a more mature phenotype. Although more T cells were observed in porcine IPP follicles than in sheep IPP, CD3+ cells comprised less than 5% of the IPP follicular lymphocytes. Thus, the results clearly indicate that pig IPP is equivalent to sheep IPP.     

Segregation of B lymphocytes into stationary apoptotic and migratory proliferating subpopulations in agglomerate cultures with ileal epithelium

The B lymphocyte-epithelial cell interactions that define the microenvironment of the ileal Peyer's patch, the primary B lymphocyte organ of the fetal lamb, have been replicated in tissue culture. Mixed suspensions of ileal epithelial cells, lymphocytes and fibroblasts from fetuses of 63-103 days of gestation organized into macroscopically visible agglomerates within 72 h. These agglomerates contained translucent spherical cavities and were enclosed within a marginal cell layer and surrounded by an expanding corona of emigrating cells. The lining of the cavities and the marginal layer consisted of well-differentiated, polarized columnar ileal epithelial cells. One population of B lymphocytes in the initial mixed suspension differentiated into two discrete populations reproducing the characteristics of intact fetal ileal Peyer's patches. B cells apposed to follicle-associated epithelium (FAE) within agglomerates underwent apoptosis. The other population of emigrant B cells proliferated and expressed the BAQ44A differentiation marker. Differentiation of ileal epithelial cells into FAE, typical of Peyer's patches, was markedly accelerated. The mutually inductive influences of intestinal epithelial cells and B lymphocytes in these agglomerates replicate normal mid-gestational fetal development of the mucosal immune system and afford new opportunities for its further investigation.