The first two authors contributed equally to this work.
Cellular immune response
Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes
Article first published online: 3 DEC 2004
Copyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 35, Issue 1, pages 98–107, January 2005
How to Cite
Pabst, O., Herbrand, H., Worbs, T., Friedrichsen, M., Yan, S., Hoffmann, Matthias W., Körner, H., Bernhardt, G., Pabst, R. and Förster, R. (2005), Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes. Eur. J. Immunol., 35: 98–107. doi: 10.1002/eji.200425432
- Issue published online: 16 DEC 2004
- Article first published online: 3 DEC 2004
- Manuscript Accepted: 29 OCT 2004
- Manuscript Revised: 27 SEP 2004
- Manuscript Received: 29 JUN 2004
- Intraepithelial lymphocytes;
- Gut-associated lymphoid tissue;
- Isolated lymphoid follicles
In comparison to secondary lymphoid organs, gut-associated lymphoid tissues such as isolated lymphoid follicles (ILF) and cryptopatches (CP) have been less intensively studied. To gain a better insight into processes regulating organization and function of these structures, which are believed to participate in immune responses and extrathymic T cell development, we characterized the lymphoid structures of the murine small intestine in more detail. The size and cellular composition of small intestinal lymphoid aggregations were analyzed in C57BL/6 and BALB/c wild-type and lymphotoxin (LT)-deficient mice, by flow cytometry, histology and automated multi-color immunofluorescence microscopy evaluating large coherent areas of the intestine. These evaluations demonstrate that aggregated lymphoid structures in the small intestine vary in size and cellular composition, with a majority of structures not matching the current definitions of CP or ILF. Accordingly, significant variations depending on species, age and mouse strain were observed. Furthermore, small bowel transplantation revealed a rapid exchange of B but not T cells between host and grafted tissue. Moreover, LT-deficient animals lack any intestinal lymphoid aggregations yet possess the complete panel of intraepithelial lymphocytes (IEL). In summary, our observations disclose intestinal lymphoid aggregations as dynamic structures with a great deal of inborn plasticity and demonstrate their dispensability for the generation of IEL.
Gut-associated lymphoid tissue
Isolated lymphoid follicles
Lamina propria lymphocytes
Mesenteric lymph nodes
The intestine is continuously exposed to foreign material in the form of harmless food antigens and commensal organisms, as well as of potential pathogens. Protection against pathogens on the one hand and maintenance of tolerance to innocuous antigens on the other hand is accomplished by the gut-associated lymphoid tissue (GALT). GALT is composed of highly specialized lymphoid organs such as Peyer's patches (PP) and small lymphoid aggregations. Beside GALT, dispersed immune cells such as intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) reside throughout the intestine.
In the murine jejunum, IEL can be found at a frequency of about 16 IEL per 100 enterocytes, resulting in a remarkably numerous population of these cells, considering the size of the entire organ 1. IEL are effector T cells that also maintain the integrity of the mucosal epithelium 2. The majority of intestinal IEL are CD8+, with significant numbers expressing the CD8αα homodimer and frequently employing the TCRγδ instead of the TCRαβ 3. Contradictory results have been reported regarding the origin of IEL, suggesting that CD8αβ+ IEL are of thymic origin whereas CD8αα+ IEL develop locally in the gut 3–5. Cryptopatches (CP), numerous small and randomly distributed clusters of lymphoid cells in the basal lamina propria of the murine intestine, are composed of predominantly lineage-negative (CD3–CD4–CD8–B220–) cells expressing the stem cell factor c-kit 6 and have been hypothesized to be sites of extrathymic T cell development 6–8.
In addition to CP, two well-recognized types of aggregated lymphoid structures are present in the intestinal wall: isolated lymphoid follicles (ILF) and PP. ILF have been described in numerous species including human 9, rabbit 10, guinea pig 11 and mouse 12. In the mouse, approximately 100–150 ILF, very much like PP, colonize the small intestine along the antimesenteric wall. The cellular composition of ILF has been reported to resemble PP: both structures are predominantly filled with B cells and may possess germinal centers and a follicle-associated epithelium (FAE) containing microfold cells specialized for the uptake of antigen from the gut lumen 9, 12. Thus, ILF and PP are assumed inductive sites for intestinal immune reactions. They share common developmental traits as assembly of both structures requires IL-7Rα and LTα signaling. ILF and PP are absent in lymphotoxin (LT)α-deficient animals and drastically reduced in number in IL-7Rα-deficient mice 12.
By examining aggregated structures in the murine gut by surveying large coherent intestinal areas, we demonstrate that a clear-cut distinction between ILF and CP is not plausible, as the phenotype of lymphoid aggregations is subject to recurrent modulations reflecting age and environmental conditions. Importantly, LTα- and LTβ-deficient animals do not develop any aggregated lymphoid structures in the intestine yet possess all intestine-specific IEL subpopulations, demonstrating that intestinal lymphoid aggregations are dispensable for IEL formation.
Distribution and architecture of aggregated lymphoid structures in the small intestine
Currently, CP and ILF are thought to serve non-overlapping functions and to differ in localization, size and cellular composition. Therefore, they are usually regarded as two genuine and separate types of intestinal lymphoid aggregations 6, 12.
A systematic analysis of these small structures requires an exhausting series of consecutive sections covering large coherent areas. Therefore, we developed a technique that allows an automatic assembly of large composite images taken from successive horizontal sections of intestinal tubes that were cut open and embedded as planar sheets (Figs. 1, 2). In addition, vertical sections were performed on Swiss rolls made from longitudinally opened gut segments. The location and principal architecture of aggregated structures in the intestine are summarized in Fig. 1. The jejunum of an 8-week-old BALB/c mouse was either used for horizontal sections through the crypt zone (Fig. 1A, B, E) or for vertical sections along the proximal-distal axis of intestinal villi (Fig. 1C, F–H). In horizontal sections, prominent lymphoid aggregations confined to the anti-mesenteric wall appear as dense aggregations of cells with an average diameter of 150 µm in the crypt zone (Fig. 1A, B). In the vertical sections, most of these structures have the shape of a single dome (Fig. 1C). These large structures can also be identified from the mucosal side using a stereomicroscope (Fig. 1D). Thus, based on morphological criteria, these structures fit the current definition of ILF 12. Beside these ILF, barrel-shaped lymphoid aggregations are present in the intestinal wall, localizing to the lower third of “normal-sized” villi (Fig. 1F). These structures have an average diameter of 80 µm (Fig. 1E), thus fitting the current definition of CP. In contrast to the dome-shaped ILF, villi harboring CP cannot be distinguished from neighboring villi by stereomicroscopy as described above (Fig. 1D). However, neither anti-mesenteric localization nor the size of an individual structure suffices for a definitive classification into either category as judged by large-scale analysis of horizontal sections. Small-sized structures can be found along the anti-mesenteric axis, and vice versa, large aggregations of cells can be identified at locations other than the anti-mesenteric wall (compare the localization of structures shown in Fig. 2A and 2B1–B6). Moreover, in approximately 50% of all lymphoid structures identified on vertical sections, analysis of consecutive sections revealed that the villi seem to bulge out just above the crypt zone. Thus, these aggregations resemble an intermediate structure between ILF and CP (Fig. 1G). Moreover, when analyzing the size of all lymphoid formations independent of their localization, we failed to identify distinct classes of small- and large-sized structures (Fig. 1I).
In a next step, we compared size and number of lymphoid aggregations in the intestine of young and old C57BL/6 mice with those of young BALB/c mice. In all animals analyzed, we found comparable numbers of aggregated lymphoid structures in the small intestine (Table 1; approximately 50 structures/cm2, resulting in a total of about 1,000 structures/small intestine; data not shown). In contrast, the average size of lymphoid aggregations differed between young and old C57BL/6 mice, as well as between C57BL/6 and BALB/c mice. Old mice possess significantly larger structures than young mice (Table 1). Furthermore, in older animals, the percentage of large-sized structures found outside the anti-mesenteric axis is considerably increased (data not shown). Thus, although some of the structures present in the intestinal wall fit into the definitions of CP and ILF, an unambiguous classification based on morphological criteria is not possible in most cases.
|Size in μm2||Number of structures/cm2||Structure-area as % of total section-area|
|C57BL/6 (8 weeks)||8,800||58||0.51|
|C57BL/6 (40 weeks)||19,500||54||1.05|
|BALB/c (8 weeks)||13,200||46||0.61|
Cellular composition of lymphoid aggregations in the small intestine
Based on the analysis of hematoxylin (HE) staining, consecutive sections were selected for three-color immunofluorescence microscopy, with various combinations of antibodies directed against IL-7Rα, CD117 (c-kit), IgD, B220, CD3 and Ly5.2, in addition to a DAPI nuclear stain. For a systematic analysis of the cellular composition of lymphoid aggregations, composite images were automatically assembled (Fig. 2). The cellular composition of individual aggregates was not dependent on their position in the intestinal wall, as exemplified in Fig. 2. Interestingly, large and small structures at any position contained significant fractions of IL-7Rα+ cells and c-kit+ cells (Fig. 2A, B). Furthermore, only few structures were devoid of mature B and T cells, as judged by the presence of B220+IgD+CD3– and CD3+IgD– cells, respectively, and would thus match the definition of CP (Fig. 2B).
We then determined the relative frequency of B cells, T cells and c-kit+ stem cells in C57BL/6 mice. For this analysis only structures that were cut through their core were included (Fig. 3). Although the average content of B220+ cells increased as the size of these aggregations increased, the B220+ content varied considerably. Consequently, we identified comparably small aggregations with B220+ cells prevailing. Conversely, a noticeable number of large aggregations lacked significant numbers of B or T cells, but instead harbored large numbers of IL-7R+ and c-kit+ cells (Fig. 3 and data not shown).
To confirm these results, we analyzed by flow cytometry the cellular composition of individual structures dissected from BALB/c intestines under a stereomicroscope. This method only allows the isolation of the larger, dome-shaped structures (compare Fig. 1D). Flow cytometrical analysis revealed that these structures, but not mesenteric lymph nodes (MLN) or PP, contain a population of non-B, non-T cells representing approximately 25% of the entire cellular count (Fig. 4). A significant fraction of these cells did not stain for any lineage marker but expressed the stem cell factor receptor c-kit (Fig. 4 and data not shown). These cells most likely represent precursor cells of hematopoietic origin. These results are in line with previous reports observing that not only CP but also structures fitting the current definition of ILF contain a significant population of lineage-negative cells 12.
These studies corroborated our morphological observations (see previous section) that most lymphoid aggregations residing in the small intestine are distinguished by a phenotype with characteristics of both, ILF and CP. Even though both CP and ILF could be identified, they seem to represent only a minor fraction of the solitary intestinal lymphoid tissue. It is conceivable that only a large-scale study is capable of revealing the surprising heterogeneity among solitary intestinal lymphoid tissue that escaped detection in earlier reports.
Lack of cryptopatches in human, rat and porcine small intestine
In contrast to ILF, the presence of CP has so far only been reported in mice. Therefore we evaluated HE-stained sections of human, pig and rat intestines at different ages and under different breeding conditions for the presence of small lymphoid aggregations. In human intestines (n=6; age: 6.0±4 months), no structures fitting the current definition of murine CP could be found (data not shown). The same result holds true for pig intestines (n=22, 6–10 months; n=22, 8–12 months; n=4, 2–3 years; all conventionally reared Göttinger Minipigs) that were kept under either germfree or specific pathogen-free conditions. Interestingly, in sections of rat intestine, ILF could be identified on about 60% of the analyzed sections, while villi filled with lymphocytes as observed in human 9 could be detected on about 20% of the sections (n=18, 5-month-old females, Sprague Dawley; data not shown). Of note, sections analyzed from all three different species, human, pig and rat, consistently did not reveal any CP-like structures.
The phenotype of aggregated lymphoid structures in the intestinal wall is highly dynamic
The observed heterogeneity of intestinal lymphoid aggregations may relate to a dynamic behavior of cells that constitute these structures. In order to test this hypothesis, we performed small bowel transplantations. Wild-type Ly5.1 intestinal tubes were transplanted together with the draining MLN into congenic Ly5.2 mice. The transplanted intestine was almost devoid of the intestinal load of nutrients and bacteria present in the host intestine. This, we hypothesized, might result in different cellular compositions of aggregated structures in host and grafted intestines.
Lamina propria cells from host and graft intestine were isolated 4 days and 14 days after transplantation and investigated by flow cytometry. PP were excluded from cell preparations, whereas all other lymphoid structures were included. Interestingly, the redistribution pattern of T cells differed from that of B cells. Very few T cells from the host were present in the grafted tissue (Fig. 5A and data not shown), and vice versa, only a few hundred Ly5.1-positive T cells from the graft were present in the lamina propria of the host. These results are in line with previous reports showing a very slow exchange of intestinal T cells in parabiotic mice 13. In contrast, we observed a rapid influx of B cells from the host tissue into the grafted intestine. Already 4 days after transplantation, more than 60% of all B cells present in the graft lamina propria were of host origin (Fig. 5A). Likewise, a significant influx of graft cells into the host lamina propria was observed. Importantly, a detailed analysis of more than 250 aggregated structures from six transplanted mice revealed a clear shift in the size distribution towards large structures in the graft (Fig. 5C), which may mainly trace back to immigrated B cells.
Four-color immunofluorescence microscopy with anti-Ly5.2, anti-B220 and anti-CD3 antibodies, in combination with a nuclear DAPI stain, confirmed a considerable expansion of B220+ cells of host origin in the majority of the lymphoid aggregations in the graft tissue (Fig. 5B, D). Notably, immature IL-7Rα-expressing lymphocyte precursor cells in the grafted tissue were of graft and not of host origin. These results clearly demonstrate that the phenotype of aggregated structures in the intestinal wall is dynamic and adapts rapidly to altered environmental conditions.
Cryptopatches are dispensable for IEL generation
Previous reports suggest a close interrelation between CP and the generation of IEL. To address whether aggregated structures in the intestinal wall are required for the generation of intestinal IEL, we analyzed LTα- and LTβ-deficient mice that are devoid of ILF yet posses an almost wild type-like composition of IEL 14. Most interestingly, our analysis revealed that these mutants lack all lymphoid aggregations of whatever size along the entire small intestine (Table 1, Fig. 6A, and data not shown), whereas a considerable number of scattered c-kit+ cells is present in the crypt region of LT mutants. Thus, aggregated structures in the intestine are completely dispensable for the generation of IEL under conditions where LT signaling is absent. Comparison of IEL subpopulations from C57BL/6 wild-type mice and LTα mutants confirmed that TCRγδ+ as well as CD8αα+ cells, which are currently assumed to derive from CP, are present in LTα mutants (Fig. 6B, C). Notably, the absolute number of all IEL subpopulations was considerably increased in LTα-deficient mice when compared to wild-type animals. This finding might reflect that in the absence of LT signaling and all peripheral lymphoid organs, lymphocytes are atypically distributed in this organism. Alternatively, increased numbers of IEL in LTα mutants might relate to the overall susceptibility of these animals to autoimmune diseases, including inflammatory bowel disease 15. However, despite these abnormalities in the number of IEL, all subtypes were generated independently from the presence of aggregated structures in the intestinal wall.
A variety of lymphoid aggregations in the murine small intestine has been described in the literature. While organization and function of PP are quite well understood, knowledge about ILF and CP is far less validated. Each compartment has been reported to display a characteristic phenotype and to serve distinct functions.
Analysis of aggregated intestinal structures is increasingly hampered with decreasing size. To minimize technical limitations and to warrant the comparability of results, we developed a standard technique that allowed us to analyze statistically significant numbers of structures on serial sections. Applying this technique to conventionally reared wild-type mice, we were surprised to note that the intestine is not simply decorated with ILF and CP, obeying current definitions. The assembly of lymphoid aggregations is rather inhomogeneous in size, location and cellular content, and most lymphoid structures do not fall into either category, ILF or CP. This impressive heterogeneity favors a model of interconvertible lymphoid structures over a fixed concept of ILF and CP. Such a concept receives support from data obtained from different species or mouse strains and from mice of different ages as well as under different environmental influences deriving from nutrition and breeding conditions.
The existence of significant mouse strain-dependent differences was already implicated by previous reports. Initially, Hamada et al. 12 analyzed BALB/c and C57BL/6 mice by immunohistochemistry on frozen sections and observed well-developed ILF in both strains. In contrast, Lorenz et al. 16 could not at all identify ILF in the proximal jejunum of conventionally reared C57BL/6 mice by gross examination using low power stereomicroscopy, although they could locate these structures in the ileum. In the present study, we could show that differences in the size of aggregated lymphoid structures depend on mouse strain and age of the animals: lymphoid aggregations in 6–8-week-old BALB/c mice are considerably larger than in age-matched C57BL/6 animals. Moreover, in C57BL/6 mice 4–6 months of age, these structures are about twice as large with respect to the diameter as in 6–8-week-old animals (Table 1).
In other species such as sheep, pigs and dogs, a second type of PP is present in the distal ileum, sharing many features with a primary lymphoid organ 17. Diversity in GALT composition is also reflected by our observation that mice, but not humans, pigs or rats, possess CP. As ILF and CP are known to develop postnatally in mouse 12, we may have failed to detect CP in other species due to age specifications of the samples. In general, it might be difficult to directly compare data obtained from different species at different ages, as there are considerable species-specific variations in the developmental speed. Nevertheless, as sections analyzed from all three different species, human, pig and rat, consistently did not reveal any CP-like structures, this adds evidence to the idea that murine CP represent a polarized” aspect of solitary intestinal lymphoid tissue. Despite these differences concerning its organization, GALT has to fulfill principally the same tasks in all species. The presence of a structure that is phenotypically species specific might therefore reflect evolutionary history; however, phenotypically distinct structures might still perform similar functions in different species.
A key feature of dynamic and interconvertible small GALT relates to a well-known trait of many immunologically competent cells: migration and recirculation among different compartments and inside lymphoid organs. From this perspective, the size of a lymphoid structure is considerably influenced by population dynamics. A dynamic behavior of aggregated structures can be concluded from the results of the bowel transplantation experiments performed in this study. The overall enlargement of the aggregated structures in the graft and the increased proportion of B cells demonstrate a dynamic behavior of at least the B cell subpopulation in response to environmental cues. Furthermore, lymphoid aggregations in germfree mice have been reported to contain reduced numbers of lineage-positive cells, while colonization of these animals with microorganisms leads to an increase in the proportion of lineage-positive cells 12, 16. Interestingly, a recent report proposed to distinguish immature and mature ILF in mice. Lorenz et al. 16 observed that ILF could be reconstituted postnatally in LTα mutants by bone marrow transplantation from wild-type donors. Reconstituted ILF either resembled typical ILF, with a prevailing B cell core and anti-mesenteric localization, or appeared as randomly scattered structures with a low to intermediate B cell content. These findings are in line with the observations reported here. Aggregated structures with a non-anti-mesenteric localization may contain significant numbers of B cells, and the immature ILF proposed by Lorenz and colleagues might well correspond to the previously reported CP. These observations also suggest that mouse strain-dependent variations, age, housing conditions and other environmental factors might account for apparent contradictions among published data.
Apart from these phenotypic heterogeneities/similarities, the probably more important issue relates to the functions of these structures. One point of objection concerns the importance of microfold cells in the epithelia overlying small GALT. They might well outnumber microfold cells in the FAE covering PP. In some systems that experimentally inhibit PP formation, small GALT is not affected 12 and might therefore rescue functions normally attributed to PP.
Moreover, origin and differentiation of IEL are still a matter of debate. There is evidence that CD8αα+ CD4– IEL can develop independently of the thymus. In contrast, the thymus has been shown to be absolutely required for the generation of CD8αβ and double-positive (CD4+CD8αα) IEL 3, 5, 7. Saito et al. 8 have shown that cells isolated from murine intestinal lymphoid aggregations could generate TCRαβ CD8αα and TCRγδ CD8αα IEL when transferred into lethally irradiated SCID mice, whereas cells from PP or MLN could not. As in the course of reconstitution, the occurrence of CP has been shown to precede generation and full maturation of IEL in the overlying epithelium 18, CP are widely accepted as the major breeding places for extrathymic IEL 19, 20. The sequence of local T cell precursor differentiation in the murine gut has been established by Lambolez et al. 7, suggesting that the subpopulation of Thy1+c-kit+ lymphoid cells residing in the CP are the progenitors of gut-derived IEL. Further evidence for the in situ rearrangement of TCRγδ+ IEL in the gut was provided by Laky et al. 21. They could show that in IL-7-deficient mice, which otherwise lack TCRγδ+ IEL and have diminished CP and PP, expression of IL-7 under the control of the intestinal fatty acid-binding protein promoter could partially restore the development of TCRγδ+ IEL, as well as of CP and PP, within the intestine, but not in other tissues 21. A close interrelationship between CP and IEL was furthermore deduced from a study by Makita et al. 22 addressing the role of CP in intestinal inflammation. In a mouse strain that spontaneously develops a chronic intestinal inflammation with similarities to human Crohn's disease, the onset of the disease correlates with a decreased number of CP and CD8αα+ IEL. In contrast, other reports doubt that the development of IEL is truly thymus independent and question that CP are the site of their extrathymic differentiation 23. Athymic mice possess very low numbers of CD8αα IEL, and in utero thymectomy in sheep, as well as neonatal thymectomy in mice, results in a dramatic drop in the number of IEL, including CD8αα+ IEL, during the first span of life 24–26. Most recently, in a study of recombinase-activating gene-2 promoter activity in a transgenic mouse model, Guy-Grand et al. 23 could not detect GFP expression in the gut. In contrast, GFP expression could be detected in PP and MLN, suggesting that PP and MLN, rather than CP, might be involved in extrathymic T cell differentiation in athymic mice. In contrast to previous reports, we could not detect any intestinal lymphoid aggregations in LTα or LTβ mutants, despite the presence of increased numbers of CD8αα+ IEL. Instead, we observed c-kit+ CD3– cells, which might well represent precursor cells, dispersed throughout the tissue in LTα mutants. Thus, we cannot exclude that local IEL differentiation occurs in the absence of organized GALT. However, our findings add evidence to the notion that lymphoid aggregations in the intestine are dispensable for the generation of IEL.
In conclusion, based on the data presented here, neither morphological nor functional characteristics support a strict classification of small intestinal lymphoid aggregations into ILF and CP, as currently approved. Instead, we propose to refer to these structures in total as solitary intestinal lymphoid tissue (SILT), distinguished by plasticity and rapid responsiveness to external stimuli.
Materials and methods
Preparation of sections and microscopy
Animals were bred at the central animal facility of Hannover Medical School under specific pathogen-free conditions, or purchased from Charles River (Germany). LTα- and LTβ-deficient mice on a C57BL/6 background have been described 27. The small intestine was excised, flushed with PBS and opened along the mesenteric side. For horizontal sections, fragments of about 20 mm in length were flattened with the mucosal side downwards on filter paper, embedded in OCT compound and frozen on dry ice. For vertical sections, gut fragments of approximately 5 cm in length were washed in PBS, followed by a 50% mixture of OCT and PBS, and transferred to OCT, before Swiss rolls with the luminal side facing outwards and the proximal end locating to the centre of the roll were prepared. Cryosections (6 µm) were air-dried and fixed for 10 min in ice-cold acetone.
The following antibodies and conjugates were used in this study: anti-CD8α-bio, anti-TCRαβ-FITC, anti-TCRγδ-PE, anti-CD11b-bio (Caltag), anti-CD4-PerCp, anti-Ter119-bio, anti-CD11c-bio (BD Biosciences), anti-CD19-bio (Southern Biotechnologies), anti-CD117 (c-kit, clone ACK2; Natutec), anti-Ly5.1-FITC and anti-Ly5.2-FITC (Cymbus Biotechnology), anti-IL-7Rα (clone A7R34). Anti-CD8β (clone RM CD8), anti-CD3 (clone 17A2), anti-IgD (clone HB250) and anti-B220 (clone TIB146) antibodies were provided by Elisabeth Kremmer (GSF München, Germany). Biotinylated antibodies were recognized by streptavidin coupled to Alexa 750 (Molecular Probes) or PerCp (BD Biosciences). Unconjugated anti-IL-7Rα and anti-c-kit antibodies were recognized by mouse anti-rat Cy3 conjugates (Jackson Laboratories). FITC, Cy3 and Cy5 conjugates of anti-B220, anti-CD3 and anti-CD8β antibodies were prepared as recommended by the manufacturer (Amersham).
Immunohistochemistry was performed according to standard protocols. Briefly, sections were rehydrated in TBST (0.1 M Tris pH 7.5, 0.15 M NaCl, 0.1% Tween-20), pre-incubated with TBST containing 5% rat or mouse serum, blocked with 0.001% avidin/PBS and 0.001% biotin/PBS and stained with a cocktail of biotinylated or fluorescent dye-coupled antibodies in 2.5% serum/TBST. Biotinylated antibodies were visualized by fluorescent streptavidin conjugates. Nuclei were visualized by DAPI staining (1 µg/ml DAPI/TBST), and sections were mounted with MOWIOL. Staining with unconjugated primary antibodies (anti-IL-7Rα or anti-c-kit) and their detection with a mouse anti-rat Cy3 antibody were performed prior to the staining procedure described above. Composite images were automatically assembled using a motorized Axiovert 200M microscope (Carl Zeiss) with an autofocus module and KS300 software (Carl Zeiss).
To obtain single-cell suspensions of MLN and PP, organs were minced through a nylon mesh and washed with PBS supplemented with 2% FCS. For isolation of IEL and LPL, gut content and PP were removed before intestines were opened longitudinally. Intestines were washed twice in cold PBS and once in cold PBS/5% FCS/5 mM EDTA, and incubated twice in 25 ml RPMI 1640 medium/5% FCS/5 mM EDTA at 37°C (spun down at 150 rpm). Supernatants were pooled, filtered through a nylon mesh, pelleted and resuspended in 40% Percoll (Amersham) in RPMI/5% FCS. This cell suspension was overlaid onto 70% Percoll in RPMI/5% FCS and centrifuged at 800×g for 20 min. IEL were recovered from the interphase and washed twice in PBS/2% FCS. To isolate LPL, the remaining tissue was washed with PBS, cut into small pieces and incubated at 37°C for 30 min in RPMI/20% FCS/0.5 mg/ml collagenase A (Roche). The resulting cell suspension was enriched for LPL by discontinuous gradient centrifugation as described above.
Cells were stained using the antibodies described above. Lineage-positive cells were excluded using a cocktail of biotinylated antibodies directed against CD3, CD19, CD11b, CD11c and Ter119, recognized by streptavidin/PerCp.
Mouse vascularized small bowel transplantation was performed as described 28, with some modifications. C57BL/10 Ly5.1 mice were used as donors and C57BL/10 Ly5.2 mice as recipients. Briefly, under the combined anesthesia of Ketamine and Rompun, the donor jejunum and proximal part of the ileum were isolated with the superior mesenteric artery and portal vein attached. After luminal irrigation and vascular perfusion, the graft was stored at 4°C in Ringer's solution until implantation. The graft portal vein and superior mesenteric artery were anastomosed to the recipient's inferior vena cava and abdominal aorta, respectively, in an end-to-side fashion. The proximal end of the graft was exteriorized as a stoma. The graft secretion was drained into the host alimentary tract by an end-to-side anastomosis between graft ileal end and host jejunum.
This work was supported by a Deutsche Forschungsgemeinschaft Grant, SFB621-A1, to R. Förster.
- 14Lymphotoxin controls alphaEbeta7-integrin expression by peripheral CD8+ T cells. Immunol. Cell Biol. 2001. 79: 323–331.Direct Link: