THE ANATOMICAL RECORD 250:182–189 (1998) Appearance and Development of Lymphoid Cells in the Chicken (Gallus gallus) Caecal Tonsil M. GÓMEZ DEL MORAL, J. FONFRÍA, A. VARAS, E. JIMÉNEZ, J. MORENO, AND A.G. ZAPATA* Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain ABSTRACT Background: We have analyzed by electron microscopy, immunohistochemistry, and flow cytometry the development of chicken caecal tonsil, the largest lymphoid organ of avian gut—associated lymphoid tissue (GALT). Methods: White Leghorn chickens of different ages obtained from a local supplier were routinely processed by transmission electron microscopy. For both immunohistochemistry and flow cytometry, we tested a battery of specific monoclonal antibodies (mAbs) to chicken cell markers on caecal cryosections or cell suspensions, respectively. Results: A rudimentary caecal tonsil occurs at the end of incubation. The organ grows just after birth, reaching the adult condition 4 days later. Firstly (4 days to 2 weeks), it contains predominantly T lymphocytes, principally TcRab1 and CD41 cells, which occupy largely the named caecal diffuse lymphoid tissue. In adult tonsils (6-week-old chickens) however, B lymphocytes, mainly expressing either IgM or IgA, predominate. They occur in both the subepithelial zone and the germinal centers, in which there are also a few T cells. After 2 weeks the CD81 lymphocytes gradually become more numerous than CD41 cells. In the tonsillar epithelium CD81TcRgd1 T cells, CD81TcRgd2ab2, presumably NK cells, and a few B lymphocytes are the main cell subpopulations. Conclusions: Chicken caecum grows fast after hatching. The diffuse lymphoid tissue largely contains TcRab CD41 or CD81 cells. CD81 cells of caecal epithelium represent gd T cells or NK cells. B lymphocytes which occur in the subepithelial zone, germinal centers, and, in few numbers, the caecal epithelium predominantly express either IgM or IgA. Anat. Rec. 250:182–189, 1998. r 1998 Wiley-Liss, Inc. Key words: T- and B-lymphocytes; caecal tonsils; development; chicken (Gallus gallus) The caecal tonsils, two large lymphoid aggregates, each occurring in the caecum-rectum junction, represent the largest lymphoid organs of avian gut-associated lymphoid tissue (GALT). Their structure has been histologically analyzed in several avian species (Magnan, 1911; Looper and Looper, 1929; King and McLelland, 1975), but, despite a recent review on the chicken gastrointestinal immune system (Lillehoj and Trout, 1996), very little is known about the nature of the cell populations they contain and their development and immunological significance. Via different approaches, the subepithelial area of caecal tonsil has been considered as a bursa-dependent area (Looper and Looper, 1929; Jankovic and Mitrovic, 1967; Albini and Wick, 1974; King and McLelland, 1975), whereas the diffuse lymphoid tissue, which occupies the medium and deep regions of the lymphoid aggregates, consists mainly of T cells (Hoshi and Mori, 1973). Germinal centers, also appearing in the caecal tonsil, consist mainly of IgMr 1998 WILEY-LISS, INC. and IgG-positive cells (Jeurissen et al., 1989), but also a few T lymphocytes (CD41TcRab1) have been identified in them (Bucy et al., 1988). Some fibroblastic reticular cells and a few lymphocytes identify, the last few days of incubation (days 18–19), in the intestinal walls the presumptive caecal tonsil which will appear after birth (Looper and Looper, 1929; Jankovic and Mitrovic, 1967). In 5-day-old chickens, the caecal tonsil consists of IgM-, IgG-, or IgAexpressing B cells and a few IgM1 and IgA1 plasma cells (Jeurissen et al., 1989). First tonsillar germinal centers were histologically identified in 8-day-old chickens (Looper and Looper, 1929), but Jeurissen et al. (1989) described them only 2 weeks after birth when *Correspondence to: Agustı̀n G. Zapata, Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain. E-mail: Zapata@eucmax.sim.ucm.es Received 10 April 1997; Accepted 21 October 1997 183 DEVELOPMENT OF CHICKEN CAECAL TONSILS follicular dendritic cells, retaining immune complexes on their cell processes, could be demonstrated. Functionally, the caecal tonsils have been described as secondary lymphoid organs, equivalent to the avian Peyer’s patches (Glick et al., 1981), human tonsils (Magnan, 1911), or the mammalian appendix (King and McLelland, 1975), involved in antigen-specific humoral responses (Jankovic and Mitrovic, 1967; Jankovic, 1968; Orleans and Rose, 1970). Other authors, however, have emphasized the difficulties that antigens coming from the cloaca have to reach the avian GALT, especially the caecal tonsil (Jeurissen et al., 1991). In this regard, the immune relevance of the caecal tonsil has been correlated with the involution of the bursa of Fabricius. Previous to this involution, the bursa itself could process antigens. Caecal tonsils, however, constitute a reservoir for coccidians in infected birds (del Cacho et al., 1993; Lillehoj and Trout, 1996). In the present study, we have combined electron microscopy, immunohistology, and flow cytometry, using a battery of mAbs raised specifically against distinct chicken surface cell markers, to morphologically and phenotypically characterize the lymphoid cells of the chicken caecal tonsil as well as their appearance and development throughout embryonic and postnatal life. MATERIALS AND METHODS Animals In the present study we used white Leghorn chickens, obtained from a local supplier, of the following ages: 20 days of incubation (5 specimens), neonates (25), 4 days (20), 1 week (20), 2 weeks (10), and 6 weeks (10). The animals, grown in conventional conditions, were apparently healthy. The ultrastructural study demonstrated occassionally the presence of cryptosporidians in the caecal lumen of some specimens. Immunohistochemistry Three chickens of each stage studied were sacrificed, and the two caecal sacs were aseptically removed. Their proximal regions (caecal tonsils) were snap-frozen in liquid nitrogen and stored at 280°C until use. Cryosections of 6 µm were fixed in acetone for 10 min and air-dried for at least 1 h. Endogenous peroxidase was blocked with 1% H2O2 in methanol. After washing in PBS, the sections were incubated for 1 h with the specific mAbs listed in Table 1, and a 1/40 solution of peroxidase-conjugated rabbit anti-mouse immunoglobulins in phosphate buffered saline (PBS) (Dako, Glostrup, Denmark). The peroxidase activity was revealed using 0.05% 3, 3’ diaminobenzidine (Sigma Co., St. Louis, MO) as chromogen, diluted in PBS plus 0.01% H2O2. Sections were counterstained with methylene blue and gradually dehydrated with graded alcohol and mounted in DePex. Negative controls were carried out on successive sections that received only the second antibody. Histological sections were photographed in a Labophot (Nikon) light microscope provided with an Agfapan APX 100 film (Agfa, Leverkusen, Germany). Flow Cytometry In the flow cytometrical study, at least five chickens were used for each stage analyzed. Cells obtained from TABLE 1. Monoclonal antibodies used in this study mAb CVI-His-C7 CVI-ChIgM-59.7 CVI-ChIgG-47.3 CVI-ChIgA-46.1 CT4 CT8 TcR1 TcR2 Specificity Source Pan-leukocytes IgM IgG IgA CD4 CD8 TcRgd TcRaVb1 S.H.M. Jeurissen (Central Veterinary Institute, Lelystad, Netherlands) M.D. Cooper (University of Alabama, Birmingham) the caecal tonsils by gently pressing through a steel mesh were suspended in PBS containing 2% fetal calf serum (FCS) and 0.1% NaN3 (pH 7.2). For one-color analysis, 0.5 3 106 cells were incubated with the specific mAbs listed in Table 1 for 30 min and, after PBS washing, with an FITC-conjugated rabbit anti-mouse Ig (Dako) 1:100 diluted in PBS. For two-color analysis, one more incubation was achieved with phycoerythrin (PE)-conjugated CT8 mAb. Propidium iodide was used to exclude the dead cells. Relative immunofluorescence intensities were measured by flow cytometry with a FACScan (Becton-Dickinson, San Jose, CA). PC-Lysis software (Becton-Dickinson) was used for the analysis of the results. Electron Microscopy Small pieces of tissue from the caecal tonsils aseptically isolated either from 1-day-, 4-day-, or 1-week-old chicken (at least three animals for each analyzed stage) were fixed by immersion in 2.5% glutaraldehyde, buffered to pH 7.3 with Millonig’s fluid, postfixed in 1% osmium tetroxide in the same buffer, and dehydrated in acetone for embedding in AralditeTM. Ultrathin sections were obtained with a Reichert OM-U3 ultratome, double-stained with uranyl acetate and lead citrate, and examined with JEOL (Peabody, MA) 100B and Zeiss (Thornwood, NY) EM 902 electron microscopes. RESULTS Twenty-Day-Old Embryonic Chickens The tonsillar lymphoid tissue of adult chickens is organized in a named diffuse lymphoid tissue (see Hoshi and Mori, 1973; Glick et al., 1981), which occupies the medium and deep regions of lymphoid aggregates in which numerous germinal centers also occur. In addition, numerous lymphoid and nonlymphoid cells infiltrate the caecal epithelium of both villi and branching crypts and appear in the subepithelial region, named dome by other authors (Glick et al., 1981), which corresponds to the more superficial region of lymphoid aggregates. At the end of incubation, the caecal sacs or caeca represented two blind diverticula arranged ventrolaterally to the ileo-colonic junction. By using the mAb His-C7 raised specifically to chicken leukocytes, we could distinguish small cell aggregates in the lamina propria of both proximal and distal areas of the caecum. Moreover, a few positive cells appeared randomly scattered in the chorion and in the caecal epithelium (Fig. 1a). Some of these cells were identified as macrophages or gd T cells (TcR-11 cells). 184 M. GÓMEZ DEL MORAL ET AL. Fig. 1. a: Small group of leukocytes (His-C71 cells) in the proximal region of the caecum of a 20-day-old embryonic chicken (arrowheads). Note the existence of intraepithelial leukocytes (arrows). 350. b: His-C71 cells in proximal caecum of a 1-day-old chicken (arrows). Note the increased numbers of leukocytes occurring after birth. 350. c: IgM1 cells (arrows) occupy mainly the subepithelial region of the proximal caecum of a 1-day-old chicken. 350. d: Cell aggregates of CD41 T lymphocytes (arrowheads) in the proximal caecum of a neonatal chicken. 350. e: TcR2 (ab)-expressing cells (arrows) in the lymphoid cell aggregates of the caecal tonsil of 1-day-old chickens. Note that their distribution resembles that exhibited by the CD41 lymphocytes. 320. f: Small groups of CD81 cells (arrows) in the proximal caecum of a 1-day-old chicken. 350. g: TcR1 (gd)-expressing cells (arrows) in the proximal caecum of a 1-day-old chicken. 350. h: Migrating lymphocytes (L) in the epithelium (Ep) covering the proximal caecum of a 1-day-old chicken. Note the little development of the epithelial cell microvilli (arrows) and the abundance of mitochondria (m). 31,600. i: Parasitic oocytes (arrows) adhered in the surface of dark epithelial cells of the proximal caecum of a 1-day-old chicken. 33,500. j: Electron-dense cells containing engulfed parasitic oocysts (arrows) among the tonsillar lymphoid tissue of a 1-day-old chicken. Medium lymphocytes (L). 33,500. DEVELOPMENT OF CHICKEN CAECAL TONSILS 185 One-Day-Old Chickens Numerous His-C71 leukocytes marked in the lamina propria showed the location of the future caecal tonsil in 1-day-old chickens (Fig. 1b). In agreement with the increased numbers of lymphoid cells which appeared in the caecal tonsil in the first days of postnatal life, the ultrastructural study demonstrated lymphoid infiltrations in the epithelium (Fig. 1h) and among the connective tissue elements of the lamina propria. Rarely we identified parasites, presumably cryptosporidians, in both the caecal lumen, adhered to the lining epithelial cells (Fig. 1i), and in the cytoplasm of very electrondense cells which appeared in the incipient tonsillar aggregates (Fig. 1j). The lymphoid cells consisted mainly of small and medium lymphocytes but also lymphoblasts, immature plasma cells, frequently in division, and occasionally interdigitating cells. A few IgM, but neither IgG nor IgA, positive cells occupied the subepithelial region (Fig. 1c). The majority of lymphoid cells corresponded, however, to T lymphocytes which expressed CD4 (Fig. 1d) and/or TcRab cell markers (Fig. 1e). Both cell populations appeared in the same locations in the lymphoid aggregates and were not found in the tonsillar epithelium. A few CD81 cells appeared also in the caecal tonsils of 1-day-old chickens, occupying different locations in the lymphoid aggregates than CD41 lymphocytes (Fig. 1f). Moreover, CD81 lymphocytes appeared in the subepithelial zone and in the epithelium. A similar distribution was found for gd T cells (Fig. 1g). According to the flow cytometrical study, the mononuclear cell fraction obtained from the proximal region of the caecum of 1-day-old chickens consisted of about 15% leukocytes (His-C71 cells), including 7% IgM1 B cells and 6% T lymphocytes (Fig. 2a). On the other hand, the percentages of CD41 cells and CD81 cells were similar, increasing to around 4% (Fig. 2b). As suggested above by the immunohistological results, the TcRab-expressing cells corresponded principally to CD41 lymphocytes rather than to CD81 cells (Fig. 2c). Four-Day-Old Chickens Increased numbers of His-C71 cells appeared 4 days after birth in the caecal tonsils, as shown also by the EM study (Fig. 3a). Ultrastructural images of both lymphoblasts and immature plasma cells in division (Fig. 3b) and circulating lymphocytes crossing the blood vessel walls of caecal tonsil (Fig. 3c) suggested that the increased cellularity of the caecal tonsil in this stage was due to both in situ proliferation and presumably cell migration from blood circulation. Likewise, the numbers of B cells increased, occupying mainly the subepithelial zone (Fig. 3d), although a few cells occurred intraepithelially and in the lymphoid follicles. In this stage, both IgA1 and IgG1 cells were detected for the first time (Fig. 3e). These latter occupied the subepithelial zone but also occurred in the center of lymphoid follicles, presumably associated to the cell processes of a few follicular dendritic cells in incipient germinal centers (Fig. 3e). As in the previous stages analyzed, ab1 T cells and CD41 cells colocalized together, filling the vast majority of lymphoid follicles. Meanwhile, the CD81 lymphocytes predominated in the subepithelial zone and inside the tonsillar epithe- Fig. 2. a: Postnatal evolution of the percentages of distinct lymphoid cell subsets of chicken proximal caecum determined by flow cytometry using specific mAbs. b: Postnatal evolution of the percentages of either CD4- or CD8-positive cells of chicken proximal caecum. c: Postnatal evolution of the percentages of either TcR1 (gd)- or TcR2 (ab)expressing lymphocytes of chicken proximal caecum. The values represent the mean 6 SEM of at least five independent experiments. lium, although there were also positive cells in the lymphoid follicles (Fig. 3f). An important increase of the frequency of total leukocytes, which mainly corresponded to that of T lymphocytes, occurred in this stage (Fig. 2a). In fact, CD41 cells and CD81 cells increased in parallel (Fig. 2b), although the former represented a 186 M. GÓMEZ DEL MORAL ET AL. Fig. 3. a: Abundant lymphoid tissue occupies the lamina propria of the proximal caecum of 4-day-old chickens. Note the presence of lymphoid cells in division (arrows), lymphocytes (L), lymphoblasts (LB), and reticular cells (R). 3600. b: Lymphoblasts (LB) in division in the caecal tonsil of a 4-day-old chicken. L, lymphocytes. 32,200. c: Lymphocytes (L) migrating through the blood vessel endothelial cells (En) in the tonsillar lymphoid tissue of a 4-day-old chicken. 31,500. d: IgM1 cells (arrows) in the subepithelial region (SE) of the caecal tonsil of a 4-day-old chicken. 350. e: Distribution of IgG1 cells in the tonsillar lymphoid tissue of a 4-day-old chicken. Note the existence of a reticular pattern of staining (arrows) in areas with incipient germinal centers. 350. f: CD81 cells (arrows) in the lymphoid aggregates of the proximal caecum of 4-day-old chickens. 320. g: Cell suspensions obtained from 4-day-old caecal tonsils were analyzed by single-color flow cytometry analysis for the expression of CD4 (left) and CD8 (right). Open histograms correspond to labeling with the relevant mAb. Solid histograms correspond to negative control labeling with an isotype-matched irrelevant mAb. more homogeneous cell subpopulation than that of CD81 cells (Fig. 3g). Also, both TcR1- and TcR2expressing T cells increased (Fig. 2c). However, whereas the TcR-21 cells underwent a very important increase between 1 and 4 days and later at 2-4 weeks, the percentage of TcR1-expressing cells showed few variations after 4 days, except for a modest increase between 2 and 6 weeks (Fig. 2c). appearance of germinal centers and the important increase observed in the percentage of CD81 cells (Fig. 2b), there were no significant quantitative or qualitative changes in 1–2- week-old chickens compared to the condition observed 4 days after birth. The increased numbers of CD81 in 2-week-old chickens correlated well with the percentage of double-positive cells which expressed CD8 and TcR2 (Fig. 4a). In 1-week-old chickens, the numbers of both IgA1 and IgG1 B cells, according to our in situ immunohistochemical study, also underwent an increase, but, unfortunately, in our hands the mAbs used to identify these B cell subpopula- One-Week- and Two-Week-Old Chickens Apart from the establishment of the histological organization of adult caecal tonsil, with the definitive DEVELOPMENT OF CHICKEN CAECAL TONSILS Fig. 4. a: Two-color flow cytometry analysis of CD8 and TcR2 (ab) expression was examined on cell suspensions obtained from 2-weekold caecal tonsils. The percentage of CD81TcR21 cell subpopulation is listed in the corner of the quadrant. b: IgA1 cells in a germinal center of the tonsillar lymphoid tissue of a 6-week-old chicken. 3200. c: A few CD41 cells (arrows) in a germinal center (GC) of the tonsillar lymphoid tissue of a 6-week-old chicken. 3200. d: CD81 cells (arrows) in tonsillar germinal center (GC) of a 6-week-old chicken. 3200. tions immunohistologically did not work in flow cytometry. Six-Week-Old Chickens A new numerical increase in the cellularity of the caecal tonsil occurred in 6-week-old chickens. This increase was presumably due to the increase undergone by B lymphocytes, whereas T cells did not undergo important modifications with respect to previous stages studied (Fig.2a). Furthermore, because the total number of B lymphocytes was determined by using only the numbers of IgM1 B cells and the immunohistological study also demonstrated important increases in IgGand, mainly, IgA-expressing B cells (Figs. 2a, 4b), we could conclude that in adult caecal tonsil the B lymphocytes predominate quantitatively over the T cell population. On the other hand, apart from the increase in IgA1 cells and their presence in the germinal centers (Fig. 4b), the immunohistological study demonstrated the presence of some TcRab1, CD41 (Fig. 4c), and CD81 cells (Fig. 4d) but not TcRgd1 cells in the germinal centers. Moreover, the TcRab1 and CD41 T lymphocytes predominate in the lamina propria of tonsillar villi, whereas TcRgd1 and CD81 cells appeared in both the subepithelial zone and the epithelium, as previously indicated. According to the flow cytometrical results, although the number of TcRgd-expressing cells had increased in respect to previous stages, the TcR21 cells were the most abundant T lymphocytes in the adult chicken caecal tonsil (Fig. 2c). Also the numbers of CD81 cells remained higher than those of CD41 lymphocytes (Fig. 2b). DISCUSSION Our immunohistochemical results confirm the existence of a rudiment of the caecal tonsil at the end of embryonic life, previously described as small clusters of lymphocytes and reticular cells in the caecal walls (Looper and Looper, 1929; Jankovik and Mitrovic, 1967; Hoshi and Mori, 1973). These clusters consist of His-C7– 187 positive cells which presumably include, apart from macrophages, B lymphocytes and a few ab T cells, although the time of appearance of these later cells in the chiken embryonic gut is a matter of discussion. Bucy et al. (1990) reported that they migrate into the intestine on day 20 of embryonic life, whereas Vainio et al. (1988) described that only after hatching was there a substantial migration of TcRab-expressing cells to the chicken peripheral lymphoid organs. Furthermore, some early gd1 T cells also appear in the embryonic caecal epithelium. They probably correspond to a first wave of gd T cells which colonize the chicken gut 2 days after their appearance in the thymus, since the second one occurs just after hatching (Dunon et al., 1993), in agreement with the postnatal increase of caecal gd T cells observed in the current study. Despite its appearance at the end of embryonic life, the chicken caecal tonsil grows principally just after hatching. Our EM analysis demonstrates that, 24 h posthatching, the proximal zone of chicken caecal tonsil contains all the lymphoid elements, including intraepithelial lymphocytes, immature plasma cells, and blast cells, as well as macrophages and interdigitating cells, observed in adult caecal tonsil. In the following days, the caecal lymphoid tissue grows quickly, reaching the adult histological condition 4 days after hatching in a process which apparently involves both in situ cell proliferation and massive migration of circulating lymphocytes. It continues to grow, nevertheless, at least until the sixth week. In agreement, Hoshi and Mori (1973) indicated that the caecal tonsil is fully developed in 10-day-old chickens, although the number of germinal centers is still low. The occasional observation of cryptosporidians in some specimens used in the current study could explain the fast development of the tonsillar lymphoid tissue. The influence of intestinal antigens in the postnatal development of chicken caecal tonsil is generally accepted (Boyd and Ward, 1984; Jeurissen et al., 1991), although there is some controversy about the accessibility of antigens to the caecum during postnatal life (Sorvari et al., 1977; Befus et al., 1980; Jeurissen et al., 1991). Research in progress on the development of caecal tonsil in germ-free chickens could clarify this subject. In respect to the lymphoid cell populations phenotypically identified in the developing chicken caecal tonsil, both T and B lymphocytes (IgM- but not IgG- or IgA-expressing cells) occur in the incipient caecal tonsils of 1-day-old chickens. Boyd and Ward (1984), however, did not find B lymphocytes identified by an anti-chicken mature B cell alloantiserum either in the caecal tonsil or in the small intestine of 1-day-old chickens, and Bucy et al. (1988) indicated that the first T lymphocytes appear in the gut of 3-day-old chickens. The number of TcRgd1 T cells remains low at this stage, increasing in 4-day-old chickens. There are, nevertheless, certain discrepancies between our own immunohistochemical and flow cytometrical results, presumably related to technical problems in isolating the cell populations, especially those such as TcRgd T cells, which mainly occupy the caecal epithelium. In the following days, although T cells remain the predominant lymphoid cell subpopulation, the number of B lymphocytes increases in the caecal tonsil. In agreement, Jeurissen et al. (1989) described the exis- 188 M. GÓMEZ DEL MORAL ET AL. tence of IgM1, IgA1, and IgG1 blasts as well as mature plasma cells in the caecal tonsil of 5-day-old chickens. These B cell subsets are mainly found in the subepithelial region, as previously reported by other authors (Jankovic and Mitrovic, 1967; Hoshi and Mori, 1973; Fonfrı́a, 1992), whereas TcRab1 CD41 T cells occur in the central and deep zones of the tonsil (Lillehoj and Trout, 1996). These areas, named diffuse lymphoid tissue, were identified as T-dependent regions (Jankovic and Mitrovic, 1967; Hoshi and Mori, 1973; Fonfrı́a, 1992), which in adult chicken tonsils contain TcRabexpressing lymphocytes (Vainio et al., 1988). On the contrary, they are rare in the lamina propria or in the epithelium, as previously reported in other intestinal locations (Bucy et al., 1990). Both CD81 lymphocytes and TcRgd-expressing cells predominate, however, in the intraepithelial lymphoid cells as well as in the subepithelial zone, although there are a few CD81 cells in the diffuse lymphoid tissue which presumably expresses the ab T cell receptor. Although well-organized germinal centers were not histologically identified until the second week of postnatal life, in agreement with other reports (Jeurissen et al., 1989; Jankovic and Mitrovic, 1967), our immunohistochemical study demonstrates areas in the 4-day-old caecal tonsil which are presumably similar to the cell clusters of large lymphoblasts and reticular cells identified in 8-day-old chickens as precursors of the tonsillar germinal centers by Hoshi and Mori (1973). In mammals, the retention of immune complexes on the surface of follicular dendritic cells is a key process for the development of germinal centers (Liu et al., 1992). Both IgM1 and IgG1 cells but not IgA1 cells appear in the first germinal centers observed in 2- week-old chickens, a fact previously reported by Jeurissen et al. (1989). They also contain a few CD41 TcRab1 T cells and even fewer CD81 cells but not TcRgd cells. The existence of T cells in the germinal centers has been described in both birds (Bucy et al., 1988) and mammals (Kosco and Gray, 1992), and thymectomy decreases the numbers of germinal centers in the caecal tonsils and other peripheral lymphoid organs of chickens (Jankovic and Mitrovic, 1967; Hoshi and Mori, 1973). In the last stage studied (6 weeks), there is a remarkable increase of B lymphocytes which reach higher values than those of T cells, confirming previous results using specific alloantisera (Albini and Wick, 1974). In parallel, IgA-expressing cells undergo an important increase, appearing, apart from in the subepithelial zone, in the germinal centers. The information available on Ig isotypes expressed by chicken gut B lymphocytes is controversial. Whereas some authors have noticed a higher number of IgG1 cells than IgAexpressing cells in adult caecal tonsil (Jeurissen et al., 1989; del Cacho et al., 1993; Lillehoj and Trout, 1996), Peyer’s patches (Burns, 1982), and intestinal mucosae (Arnaud-Battandier et al., 1980), others, in agreement with our own results, detected few IgG1 cells in the chicken GALT (Boyd and Ward, 1984). In the mammalian GALT, the number of IgA1 cells greatly exceeds that of IgG1 B lymphocytes (Crabbe and Heremans, 1966). Finally, from the first few days of development, CD81 lymphocytes and gd T cells, as in other gut locations (Bucy et al., 1988), comprise the main cell population which appears in the epithelium covering the caecal tonsil. Because, as confirmed by our own flow cytometrical studies, more than 65% of chicken gd T cells express CD8 (Bucy et al., 1990; Lillehoj and Trout, 1996), most intraepithelial lymphocytes found in the developing caecal tonsil are CD81gd1 cells. The remaining intraepithelial CD81 cells might be TcR0 cells, a cell population found in chickens which appears early in ontogeny, expressing cytoplasmic CD3 but neither TcRab nor TcRgd, and it is functionally related to NK cells (Chen et al., 1990). In adult chickens, this cell population represents 30–40% of the total cytoplasmic CD31 cells present in the intestinal epithelium (Chen et al., 1990). CD41 cells were never detected, and only a few TcRab lymphocytes were occasionally identified in this location. Moreover, in agreement with pioneer results from thymectomized and/or bursectomized chickens (Back, 1970a,b), the number of T cells is higher than that of B lymphocytes in the gut epithelium. More recently, the presence of B lymphocytes in the chicken gut epithelium was immunohistochemically confirmed using specific mAbs (Jeurissen et al., 1989). In both mice and humans, intraepithelial lymphocytes are also mainly T cells, although these are phenotypically more heterogeneous than those described in chickens (for review see Guy-Grand and Vasalli, 1993). 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