Changes in Human Mucosal γδ T Cell Repertoire and Function Associated with the Disease Process in Inflammatory Bowel Disease

Changes

in

Human

Mucosal

y6

T

Cell

Repertoire and Function

Associated

with

the

Disease Process in

Inflammatory

Bowel Disease

Laila

D.

McVay,*

Baiqing Li,*

Renee

Biancaniello,*

Mary Anne

Creighton,* Dale

Bachwich,A

Gary

Lichtenstein,*

John L.

Rombeau,§

and

Simon R.

Carding*

*Department

of

Microbiology, tDivision of Gastroenterology, and

§Department

of Surgery,

University of Pennsylvania School of

Medicine,

Philadelphia, Pennsylvania 19104-6021, U.S.A.

ABSTRACT

Background: Although y6T cells are amajor compo-nentofthe human intestinal mucosa, it is not clear what role they play in mucosal inimunity or ifthey are in-volved in the disease process of inflammatory bowel disease (IBD).

Materials and Methods: Flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) as-says were used to identify quantitative and qualitative changesinthe repertoire of-y8Tcells presentinsurgical and/or biopsy samples of normal and inflamed colon from individual patients with ulcerative colitis (UC) or Crohn's disease (CD). Cytokineproductionand the abil-ity toadhere to and interact withcolonicfibroblasts were used tocompare the functionalproperties of yST cells isolated from the normal and diseased colonic mucosa. Results: Increased numbers of yS T cells localized in areasof inflammation andtissue injurywere found in

the majority of patients, irrespective of the typeofIBD present.This expansion was attributable to an increase in V61+ cells expressing a V5I-(D63)-J&1-encoded T cell receptorand was seen in patientswithseveredisease as well asthosewith newly diagnosedorlesssevereforms of IBD. Among T cells present in the inflamedmucosaof patientswith CD, y8Tcells, particularlyV81+cells, were a major source of the proinflammatory cytokine inter-feron--y and couldinteractwith colonic fibroblasts. Conclusions: Ourresults demonstrate thatthe chronic inflammatoryimmune responsecharacteristic of IBD is associatedwith distinctchanges in thenumber, distribu-tion, composidistribu-tion, and function of mucosal 'yS T cells. Through the production of cytokines andphysical inter-actionwithothercells, y8Tcells canperforman immu-noregulatory function and contributetothe pathophys-iology of IBDs.

INTRODUCTION

Inflammatory bowel disease (IBD) represents a complex group of idiopathic chronic inflamma-torydisorders of which Crohn's disease (CD) and ulcerative colitis (UC)arethe majorforms. While thereareseveralfeatures that distinguishCDand

Addresscorrespondence and reprint requests to:SimonR.

Carding,Department ofMicrobiology, 802FAbramson

Center, 34thStreet & Civic CenterBlvd.,Philadelphia, PA

19104, U.S.A.Phone:215-573-3022; Fax:215-573-9068;

E-mail: [email protected]

© 1997,THEPICOWERINSTITUTE PRESS. Allrightsreserved.

MolecularMedicine,Volume 3, Number 3, March 1997 183-203

UC, there aremanyepidemiological, clinical,

ra-diologic, endoscopic, and histologicfeatures that are shared between these two disorders.

Several immunologic and histopathologic

features of UC and CD can be explained as a

defect in mucosal immunoregulation, and as a direct or indirect consequence ofpersistent mu-cosal T cell activation (1-4). Experimental evi-dence for a direct role for T cells in IBD comes

from various animal models, especially "gene

knock-out" strains that spontaneously develop

bowel lesions. In particular, the presence of in-testinal inflammation in transgenic mice defi-cient inaC3Tcells (5) orTcell-derived cytokines

such as interleukin 2 (IL-2) (6) suggest that T

cells are responsible for the homeostatic

regula-tion of mucosal immune responses.

Inthehuman intestine,

y&'

cells areamajor component (30-40%) of theTcellsinthe

intra-epithelial spaces and the majority (>70%)

ex-press V6I-encoded receptors (7). Inview of the predominance of V61+ cells within the human

intestinal mucosa and the possibility that they have similar effector functions to their murine

counterparts, it seems likelythat they may play

an important role in the generation and/or

reg-ulation ofinflammatory responsesinthe

intesti-nal mucosa. The ability of yST cells to interact

with and influence the activity of a variety of

stromal cells (8-10), including epithelial cells

(8,1 1), and thefindingthat the severity ofIBDin Tcell receptor (TCR)-a/3 deficient miceis signif-icantly reduced when crossed withmice lacking ,y6 T cells (5) are all consistent with an immuno-regulatory role for mucosal 'y6T cells.

Whereas several groups of investigators have described an increase in the number of V81+

intraepithelial (IE) y8Tcells within the intestine of patients with coeliac disease (12-16), the

im-munohistochemical analyses of mucosal yB T

cellsinpatientswithIBDhave generated

contra-dictory results (16,17). Although one group has shown that thedistribution and relative number of TCR-5+ cells in sections of colonic mucosa

obtained fromIBDpatients was similar to thatin

sections of normal colonic mucosa (16), the re-sults of another study showed that there were fewer TCR-6+ cells in the inflamed mucosa of patients with IBD, and particularly, those with

UC (17). Thus, evidence for the involvement of

y8Tcells inIBDremains inconclusive. To inves-tigate furtherthe possible involvement of muco-saly6TcellsinIBD wehave useda combination ofsensitivecellular and molecular techniques to determine if thereareany qualitative or

quanti-tative changes in y8 populations in the colonic mucosa of patients with CD and UC.

The results of our studies identify changes in the number, distribution, and repertoire of

y6

T cells that are present within the lesions of the inflamedintestine inboth UC and CD. The finding

that,incontrastto

y8

Tcellspresentinthehealthy, noninvolvedmucosa, yScellsintheinflamed

mu-cosaconstitutively produce theinflammatory cyto-kine interferon y (IEFN-,y) and selectively interact with colonic stromal (fibroblasts) cellssuggest that

mucosal

y6

Tcells can contribute to the

develop-ment of thedisease processin IBD.

MATERIALS

AND

METHODS

ProcurementandProcessing of

Patient

Samples

Surgical resectioned segments of intact colon

were obtained from patients with UC, CD, or

diverticular disease (diverticulitis) atthe time of elective surgery. Histologically normal,

nonin-volved, mucosa was obtained from the

margins

of

surgical

resectioned colon that were atleast a

distance of 15 cm from any lesions. Mucosal

biopsies

were obtained with the approval of the

HumanInvestigations Committee of the

Univer-sity of Pennsylvania School of Medicine. The

origin andnature of the patient samples used in this studyare compiled in Tables 1 and 2. When

available, peripheral blood mononuclear cells (PBMCs) from the same patient were used as

control samples. Patient-derived samples were

used for RNA extraction, reverse transcriptase-polymerasechain reaction (RT-PCR) analysis, in situ hybridization, and/or cell isolation for flow

cytometric andfunctional analyses.

CellIsolation

Peripheral blood mononuclear cellswereisolated from 5 to 10 ml of peripheral blood by density gradient centrifugation using Ficoll-Hypaque (Organo-Teknika, Durham, NC) and washedone totwotimes withPBS. Established human fibro-blast cell lines of skin (CRL-1509), lung

(CCD-1llLu), or colon (CCD-18Co) origin were ob-tained from the American Type Culture Collection (ATCC, Rockville,MD). Aprimary

co-lonic fibroblast cell line was derived from the involved mucosal tissue fragments of a patient with ileocolonic CD (CD-11, Table 1) as de-scribed previously (18). This linewasmaintained inDulbecco's modified Eagle's medium (DMEM) containing 10%fetal calfserum(FCS) and penicil-lin/streptomycin/bactrim/amphotericin B. Priorto

reachingconfluency, cells weretrypsinized and di-luted 1:5 in complete media and platedinto tis-sue culture-treated 20-mm petri dishes.

Adher-ence assays were performed with primary

fibroblasts that had beenpassaged twice.

IEand Lamina Propria (LP) T Cells

A modified version of a previously published procedure (19) was used to isolate IE and LP

lymphocytes (L). Briefly, after removal of all

as-sociatedmesentery, fat, and underlying

L. D. McVayet al.: y6TCells andInflammatoryBowel Disease

and cut into 2- to 3-cm pieces. IE lymphocytes

wereeluted by 3to4sequentialincubations with

Hank'sbalanced salt solution (HBSS) containing

0.1 mM EDTA at 37°C each for 15 min with

constantmagnetic stirring. The eluted cells were

then subjectedtotwo-stepdiscontinuous Percoll

(Pharmacia-LKB, Uppsala, Sweden) gradient centrifugationat 600 X gfor 20min using 70%

and 40% Percoll. IEL were removed from the

interface between the70% and40% Percoll

lay-ers. The remaining tissue fragments were incu-bated with HBSS containing collagenase typeIV

(Sigma, St. Louis, MO) at 370C for 4-6 hr with constant stirring. The recovered LP cell

suspen-sion was then subjected to Ficoll-Hypaque den-sity gradient centrifugation to isolate viable cells from which LP lymphocytes were obtained by

Percoll gradient centrifugation as described

above for IEL isolation. The yield of LP T cells

(CD3+ cells) from the noninvolved mucosa and

fromthe involved mucosa was calculatedas the

number of cells per gram of tissue used for

iso-lation, and this yield was routinely comparable to that reported by others (20,21).

IMMUNOMAGNETIC SELECTION OF MUCOSAL IEL AND LP

7y+

TCELLS. Anegative-immunomagnetic

selec-tionprocedure (22) was used to obtain

homog-enous populations of

TCR-,yS+

or TCR-aIB+ IEL

andLP cells from theinvolved and noninvolved

mucosa of colonic surgical specimens. Briefly,

total LP and IEL cell samples were sequentially

incubated with a mouse anti-human TCR-ac

(clone BW242/412; T CellDiagnostics, Woburn,

MA) or a mouse anti-human TCR--y6 antibody

(11F2, Becton Dickinson) and a rabbit

anti-mouseimmunoglobulin G (IgG) antibody

conju-gatedto supramagnetic particles (BioMagBeads,

Perseptive Diagnostics, Cambridge, MA). The

cells were then transferred to a 25-cm2 tissue

culture flask and placed on a plate magnet and

the "nonmagnetized" cells were removed. The

purity of eachT cellpopulation wasdetermined

by flow cytometry, and cell viability, by trypan blue dye exclusion.

Flow Cytometry

Aliquots (0.2-1.0 X 106) of bulkorfractionated

IEL and LP mononuclear cells were stainedwith

fluorochrome-conjugated mouse anti-human

CD3- (clone S4.1; Caltag Labs., San Francisco,

CA), CD8a- (3B5, Caltag), TCR-y8- (l1F2,

Bec-ton Dickinson), TCR-Vy9- (T Cell Diagnostics), TCR-V81- (TS8.2; T Cell Diagnostics),

TCR-V61-J31- (8TCS-1; T Cell Diagnostics) and/or

TCR-V52- (23) specificantibodies, fixed andanalyzed

on a FACScan (Becton Dickinson) using Lysis

IIor CellQuest softwareasdescribedpreviously (23).Fluorochrome-conjugated isotype-matched

mouse antibodies or irrelevant specificity (Caltag)

and anti-human CD45-specificantibodies (Dako

Corporation, Carpinteria, CA) wereusedto

con-trol for nonspecific reactivity and as fluoro-chrome-compensation controls, respectively.

RT-PCR Analysisof the TCR-VS

Repertoire

Total cellular RNAwas preparedfrom intestinal tissueor PBMC byCsCl density gradient centrif-ugation (24). RNA pellets were extracted with acidic phenol once, with phenol:chloroform

(1:1) once, and twice with chloroform prior to

ethanol precipitation. Approximately 10 ,tg of total cellular RNA wasused to synthesize cDNA

using random hexanucleotides as described

pre-viously (25). Anested PCR assay (24) was used

to analyze the

V8-J6

rearrangements expressed by

y6

T cells in colonictissue and blood

mono-nuclear cell RNA/cDNA samples. Briefly, oligo-nucleotide primers specific for V81, V82, or V53

in combination with a CS gene segment primer

were used to amplify cDNA. The products of

amplification were electrophoresed and

TCR-PCR products gel purified and quantitated by

DNA fluorimetry. Equivalent amounts of each

cDNA were usedas a template for a second am-plificationusingthe same forward VS primer and

a reverse-primer specific for each of the four human JS genes. The products of amplification

were electrophoresed on 1.8% agarose gels, Southern blotted, and hybridized with

[y32p]

-ATP end-labeled VS1, V82, or V83 oligonucleo-tide probes containing sequence internal to the VA primers used for PCR. Individual V1I-JS1-4,

V62-J61

-4, and

V83-J61

-4receptor cDNAclones

were usedto determine the optimum conditions for theproportional amplification ofVS-JS genes with no cross-priming. For analysis of mucosal biopsy specimens, 5 to 6 individual pieces of

tissue were pooled and total cellular RNA was

isolated as described above. After generating cDNA,aliquotswereanalyzedforV 1-J5

expres-sion by PCR amplification and Southern blot

analysis asdescribed for the surgical samples.

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-TABLE 2. Biopsysamples ofintestinal tissue

Sample Tissue Sample

Case Duration

No. Code No. Age (Sex) Diagnosis (years) N I PB

1 Bl-UC 82494 26 (M) UC 15, recurrence + +

2 B2-UC 21495 25 (M) PanUC 12, recurrence + +

3 B3-UC 11295 37 (M) Nonspecific colitis 1 week + + +

4 B4-UC 72194 (M) UC 5, recurrence - +

5 B5-UC 12795 47 (F) Active UC 5-6, recurrence - +

6 B6-UC 9894 66(M) Noninvolved UC 13, recurrence +

7 B7-UC 3895 28 (M) UC 5, recurrence - + +

8 B8-CD 72794 (F) CD 2, recurrence - +

N,noninvolved; I, involved;PB,peripheralblood;UC, ulcerativecolitis;CD, Crohn'sdisease.

In Situ Hybridization

In situ hybridization was used to identify and localize populations of y8 T cells and IFN-,y-pro-ducing cells within frozensections ofintestine as

described previously (22,26,27). Briefly, 1- to

2-cm2 pieces of tissue from involved or

nonin-volved areas along the length of the resected intestine were snap frozen inTissue Tek (Miles, Elkhard, IN) and stored at -860C. Six-10 ,um

sections were cut from tissueblocks usinga cry-ostat, fixed in4% paraformaldehyde inPBS, pH 7.4,for20 min at roomtemperature, then stored

in 70% ethanol at 4°C in airtight containers.

35S-labeled RNAprobes (0.2 ng

RNA/,li;

specific activity >109 cpm/,tg) complimentary to the

coding (antisense) or noncoding (sense)

se-quences human IFN-,y (26) genes were used for detection of TCR- and

IFN--y-mRNA+

cells.

IFN-y Assay

IFN-'y protein production was analyzed from

bulk, nonfractionated T cells and purified ac3 T

cells, and y5 T cells isolated from segments of noninvolved or involved mucosa from two

pa-tients with ileocolonic CD and one with colonic

CD. Two hundred thousand cells were cultured

in triplicate in microtiter plate wells containing either mediaalone orwells coated withan

anti-CD3 antibody (OKT3). After 96 hr, the culture supernatantwasharvestedandassayed for IFN-,y usingacommercial radioimmunoassay (RIA) kit (Centocor, Malvern, PA). The amount ofIFN-y present was determined by reference to a

stan-dard curve generated from samples containing knownamounts of rIFN-'y assayed inparallel.

Fibroblast Adherence Assay

T cells (3 X 106) from peripheral blood and/or

normal orinvolved colon from four patients with ileocolonic CD were allowed to adhere to

con-fluent monolayers of established or primary fi-broblast cell lines in20-mm petridishes at 370C

for30 min. Nonadherent cellswere removedby gentle washing with prewarmed media and

ad-herent cells removedby brief (3-5 min) trypsin

treatment. The number of adherent cells was

determined andtheTcell composition evaluated by flow cytometry. The degree of enrichment for

'y6Tcells after adherence tofibroblast

monolay-ers was determined by the number of

'y

T cells presentinthe inputand adherent cells. The pro-portionof mucosal V1 + y6Tcells abletoadhere

to fibroblasts was determined from the number ofV61+ cells present in the input and adherent cell populations.

RESULTS

Insitu hybridization wasusedinitially to visual-ize and localize TCR-,y5+ cells infrozen sections of normal and diseased colonic mucosa.

Com-pared with sections of normal colonic mucosa,

sections ofdiseased tissue (CD) contained many

L. D. McVay etal.: y6 TCellsandInflammatoryBowel Disease

the muscularis mucosa and serosa (data not

shown). This distributionwasconsistent withthe

transmural pathology of CD. Inorderto analyze the distribution of -yS T cells in the normal and inflamed colon in more detail, flow cytometry

wasused to analyze cells directly after their

iso-lation from the colonicmucosa.

Changes in the Number, Distribution, and

Composition of Isolated Mucosal TCR-y6+

TCells in Noninvolved and Involved

Tissue from the SamePatient

Three-color flowcytometrywasusedtocompare

thenumber, distribution, and compositionof -y8

TcellsintheIE spaceandLPof the noninvolved

and involvedmucosa of individualIBD patients.

This approach was preferred to

immunohisto-chemical detection of T cells in intestinal tissue since this technique is more tedious and labor

intensive, it is difficult to perform, and it is a

relativelyinsensitivemeansof detectingantigens

such as the y6 TCR that are expressed at low

levels (28,29). For our study, surgically

resec-tioned segments of colon from a total of seven

patientswithcolonic CDorUCwereused for this

study. It should be noted that in contrast to the smallbowel which is rich in IELs, the colon has

relatively few IELs. Peripheral blood samples

from these patients were analyzed inparallel.

The cytofluorographs ofthe analysis of one

UC patient sample (Fig. LA) and the compiled analysis of allseven IBDpatients (Fig. 1B)

iden-tifydifferences in the distribution, number, and

composition of y6 T cells inthe blood and

intes-tine. In the blood, -y5 T cells comprised a small

proportion of T cells (3-4%), of which >95%

were CD4-, CD8- (data not shown) and

ex-pressedaTCRencodedbythe Vy9 andV52gene segments (Fig. iB). By contrast, inthecolon, ya

T cellsmade upalarger proportion of theTcells

presentinthe colonicmucosa,particularlyinthe

IE space (20-30%). Less than 10% of the IEL

population were CD19+, demonstrating that the

IELpreparationscontainedlow levels of

contam-inating LP lymphocytes. The 10-20% of

CD3-cellspresent inthe IEL preparationswereCD45

(datanotshown), whichis consistent with them

being contaminating epithelial cells.

In the involved mucosa of all of the IBD

samples analyzed, ySTcells comprisedamajorT

cell population in the LP of CD (mean, 43%;

range, 23-63%) and UC (mean, 64%; range,

45-83%) patients. Assumingthat the mucosalT

cell isolation procedure did not result in the

se-lective loss of ySTcells (seeMaterials and

Meth-ods), it was possible to determine the relative

number of y8Tcellspresentinthe involvedand

noninvolved mucosa. The averageyieldof T cells

(CD3+) recovered from the LP was 5.9 ± 0.4 X

1o6cells/g fromthe noninvolved colonic mucosa and 2.4 ± 0.6 X 107 cells/g from the involved mucosa, which is comparable to the values

re-ported previously (20,21). The proportion of

TCR-af3+ andTCR-,y8+ cells inthe noninvolved mucosa was 74% ± 10% and 26% ± 9%,

re-spectively (data not shown). Thus, noninvolved

colonicmucosasamples containedanaverage of

1.6 ± 0.3 X 106 -y Tcells/g tissue. Since in the

involved mucosa TCR-af3 and TCR-,yB repre-sented 54% ± 15% and 46% ± 22%, respec-tively, this tissue contained on average 6.9 ± 0.3 X 106 y6 cells/g tissue, representing an al-most 5-fold increaseover thatfound inthe

nor-mal mucosa.

Furthercharacterization of the mucosal y6T

cells in the normal and inflamed colon using

TCR-V,y/8-(J6)-specific antibodies revealed some interesting differences in the y6 T cell subsets present (Fig. iB). In the noninvolved colonic mucosa, V81+ ySTcells comprisedamajor

sub-set (43-52%) of IE 'y8 T cells. Of these cells,

V61-J8I-encoded TCR-8 chains predominated (63-76% of V81+ cells). By contrast, in the LP, V51+ y8T cells represented a minor subset (6-10%) of y8Tcells; as seen among IEL, VI+-J6I

wasthepredominant (43-58%)TCR-6 chain

ex-pressed by these cells. Thus, VSI-JSI-encoded receptorspredominate amongV61+ yS T cells in both the IE space and LP of the normal colonic

mucosa. Vy9/VS2+

y6

T cells that make up the largest-y5Tcell subsetinthe blood (Fig. IB) had

adistributioninthe normal colonic mucosa that

wasreciprocaltothat ofthe V61 subset. Whereas they representeda minor subset (13%) of

TCR-y75+

IEL, they were the major

y6

T cell subset in theLP (67-79% ofTCR--y6+ cells).

Incomparing they6Tcell subset distribution

in the involved and noninvolved mucosa, the

most striking difference was the increase in the proportion of VS1+ -y6 cells in the LP of the involved mucosa.Inboth theUCand CD patient groups, VS1+ cells represented the major (57-76%) population of yS T cells, of which most

(76-81%) expressed a V8I-J8I-encoded TCR.

By contrast, there was no significant difference

inthe proportion ofV 1-bearing y5Tcellsinthe

A

Non-Involved

I

Involved

IEL

0

7

.1 O l 1 l2 103 4

B

%CD3+ Cells %y8+Cells %V61+Cells

Patient

T cell source %CDl9+ 'l'+ V-9+/V82+ V81+ -J81+ CD8+

CD8-Colonic CD(n=3)

Blood ND 3 ± 2 78±8 10±8 ND ND ND

EEL N 5 4 21±10 13±7 43±13 76±12 75±6 21±4

I 10±3 28±13 27±12 67±21 74±11 33±4 68±7

LP N ND 5±4 67±11 10±5 58±8 42±6 63± 7

I ND 43 ±20 51±14 76±13 81±13 15± 3 86±5

TotalUC(n=4)

Blood ND 4 ±2 83±11 12 6 ND ND ND

EL N 4±3 22±9 13±10 52±14 63±19 83±5 13±4

I 11±4 26±8 18±9 61±23 60±14 40±2 62±4

LP N ND 15±7 79±16 6±4 43±10 ND ND

I ND 64±19 29±9 57±17 76±18 ND ND

IEL.inrepithelial lymphocytes;LP.laminapropria; N. non-involved colonic mucosa; I. involved colonic mucosa;

ND,notdetermined.

FIG. 1. Distribution of

'y6

Tcells inthenormalandinflamed intestinalmucosa

(A) Representative analysis of y6Tcells present within the intraepithelialspace (IEL) and laminapropria (LP) of the colonic mucosa from apatient with total colitis. (B) CD8 expression andTCR-Vy/8usagebyIE- and

LP-y8

T

cells inthe blood, noninvolved (N) and involved (I) intestinalmucosa ofpatients withcolonic CD and totalcolitis. ND, not determined.

the intestinal mucosa ofIBD patients is

charac-terized, therefore, by changesinthe distribution of y6Tcellpopulations andby the accumulation of VS1+ cells in the LP. Although we do not

know if thisincrease in y8Tcells and V61+ cells represents expansion of resident mucosal cells or

cells infiltrating from the blood, differences in

the phenotype of V61+ cells in the LP (and IE

spaces) of normal colonic mucosa (majority

CD8+) versus involved mucosa (majority CD8 )

suggests they are mainlyofperipheral origin.

Bias inExpression of V81-encoded

Receptors by yST Cells in Surgical

Resected Tissue Specimens of IBD

Patients as Demonstrated by

RT-PCRAnalysis

Repertoireanalysis usingantibodies and flow cy-tometry is limited by the size of mucosal tissue samples, the number of cells that can be

recov-ered fromeachsample, andbytheavailability of TCR-V(J)-specific antibodies. Also, since the

LP

.

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F..-:

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L. D. McVay etal.: ySTCells andInflammatoryBowelDisease

ILEUM TERMINAL

VIl- J3J2 33 1123334

L.JLJW-i-

L.JL.AJ"L-NL-1 NL-2 NL-3

UC-I

UC-2

UC-3

UC-4

UC-5

uc-8

UC-9

uc-10

UC-12

UC-15

ASCENDING TRANSVERSE DESCENDING

COLON COLON COLON

Jt32J2 4 312J3 4, JilJ2 34

L JJL- _JL-JLJi L-LMJ

CD-1 CD-2

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CD-S

CD-7

CD- II CD-12

DIV-1

DIV-2

DIV-3

not d)NA

FIG. 2. RT-PCRanalysis ofV61-J6 TCR expressioninsurgical samples ofnormal,noninvolved, and

inflamed, involved intestine ofindividual patients withCD,UC, diverticulitis (DIV),ornormal (NL)

mucosafromvarious locations along thelength oftheintestine

Mucosal cDNAsampleswere amplifiedfirst with primersspecific for V81 and CS andtheamplified products were gelpurified then reamplifiedin asecondPCRreaction withthe sameV61 primer used for the first amplification

and witha reverseprimerspecific foreachof the four J6 genes. V51-J5PCRproducts wereelectrophoresed

through 2% agarose gels, blotted, andprobedwithaninternal 32P-labeledV81-specificoligonucleotide probe. Autoradiography wascarried out for 24 hr. Asterisksindicate V6-J$ profile ofnoninvolvedcolonicmucosa.

V61-J61-specific antibody may have some

reac-tivity with J6-encoded receptors other than J61 (30), it was important to corroborate the results obtained by flow cytometry using another

inde-pendent approach. A sensitive RT-PCR based

method thatwehavepreviouslyused for

analyz-ing TCR-,y5 repertoire analysis in human fetal tissues (24,25) was used for this purpose. The

assay uses oligonucleotide primers specific for

V61, V62,orV83,incombinationwithaCSgene

segmentprimertoamplifycDNAgeneratedfrom

mRNAsamples usingreversetranscriptase.After

gelelectrophoresis the products of amplification

were excised from the gel, and equivalent

amounts usedas a template for a second

ampli-fication using thesame forward VSprimeranda

reverse-primer specific for each of the four

hu-manJ8 genes. In establishingthisassay,

individ-ualV81-JT1-4, V82-J51-4, and V63-J61-4

recep-tor cDNA clones were used to determine the

optimum conditions for the proportional

ampli-fication ofV5-J5genes; therewas no

cross-prim-ing. The products of this second amplification

werethenelectrophoresedon 1.8%agarosegels,

Southern blotted, and hybridized with [y32p]_

ATP end-labeled V51, V62, or V83

oligonucleo-tide probes containing sequence internal to the

VS primers used for PCR.

This assay wasfirst used to analyze samples

of intestine from noninvolvedandinvolved

seg-SIGHOID/_RECTUMX

JL J2J3 .

PERIPHERAL,

BIAWD J3 J2 3 4

L-JLJL-JL-i

INVOLVED NON-INVOLVED PERIPHERALBLOOD

V62- Jl J2 J334 JJ2334 J1 J2 J3 J4

t .,_ I__ LI L-J XtJ*.I

NL-lI NL-2

NL-3

UC-2

UC-4

UC-IO

UC-11

UC-'1

CD-I

CD-2

CD-4

CD-8

CD-7

CD-6

INVOLVED

V63- J3 J2 J3 J4

L.JLJiL- L-J

NL-I

NL-2

NL-3

UC4

-UC-II

-UC-12

-UC-IO

CD-I

CD-5

CD-12

CD-8

CD-7

CD-6

NON-INVOLVED PERIPHERALBLOOD

JI J2_{L_JL__J.}J3 J4 J3 J2 J3 J4

I L- _JL_J LI.JLIJ

FIG. 3. Profile ofV62-J6 andV63-J6TCRexpression in surgical samples ofnoninvolved andinvolved intestine of individualpatients with CD, UC, or normal (NL) mucosa from colon cancer patients RT-PCR was carried out as described in Fig. 2-using 32P-labeled oligonucleotide probes complimentary toV62 or V63. Autoradiography wascarried out for 24 hr.

ments of tissue obtained from 20 patients with

IBD for Va-Jl receptor expression. Ten patients

diagnosed withUC (1-16inTable 1), sevenwith

CD (17-28 in Table 1), and three patients with

diverticular disease (diverticulitis, 29-31 in

Table 1) were used as a source of involved and noninvolved mucosalmRNA. In addition, tissue

from the normalmarginsofcolonicmucosafrom three colon cancer patients (32-34 in Table 1) were also used as noninvolved, "normal" con-trols. The results of this analysis shown inFig. 2

identify changes inthepatternofVS1

-J6

expres-sion in the involved mucosa compared to the noni-nvolved mucosa and blood of IBD patients.

TheV8-J5profile of the noninvolved colonic mu-cosa (* profiles in Fig. 2) was characterized by

expression of receptors encoded by V81-J61, 2,

and 3, and by very low levels or an absence of

V61-J84-encoded receptor chains. This profile

was distinct from that of theperipheral bloodin

whichallfourJ6-genesegmentswereutilizedby

V61-encoded receptors. By contrast, a single Va1-encoded receptor predominated in the

in-volved colonic mucosa. Regardless of whether the involved intestinal tissue was derived from

patients with UC, CD, or diverticulitis, V61-J61

receptor expression predominatedand itwasthe

only V81-encoded receptor detected in 13/17 IBD patients and 3/3 patients with diverticular disease. Importantly, thisfinding agreeswith the results obtained by flow cytometry in which

V51-J61 cells were the major

V61'

yb T cell

population in the involved colonic mucosa

(Fig. 1), and it also demonstrates the utility of this procedure for TCR--ya repertoire analysis.

DNA sequence analysis of randomly selected

V1-Ca cDNAs from involved and noninvolved

mucosal mRNAsamples from a patient with UC did not identify any V51 receptors that were uniquetothe inflamed colonic mucosa (datanot

shown). All of the Val-TCR-cDNAs encoded

functional, in-frame receptors consistent with the receptor cDNAs detected by RT-PCRanalysis being derived from productively rearranged

genes. The V51-Ca cDNAs from noninvolved

mu-L. D. McVayetal.: ySTCellsandInflammatory Bowel Disease

INVOLVED

V61- JI J2 J3 J4

L-)LJ j

NON-INVOLVED

JI J2 J3 J4

LA *_jI DURATION

15y

12y

1wk

5y

V52-J41 waspredominantor wastheonly

recep-torexpressed. Likewise,inraresamples (i.e.,

UC-10) a bias in the V63-J8 expression was also evident. Taken together, our analysis of the

TCR-V6 repertoire in the colonic mucosa

dem-onstratesthata single VSreceptor, V 1-J$1,

pre-dominates in the inflamed mucosa of patients

with CD and UC.

5-6y

13y

5y

2v

FIG. 4. Profile ofVei-Je TCRexpression in

mucosalbiopsysamples of noninvolved and

involved intestine ofindividualpatientswith colonicUC

PCRwas carried out asdescribed in Fig. 2.

Autora-diography

was carried outfor24 hr.

cosa utilized the J61 gene segment, which is consistent with the results fromboth flow cyto-metricandRT-PCRanalyses.Inaddition,asingle

D-gene segment, D63, was exclusively used by

all of the VSI-receptor chains expressed in the noninvolved (33/33) and involved (22/22) mu-cosa.

Lack ofBias inthe Expressionof V62- and

Ve3-encoded Receptorsin Surgical

Resected Tissue Specimens of

Patients with IBD

Wewereinterestedinknowing whether the V82

and V63 receptors, which are similar to V81-encoded receptors, were also biased in JS gene

usage. Figure 3 shows the V82/V83 repertoire

analysis of 10 IBD patients representative of a

total of20patient samples analyzed. Inthe

nor-mal intestine, V62 receptors primarily utilized J61 and J63 whereas V83 receptors utilized all fourJ6 genesegments.Thisprofile ofV62-J6 and V63-J6 expressiondidnotchange significantly in the noninvolved intestine or in tissue samples

obtained from different regions of the inflamed intestine (data not shown). In2/16 CD samples

and 3/35 UC samplesfromtheinflamedmucosa,

Bias inExpression ofV61-encoded

Receptors by yST CellsinBiopsy

Tissue Specimens

TheRT-PCRassaywasnextappliedtotheanalysis

of colonic mucosal tissuebiopsy samples to

deter-mine ifchanges in yST cell repertoire detected in patients with severe disease were present in

pa-tients with newly diagnosed and/or less severe

forms ofIBD (Table 2).Becauseofthe small size of the mucosal tissuesamples procured by

sigmoidos-copyandcolonoscopy, itisnotnormallypossibleto

isolatesufficient numbers ofcellsfor

fluorescence-activatedcellsorting (FACS)analysis.Thesamples

obtained from eight UC patients (Table 2) were

therefore processed forRNAisolationandRT-PCR

analysis of TCR-V6-JT expression. The results of this analysis shown in Fig. 4 clearly identify V61-J61 receptorexpression asbeing the predominant

receptorexpressed inallof thepatientsamples.In

five of eight patient samples itwastheonlyV61-J6

TCR-8chainexpressed. Interestingly, this receptor was also predominant in the mucosa of a newly

diagnosed UC patient who had symptoms of

dis-easeforless than 2weeks. Theexpressionofother

V81-Jo-encodedreceptorsin noninvolved samples strongly suggests that the predominance of

V81-J61 receptor expression in the involved mucosa

was notdueto a PCR-relatedartifact.

In summary, our cellular and molecular

analyses of y6 T cells in the colonic mucosa of

patients with IBD has shown that these cells

accumulate in large numbers at the site of

in-flammation and tissue injury and canbe

distin-guishedbyexpression ofaTCR-8chainencoded

by V61-J61 gene segments. These changes were

apparent in all the IBD patients we analyzed,

irrespective of the form orstage and severity of

disease. To begin to understand the significance ofthese changes in the mucosal y8 T cell

reper-toire for the disease process inIBD, subsequent

experiments were designedtoanalyzethe effec-torfunction of mucosaly5T cells.

B-I

B-2

B-3

B4

B-5

B-6

B-7

B-8

A l 5B | ihi i

,... ..,.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

, 1_

M_

~

~

~

~

~

~

~

~

M

FIG.5.In situ hybridizationanalysis ofIFN-ygene expressionin sections ofnoninvolved. r Fed

areasof colon from CD patient, CD-i

Lo

_;1L5...

^_S

7:t.;..':'4'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

.

....

II s;4sAl~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~31r -5-':::.:

w --~~~~d..._

(A). 5Nonsinvolved issu sen Rn probe. (B Noninvole d tiss, aontinsenseiRnA probe(ninvolved tiss.e,

sense RNAprobe. (D-F) Involvedtissue, anti-sense RNAprobe. L, lumen;M, mucosa; S, submucosa; By, blood

L. D. McVayetal.: ySTCells andInflammatoryBowel Disease

TABLE 3. IFN-,yproductionbylaminapropriaTcells in the normal and diseased colon

U/ml TCell

Populationa %mRNA+ Cellsb Media anti-CD3

CDPatient #1 (Ileocolonic)

Total N 12 ±4 <5 39 ±8

I 39 ± 5 5 99± 10

a,+ T cells N 10±2 10 ±2 38 10

I 46± 10 9±3 53± 14

-y5+ T cells

(9%VS1+) N 16±4 7 ± 6 19 ±2

(35% V$1+) I 39 ± 5 12 ±4 109± 5

CDPatient#2(Ileocolonic)

Total N 15 ±3 8 ±9 13 ±2

I 61 ±6 7 ± 7 78 ±9

a,l3Tcells N 11±5 6±6 23±5

I 56 ±8 13 11 57 ±8

,yS+ Tcells

(12% V81+) N 17 9 11 8 18± 13

(53% V61+) I 38 7 24 10 165± 11

CDPatient#3 (Colonic CD)

Total N 11 6 <5 59 ± 17

I 35 10 43 19 78± 21

ap+

Tcells N 29±5 18 9 39± 6

I 39 7 33 11 54± 10

,yb+ Tcells

(15%V61+) N 9±3 12±8 35± 12

(43% V$1+) I 66 8 34 15 177 ± 21

aNonfractionated (total)Tcells andpurified populations of

af3+

and-yS+ cells were isolated from segments of noninvolved (N)

and involved(I) colonicmucosa.Frequency ofV8I-expressing cellsin y6Tcell preparations is shown in parentheses.

bFrequencyof cellsthat afterhybridizationinsituwith anti-sense probe were overlayed with more than 5 grains.

Mucosal ySiT Cells Area Major Sourceof IFN-,yProduction in IBD

Since IFN-y production appearstobe an

impor-tant mediator of the inflammatory process and tissue injury in patients with IBD (31) and is involvedinthepathogenesis of intestinal

inflam-mation inanimal models ofIBD (32), we inves-tigated whether

y8

T cells in the intestinal mu-cosaof patients withIBD were a sourceof IFN-,y.

In situ hybridization analysis of

IFN-'y

mRNA

expression in tissue sections from involved or

normal intestinal mucosaltissue (Fig. 5) showed that the

IFN-'y

genewasconstitutively expressed

atlowlevelsby cellsinthenormal, noninvolved,

mucosa. By contrast, in sections ofinvolved co-lonic tissue froma CD patient (CD-1), the num-berof IFN-'y-mRNA+ cells wasgreatly increased (Fig. SD). Nohybridization signal was detected in

sectionsprobed withacontrol senseprobe (Fig.5A

andC). The distribution of IFN-,ymRNA+ cells in

diseased mucosa (Fig. SD) was similar to that of

TCR-S+ mRNA-expressing cells (data not shown), suggestingT (y6) cells as alikely source of IFN-,y production. Inaddition, the identification of IFN-,y

rnRNA+ cells in blood vessels in the submucosa (Fig. SE) and muscle (Fig. SF) was consistent with

A

r-

F-c_ 0

._

0

0

B

0 0

-._

0

E

0

._ 3

Po

Colon Skin Lung

Fibroblast Cell Lines

N I N I N I N I

CD7-ML CD8-ML CD9-ML CD10-ML

FIG. 6. Enrichment ofy6Tcells amongperipheral blood (PB) and colonic mucosal (ML)Tcellsby adherence tofibroblast cell lines

(A) Selectiveadherenceofy6Tcellstofibroblast celllinesof intestinalorigin. Tcells isolated from the blood of normal healthy donors (N-PB) andCDpatients (CD-PB) and from segments of inflamed(involved) colon fromCDpatients

(CD-ML) wereco-cultured with different established fibroblast cell lines for30minat370C.The fold-enrichment ofys

Tcells among theadherentcells was determinedby comparing the number ofySTcellsintheinputandadherent frac-tion, the values for whichwereobtainedbystaining with anti-TCR antibodies and flow cytometry. (B) Preferential ad-herence of mucosal 'ySTcellstocolonic fibroblasts. ColonicTcellsisolated from segments of involved (I)and nonin-volved (N) colon from thesame CDpatient werecultured withthe colonic fibroblast cellline,CCD-18Co,for30min. Fold-enrichment of adherenta(3and ySTcellswasdetermined byflow cytometry.

the transmural distribution of

TCR-8-mRNA'

cells (datanotshown).

To identify the cellular source(s) of IFN-,y production in the colonic mucosa, IFN-y-gene expressionand protein secretionwasanalyzedin

isolated populations of

y6

and aj3 LP T cells. These T cell populations were isolated from the mononuclear cell fraction of noninvolved and involved mucosa from three patients with CD using immunomagnetic selection. The isolated populationswereroutinely >95% CD3+and the degree of contamination of the

y8

fraction by

aI3+ cells and the af3 fraction by y6+ cells was routinely <10% (data not shown). As seen in ourpreviousflow cytometricanalyses of mucosal

Tcells (Fig. IA), VS1 + cells were the major y6T

cell subset (67 ± 8%) in the involved mucosa compared with the noninvolved mucosa (12 ±

2%).Theincrease inthe number ofTcellsinthe

LPof involvedmucosa(seeabove)wasparalleled by an increase in the frequency of cells that

constitutively expressed IFN-,y-mRNA (Table 3). Thiswasparticularly apparent for

y6

Tcells that contained a higher proportion of mRNA+ cells than af3 T cells. The analysis of

IFN-'y

secretion

was consistent with the mRNA analysis and

demonstrated that

y8

T cells, including V81 +

cells, were a major source of this

proinflamma-tory cytokine in the involved mucosal CD

sam-ples analyzed.

Selective Interaction of 'y6 T Cells with Colonic Fibroblasts

Inviewofthe accumulation of y6Tcells, partic-ularly VS1+ cells, in the LP of the involved

in-testinal mucosa of CD patients and results from previous studies demonstrating that y8 T cells

can interact with stromal cells in other tissues

suchasskin (9,33),wedetermined if mucosal y6

Tcells, particularly VS1+cells, from patients with

IBD could adhere to and interact with colonic stromal cells, and fibroblasts in particular. To accomplish thiswe took advantage of the

avail-ability of established and well-characterized fi-broblast cell lines of different mucosaltissue

or-igin and the y8 T cell-skin fibroblast adherence assaydescribedby White and co-workers (9).To

date, the possibility that

y6

T cells can interact with colonic stromal cells suchas fibroblasts and how this interaction might be affected during

IBDhasnotbeeninvestigated. Bulkpopulations of colonicT cells isolated from segments of

nor-mal and involved tissue of individual IBD

pa-tients were briefly incubated with confluent

L. D. McVay et al.: ya TCells and InflammatoryBowel Disease

B

U,

.-A (U

0

WI-U,

.0

80

60

40

-20

0-CD7-ML CD8-ML CD9-ML CD10-ML CD-11-ML

FIG. 7. Predominance of fibroblast-adherent VS1I 'yi Tcells in the inflamed colonic mucosa

Colonic Tcells isolated from segments of involved and noninvolved colon of CD patientswereco-cultured for 30 minwith the established colonic fibroblast cell lines CCD-18Co (A) ora primary colonicfibroblast cell line ob-tainied frorn involved mucosa of CD Patient 11 (B). Theproportion ofV61+ fibroblast-adhering cells was deter-mined from the nluLmber of V6l+ cells in the inpLutand adherent cell populations by flow cytometry.

and phenotype of adherenit and nonadherent

cells determiined by flowcytometry.

As seein in Fig. 6A, mucosal y6 T cells from

one of two patienits with CD (CD8) were

consid-erablyenriched for after co-culture with colonic fibroblasts. Whereas co-culture with skin- or

luIng-derived libroblasts produced between 1.5-and 3.5-fold enlrichment of -y6 T cells, culture

with the colonic fibroblast cell line CCD-18Co resulted in an almost 10-fold enrichment of y6T

cells. A similar level of y6 Tcell enrichment was

also seenusinganothercolonicfibroblast cell line

(CCD- 12Con) (data not shown), excluding the

possibility that the interaction with the CCD-18Co cell line was due tosomepeculiarproperty

of this cell line. Analysis of nonadherent T cells from cultures with colonic fibroblasts showed a

reciprocal decrease in the y6 T cells (data not

shown), excluding the trivial explanation that theirenrichmeintwas dueto preferential survival duringorafter culture.Thespecificityof mucosal

-y T cell-fibroblast interaction was also

demon-strated by the failure to see a similar enrichment

of y6 T cells tisinlg peripheral blood from two of

thesame CDpatients, orusingfibroblasts derived

from sources other than the colon. Enrichment

of o43 T cells am-ong adherent cells after

co-cul-ture with colonic fibroblasts, or fibroblasts of other tisstic origin (data not shown), was 2-lold or less (Fig. 6B), which suggests that increased

adhesiveness of -y T cells reflects an inherent

p)roperty of these cells rather than a function of

the fibroblastmonolayer. Slightly higherlevelsof

-y6 T cell enrichment were seen usinlg

lympho-cytesfrom the involvedversus noninvolved

mu-cosa with three of the four patienits with CD

(Fig. 6B).

Analysis of the fibroblast-adherent -y5Tcells

showed that the majority of those isolated

from the noninvolved (75%0/ + 8%) and

in-volved (84%±+ 9%) mucosa of four patients

with CD (#7-10; Fig. 6B) were V61+. By

com-paring thenumberofV61+ cellsinthe input and adherent cell populations it was possible to

de-termine what proportion of these cells in the

noninvolvedversusinvolvedmucosaofthesame

patient were fibroblast-adherent (Fig. 7A).

Whereas in the noninvolved mucosa

fibroblast-adherent V61 cells represented <40% (mean of 310% with a range of 24% to 390/o) they

repre-senited almost 80% (mnean of 78% with a range

of 63% to 92%) of V61+ cells in the involved

mucosa. Thus, the accumulation of V61+ -y T

cells in theinflamedmucosaofpatientswithIBD

wasassociated withan increase intheirabilityto

interactand adheretocolonicfibroblasts. Usinga

primary colonic fibroblast cell line derived from mucosal tissue sample from a patient with pri-mary colonic CD (Fig. 7B), we have reproduced

the resLilts obtained using established colonic fi-A

100

80

60

40

20

cn

0

-W

U,

0

O Noninvolved * Involved

CD12-ML

nn

broblast cell lines. In particular, using either do-nor-matched or allogeneic LP lymphocytes from noninvolved or involved mucosa as input cells, a higher proportion of V81+ cells from the in-volved mucosa (88% ± 6%) were fibroblast-adherent compared with those from the nonin-volved mucosa (47% ±- 5%).

DISCUSSION

We have compared the phenotypic and func-tional characteristics of ySTcells within the nor-mal and inflamed colonic mucosa of a large number of patients with IBD. Our results

dem-onstrate that the chronic inflammatory immune responsethat occurs inthecolon of IBD patients is associated with distinct changes in the

num-ber, distribution, composition, and function of mucosal y6 T cells. The size of the y6 T cell

population in the diseased mucosa was signifi-cantly increased and could be attributed to an

increase in V81+ y8 T cells, among which, cells thatexpressedaTCR8-chain encodedbyV1-J81 gene segments predominated. These changes in

mucosal ySTcells were notrestrictedto individ-ual patientsor aparticulargroupof patientswith

IBDbutwerepresentinthe majorityof UC, CD, and diverticulitis patient samples that we

ana-lyzed. The predominance of V81-J81

receptor-bearingcells was seen inpatientswith advanced forms of disease who required surgery and in

biopsy samples obtained from patients with less

severe forms of IBD, as well as in apatient that had clinical signs of disease for less than2weeks. AmongTcells presentintheinflamedmucosaof patients with CD, y6Tcells were a major source

of theproinflammatory cytokineIFN-,yandwere

enriched for V81+ yBTcells that couldselectively

interactwithcolonicfibroblasts. Althoughit was notpossible to determine ifthese changes inthe properties of mucosal

y6

cells were primary ab-normalities of these disorders, they are

consis-tent with, and suggest that they contribute to,

thepathophysiology of IBD.

The results of our studies contrast with those obtained previously from the immunohisto-chemical analysis of y8 T cells in colonic tissue sections obtained from IBD patients in which a

decrease (17) ornochange (16)inthenumber of

TCR-6+

cells was reported. A likely explanation

for the apparent discrepancy between thesetwo

studies and our own is the different techniques used. The immunohistochemical detection of T

cells inintestinal tissueisdifficult to perform and is a relatively insensitive means of detecting an-tigens such as the yS TCR that are expressed at

low levels (28,29). In addition, since therelative number ofy8Tcells within the intestinal mucosa has been shown to vary greatly, not only be-tween different sections of the same tissue but also between sections of the samesiteoriginating from different subjects (29), estimates of their relativenumberbased upon this technique alone

are unlikely to be accurate. Finally, since the

degree ofinflammatory activity may also

signif-icantly influence the distribution of y6T cells in

the diseased colon, variation among individual patients withIBD may also accountfor some of the contrasting results obtained fromthe

differ-ent studies.

The increase in

V61-expressing y8

T cells in

theLPof the inflamed colonofIBDpatients may represent the expansion ofresident and/or infil-trating cells fromthe blood. Since there are

cur-rently no phenotypic markers, including CD8, that can unambiguously discriminate between intestinal and blood-derived V1

y8

T cells, itis not possible to definitivelyidentify the origin of V61+ cells within the LP of patients with IBD.

However, several pieces of evidence suggest that

alarge number of these cellsarederived from the

peripheral blood. The phenotype of LP V61+

(CD8 ) cells in IBD patients is similar to that of those normally found in theblood (34) and

re-sembles the phenotype of blood-derived

y$

T

cells that possess transendothelial migratory properties andaccumulate within tissuesatsites of inflammation (10). Interestingly, Chowers andcolleagues (3 5) haverecentlyshownthat,on

the basis of the presence of unique junctional

sequences of TCRs expressed by intestinal

y6

T

cells, it is possible to distinguish between V81+

cells normally resident in the blood and intes-tine.

Irrespective of the origin of the V81+

-y6

T

cells that accumulate in the colonic mucosa of

IBDpatients, animportantquestionthatremains tobeaddressedis thenature of thestimulus that drives their expansion. The oligoclonal nature

and limited structural diversity of the intestinal

VMI

repertoire in the normal intestine (35) sug-gestthat thearrayofligands recognized bythese receptors is limited. There are three possible

sources ofthese ligandsinthe inflamed colon of

IBD patients. The first is that they are enteric

(bacterial and food) antigens that mucosal V81+

y8

Tcells could beexposedtoas aresult of them

L. D. McVayet al.: y6TCellsandInflammatory Bowel Disease

intestinal wall, which is disrupted as part ofthe

disease process in IBD. The recent observation

that E. coli and P.

aeruginosa,

which normally inhabit the gastrointestinal tract, can stimulate V51-bearing peripheral blood y6 T cells (36) is

consistentwith thispossibility. Furthermore, the

finding that gut T cells from patients with IBD

proliferate vigorously to preparations of their ownflora (37) suggests that abreakdown inthe mechanismsthatnormally operateto maintain T

cellhyporesponsiveness ("oral tolerance")to en-teric antigens is an important pathogenetic mechanism of IBD. A second possibility is that the antigens are endogenously derived and are

expressed by stressed ordamaged epithelial cells. The selective accumulation of V81+ y6 Tcells in

the IE spaces of the intestine has long been

in-terpreted as evidence for an immune surveil-lance role for thesecellsinwhich they recognize and react with a common antigen, such as heat shock proteins (HSP), expressed by epithelial cellsas aresult ofinfection, damage, or transfor-mation (38). To date, however, recognition of

self-antigens such as HSP by y6 T cells has only been demonstrated for cells that utilize a

recep-torencoded by the V82 and Vy9 gene segments (39,40). Extensive epithelial cell damage could, however, enable IEL such asVMI+ cells to"leak" intothe LP, thus contributing to the increase in the number of V81+ cells seen inthe LP of IBD patients. The third possibility is that the

activa-tionofV61-expressing ySTcells is antigen-inde-pendent and does not involve the TCR/CD3

complex. The existence of alternative, antigen-independent pathways of mucosal T cell

activa-tion is suggested by the finding that LP T cells, unlikeperipheral bloodTcells, from normal and

IBD patients exhibit vigorous proliferative re-sponses via the CD2/CD28 signaling pathway (41-45).The presenceinthe inflamed mucosa of

large numbers of activated T cells that produce

cytokines (46-49)couldalso serve to activate,in a"bystander" manner, V61+cells (50), as well as

tomaintain apopulation of chronically activated

T cells within the mucosa.

The

y8

T cells that accumulate in the in-flamed mucosa of patients with CD possess two functional characteristics that are relevant to the

pathophysiologyof thisIBD. The first is that they

are a majorsource oftheproinflammatory cyto-kine IFN-y, which has long been shown to me-diate epithelial cell injury (51). Although the

constitutive production of

IFN-'y

by colonic lym-phocytes from IBDpatients has been previously reported (44,49,51), our analysis identifies yS T

cells, including V61 + cells, as a source of this

cytokine in situ in the inflamed mucosa, and

suggests a meansby which they might contribute

to the chronic inflammatory immune reaction andtissueinjuryin CD.Thesecondcharacteristic

isthe selective adherenceand interaction of

mu-cosal y6 T cells, particularly VS1+ cells, with

in-testinal-derived fibroblasts. Although uncon-trolled fibroblastproliferation, excessivecollagen secretion,and fibrosis cause severe tissuedamage

and are important complications of CD (52,53),

theprocesses regulating normal fibroblast

activ-ity arepoorly understood. Onewayinwhichthe

activity of fibroblasts and other stromal cells could be regulated and coordinated is through direct interactionswith othercells, such asVS1+

y6 Tcells. Theproduction by -yBT cells of tumor necrosis factora (TNF-a), ILI-f, and

transform-ing growth factor ,B (TGF-,B) (54) that can pro-mote fibroblast growth and collagen synthesis (55), and the observation that

epithelial-associ-ated y8 T cells in the skin can influence the

activity of keratinocytes by the production of

keratinocyte cellgrowth factor (8) areall consis-tent with this hypothesis. This immunoregula-torypropertyofySTcellsisfurther substantiated by the results fromavarietyof animal models of infectious disease in which the ability of -yS T cells toinfluence the activity of other cell pop-ulations and the outcome of inflammatory im-mune responses has been demonstrated (56-61). Since ithaspreviously been shown that fibroblasts can prolong the survival ofT cells in

culture after the removal of IL-2 and antigen (62), fibroblast adherence may also provide a

means of localizing or retaining activated, pe-ripheral blood-derived, V51+ cells in the in-flamed colon. Although we do not yet have an explanation for the differences in 'y$ T cell

sub-sets adhering to colonic fibroblasts, it could be related to quantitative and/or qualitative

changes in the expression of adhesion/integrin

molecules such as LFA-1 and VLA-5 by V81+

cells (9,33,63) and their reciprocal receptors,

ICAM-1 and fibronectin, respectively, by fibro-blasts (9,33). The preferential association of VS1+ y8Tcells withfibroblasts provides a possi-ble explanation for their accumulation in the lungs (64) and joints (65-69) insarcoidosis and rheumatoid arthritis patients, respectively. Inter-estingly, inflammatory disorders of the lung, joints,skin,andeyeareoften presentas

where, asa result of theirabilityto interactwith fibroblasts or other connective tissue elements,

they accumulate and can contribute to the

in-flammatory immune reaction and inflammation atthese sites.

Insummary, we have demonstrated that the chronic inflammatory reaction and tissue injury that occurs in the colon of patients with IBD is associated with distinct changes in the number, distribution, composition, and effector function of mucosal

'y

Tcells,particularlyV81-expressing cells. These findings suggest that these cells

con-tribute to at least some of the immunological and

pathological changes presentin the inflamed

mu-cosa of patients with IBD, andparticularly CD.

ACKNOWLEDGMENTS

We aregrateful to Ms. Linda Hurd for compiling

pertinentdataon IBDandnormal patientsinthis study. We thank William G. Crowley for expert technical assistance.This work wassupportedby grants from the American Cancer Society (JFRA 399) and the National Institutes of Health (Al 31972) toS.R.C. L.D.M. wassupported in part by

a National Institutes of Health Training Grant (5-T32-CA-09140-20) and a Crohn's and Colitis of America (CCFA) Fellowship.

REFERENCES

1. Strober W, James SP. (1986) The immuno-logical basis of inflammatory bowel disease.

J. Clin. Immunol. 6: 415-433.

2. Shanahan F, Targan S. (1989) In: Shaffer E, Thompson ABR, (ed). Modern Concepts in Gas-troenterology. Plenum, New York, pp. 291-310.

3. Strober W, Ehrhardt RO. (1993) Chronic

in-testinal inflammation: An unexpected out-come in cytokine or T cell receptor mutant mice. Cell 75: 203-205.

4. MacDonald TT, Spencer J. (1988) Evidence that activated mucosal T cells play a role in the pathogenesis of enterpathy in human smallintestine.J. Exp. Med. 167: 1341-1349.

5. MombaertsP,Mizoguchi E, Grusby MJ,etal.

(1993) Spontaneousdevelopment of inflam-matory bowel disease inT cell receptor mu-tant mice. Cell 75: 275-282.

6. Sadlack B, Merz H, Schorle H, et al. (1993) Ulcerative colitis-like disease inmice with a

disrupted interleukin 2 gene. Cell 75: 253-261.

7. Deusch K, Luling F, Reich K, etal. (1991) A major fraction of humanintraepithelial lym-phocytes simultaneously express the y6 T cell receptor, the CD8 accessory molecule

and preferentially uses the VS1 gene seg-ment. Eur. J. Immunol. 21: 1053-1059. 8. Boismenu R, Havran WL. (1994)

Modula-tion ofepithelial cell growth by intraepithe-lial y6 T cells. Science266: 1253-1255. 9. White B, Korn JH, Piela-Smith TH. (1994)

Preferential adherence of human y8, CD8+ andmemoryTcellstofibroblasts.J.Immunol. 152: 4912-4918.

10. Galea P, Brezinschek R, Lipsky PE, Oppen-heimer-Marks N. (1994) Phenotypic charac-terization ofCD4U/a TCR+ and y8 TCR+ T

cells with a transendothelial migratory

ca-pacity. J. Immunol. 153: 529-542.

11. Kagnoff MF. (1996) Mucosal immunology:

Newfrontiers. Immunol. Today 17: 57-59.

12. Halstensen TS, Scott H,BrandtzaegP. (1989)

Intraepithelial Tcells of the TCR

y6+

CD8

and V61/J81+ phenotypes are increased in

coeliac disease. Scand. J. Immunol. 30: 665-672.

13. Spencer J, Isaacson PG, Diss TC, MacDonald

TT. (1989) Expression of disulfide-linked

and non-disulfide-linked forms of the T cell receptor y8 heterodimerinhumanintestinal intraepithelial lymphocytes.Eur. J. Immunol.

19: 1335-1338.

14. Spencer J, Isaacson PG, MacDonald TT, Thomas AJ, Walker-Smith JA. (1991) Gam-ma/delta Tcells andthe diagnosis of coeliac disease. Clin. Exp. Med. 85: 109-113.

15. Rust C, PenaS,KluinP, KoningF. (1990) y8

T cells in coeliac disease. Res. Immunol. 141:

668-671.

16. Trejdosiewicz LK, Calabrese A, Smart CJ, Oakes DJ, Howdle PD, Crabtree JE, Losowsky MS, Lancaster F, Boylston AW.

(1991)

y6

Tcell receptor-positive cells of the human gastrointestinal mucosa: Occurrance

and Vregion gene expression inHeliobacter pylori-associated gastrtis, coeliac disease and

inflammatoryboweldisease. Immunology84:

440-444.

17. Fukashima K, Masuda T, Ohtani H, Sasaki I, Funayama Y, Matsuno S, Nagura H. (1991) Immunohistochemical characterization,

dis-tribution, andultrastructure oflymphocytes bearing T-cell receptor y/8in inflammatory

L. D. McVay et al.: y6TCells and Inflammatory Bowel Disease 201

18. Stallmach A, Schuppan D, Riese HH,

Mat-thes H, RieckenEO. (1992) Increased colla-gen type III synthesis by fibroblasts isolated from strictures of patients with Crohn's

dis-ease. Gastroenterology 102: 1920-1929. 19. Bull DM, Bookman MA. (1977) Isolation

and functional characterization of human intestinal mucosallymphoid cells. J. Clin. In-vest. 59: 966-974.

20. Bookman MA, Bull DM. (1979)

Character-istics of isolatedintestinal mucosalymphoid

cells in inflammatory bowel disease.

Gastro-enterology 77: 513-520.

21. Selby WS, Janossy G, Bofill M, Jewell DP. (1984) Intestinal lymphocyte

subpopula-tions in inflammatory bowel disease: An

analysis byimmunohistological and cell

iso-lation techniques. Gut 25: 32-40.

22. Carding SR, Kyes S,Jenkinson EJ, Kingston R, Bottomly K, Owen JJT, Hayday AC. (1990) Developmentally regulated fetal

thy-micandextrathymic T-cell receptor

y6

gene expression. Genes Dev. 4: 1304-1315. 23. Li B, Rossman MD, Imir T, Oner-Eyuboglu

AF, Lee CW, Biancaniello R, Carding SR. (1996) Disease-specific changes in

-yS

T cell

repertoireandfunctioninpatientswith

pul-monarytuberculosis. J.Immunol. 157: 4222-4229.

24. McVay LD, Carding SR. (1996) Extrathymic

generation of

y8

T cells bearing invariant antigen receptors during fetal development.

J. Immunol. 157: 2873-2882.

25. McVay LD, Carding SR, BottomlyK,Hayday

AC. (1991) Regulated expression and struc-ture ofTCR gamma delta transcripts in

hu-manthymic ontogeny. EMBOJ. 10: 83-91. 26. Buschle M, Campana D, Carding SR,

Rich-ards C, Hoffbrand AV, Brenner MK. (1993)

Interferonyinhibitsapoptoticdeathin B cell chronic lymphocytic leukemia. J. Exp. Med.

177: 213-218.

27. CardingSR, McNamara JG, Pan M, Bottomly

K. (1990) Characterization of

y6

Tcellclones isolated from human fetal liver andthymus.

Eur. J. Immunol. 20: 1327-1335.

28. Borst J, van Dongen JJM, Bolhuis RLH, Pe-ters PJ, Hafler DA, deVries E, van de Griend

RJ. (1988) Distinct molecular forms of

hu-man Tcell receptor

y/6

detectedonviableT cells byamonoclonal antibody. J. Exp.Med.

167: 1625-1644.

29. Vroom T, Scholte G, Ossendorp F, Borst J. (1991) Tissue distribution of human y5 T

cells: No evidence forgeneral epithelial tro-pism. J. Clin. Pathol. 44: 1012-1017.

30. Porcelli S, Brenner MB, Band H. (1991) Bi-ology of the human y6 T-cell receptor.

Im-munol. Rev. 120: 137-183.

31. Fiocchi C. (1994) Cytokines. In: Targan SR,

Shanahan F (eds). Inflammatory Bowel Dis-ease, from Bench to Bedside. Williams and

Wilkins, Baltimore, pp. 107-122.

32. Powrie F, Leach MW, Mauze S, Menon S,

CaddleLB, CoffmanRL. (1994) Inhibitionof

Thl responses preventsinflammatory bowel disease in scid mice reconstituted with

CD45RBhi CD4+ T cells. Immunity 1: 553-562.

33. NakajimaS, RoswitWT, LookDC, Holtzman

MJ. (1995) A hierarchy for integrin expres-sion and adhesiveness among T cell subsets thatislinkedtoTCR geneusageand

empha-sizes V81+ -y5 T cell adherence and tissue retention. J. Immunol. 155: 1117-1131. 34. GrohV, PorcelliS,FabbiM,Lanier LL,Picker

J, Anderson T, Warnke RA, Bhan AK,

Strominger JL, Brenner M. (1989) Human

lymphocytes bearing T cell receptor y/1 are

phenotypically diverse and evenly distrib-uted throughout the lymphoid system. J. Exp. Med. 169: 1277-1294.

35. Chowers Y, Holtmeier W, HarwoodJ,

Mor-zycka-Wroblewska E, Kagnoff MK. (1994)

The VS1 receptorrepertoire in human small

intestine and colon. J. Exp. Med. 180: 183-190.

36. KerstenCM, McCluskey RT, BoyleLA,

Kur-nickJT. (1996)Escherichia coli and

Pseudomo-nasaeruginosa induce expansion ofV82 cells

inadult peripheral blood, but of V81 cells in

cord blood. J. Immunol. 157: 1613-1619. 37. Duchman R, Kaiser E, Hermann E, Mayet

W, Ewe K, Meyer-Zum K-H. (1995)

Toler-ance exists towards resident intestinal flora but is broken in active inflammatory bowel disease. Clin. Exp. Immunol. 102: 448-455. 38. Janeway CA, Jr, Jones B, Hayday A. (1988)

Specificityandfunction ofTcellsbearing

y6

T cellreceptors. Immunol. Today9: 73-77. 39. Munk ME, Schoel B, Modrow S, Karr RW,

Young RA, Kaufmann SHE. (1989) T

lym-phocytes from healthy individuals with specificity to self-epitopes shared by

myco-bacterial and human 65-kilodalton heat shock protein. J. Immunol. 143: 2844-2849. 40. Haregewoin A, Soman G, Hom RC, Finberg

mycobacterial heat-shock protein. Nature 340: 309-312.

41. Ebert EC. (1989) Proliferative responses of

human intraepithelial lymphocytes to vari-ous Tcell stimuli. Gastroenterology978: 1372-1381.

42. Pirzer UC, Schurmann G, Post S, Betzler M, Meuer S. (1990) Differential responsiveness

to CD3Ti vs. CD2-dependent activation of

human intestinalT lymphocytes. Eur. J.

Im-munol. 20: 2339-2342.

43. Qiao L, Schurmann G, Betzler M, Meuer S. (1991)Activationandsignalingstatusof hu-man lamina propria T lymphocytes.

Gastro-enterology 101: 1529-1536.

44. De Maria R, Fais S, Silvestri M, Frati L, Pal-lone F, Santoni A, Testi R. (1993) Continu-ous invivo activation and transient hypore-sponsiveness to TCR/CD3 triggering of human gut lamina propria lymphocytes.

Eur. J. Immunol. 23: 3104-3108.

45. Targan SR, DeemRL, LiuM,WangS, NelA. (1995) Definition of a lamina propriaT cell

responsive state:Enhanced cytokine respon-siveness of T cells stimulated through the

CD2 pathway. J. Immunol. 154: 664-675. 46. Fais S, CapobianchiMR,PalloneF, Di Marco

P,BoirivantM,DianzaniF,TorsoliA. (1991) Spontaneous release of interferon-y by

in-testinal lamina propria lymphocytes in

Crohn'sdisease. Kineticsofinvitroresponse to interferon 'yinducers. Gut 32: 103-107. 47. Mullin GE, Lazenby AJ, Harris ML, Bayless

TM, JamesSP. (1992)Increased IL2-messen-gerRNA in the intestinal mucosal lesions of Crohn's disease but not ulcerative colitis. Gastroenterology 102: 1620-1627.

48. Brynskov J, Tvede N, Andersen CB, Vrlien

M. (1992) Increased concentrationsof

inter-leukin 1,3, interleukin 2 and soluble

inter-leukin 2 receptors in endoscopical mucosal

biopsy specimens with active inflammatory

bowel disease. Gut 33: 55-58.

49. Fuss IJ, Neurath M, Boirivant M, Klein JS,

de laMotte C, Strong SA, FiocchiC, Strober

W. (1996) Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in

in-flammatory bowel disease: Crohn's disease

LP cells manifest increased secretion of

IFN-,y, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J.

Im-munol. 157: 1261-1270.

50. BouillierS, CochetM,Poccia F, GougeonML. (1995) CDR3-independent y5 V81+ Tcell

ex-pansion in the peripheral blood of HIV-in-fected persons. J. Immunol. 154: 1418-1431.

51. Deem RL, Shanahan F, Targan SR. (1991)

Triggeredhumanmucosal Tcells release

tu-mor necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells. Clin. Exp. Med. 83: 79-84.

52. Graham MF, Diegelmana RF, Elson CO,

Lindblad WJ, Gotschalk N, Gay R. (1988) Collagencontentand typesinthe intestineal

stricturesof Crohn's Disease. Gastroenterology 94: 257-265.

53. Mathew RC, Weinstock RJ. (1994)

Granu-loma formation. In: Targan SR, Shanahan F

(eds). Inflammatory Bowel Disease, from Bench to Bedside. Williams and Wilkins, Baltimore, pp. 151-170.

54. Haas W, PereiraP,TonegawaS. (1993) y/8T

cells.Annu. Rev. Immunol. 11: 637-685. 55. Sartor, RB. (1994) Cytokines in intestinal

inflammation: Pathophysiological and clini-cal considerations. Gastroenterology 106: 533-549.

56. DohertyPC, Allan W,Eichelberger M, Card-ing SR. (1992) Roles of

af3

and yS T cell subsets inviral immunity. Annu. Rev.

Immu-nol. 10: 123-151.

57. Hiromatsu K, Yoshikai Y, Matsuzaki G, Ohga S,MuramoriK,MatsumotoK,Bluestone JA,

Nomoto K. (1992) Aprotectiverole of y/8T

cells inprimaryinfection with Listeria mono-cytogenes in mice. J. Exp. Med. 175: 49-56.

58. RosatJP,MacDonald HR,LouisJA. (1993)A

role for y8+ T cells during experimental in-fection ofmicewith Leishmania major. J. Im-munol. 150: 550-555.

59. Skeen MJ, Zeigler HK. (1993) Induction of

murineperitoneal

y6

Tcells and their role in resistance to bacterialinfection. J. Exp. Med.

178: 971-984.

60. Tsuji M, Mombaerts P, LeFrancois L, Nus-senzweig RS, Zavala F, Tonegawa S. (1993)

-y6Tcellscontribute to immunity against the liver stages of malaria in aB3 T cell deficient

mice. Proc.Natl. Acad. Sci. U.S.A. 91: 345-349. 61. Carding SR, Allan W, McMickle A, Doherty

PC. (1993) Activation of cytokine genes in T cells during primary and secondary murine influenza pneumonia.J. Exp.Med. 177:

475-482.

62. Scott S, Pandolfi F, Kurnick JT. (1990)

Fi-broblasts mediateTcell survival:Aproposed mechanism forretentionofprimedTcells.J.

Exp. Med. 172: 1873-1876.

Ad-L. D.McVay etal.: y6TCells and Inflammatory BowelDisease 203

hesion and costimulation of proliferative

re-sponses ofhumany6Tcellsby interaction of

VLA-4and VLA-5 with fibronectin. Immunol.

Lett. 35: 101-105.

64. Forrester JM, Newman LS, Wang Y, King

TEJ, Kotzin BL. (1993) Clonal expansion of lung V81+ T cells inpulmonary sarcoidosis.

J. Clin. Invest. 91: 4291-4302.

65. Soderstrom K,Halapi E, Nilsson E, Gronberg A, van Embden J, Klareskog L, Kiessling R.

(1990) Synovial cells responding to a 65-kD

mycobacterial heat shock protein have a

high proportionof a TCRy8 subtype

uncom-mon inperipheral blood. Scand. J. Immunol.

32: 503-515.

66. Smith MD, Broker B, Moretta L, Ciccone E,

Grossi CE, Edwards JCW, Yuksel F, Colaco

B, Worman C, MacKenzie L, Kinne R,

Weseloh G, Gluckert K,LydyardPM. (1990)

T y6 cells and their subsets in blood and synovialtissuefrom rheumatoid arthritis

pa-tients. Scand. J. Immunol. 32: 585-593.

67. Sioud M, K7jeldsen-Kragh J, Quayle A,

Kal-venes C, Waalen K, Forre 0, Natvig JB. (1990) The VS gene usage by freshly isolated

Tlymphocytes fromsynovial fluids in rheu-matoid synovitis: A preliminary report. Scand. J. Immunol. 31: 415-421.

68. Kjeldsen-Kragh J, Quayle A, Kalvenes C, Forre 0, Sorskaar D, Vinje 0, Thoen J, NatvigJB. (1990)Ty5cells injuvenile

rheu-matoid arthritis and rheumatoid arthritis. ScandJ. Immunol. 32: 651-659.

69. Bucht A, Soderstrom K, Hultman T, Uhlen

M, Nilsson E, Kiessling R, Groenberg A.

(1992) Tcell receptor diversity and

activa-tion markers in the V delta 1 subset of rheumatoid synovial fluid and peripheral blood T lymphocytes. Eur. J. Immunol. 22: 567-574.

70. Spiro HM. (1993) Complications of colitis: Extracolonic complications. In: Spiro HM (ed). Clinical Gastroenterology. McGraw-Hill,

New York, pp. 746-759.