Veterinary Immunology and Immunopathology, 31 (1992) 347-360 Elsevier Science Publishers B.V., Amsterdam

347

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens Hyun S. Lillehoj 1 and Kyeong S. Chung Protozoan Diseases Laboratory, Livestock and Poultry Sciences Institute, US Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705 USA (Accepted 15 April 1991 ) ABSTRACT Lillehoj, H.S. and Chung, K.S., 1992. Postnatal development ofT-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens. Vet. Immunol. Immunopathol., 31- 347360. Postnatal development of various T-lymphocyte subpopulations expressing CD3, CD8, CD4, and antigen-specific TCR heterodimers aft (TCR2) or y~ (TCR1) was investigated in two different inbred chicken strains, SC and TK. The ratios of jejunum T-cells expressing TCR1 to TCR2 in the intraepithelium of SC and TK strains gradually increased after hatching and were 3.40 and 4.28 by 12 weeks in TK and SC chickens respectively. The ratios of TCR 1+ to TCR2 +-cells in intraepithelium and the lamina propria in SC chickens were 0.96 and 1.23 at 8 weeks and 4.29 and 2.15 at 12 weeks, respectively. Jejunum intraepithelial lymphocytes expressing the CD8 antigen increased gradually until 4-6 weeks of age and subsequently declined as chickens aged. CD4+-cells represented a minor subpopulation among the intestinal lymphocyte subpopulations. Therefore, the composition of various T-cell subpopulations in the intestine depended upon host age, the regions of the gut examined and host genetic background. These results suggest that changes in T-cell subpopulations in the intestine may reflect age-related maturation of the gut-associated lymphoid tissues.

ABBREVIATIONS FACS, fluorescence activated cell sorter; GALT, gut-associated lymphoid tissues; IEL, intraepithelial lymphocytes; IF, immunofluorescence; LPL, lamina propria lymphocytes; mAb = monoclonal antibodies; PE, phycoerythrin.

INTRODUCTION T h e i n t r a e p i t h e l i a l l y m p h o i d p o p u l a t i o n is a c o m p o n e n t o f t h e g u t a s s o c i ated immune system that has evolved unique features including antigen pres e n t i n g , i m m u n o r e g u l a t o r y , a n d e f f e c t o r cell t y p e s t h a t a r e d i s t i n c t f r o m t h e i r c o u n t e r p a r t s i n t h e s y s t e m i c i m m u n e s y s t e m . It is e v i d e n t t h a t c o n s i d e r a b l e ~Author to whom correspondence should be addressed. © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

348

H.S. LILLEHOJ AND K.S. C H U N G

heterogeneity exists in the intestinal T-lymphoid cell population (Befus et al., 1980; Ernst et al., 1986). Two major lineages of T-cells have been defined with regard to the expression of the CD3-associated TCR heterodimer: those composed of aft (TCR2) polypeptide chains and those with 7~ (TCR1) chains (Allison and Lanier, 1987; Brenner et al., 1986, 1987b). Recent studies in mice have indicated that systemically distributed T-cells express TCR2 (Allison and Lanier, 1987) whereas those restricted to the skin (Koning et al., 1987; Kuziel et al., 1987; Stingl et al., 1987 ) and gut (Bonneville et al., 1988; Goodman and Lefrancois, 1988) preferentially express TCR1. These observations led to a view that anatomical dichotomy in the tissue distribution of these two T-cell lineages is associated with unique functions (Janeway et al., 1988 ). Similar results have also been reported in avian species (Bucy et al., 1988 ) and humans (Bucy et al., 1989) where immunohistological examination has shown that TCR1 + T-cells are localized in the epithelium whereas T-cells expressing TCR2 are found in lamina propria. Furthermore, TCR 1÷cells in intraepithelial lymphocytes (IEL) were exclusively shown to be CD3 + C D 4 - C D 8 +. However, further in situ and flow cytometric analyses of isolated human IEL provided a contrasting view since the majority (70-90%) of the CD3 ÷ CD8 ÷-cells expressed the o~flheterodimer and less than 8% were ~ + T-cells (Cerf-Bensussan et al., 1987; Brandtzaeg et al., 1989b). These observations suggested that species differences in the expression of TCR genes as well as other lymphocyte surface markers in the intestinal mucosal lymphoid system are greater than generally appreciated. This study investigated the postnatal development of chicken T-cells expressing TCR 1 and TCR2 using a newly defined set of well characterized mAb. The results described herein reveal differences in the T-cell composition of IEL and lamina propria lymphocytes (LPL) isolated from anatomically diverse regions of the intestines of SC and TK chickens as well as strain differences in maturation of intestinal T-cell subpopulations. MATERIALSAND METHODS

Chickens Embryonated eggs of SC(B2B 2) and TK(B15B z~) inbred chickens were purchased from Hy-line International Production Center, Dallas Center, IA. Healthy chickens of both sexes were housed in clean cages and provided with water and food ad libitum. Special care was taken to avoid exposure to specific pathogens such as Marek's disease virus, herpes virus, mycoplasma or Eimeria spp.

POSTNATAL DEVELOPMENT OF CHICKEN INTESTINAL T-CELLS

349

Monoclonal antibodies (mAb) used The following mAbs were used: anti-TCR1 and anti-TCR2 (Chen et al., 1988); CT4 (anti-CD4) and CT3 (anti-CD3) (Chan et al., 1988); 13-3A1 (anti-human T-cell) (ATCC, Rockville, MD ); CTLA3 (anti-CD8) (Lillehoj et al., 1988); K-55 (anti-pan lymphocytes (Chung et al., 1990). The optimum dilutions of all the mAb were predetermined.

Preparation of intestinal IEL and LPL IEL were prepared as described (Tagliabue et al., 1982; Chai and Lillehoj, 1988 ) with some modifications. In brief, the jejunum (from below the duodenal loop to just before Meckel's diverticulum), duodenal c-loop, or caeca were removed from ten chickens, cut longitudinally, washed in several changes of ice-cold Ca 2+ and Mg 2+ free HBSS supplemented with 5% FCS (Hyclone Lab. Inc., Logan, UT) (5% CMF-HBSS) and cut into 1-2 cm pieces. In some experiments, the longitudinally open sections were clamped and rubbed with fingers in 5% CMF-HBSS to mechanically remove IEL (Arnaud-Battandier et al., 1980) and thereafter cut into smaller pieces. Intestinal mucus was removed by sequential treatments with 10 mM dithiothreitol (Sigma, St. Louis, MO) in 5% CMF-HBSS for 7-10 min at room temperature and 10 - 4 M EDTA in 5% CMF-HBSS for 15-20 min at 41°C. Cells in the supernatant were washed two or three times, and passed through nylon-wool (Robbins Scientific Co., Mountain View, CA) to remove dead cells and cell aggregates. The nylon wool column-treated cells consisted of small, round, viable leukocytes and larger epithelial cells. LPL were prepared as described (Tagliabue et al., 1982) with some modifications. In brief, the intestinal pieces that had rendered the IEL were extensively washed with ice-cold 5% CMF-HBSS and incubated in 5% CMF-HBSS containing 10 -4 M EDTA for 15-20 min with frequent gentle agitation. The cell-containing supernatant was discarded to minimize contamination with IEL. The remaining pieces were extensively washed, cut to 5-mm pieces and incubated with 1.2 U ml-1 of Dispase (Bohringer Mannheim, Indianapolis, IN) in 5% CMF-HBSS at 37°C with frequent gentle swirling. The supernatant fluid containing LPL was washed three times, passed through nylon wool and further purified by either centrifugation on a discontinuous Percoll density gradient as described (Chai and Lillehoj, 1988), or by the mechanical method described (Arnaud-Battandier et al., 1980). In the latter case, intestinal pieces were washed twice with 5% CMF-HBSS supplemented with 10 - 4 M EDTA until the supernatant fluid was clear, the lamina propria scraped from the muscularis mucosae with a surgical blade, homogenized with a glass tissue grinder, the tissue homogenate washed twice and passed through nylon wool.

350

H.S. LILLEHOJ AND K.S. CHUNG

In some experiments, IEL and LPL were further purified by centrifugation on a discontinuous Percoll density gradient by resuspension in 30% Percoll and centrifugation at 500 X g for 20 min at 20 oC. The epithelial cell-containing supernatant was discarded and the cell pellet, which consists mostly of leukocytes, was resuspended in 30% Percoll to 2 × 107 cells ml-1, layered on the top of Percoll layers of 70, 60, 50 and 40%, and centrifuged again. The cells accumulating at the interfaces of 40/50% and 50/60% were pooled and washed three times. Cell viability was greater than 90% as determined by trypan blue exclusion.

Preparation of spleen and caecal tonsil lymphocytes Spleen and caecal tonsil lymphocytes were prepared as described (Lillehoj et al., 1988 ). Freshly removed spleens or caecal tonsils were homogenized by gently pressing them through a stainless steel mesh. The cells were washed twice, quickly passed through a nylon wool column, centrifuged, and resuspended in immunofluorescence (IF) staining buffer (Eagle's balanced salt solution without phenol red supplemented with 3% FCS and 0.1% sodium azide ) to 1 X 107 cells m l - 1.

Conjugation of mAb with biotin For two color IF analysis, mAb CTLA3 was biotinylated as described (Bayer et al., 1979) with modifications. The mAb was precipitated with saturated ammonium sulfate and dialyzed first against distilled water and then 0.1 M sodium bicarbonate. Two hundred and fifty micrograms of biotinyl-N-hydrosuccinimide ester (1 mg ml-~ in dimethylsulfoxide) was added to 2 mg of the mAb solution and incubated at room temperature for 1.5 h. The biotinconjugated antibody was desalted with Sephadex G-25 (Pharmacia Biotechnology, Uppsala, Sweden ) according to the manufacturer's specifications and titrated to determine the optimal working dilution.

IF and fluorescence activated cell sorter (FACS) analysis Indirect IF was carried out as described (Lillehoj et al., 1988 ). Cells were resuspended in the staining solution. 1 × 106 cells were incubated with mAb followed by FITC-conjugated goat anti-mouse IgG (Sigma). For two-color IF, cells stained with mAb and FITC-conjugated goat anti-mouse IgG were incubated with biotin-conjugated mAb CTLA3 followed by phycoerythrin (PE)-conjugated streptavidin (Becton Dickinson, Mountain View, CA). Unless stated otherwise, incubations were performed on ice for 30 min, washed two or three times with staining buffer after each incubation, and analyzed

POSTNATALDEVELOPMENTOFCHICKENINTESTINALT-CELLS

351

using an EPICS PROFILE II (Coulter Corp., Hialeah, FL) flow cytometer. Data were collected on a total of 1 × 104 viable lymphocytes.

Statistical analysis Statistical evaluation of all results was performed by testing the significance of group difference using the Student's t-test or by analysis of variance. RESULTS

Comparison of different methods of lEL and LPL preparation Because mechanical and chemical isolation techniques have been used most commonly to prepare intestinal lymphocytes in mammalian systems, single cells isolated by these methods from the intraepithelium and lamina propria of the chicken were compared. The composition of intestinal cells varies depending upon the methods used to isolate them (Ernst et al., 1987 ). As shown in the Table l, the overall cell yield varied according to the method employed. Preparation of IEL by incubation with EDTA or LPL by dispase treatment produced significantly more viable intestinal cells when compared with the mechanical preparation methods ( P < 0.05 ). The percentages of cells stained with mAb against CD3, or TCR1 in LPL prepared by different methods were statistically different ( P < 0.05 ) but the percentages of cells stained with mAb against CD3, TCR1, TCR2 or CD8 in all the other preparations were not statistically different ( P > 0.05 ). For the experiments described below, IEL were prepared by EDTA and LPL by dispase procedures. TABLE1 Lymphocyte composition oflEL and LPL preparations separated by mechanical and chemical methods ~ Antigen

CD3 TCR1 TCR2 CD8

IEL

LPL

Rubbing ~ (5.4X 106) 2

EDTA ( 10.8X 106)

Scraping (24.8 X 106 )

Dispase (62X 107 )

69.53 31.6 23.7 33.2

71.4 38.1 24.1 29.4

68.7 35.7 22.5 29.5

84.2 51.0 21.0 30.9

aPrepared as described in Materials and Methods. 2Leucocyte numbers obtained. 3% Positive cells compared to total number of K-55 +-cells.

352

H.S.

LILLEHOJ

AND

K.S. C H U N G

Postnatal changes in the IEL subpopulations To investigate the dynamics of intestinal lymphocyte subpopulations, jejunum IEL from SC and TK chickens ( 5 - 1 0 chickens per group) were examined for the expression of various T-cell surface antigens between 1 and 15 weeks after birth. In SC chickens, the percentage of CD3 +-cells increased from 24.4% of total lymphocytes (K-55 + ) at 1 week to 42.0% at 2 weeks and 62.6% at 4 weeks. Between 6 and 15 weeks, CD3+-cells represented about 70% of the total lymphocytes. The percentages of T-cells expressing the TCR 1, TCR2, CD4 or CD8 antigens were calculated with respect to the total number of CD3+-cells. As shown in Fig. 1A, the fraction of TCR1 +-cells gradually increased from 16% of the total CD3+-cells at 1 week to 78% at 15 weeks. In contrast, TCR2+-cells, which represented 96% of the CD3 + population at 1 week, gradually decreased to 16% of CD3 +-cells at 15 weeks. CD8 +-cells constituted 43% of the CD3 +-cells at 1 week, increased to 72% by 4 weeks, and gradually decreased to 34% of CD3+-cells at 15 weeks. CD4+-cells represented a relatively minor subpopulation of CD3 +-cells through 15 weeks of age, declining from 15% at 2 weeks to 3% at 15 weeks. T-cell subpopulations in jejunum IEL of TK chickens showed maturation patterns that are different from that seen in SC chickens. CD3+-cells in TK B

1O0 "

H

TCR1

r]--.~TCR2

~ co8

~0 O

80"

80 t = =TCR1 TCR2 70 ~ CD8 60

+

0360 Q 0

4O I

~

40

aol=

E 0

2° 1

0-20

=

lo4 0

)

54

;

;l?"S

0

1

i

2

i

4

i

6

1'2

Age in Weeks

Fig. 1. Postnatal change of T-cell subpopulations in the intestinal epithelium of SC chickens (A) and TK chickens (B). The cells were isolated from jejunal epithelium, IF stained with mAbs CT3, CT4, CTLA3, TCR1 and TCR2, which react with avian homologues of mammalian CD3, CD4, CD8, TCR1, and TCR2, and then analyzed using EPICS II Profile flow cytometer. The percentage of each subpopulation was calculated by taking that of CT3+-cells as 100%. Each data point represents the value obtained from the cell preparation from more than five chickens.

POSTNATALDEVELOPMENTOF CHICKEN INTESTINALT-CELLS

353

chickens reached the adult level m u c h earlier than in SC chickens (data not shown ). As early as 1 week, the level of CD3 +-cells ( 44% of K-55 +-cells ) was higher than that of SC chickens, stabilized at about 67% from 2 through 6 weeks and then increased to 93% at 12 weeks. As shown in Fig. 1B, the percentage o f T C R 1 +-cells in the total CD3 +-cell population in TK chickens also started at higher level (27% at 1 week) compared to SC chickens and increased to 69% at 12 weeks. TCR2, the major T C R type at 1 week (56% of total CD3 +-cells ), slowly decreased and represented 20% of total CD3 +-cells at 12 weeks. CD8 +-cells increased from 36% of CD3 +-cells at 1 week to 66% at 6 weeks but thereafter decreased to 39% at 12 weeks.

D&tribution of TCR1 and TCR2-lymphocytes in intraepithelium and lamina propria To assess the tissue distribution of T C R 1 + and TCR2 +-cells in the two main compartments of intestine, IEL and LPL from SC chickens of different ages were analyzed by FACS with mAb against these antigens (Table 2 ). At 8 weeks of age, TCR1 + and TCR2+-cells were present in approximately equal numbers in IEL and LPL. However, TCR1 +-cells in the intestinal intraepithelium increased significantly above TCR2 +-cells at 9 weeks and showed a fourfold increase over TCR2 +-cells at 12 weeks ( P < 0.05 ). The frequency of T C R 1 +-cells in the lamina propria was also significantly higher than that of TCR2+-cells at 9 and 12 weeks of age ( P < 0 . 0 5 ) . A similar trend was observed in TK chickens (data not shown). The ratios of T C R 1 + and TCR2 +cells in the spleen and caecal tonsil in 8-week-old SC chickens were 1.14 and 0.25 respectively.

Comparison of lEL and LPL T-lymphocyte subpopulations from different anatomical regions of the intestine IEL and LPL obtained from the d u o d e n u m , jejunum, caeca, or caecal tonsils of 6- to 8-week-old SC chickens were examined for their constituent Tlymphocyte subpopulations. Although the percentages of TCR 1+ and TCR2 +TABLE2 Ratios of TCR 1+ to TCR2 +-cells in IEL and LPL from the jejunum of SC chickens of different ages Age (weeks)

IEL

LPL

8 9 12

0.961 1.58 4.29

1.23 2.43 2.15

~The ratio was calculated by dividing the percentage positivity of the cells to mAb TCR1 with that of TCR2 in the same cell preparation pooled from two or more chickens.

354

H.S. LILLEHOJ AND K.S. CHUNG

TABLE 3 Percentage composition of intestinal IEL and LPL and caecal tonsil lymphocytes of 6- to 8-week-old SC chickens Antigen

TCR 1 TCR2 CD3 CD8 CD4

IEL ~

LPL ~

Caecal tonsil

Duo.

Jej.

Caeca

Duo.

Jej.

Caeca

32.72 32.1 72.1 36.8 N.D.

33.3 33.2 69.0 24.5 7.4

37.2 19.0 61.5 27.5 N.D.

32.9 36.7 79.5 N.D. N.D.

45.8 26.0 83.9 32.3 7.8

52.9 19.6 78.3 N.D. N.D.

3.9 12.7 22.0 7.3 7.8

~Cells were isolated as described in Materials and Methods. 2Each data represents the mean value from two or more analyses done on the cell preparations from two or more chickens. Percent positive cells compared to total number of K-55 +-cells. N.D., not done.

cells in these regions were not significantly different from each other ( P > 0.05 ), they were all significantly greater ( P < 0.05 ) than the fraction of the lymphocytes in the caecal tonsil expressing these antigens. The percentages of duodenum, jejunum and caecum TCR1 +-cells were 33%, 33%, and 37% respectively in the intraepithelium and 33%, 46% and 53% in the lamina propria. TCR2+-cells represented 32%, 33% and 19% in the duodenum, jejunum and caecum respectively and 37%, 26% and 20% in the lamina propria (Table 3). CD3 + IEL varied between 62% and 72% while the fraction of CD3 + LPL ranged from 78% to 84% of the K-55 + population in these regions of the intestine. In contrast, 22% of the total caecal tonsil lymphocytes were CD3 +. The percentages of CD8 + T-lymphocytes ranged from 25% to 37% in IEL and constituted 32% in jejunum LPL and 8% of the caecal tonsil lymphocytes. CD4+-cells represented 7% in jejunum IEL and 8% in LPL and caecal tonsil lymphocytes. Interestingly, only 30-40% of the total jejunum T-lymphocytes (TCR1 + or TCR2 + ) expressed either CD4 + or CD8 +. Surface IgM + and IgG+-cells constituted minor subpopulations representing 1.6% and 1.2% of jejunum IEL, and 3.7% and 4.1% of jejunum LPL respectively. The majority of the caecal tonsil lymphocytes were immunoglobulin expressing cells with IgM as a predominant isotype (data not shown).

Coexpression of CD8 with TCR1, TCR2, or CD4 IEL isolated from the jejunum of SC chickens were analyzed by two-color IF for expression of CD8 and TCR1, TCR2 or CD4. As shown in Fig. 2, 26% and 43% of these T-cells expressing TCR 1 and TCR2 respectively were stained with the anti-CD8 mAb. In contrast, 73% and 44% of splenic T-cells expressing TCR1 and TCR2 were CD8+-cells while 54% and 24% from the caecal

POSTNATALDEVELOPMENTOF CHICKENINTESTINALT-CELLS

Spleen

IEL A 2

I

355

C.T.

C

B

a

0

_a

3

O

~

D

I

F

G

H 7

I

I

C~I

n" O

F

3

:

,,-

4

"~

CD8-Red

3::--:

4

Fluorescence

Fig. 2. Two-color IF analysis of CD8 antigen expression on T-ceils expressing TCR ] and TCR2. The cells were first stained with mAb TCRI, TCR2, and CT4 followed by FiTC-conjugated goat anti-mouse IgG, and then followed by biotin-conjugated CTLA3 and PE-conjugated streptavidin. The percentage of the cells in each region is depicted in the Table below.

Group

A B C D E F G H I

Percentage of cells in region 1

2

3

4

18.9 10.6 23.4 10.5 16.7 1.6 14.2 8.3 14.6

0.4 0.1 0.3 28.5 5.5 1.9 11.1 5.5 4.5

35.5 78.5 68.4 45.5 77.2 91.9 41.2 77.2 77.0

45.2 10.8 7.9 15.6 9.0 5.4 33.5 9.0 3.9

356

H.S. LILLEHOJ AND K.S. C H U N G

tonsils expressed TCR 1 and TCR2-cells in conjunction with CD8 +. In all of these cell preparations, CD4 + and CD8 +-cells were mutually exclusive. DISCUSSION

The results of this study indicate that changes in the composition of Tlymphocyte subpopulations in the intestine reflect an age-related maturation of gut-associated lymphoid tissues (GALT). This conclusion is based upon the following observations: ( 1 ) The ratio ofT-cells expressing TCR1 to TCR2 in the jejunum intraepithelium increased with age. (2) CD8 +-cells gradually increased in number with age, reaching the adult level at 4 weeks with a subsequent decline at 15 weeks. (3) CD4+-cells decreased with age. Furthermore, significant differences in the composition of various subpopulations of lymphocytes existed, depending upon the genetic make-up of the host and the region of the intestine from which the IEL were isolated. Other age-related changes in the maturation of the intestinal lymphoid system have been described (Befus et al., 1980; Jeurissen et al., 1988). The factors that influence the shift in T-cell composition are not clear but it is probably heavily influenced by environmental antigen exposure. As chickens age, intestinal lymphoid aggregates undergo involution and the distribution of lymphoid follicles become less distinct and fewer in number. There appears to be a relative depopulation of the subepithelial zone in both caecal tonsil and peyers patches around 20 weeks of age (Befus et al., 1980). The number of germinal centers are also sparse in the newborn animal, and rise in number as the animal is exposed to environmental antigens (Jeurissen et al., 1988). This suggests that the presence of IEL cells in the small intestine is a function of intraluminal antigenic stimulation. SC and TK chickens are easily available commercially and provide a valuable experimental model to study the ontogenic acquisition of immune response. The present study shows that a strain difference exists in postnatal development of intestinal T-lymphocytes. Whether the difference seen in these two chicken strains is due to a host genetic factor or not needs to be established. In SC and FP chickens, a strain closely related to TK, a difference in T-lymphocyte complement was reported (Goidl and Thesis, 1985 ). Furthermore, SC and FP chickens show different disease susceptibility to Marek's disease virus (Hudson and Payne, 1973 ) and to Eimeria infections (Lillehoj, 1987). Early in chicken fetal development, precursor thymocytes pass through the thymus and acquire separate functional activities (Vainio and Lassila, 1989; Sowder et al., 1988). Ontogenic analysis of avian thymocytes expressing the antigen specific TCR has revealed that TCR 1 is expressed on a small percentage of cells by embryonic Day 11, increases to 30% by Day 15, and then declines to about 5% by hatching (Sowder et al., 1988). CD3+-thymocytes

POSTNATAL DEVELOPMENT OF CHICKEN INTESTINAL T-CELLS

3 57

bearing TCR2 appear after Day 15 of fetal development and then quickly increase in number to exceed the level of T-cells expressing TCR I. Thymocytes expressing the CD4 antigen appear on embryonic Day 13 and increase rapidly to 90% by birth (Chan et al., 1988 ). The appearance of CD8 + T-cells during embryonic development is very similar to that of CD4+-cells, and by the end of embryogenesis, most thymocytes express both of these molecules. In the periphery, however, CD4+-cells can be observed in substantial numbers only after hatching and by the end of the first month, adult levels have been reached: 20% of spleen cells and 40-45% of blood lymphocytes (Chan et al., 1988 ). Furthermore, these CD4+-cells occupy characteristic histological locations, periarteriolar sheaths of the spleen and lamina propria of the intestine. A previous study reported that chicken TCR 1 + and TCR2+-cells occupy distinct histological microenvironments with the majority of intestinal IEL being TCR1 + (Bucy et al., 1988). In contrast, we found that at 1-4 weeks, TCR1 +-cells only constituted a minor fraction (16%-27%) while TCR2 +cells were the major subpopulation (48%-96%). Over time, the percentage of IEL TCR 1+-cells increased and TCR2 +-cells decreased such that at 6 weeks, TCR 1+-cells predominated. These observations suggest that the changes in TCR subpopulations occur as a result of an age-dependent maturational process. In contrast to a published result (Bucy et al., 1988 ), throughout postnatal development we observed both TCR 1+ and TCR2 +-cells to be present in the intraepithelium and lamina propria. Two color IF indicated that both TCR 1+ and TCR2 +-cells coexpressed the CD8 antigen although the percentage of cells expressing TCR 1 and CD8 or TCR2 and CD8 varied with the time source. Furthermore the present study shows for the first time that a significant proportion of intestinal T-lymphocytes in chickens do not bear CD4 or CD8 antigen. The differences seen between this and the previous study (Bucy et al., 1988 ) may be due to a different methodology (in situ immunohistology vs. flow cytometric analysis of isolated IEL and LPL) or host related factors such as age, strain or intestinal region examined. In humans, TCR2 +-cells represent 70%-92% of CD3 +CD8 + jejunum IEL whereas TCR 1+-cells represent below 8% (Brandtzaeg et al., 1989b). CD8 +cells represented 84%, 87% and 87% in jejunum, ileum, and colon IEL and 31%, 34% and 36% in jejunum, ileum, and colon LFL respectively (Smart et al., 1988). Most IEL expressed the CD8 antigen and 20% of IEL expressed neither CD4 or CD8 (Brandtzaeg et al., 1988 ). In contrast to these results, a recent study showed a preferential location of human Tyc~-cells (80% of intestinal TCR 1 ) in the intestinal epithelium and Taft-cells in the lamina propria (Bucy et al., 1989) as stained by the mAb flF1 (Brenner et al., 1987a). In mice, IEL consisted of 80% Lyt-2 whereas LPL consisted of 32% Lyt-2 cells (Ernst et al., 1986). Virtually all ( > 80%) of the IEL expressed the CD8 an-

3 58

H.S. LILLEHOJ AND K.S. CHUNG

tigen and less than 5% were C D 4 + C D 8 - (Bonneville et al., 1988). CD3 ÷ IEL expressed p r e d o m i n a n t l y y8 T C R (Bonneville et al., 1988; Klein, 19 86 ). The recently proposed concept of a d i c h o t o m y in the distribution of TCR2 and TCR1 subsets in mice (Janeway et al., 1988) m a y not hold true in m a n (Brandtzaeg et al., 1989a) or chickens. Whether this discrepancy truly reflects a major species difference remains to be clarified. Despite the suggestion that T C R 1 +-cells are different in ontogeny and antigen specificity from TCR2+-cells (Tonegawa et al., 1989), the function of these cells in the GALT i m m u n e system is not clearly understood. Two as yet unproven functions have been ascribed to the intestinal IEL, MHC-restricted or non-restricted cytotoxicity (Borst et al., 1987; Moingeon et al., 1987 ). In chickens, the IEL isolated from the small intestine had N K cell activity against the avian t u m o r target LSCC-RP9 (Chai and Lillehoj, 1988) and this IEL N K activity significantly increased during the early phase of secondary infection by Eimeria (Lillehoj, 1989). Further investigation of the function o f l E L T C R 1 ÷ and T C R 2 ÷-cells in normal and disease states will provide enhanced understanding of their i m m u n o r e g u l a t o r y functions in the gut. ACKNOWLEDGMENTS The authors thank Marjorie Nichols for excellent technical assistance. Critical review of the manuscript by Drs. L. Bacon, K. M a d d e n and L. Keller is deeply appreciated. Generous gift o f monoclonal antibodies from Dr. ChenLo Chen ( T C R 1, TCR2, CT3 and CT4) is deeply appreciated. This work has been supported in part by the U S D A Competitive Grant 86-CRCR-1-2209. Dr. K.S. Chung was on Sabbatical leave from Chung N a m National University, Tae-jon, Korea.

REFERENCES Allison, J.P. and Lanier, L.L., 1987. Structure, function, and serology of the T-cell antigen receptor complex. Annu. Rev. Immunol., 5: 503-540. Arnaud-Battandier, F., Lawrence, E.C. and Blaese, R.M., 1980. Lymphoid populations of gut mucosa in chickens. Dig. Dis. Sci., 25: 252-259. Bayer, E.A., Skutelsky, E. and Wilchek, M., 1979. The avidin-biotin complex in affinity cytochemistry. Methods Enzymol., 62:308-315. Befus, A.D., Johnston, N., Leslie, G.A. and Bienenstock, J., 1980. Gut-associated lymphoid tissue in the chicken. I. Morphology, ontogeny, and some functional characteristics of Peyer's patches. J. Immunol., 125: 2626-2632. Bonneville, M., Janeway, C.A., Jr., Ito, K., Haser, W., Ishida, I., Nakanishi, N. and Tonegawa, S., 1988. Intestinal intraepithelial lymphocytes are a distinct set of y~ T cells. Nature, 336: 479-481. Borst, J. V., Van de Griend, R.J., Van Oostveen, J.W., Ang, S.L., Melief, C.J., Seidman, J.G.

POSTNATAL DEVELOPMENT OF CHICKEN INTESTINAL T-CELLS

3 59

and Bolhius, R.L.H., 1987. A T-cell receptor ~,/CD3 complex found on cloned functional lymphocytes. Nature, 325: 683-688. Brandtzaeg, P., Sollid, L.M., Thrane, P.S., Kvale, D., Bjerke, K., Scott, H., Kett, K. and Rognum, T.O., 1988. Lymphoepithelial interactions in the mucosal immune system. Gut, 29: 1116-1130. Brandtzaeg, P., Halstensen, T.S., Scott, H., Sollid, L.M. and Valnes, K., 1989a. Epithelial homing ofy~ T cells? Nature, 341:113-114. Brandtzaeg, P., Bosnes, V., Halstensen, T.S., Scott, H., Sollid, L.M. and Valnes, K.N., 1989b. T lymphocytes in human gut epithelium preferentially express the ot/fl antigen receptor and are often CD45/UCHLl-positive. Scand. J. Immunol., 30: 123-128. Brenner, M.B., McLean, J., Dialynas, D.P., Strominger, J.L., Smith, J.A., Owen, F.L., Seidman, J.G., Rosen, F. and Krangel, M.S., 1986. Identification of a putative second T-cell receptor. Nature, 322: 145-149. Brenner, M.B., McLean, J., Scheft, H., Warnke, R.A., Jones, N. and Strominger, J.L., 1987a. Characterization and expression of the human y~ T cell receptor by using a framework monoclonal antibody. J. Immunol., 138:1502-1509. Brenner, M.B., McLean, J., Scheft, H., Riberdy, J., Ang, S.A., Seidman, J.G., Devlin, P. and Krangel, M.S., 1987b. Two forms ofT cell receptor protein found on peripheral blood cytotoxic T lymphocytes. Nature, 325: 689-694. Bucy, R.P., Chen, C.-L.H., Cihak, J., Losch, U. and Cooper, M.D., 1988. Avian T cells expressing y~ receptors localize in the splenic sinusoids and the intestinal epithelium. J. Immunol., 141: 2200-2205. Bucy, R.P., Chen, C.-L.H. and Cooper, M.D., 1989. Tissue localization and CD8 accessory molecule expression of T cells in humans. J. Immunol., 142: 3045-3049. Cerf-Bensussan, N., Jarry, A., Brousse, N., Lisowska-Grospierr, B., Guy-Grand, D. and Griselli, C., 1987. A monoclonal antibody (HML-1) defining a novel membrane molecule present on human intestinal lymphocytes. Eur. J. Immunol., 7: 1279-1285. Chai, J.-Y. and Lillehoj, H.S., 1988. Isolation and functional characterization of chicken intestinal intraepithelial lymphocytes showing natural killer cell activity against tumour target cells. Immunology, 63:111-117. Chan, M.M., Chen, C.-L.H. and Cooper, M.D., 1988. Identification of the avian homologues of mammalian CD4 and CD8 antigens. J. Immunol., 140" 2133-2138. Chert, C.H., Cihak, J., Losch, U. and Cooper, M.D., 1988. Differential expression of two T cell receptors, TCR1 and TCR2, on chicken lymphocytes. Eur. J. Immunol., 18: 539-543. Chung, K.S., Lillehoj, H.S. and Jenkins, M.C., 1991. Avian leucocyte common antigens: tissue distribution and biochemical characterizations using new monoclonal antibodies. Vet. Immunol. Immunopathol., 28: 259-293. Ernst, P.B., Clark, D.A., Rosenthal, K.L., Befus, A.D. and Bienenstock, J., 1986. Detection and characterization of cytotoxic T lymphocyte precursors in the murine intestinal intraepithelial leucocyte population. J. Immunol., 136: 2121-2126. Ernst, P.B., Befus, A.D., Dyck, N., Lee, T.D.J. and Bienenstock, J., 1987. Separation and characterization of leucocytes from the intestine. In: T.G. Pretlow, II and T.P. Pretlow (Editors), Cell Separation. Academic Press, New York, pp. 141-162. Goidl, E.A. and Thesis, G.A., 1985. Delayed maturation of the antibody response to type 2 thymus-independent antigen in a partially inbred strains of chickens. J. Immunol., 134: 22652267. Goodman, T. and Lefrancois, L., 1988. Expression of the 7J T cell receptor on intestinal CD8 + intraepithelial lymphocytes. Nature, 333: 855-858. Hudson, L. and Payne, L.N., 1973. An analysis of the T and B cells of Marek's disease lymphomas of the chickens. Nature, 241: 52-57.

360

H.S. LILLEHOJ AND K.S. C H U N G

Janeway, C.A., Jr., Jones, B. and Hayday, A., 1988. Specificity and function of T cells bearing yOreceptors. Immunol. Today, 9: 73-75. Jeurissen, S.H.M., Janse, E.M. and Koch, G., 1988. Meckel's diverticulum: a gut associated organ in chickens. In: S. Fossum and B. Rolstad (Editors), Histophysiology of the Immune System. pp. 599-605. Klein, J.R., 1986. Ontogeny of the Thy-1-, Thy-2 ÷ murine intestinal intraepithelial lymphocyte. J. Exp. Med., 164: 309-314. Koning, F., Stingl, G., Yokoyama, W.M., Yamada, H., Maloy, W.L., Tschachler, E., Shevach, E.M. and Coligan, J.E., 1987. Identification ofa T3-associated - T cell receptor on Thy-+ 1 dendritic epidermal cell lines. Science, 236: 834-837. Kuziel, W.A., Takashima, A., Bonyhadi, M., Bergstresser, B.R., Allison, J.P., Tigelaar, R.E. and Tucker, P.W., 1987. Regulation of T-cell receptor ~,-chain RNA expression in murine Thy1 + dendritic epidermal cells. Nature, 328: 263-266. LiUehoj, H.S., 1987. Effects of immunosuppression on avian coccidiosis:Cyclosporin A, but not hormonal bursectomy abrogates host protective immunity. Infect. Immun., 55:1616-1621. Lillehoj, H.S., 1989. Intestinal intraepithelial and splenic natural killer cell reponses to Eimeria infections in inbred chickens. Infect. Immun., 57:1879-1884. Lillehoj, H.S., Lillehoj, E.P., Weinstock, D. and Schat, T.S., 1988. Functional and biochemical characterization of avian T lymphocyte antigens identified by monoclonal antibodies. Eur. J. Immunol., 18: 2059-2063. Moingeon, P., Jitsukawa, S., Faure, F., Troalen, F., Triebel, F., Graziani, M., Forestier, F., BelIer, D., Bohuon, C. and Hercend, T., 1987. A ~ chain complex form a functional receptor on cloned human lymphocytes with natural killer-like activity. Nature, 325: 723-726. Smart, C.J., Trejdosiewicz, L.K., Badr-el-din, S. and Heatley, R.V., 1988. T-lymphocytes of the human colonic mucosa: functional and phenotypic analysis. Clin. Exp. Immunol., 73: 6369. Sowder, J.T., Chen, C.-L.H., Ager, L.L., Chan, M.M. and Cooper, M.D., 1988. A large subpopulation of avian T cells express a homologue of the mammalian gamma/delta receptor. J. Exp. Med., 167: 315-322. Stingl, G., Gunter, K.C., Tschachler, E., Yamada, H., Lechler, R.I., Yokoyama, W.M., Steiner, G., Germain, R.N. and Shevach, E.M., 1987. Thy-1.+ dendritic cells belong to the T-cell lineage. Proc. Natl. Acad. Sci., 84: 2430-2434. Tagliabue, A., Befus, A.D., Clark, D.A. and Bienenstock, J., 1982. Characteristics of natural killer cells in the murine intestinal epithelium and lamina propria. J. Exp. Med., 155:17851796. Tonegawa, S., Berns, A., Bonneville, M., Farr, A.G., Ishida, I., Ito, K., Itohara, S., Janeway, C.A., Jr., Kanagawa, O., Katsuiki, M., Kubo, R., Lafaille, J.J., Mombaerts, P., Murphy, D.B., Nakanishi, N., Takagaki, Y., Van Kaer, L. and Veebeek, S., 1989. Diversity, development, ligands and probable functions of ~ T cells. In: F. Melchers et al. (Editors), Progress in Immunology. VII. Springer-Verlag, Berlin, Germany, pp. 243--257. Vainio, O. and Lassila, O., 1989. Chicken T cells: Differentiation antigens and cell-cell interactions. Crit. Rev. Poult. Biol., 2: 97-102.

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and lamina propria in chickens.

Postnatal development of various T-lymphocyte subpopulations expressing CD3, CD8, CD4, and antigen-specific TCR heterodimers alpha beta (TCR2) or gamm...
807KB Sizes 0 Downloads 0 Views