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THE ANATOMICAL RECORD 250:182–189 (1998)
Appearance and Development of Lymphoid Cells in the Chicken
(Gallus gallus) Caecal Tonsil
Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain
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.
Received 10 April 1997; Accepted 21 October 1997
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
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.
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
S.H.M. Jeurissen
(Central Veterinary
Institute, Lelystad,
M.D. Cooper (University of Alabama,
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.
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
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).
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.
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
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
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).
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–
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-
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,
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).
In summary, our combined immunohistochemical
and flow cytometrical results suggest the following: 1)
although B lymphocytes presumably occur already in
the rudiment of the caecal tonsil identified at the end of
embryonic life, T cells, mainly TcRab1 and CD41 cells,
predominate in the chicken caecal tonsil until 2 weeks
of postnatal life; 2) later B lymphocytes, which predominatly express IgM or IgA, constitute the main lymphoid
cell subpopulations; 3) likewise, CD81 cells, which
gradually increase after 2 weeks, become more numerous in the chicken caecal tonsils than CD41 lymphocytes; and 4) this cell subset is not, however, homogeneous, comprising, apart from TcRab-expressing cells,
gd T cells and presumably NK cells, these two latter cell
populations principally occuring in the tonsilar epithelium.
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