Immune cell distribution in the small intestine of the pig: immunohistological evidence for an organized compartmentalization in the lamina propria

t etermarv Immunology and lmmunopathologv, 37 ( 1993 ) 4 9 - 6 0

49

Elsevier Science Pubhshers B V , A m s t e r d a m

Immune cell distribution in the small intestine of the pig: immunohistological evidence for an organized compartmentalization in the lamina propria* M A V e g a - L 6 p e z ~, E T e l e m o e, M

Bailey, K Stevens and C R Stokes

UntI'ersttv of Brtstol, Department o/I etermar~ ~Iedl~me LangJord Houw Lang/ord Brt~tol BSI8 7DU, Uh (Accepted 15 July 1992 )

ABSTRACT Vega-Lopez, M A , Telemo, E , Bailey, M , Stevens, K and Stokes, C R , 1993 Immune cell distribution in the small intestine of the pig ~mmunohlstologlcal evidence for an organized compartmentahzatlon in t h e l a m m a proprm ¢et Immunol lmmunopathol 37 49-60 Using monoclonal antibodies m lmmunohlstochemlstry the distribution of the cells with the following surface antigens was studied m samples of proximal and distal small intestine office 6-monthold pigs CD2, CD4 (helper/reducer T-cells) CD8 (suppressor/c~totoxtc T cells), accessor3 cell marker (monocyte/granulocyte), MHC Class 11 ( DRw ), and lnterleukln 2 ( II-2 ) receptor CD2 + cells were found m high numbers m both the epithelium and the lamina proprla More cells were demonstrated in vllhs than m crypts (proportton ~ 4 1 ) ~t least two subpopulatlons of mtraeplthehal lymphocytes were identified aplcally m the eplthehum there were CD2 + C D 4 - C D 8 (double negative ) cells whereas cells expressing CD8 marker were concentrated around the basement membrane C D 4 + cells were locahzed m the lamina proprla towards the ~dlus core Accessory cells were distributed m crypts and the wllus base and more cells were found in ileum than in duodenum In contrast, MHC Class II + cells were located predominantly in ~ llh just underneath the basement membrane, forming a sheath of cells between the CD8 + and the CD4 + cells Cells expressing IL-2 receptor were sparse but widely distributed m both the lamina propna and the cplthehum This organized cell distribution may be related to the physiology of the mucosal immune s~stem in the gut ABBREVIATIONS APC, antigen presentmgcells, IEL, intraeplthehal lymphocvtes IL-2, mterleukln 2, IL-2R lnterleukm 2 receptor, LP, lamina proprm, Mabs, monoclonal antibodies

Correspondence to M Badey, D e p a r t m e n t o f Veterinary Medicine, Umverslt3 o f Bristol Langford House, Langford, Bristol BS18 7DU, U K q N I F A P - S A R H , Mexico, and FES-Cuautltlan-UNAM Mexico 2Current address Chmcal Immunology D e p a r t m e n t Um~erslt~ Hospital Goteborg, Sweden *This work was supported by a linked research grant, LRG 18 from the Agricultural and Food Research Councd Dr Vega-Lopez is m receipt o f a grant (54191) from the Science and Technology National Councd ( C O N A C y T )-Mexico

© 1993 Elsevier Science Pubhshers B V All rights reserved 0 1 6 5 - 2 4 2 7 / 9 3 / $ 0 6 00

50

M A VEGat-LOPEZ ET AL

INTRODUCTION

The gut-associated lymphoid tissue 1s one of the major subdivisions of the i m m u n e system The small intestine contains the largest accumulation of immunological cells in the b o d y (Trepel, 1974) Lymphocytes and plasma cells, together with macrophages, eoslnophlls, mast cells and various types of antigen presenting cells ( A P C ) populate the loose connective tissue of the lamina propria ( L P ) , lymphocytes are also present between intestinal epithelial cells (intraeplthellal lymphocytes, IEL) (Parrot, 1987) In addition, collecnons of organized lymphoid nodules (Peyer's patches, lymphoid aggregates) as well as scattered solitary lymphoid follicles line the intestinal tract Lymphocytes and plasma cells in the LP are separated from contact with unprocessed luminal contents by epithelial cells and the basement m e m b r a n e There is a d i c h o t o m y existing in the i m m u n e response of the gut On the one hand a protective local response against hazardous agents (e g virus, bacteria, parasites, toxins, etc ) is necessary On the other hand, specific local (cellular) and systemic (humoral and cellular) tolerance to potentially antigenic nutritional c o m p o n e n t s is produced ( T h o m a s and Parrot, 1974) As a step in the understanding of the regulation of mucosal responses, the ldenUfication of the possible sites and nature of contact between immunological cells within the intestine is of importance The induction of i m m u n e responses requires processing and presentation of antigens by M H C Class II + accessory cells to CD4-t- T-cells Following presentation, T-cells become activated and express receptors for lnterleukin 2 (IL-2), subsequently secreting a range of cytoklnes The anatomical relationship between M H C Class II, Tcell and activation markers may therefore provide clues as to the sites of contact with antigen Monoclonal antibodies ( M a b s ) recognizing the porcine homologues of the h u m a n C D 4 and CD8 molecules and components of the M H C have been described (Pescovltz et a l , 1984) and we have recently reported a M a b which recognizes an epltope on activated lymphocytes associated with a functional receptor for IL-2 (IL-2R) (Bailey et a l , 1992 ) In spite of studies of the pig Immune system, there is little information about the microanatomy of the diffuse intestinal i m m u n e system To further understand how mucosal responses are induced and regulated in the pig we have examreed the distribution of these cellular c o m p o n e n t s and their compartmentalizatlon within the gut MATERIALS AND METHODS

Anlmal~ Five large white pigs of both sexes, from a minimal disease and enzootlc pneumonia-free herd, maintained at the University of Bristol in accordance

IMMUNE CELL DISTRIBUTION IN PIG G U T

51

with local guldehnes, were used in this work All animals were 6 months old at the time o f slaughter The animals were killed in a commercial slaughterhouse by exsangulnatlon following electrical stunning and the abdomenal contents r e m o v e d Immedmtely

Samples Samples of proximal ( 5-15 cm away from the pylorlc junction ) and &stal ( 15-30 cm before the lleocaecal j u n c t i o n ) small intestine were taken ~mmedmtely after slaughter and snap frozen in liquid nitrogen, after being embedded in O C T c o m p o u n d (Tissue Tek II, Miles Scientific, Napervllle, IL, USA ) and stored at - 70 ° C until required

Monoclonal antibodies Monoclonal antibodies recognizing T-cell markers and accessory cells were provided by Dr J Lunney, N I H , Bethesda The clones used were MSA4, anta-CD2 (PT2, H a m m e r b e r g and SchurIg, 1986), 74-12-4, antl-CD4 and 762-11, antl-CD8 ( P T 4 and PT8, Pescovltz et a l , 1984), 74-22-15, antl-monocyte/granulocyte ( H a m m e r b e r g and Schurig, 1986), MSA3, recognizing an M H C Class II ( D R w ) determinant Clone 231 3B2, recognizing an epltope associated with the pig IL-2 and expressed by T-cells following activation, was generated in this laboratory (Bailey et a l , 1992)

Imm unohtstochemlstry Serial 4 - 6 / x m frozen sections were obtained and fixed in cold (4 °C) acetone for 15 mln, air dried for 30 min, wrapped In foil and stored at - 7 0 ° C A 5 0 / d volume of the proper dilution of M a b was used on the tissue and incubated for 2 h at r o o m temperature in a h u m i d chamber As a secondary antibody, affinity purified, biotinylated, rabbit antlmouse xmmunoglobuhn (Dakopatts, N o r w a y ) was used, for 30 mln (dilution 1 500), followed by an additional 30 mln incubation with a streptavldxn-horse radish peroxldase complex ( D a k o p a t t s ) The reaction was visuahzed using a 0 05% solution of dlamlnobenzldlne in 0 05 M TrIs-HC1 ( p H 7 6) Between steps, the shdes were thoroughly washed in TrIs-buffered saline (TBS) Endogenous peroxldase activity was quenched as described elsewhere ( H l t t m a l r and Schmld, 1989 ), and background staining was reduced using a previous incubation with 2 5% normal rabbit s e r u m / n o r m a l pig serum in TBS for 30 mln The slides were counterstalned in Harris h a e m a t o x y h n and m o u n t e d in D P X

52

M & VEGA-LOPEZET a.L

Counts of cells Cells were counted using a high resolution, semi-automatic, computer supported image analyser system ( V I D S IV, Analytical Measuring Systems, Saffron Walden, U K ) For each field the area in the LP was measured and the positively stained cells counted Results are expressed as density of cells (cells m m - 2 ) At least five randomly selected fields m each tissue sample were counted and, because of the particular distribution of cells, the villus and crypt areas were counted separately

Stattsttcs Analysis of variance and Student's t-test were used to assess the differences between groups where appropriate RESULTS

T-cell markers From the first counting of cells, it was evident that the intestinal LP cells were not evenly distributed More cells were found in vllh than in crypts ( P < 0 001 ) in both d u o d e n u m and ileum (Fig 1 ) Many C D 2 + IEL were identified (Figs 2 ( a ) and 2 ( b ) ) They accounted for up to 55% of the total C D 2 + cells in duodenal vdh and up to 59% in ileal villi C D 4 + and C D 8 + 15000***

r--i

-1

[]

I

Duodenum

#11[i

[ ] Duodenum crjpts [ ] Ileum villi [ ] I l e u m crypts

+

10000 ©

E

*p
E E Q

g

5000-

8 PT2

Mo/PMN

Class II

IL -2R

ANTIGEN

Fig 1 Quantitative distribution o f immunological cells m lamina proprla o! five conventlonall~ reared 6-month-old pigs

.

p

,~.4-'

,_y/

~"

5,/

.

" ....

iii'~ "

'~II.

x

I,illlrt •

f,i

.L lu_ II~ " ~ . , l . . , ' , ' ; > Y ' l ' , - i l ' i

'ili~i"

,,,-,

(CD8) (e f)

Fig 2 [mmunoperoxidase stained sectlonsoflntestlne from con~entlonal]) reared 6-month-old pigs longitudinal sections of~fllus (a c e) tians~erse sections of ~lllus (b d f ) Frozen sections el intestine ~eie stained ~ l i h anti-T2 ( C D 2 ) (a b) anti-T4 ( C D 4 ) (c d) and anti-T8

~,'-

~t~i,~.~

N © -1

>.

,

,

~

"~

( p a n u u u o ) ) ~ ~[4

~ c,% a, ~

ql

,,%

t

.

- ~

,

.

,a4.,/"~

,e

qg 3 Immunoperoxldase stained sections o f intestine trom con~ enllonallx reared 6-month-old pigs Frozen sectmns ot intestine were stained ~lth antl-monoc~te/glanulocxle (a) a n t > M H C Class II (b) and antl-IL-2R (arrowed) (c)

•,

b

56

M A VEG*-L(}PEZ ET AL

cells were distributed differently between the epithelium and LP Most CD4 + cells were found at the villus core, whereas the C D 8 + cells concentrated around the epithelial basement m e m b r a n e ( Figs 2 ( c ) - 2 ( f ) ) Although the major|ty of IELs were CD2 + , not all of them expressed the CD8 marker Two apparent subpopulatlons were distinguished, one composed of cells staining for CD2 only, placed aplcally w~thln the ep~thehum, and one staining also for CD8, around the basement m e m b r a n e Since only a small n u m b e r of aplcally placed IELs d~d not stain w~th the CD2 marker, th~s second population is presumably CD2 + CD8 +

Acces~oo' cells Cells expressing the m o n o c y t e / g r a n u l o c y t e marker were detected m ileum In greater numbers than m d u o d e n u m and in crypts more often than m vflh (Figs 1 and 3 (a) Their morphology recalls that of m o n o c y t e s / m a c r o p h a g e s Less than 3% of the cells stained within the LP were polymorphonucleocytes as m & c a t e d by nuclear morphology, and eoslnophlls were not stained at all, as tested by specific L e n d r u m staining (Lendrum, 1944) (data not shown )

Modulatable antigens M H C Class II ( D R w ) + cells were more numerous m vllh than in crypts ( P < 0 01 ) (Fig 1 ) They were concentrated at the villus, just underneath the basement membrane, forming a sheath of cells between the C D 4 + and the CD8 + lymphocytes (Fig 3 ( b ) ) This distribution was different from that of the macrophage/granulocyte marker, which was expressed preferentmlly w~thln the crypts and the wlh base The Class II + cells showed dendntlc-hke morphology, and ~t is noteworthy that there was no Class II staining by the ep~thehal cells, even when Mabs to D R speclfiCltles were also tested (not shown ) IL-2R expressing cells were evenly d~stnbuted in the intestinal LP and eplthehum (Fig 3 ( c ) ) Their morphology resembled blastold cells By dual staining using fluoresceln lsothlocyanate conjugated IL-2R Mab, after macrophage/granulocyte l m m u n o p e r o x l d a s e staining, ~t was confirmed that up to 30% of macrophage/granulocyte positive cells m the crypts expressed IL2R DISCUSSION

F r o m the present study ~t was clear that. at least in conventionally reared pigs, the concept of randomly scattered cells within the intestinal LP was Inaccurate Some cells were concentrated towards the vdh LP (CD2, CD4, CD8, M H C Class II ), whereas others appeared more often in crypts ( m a c r o p h a g e /

IMMUNE CELL DISTRIBUTION IN PIG G U T

57

granulocyte) In addition, within the LP the distribution ofT-cell subsets was apparently not uniform Most o f the T-lymphocytes ( C D 2 + ) were found in villi and the difference against crypts was significant ( P < 0 001 ) Among the C D 2 + cells, C D 4 + and C D 8 + subpopulations were identified occupying different sites in the vllh O f the total C D 2 + cells in villus, up to 550/o in d u o d e n u m (59% in ileum) were detected in the lntraepithehal c o m p a r t m e n t At least two subpopulatlons of CD2 + IELs were identified One o f them showed CD2 + staining in the absence of C D 4 or CD8 (double negative), and was p o s m o n e d aplcally within the eplthehum The description of these cells in circulation (Saalmuller et a l , 1989) has not given any clear indication about their role in the i m m u n e system, but their presence in lymphoid tissues may indicate a role for the C D 2 antigen in homing (Saalmuller et a l , 1990) The other IEL subpopulation was concentrated at the eplthellum's basement membrane, and apparently expressed the C D 2 + C D 4 - C D 8 + phenotype These cells may have a regulatory role in the ehcltation of suppression of the i m m u n e response (Fujihashl et a l , 1990) The majority of C D 8 + cells were found at or within the basement m e m b r a n e (Figs 2 ( e ) and 2 ( f ) ) It is conceivable that a proportion o f these cells were still in the LP, so their classification as LP cells or IELs was difficult In addition, if these cells were in a dynamic process of traffic between the LP and eplthehal compartments, that classification would be rendered inappropriate Furthermore, these CD8 + cells may well be a heterogeneous population o f cells, with further differences in their phenotype and function ( G u y - G r a n d et a l , 1991 ) that were not detected with this limited panel of Mabs In contrast to C D 8 + cells, very few C D 4 + cells were found in the epitheh u m and most o f the positive cells were localized towards the core of the villi, close to the lacteal vessel (Figs 2 (c) and 2 ( d ) ) In the pig circulation there are significant populations of double positive ( C D 2 + C D 4 + CD8 + , approximately 15%) and double negative ( C D 2 + C D 4 - C D 8 - , approximately 14%) cells (Pescovltz et a l , 1985, Lunney and Pescovltz, 1987) However, the characteristic distribution of C D 4 + and CD8 + cells in the small intestine makes a predominance of double negative or double positive cells unlikely in the LP ( c o m p a r e d with the epithelial c o m p a r t m e n t ), but dual staining would be necessary to formally demonstrate this This compartmentalization of C D 4 + CD8 - and C D 4 - C D 8 + T-cell subsets can only be explained by specific and well-enforced migratory patterns The entrance of cells, their retention and exit from the tissue might be balanced by the differential expression of receptors for structural or cell surface molecules The identification o f site specific adhesion molecules such as that postulated by K d s h a w and Murrant (1990) m a y help to elucidate this question The presence of such large numbers of T-cells, particularly of C D 4 + cells, suggests the potential for direct, local recognition of antigen This requires

58

M A VEGA-LOPEZ ET a.L

the presence of accessory cells expressing M H C Class II products and capable of antigen presentation (macrophages, dendritic cells) Cells staining for Class II, and therefore potential candidates as antigen presenting cells, appeared to occupy a position between the C D 4 + cells in the LP and the CD8 + cells at the basement m e m b r a n e (Fig 3 ( b ) ) This distribution is similar to that of analogous cells found in other species (Selby et a l , 198 l, Mayrhofer et a l , 1983, Trejdoslewicz et a l , 1987, Russel et a l , 1990) The dendritic morphology and intense staining for Class II antigen would also support a possible role in presentation of antigen In contrast to the findings in rodents (Mayrhofer et a l , 1983) and man (Trejdoslewlcz et a l , 1987, Russel et al, 1990), the intestinal epithelium in pigs did not express Class II antigens Mabs against D Q speclfiCltles (H42, Davis et a l , 1987) were also used in an attempt to identify differential expression of these antigens, but none of them stained the epithelium (data not shown) In mouse and human, Class I I + enterocytes are capable of presenting soluble protein antigens, at least in vitro (Bland and Warren, 1986, Mayer and Schleln, 1987) The absence of such cells in the pig means that Class II bearing cells in the LP therefore appear to be the only potential APC outside the organized gut-associated lymphoid tissue Their position is certainly consistent with interaction with C D 4 + cells within the LP The n u m b e r of cells expressing IL-2R in the LP of the adult pigs was low and showed no particular distribution While this indicates that some cells (macrophages or T-cells) may be activated, it provides no evidence as to the site at which presentation/activation occurs The m o n o c y t e / g r a n u l o c y t e marker is expressed by a heterogeneous population of cells ( H a m m e r b e r g and Schurig, 1986 ) and the more hmited pattern of staining In the LP with the antibody 1F4, which stains a comparable population of cells in blood, suggests more than one population of accessory cells within the LP Cells bearing the m o n o c y t e / g r a n u l o c y t e c o m m o n marker were localized in the gut LP in smaller numbers than CD2 + cells Some cells were found at the villus base and towards the core of the LP, but only occasionally were they close to the epithelium or the basement m e m b r a n e The paucity of staining at this particular location is relevant since the majority of the M H C Class II + cells were located at that site The distribution of cells with monocyte/granulocyte staining was similar to that reported for lmmunoglobulln containing cells (Brown and Bourne, 1976a,b) The role of cells expressing this antigen is unclear but could include handling, processing and presentation of antigens to T-cells H o w e v e r their predominance in the crypts makes it difficult to understand how they m a y be able to reach the antigen and to contact the T-cells In addition, these cells in the crypts did not express M H C Class II products The generation of an iccosome (immune-complex coated b o d y ) mediated mechanism for antigen delivery between the Class II + , dendrltlc-lIke cell in the villus and a monocyte/granulocyte, phagocytic cell in

IMMUNE CELL DISTRIBUTION IN PIG GUT

59

the crypts could be one mechanism of interaction between those cells (Katz, 1988, Szakal et al, 1988) Alternatively, they may be a resident population of'scavenger' cells removing cell debris among the lmmunoglobuhn-producmg cells predominant in the crypts In conclusion, we have demonstrated that the distribution ofT-cells w~thm the pig intestinal LP is not random C D 4 + T-cells localize within the vllh and are in close opposition to cells staining strongly for MHC Class II products, whereas C D 8 + cells are distributed around the epithelial basement membrane The distribution suggests preferential retention of cells at specific s~tes While this retention may be antigen driven, it implies a degree of organization of ~mmunological cells within the LP

REFERENCES

Ba~le~, M , Stevens, K , Bland, P W a n d Stokes C R , 1992 N monoclonal anl~bod~ which recognlses an ep~tope associated wtth ptg lnterleukln 2 receptors J I m m u n o l Methods 153 85-92 Bland, P W a n d Warren L G , 1986 Nntlgen presentation b~ epithelial cells o! the rat small intestine 1 Kinetics antigen speclfiClt~ and blockmg b~ ant~-Ia ant~sera Immunolog~ 58 1-7 Brown, P J a n d Bourne F J 1976a Dlstrlbut~on ol mamunoglobulln-contalnlng cells ~n the allmentar~ tract spleen and mesenterlc 15m p h nodes of the pig d e m o n s t r a t e d b5 peroxldaseconjugated a n t i s e r u m to porcine ~ m m u n o g l o b u h n s G A and M ~Xm J Vet Res 37 ~)-13 Brown, P J a n d Bourne, F J , 1976b D e v e l o p m e n t ot l m m u n o g l o b u h n - c o n t a l m n g cell populations m intestine spleen and mesenter~c l ? m p h node b? perox~dase-conjugated a n t i s e r u m s Am J Vet R e s , 37 1309-1314 Davis, W C , Maruslc, S , Lewln, H -k Sphtter G ~k Perr~man L E M c G u l r e T C and (,orham, J R , 1987 The l d e n t l f i c a u o n and anal~sls of species s p e c i f i c and cross reactive monoclonal a n t l b o & e s to leukoc:yte &fferent~at~on antigens and antigens of the major h~stocompatlbflttv complex for use m the stud\ of the ~mmunc s:ystem of cattle and other species \ et lmmunol Immunopathol,15 337-376 Fujlhash~ k , Taguchl T , M c G h e e J R Eldrldge J H Bruce M G Green D R Smgh B and Kimono, H 1990 Regulator~ function for m u r m e mtraep~thehal I~mphoc~tes T ~ o subsets of C D3 + T cell receptor-1 + lntraepltheha117 mphoc} te T cells abrogate oral tolerance J I m m u n o l , 145 2 0 1 0 - 2 0 1 9 G u ~ - G r a n d , D , Cerf-Bensussan, N , Mahssen B , Malassts-Ser~s M , Brlottet ( and Vassalh P 1991 T',~o gut mtraeplthellal CD8 + 1}mphoc~ te populations with different T cell l ecepto~s a r o l e f o r t h e g u t e p ~ t h e h u m m T c e l l d l f f e r e n t l a t ~ o n J Exp Med 173 471-481 H a m m e r b e r g , C a n d Schurlg, G 1986 Characterlsatxon of monoclonal antibodies d u e c t e d a g a l n s t s w m e l e u k o c ~ t e s Vet l m m u n o l I m m u n o p a t h o l 11 107-121 H l t t m a l r ~ a n d Schm~d, K W 1989 I n h i b i t i o n of endogenous perox~dase for the i m m u n o c~lochem~cal d e m o n s t r a t i o n of i n t e r m e d i a t e filament proteins J I m m u n o l Methods 116 199-205

60

M A VEOA-LOPEZET ~L

Katz, D R , 1988 A n h g e n presentation antigen-presenting cells a n d antigen processing ( u r r Opm lmmunol,l 213-219 Kllshaw, P J a n d Murrant, S J 1990 A ne~ smface antigen on lntraeplthchal l~mphoe~tes in the intestine Eur J I m m u n o l 20 2 2 0 1 - 2 2 0 7 kendrum A ( 1944 The staining of eosmophfl pol~morphs and e n t e r o c h r o m a f f i n cells in histological sections J Pathol B a c t e n o l , 56 441 kunne~ J K a n d Pesco~ltz, M D 1987 P h e n o D pie and functional c h a r a c t e n z a u o n ofp~g 1.~mp h o c } t e p o p u l a t l o n s Vet I m m u n o l l m m u n o p a t h o l 17 135-144 Maxer L a n d S c h l e m R 1987 E x l d c n c e f o r f u n c t l o n o f l a m o l e c u l e s o n g u t c p l t h e h a l c e l l s m m a n J Exp Med 166 1471-1483 Ma:~rhofer, G , Pugh C W and Barcla} A N 1983 The d~slmbutlon ontogenx and o n g m in the rat of la-pos~hve cells with d e n d r m c morpholog:y and of la antigen m epltheha ~ t h specml reference to the intestine Eur J I m m u n o l 13 112-122 Parrot D M V 1987 T h e s l r u c t u r e a n d o r g a n l s a t l o n o f l } m p h o l d t ~ s s u e m t h e g u l In I Brost o f f a n d S J C h a l l a c o m b e (EdHors) Food Allerg~ and Intolerance Baflllere Tmdall London, pp 3 - 2 6 Pesco~ltz, M D , L u n n % , J K a n d S a c h s , D H 1984 P r e p a r a t l o n a n d c h m a c t e r J z a u o n o t m o n o e l o n a l a n t l b o d m s r e a c h ~ e ~ l t h p o r c l n e P B L J l m m u n o l 13z; 368-375 Pescoxltz M D , L u n n % J k a n d Sachs D H 1985 M u r l n e a n t > T 4 a n d T 8 m o n o e l o n a l a n t~bodles d l s t m b u u o n and etfecls on p r o l l l e r a u o n and c \ t o t o \ l c T cells I l m m u n o l 134 37-44 Russel G J , Bhan *XK a n d W i n t e r H S , 1990 The d l s t n b u h o n o f t and B l \ m p h o c ' , t e populat,ons and M H C class I! e,~presslon ,n h u m a n fetal and postnatal intestine Pedmtr Res 27 2 3 9 - 2 4 4 Saalmuller -X, Hlrt W a n d R e d d e h a s e M J 1989 PhcnoD plc d l s c n m m a t m n between th~ m~c and extrath} mlc C D 4 - CD8 - and CD4 + CD8 + porcine T l,,mphoc.~ tes Eur I l m m u n o l 19 2 0 1 1 - 2 0 1 6 Saalmuller A , Hlrt, W and Reddehase, M J 1990 Porcine , ' / d T h m p h o c x t e subsets dflte)mg m thmr propensltx to h o m e to l y m p h m d t~ssue Eur J l m m u n o l 20 2 3 4 3 - 2 3 4 6 Selbx W S , Janoss:y, G Goldstem, G and Jewcll, D P , 1981 T lxmphoc,Ae subsets m h u m a n ,ntcstmal mucosa the d l s t n b u t m n a n d relat,onshlp to M H ( - d e n x e d antigens ( h n E',p l m m u n o l , 44 4 5 3 - 4 5 8 bzakal, 4 k Kosko, M H a n d T c w J ( J 1988 ~ n o ~ e l m ~ l x o f o l h c u l a r d e n d r l t l c c e l l - d e p c n dent lccosome-medmted m e c h a m s m tot dehxer} of ant,gen to antigen-processing cells J l m m u n o l 140 341-353 T h o m a s , H ( a n d Parrot, D M , 1974 The m d u c t m n ol tolerance to a soluble prolem antigen b } o r a l a d m m l s t r a t m n lmmunolog> 27 6 3 1 - 6 3 9 Trejdoslew~cz L K , Mallzla, G Badr-eI-Dm S Sma~l, C J Oakes D J Southgale J Howdie P D , Janossy G Poulter L W and Loso~sk,, M S 1987 T cell and m o n o n u d e a r phagocxte p o p u l a t m n s of the h u m a n small and large intestine In J Mcsteckx J R M c G h c e I B~encnstock and P L Ogra ( E d i t o r s ) Recent Adx ances m Mucosal lmmunologx Plenum Press Nh p p 4 6 5 - 4 7 3 Trepel, F 1974 N u m b e r and d l s t m b u t m n o f l ~ m p h o c y t e s m man a critical anal xs~s k h n V~ochenschr 52 511-515