Clin Exp Immunol 1993; 94:527-532

Spontaneous cytotoxicity of intestinal intraepithelial lymphocytes:clues to the mechanism

A. T. ROBERTS, S. M. O'CONNELL*, L. BIANCONE, R. E. BROLIN* & E. C. EBERT Departments of Medicineand *Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, USA

(Acceptedfor publication 15 July 1993)

SUMMARY

Human intestinal intraepithelial lymphocytes (IEL) demonstrate target cell-restricted spontaneouscytotoxic (SC) activity that is due to CD2+CD3+CD8+CD16-CD56- effector cells; they killepithelial cell (EC) tumours (such as DLD- I colon cancer cells), but not natural killer (NK)-sensitiveK-562 cells. The present study shows that the measured levels ofSC activities by IEL correlated withthose of autologous lamina propria lymphocytes (LPL), but not with those of peripheral bloodlymphocytes (PBL). Also, the susceptibilities of DLD- 1 cell clones to lysis by IEL and PBL effectorcells did not correlate, suggesting different mechanisms of lysis. Antibody blocking experimentsshowed that the main surface molecules involved in lysis depended on the effector cell type: aE#7(HML-l) on IEL and CD16 on PBL. No antibody-dependent cell-mediated cytotoxicity (ADCC)was demonstrated by IEL, even after stimulation with interferon-gamma (IFN-y). Few IELexpressed Fc receptors for IgG. This study describes further differences between the SC activities ofIEL and PBL.

Keywords natural killer activity lamina propria lymphocytesantibody-dependent cell-mediated cytotoxicity Fcy receptors colon cancer

intraepithelial lymphocytes

INTRODUCTION

Human intestinal intraepithelial lymphocytes (TEL), locatedbetween intestinal epithelial cells (EC), share many character-istics with tumour-infiltrating lymphocytes (TIL): (i) they haverare natural killer (NK) cell markers and little NK activityagainst K-562 cells [1-3]; (ii) they proliferate minimally tomitogens [4,5]; and (iii) they predominantly express aEf7 (HML-1), a surface marker found on only a few peripheral bloodlymphocytes (PBL) [6]. Thus, TEL may represent a first-line hostdefence against malignant EC.

The cytotoxic activities of TEL and PBL differ markedly. InIEL, the effectors of spontaneous cytotoxic (SC) activity areCD2+CD3+CD8+CD16-CD56-. They lyse EC tumours butnot K-562 cells, and do not show increased lytic activity withinterferon-gamma (IFN-y). In contrast, the effector cells in PBLare CD2+CD3-CD8-CD16+CD56+, are not target cell-res-tricted, and have increased cytotoxic activity with IFN-y [1].

Correspondence: Ellen C. Ebert MD, Department of Medicine,University of Medicine & Dentistry of New Jersey, Robert WoodJohnson Medical School, One Robert Wood Johnson Place-CN 19,New Brunswick, NJ 08903-0019, USA.

Unlike SC activity, lymphokine-activated killer (LAK)activity by IEL is directed against both EC tumours and K-562cells. The mechanisms of lysis are different, however, sinceantibody to aE 37 inhibits lysis of EC tumours only [7]. Thismarker, restricted mainly to epithelial lymphocytes, synergizeswith CDI la to mediate the binding of IEL to EC tumours; it isnot involved in binding to non-EC solid tumours or non-malignant monolayers [8].NK cells from the circulation have receptors for the Fc

portion of IgG and demonstrate antibody-dependent cell-mediated cytotoxicity (ADCC; [9]). Although the expression ofFc receptors on human IEL has not been previously investi-gated, their ADCC activity against the Chang (human liver) cellline using antibody raised in rabbits was found to be minimal[10].

This study examined: (i) a possible correlation between theSC activities of autologous IEL, lamina propria lymphocytes(LPL), and PBL; (ii) the susceptibility of target cell clones tolysis by effector cells in IEL and PBL; (iii) the cell surfacemarkers involved in SC activity by IEL and PBL; and (iv) thepresence of Fc receptors on IEL and their ability to mediateADCC.

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MATERIALS AND METHODS

Isolation of'lynmphocytesPBL were separated from freshly drawn whole blood using a

Ficoll density gradient (Organon Teknika, Durham, NC). IELwere separated from jejunal mucosa obtained from healthyindividuals undergoing gastric bypass operations for morbidobesity. In brief, the minced mucosa was treated for 25 min at

37 C with I mm dithiothreitol (Sigma Chemical Co., St Louis,MO) followed by three 45-min incubations in a shaking waterbath with 0-75 mm ethylenediamine tetraacetic acid (EDTA;Sigma) in calcium- and magnesium-free Hanks' buffered saltsolution (HBSS) (GIBCO, Grand Island, NY). The cells in thesupernates were collected and kept at 4 C overnight. IEL were

separated by a discontinuous Percoll (Pharmacia, Piscataway,NJ) density gradient as described previously [1,4], with theexception that the cells were resuspended in 40'S Percoll andlayered over a 60" cushion, then followed by a 30'S) layer. Aftercentrifugation, purified IEL were obtained from above the 60%,Percoll layer and contained over 90'S, lymphocytes. Most IELexpressed CD2, CD3, CD8, and T cell receptor US, but not Bl,Mo2, or CD16, consistent with the phenotype shown byimmunohistochemistry [1 ].

To isolate LPL, the treated tissue received three more 45-mintreatments with EDTA in calcium- and magnesium-free HBSS,and the cells released were discarded. The remaining tissue wasdigested for 3 h at 37 C in 20 U/ml collagenase (WorthingtonScientific, Malvern, PA) in serum-supplemented RPMI 1640,then pressed through a wire mesh cell sieve to release the trappedcells. After separation on a Percoll density gradient in themanner described, the LPL preparations were 55 + 10' CD4+and 35+11'S, CD8+. In some experiments, DRI cells were

removed by magnetic beads coated with goat anti-mouse IgG,as described previously [8].

Measurement of spontaneous Vtoto.yicitV and ADCC activitySpontaneous cytotoxicity was measured in a standard 5'Cr-release assay. Briefly, the effector cells were mixed at various

ratios with 104 target cells labelled with sodium chromate(Na2f5CrO4; New England Nuclear, Boston, MA) and incubatedtogether in conical-bottomed microwells for 4 h or 18 h at 37 C.The amount of label released from lysed targets was measuredby collecting supernates and counting radioactivity (ct/min).Spontaneous and maximal releases were obtained from targetsincubated in medium alone or in 20/ cetrimide solution,respectively.

The ct/min values from triplicate wells were averaged, andspecific cytotoxicity was calculated as:

Cytotoxicity =

experimental release -spontaneous release 100'/maximal release -spontaneous release

In some experiments, antibodies to the following markerswere added: CD2, CD3, CD8, CDI Ia (LFA-1), CD54 (ICAM-1), CD58 (LFA-3), )'Efl7 (HML-1). MHC class II (all fromAMAC, Inc., Westbrook, ME), CD16, CD45 (HLel) (BectonDickinson, Mountain View, CA), and MHC class I (Scra-lab,Sussex, UK). Any preparations containing sodium azide were

dialysed against PBS.To measure ADCC activity, 5'Cr-labelled DLD- 1 target cells

were incubated at 37 C for 18 h to allow them to adhere. The

en

co

a_

a0

70 I(a)60

50-40 -

30

20 - *%

:-J

.0

)

*a),

( b)

I .

0 -

0 10 20 30 40 50 0 10 20 30 40 50Per cent lysis by IEL

Fig. 1. The spontaneous cytotoxic (SC) activity by intraepitheliallymphocytes (IEL) against DLD-l target cells in an 18-h assay was

compared with that by peripheral blood lymphocytes (PBL) (a) and thatby lamina propria lymphocytes (LPL) (b). The results are expressed as

percentages of lysis at a 25: 1 effector-to-target cell ratio. The cytotoxicactivities by IEL and PBL did not correlate (correlation coeffi-cient=0056, P=NS). The cytotoxic activities by IEL and LPL, in

contrast, correlated well (r = 0 8, P = 00()9), if the two bracketed pointsin b were excluded as outliers [15].

medium was then removed and replaced with fresh medium or

sera (I :4 dilution) from patients with ulcerative colitis (UC) or

from normal individuals, and effector cells were added for a 4-hor 18-h assay. The amount of serum-induced cytotoxicity, or

ADCC activity, was calculated as the difference between per

cent cytotoxicity with serum and the SC activity (per centcytotoxicity without serum). In some experiments, 100 U/mlrecombinant IFN-; (Amgen Corp., Thousand Oaks, CA) were

added to the 18-h incubation of labelled targets. Serum sampleswere taken from six patients with active UC who were admittedto the hospital for a severe flare-up of disease, before steroidtherapy was instituted. Control serum was obtained fromhealthy volunteers [12,13]. All samples were frozen at -70 C,and heat-inactivated before use.

Measurement of Ec;' receptorsLymphocytes were incubated at 4 C for 30 min with Fcfragments of human IgG conjugated to biotin (AccurateChemical & Scientific Corp., Westbury, NY) followed bystreptavidin conjugated to FITC (Tago, Burlingame, CA).Fluorescence was analysed using a Coulter Profile analyticalflow cytometer with a 25 mW argon laser, as describedpreviously [14].

Statistical analivsisFor each set of data, an arithmetic mean and s.d. were

calculated. Data sets were compared with Student's t-test forpaired or independent variables. Linear regression analysis wasused to measure correlation [15].

RESULTS

The SC activities of autologous IEL, LPL, and PBL againstDLD-l target cells were measured. Cytotoxicity by PBL was

greater than that by IEL (33 7+ 13 5'S and 184± 1229'S, lysis,respectively, at a 25:1 effector-to-target cell ratio; P<0 01,n = 16), while, in turn, the cytotoxicity by IEL was greater thanthat by LPL (22 5+12 2'S, and 12 2+10 1%), respectively;P<0 05, ns= 10). The SC activities of IEL and LPL correlatedwell (r=0 8, P=0 009) when two outliers were excluded fromthe analysis (Fig. 1). There was no correlation between IEL and

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Spontaneous cytotoxicity by intestinal lymphocytes

Table 1. Effect of antibodies on spontaneous cytotoxic (SC) activity byintraepithelial lymphocytes (IEL) and natural killer (NK) activity by

peripheral blood lymphocytes (PBL) against DLD-I target cells

Effector cell

Antibody

Specificity

0.0 1-0 2-0 3.0

Lytic activity of IEL (clone/cell line)

Fig. 2. The lytic activities of peripheral blood lymphocytes (PBL) andintraepithelial lymphocytes (TEL) against DLD-l clones were tested inan 18-h assay and expressed as a ratio of lysis against the parent DLD- Iline. There was no correlation when the outlier (bracketed) was

excluded.

PBL activities (r=0 056, P=NS), however, suggesting thatintestinal and circulating effector cells have different mechan-isms of lysis. If so, then the susceptibility to lysis of any

particular cell from among the whole DLD- 1 population wouldbe expected to vary with the effector cell type. To determine ifthis was the case, clones of the DLD- I line were made by limitingdilution (0 25 cells per well), and the ability of IEL or PBL to killeach of 13 clones was tested (Fig. 2). Cytotoxicity was expressedas a fraction of the cytotoxic activity toward the parent DLD-lline, thereby correcting for variations in cytotoxic activityamong different effector cell preparations. There was no

significant correlation between the lytic activities of PBL andIEL against the DLD-I clones when one outlier was excluded,indicating that the susceptibilities to lysis depended upon theeffector cell type.

The surface molecules involved in SC activity by IEL andNK activity by PBL were identified by testing the effects ofMoAbs (IgG I or IgG2a) added to the cytotoxicity assay (Table1). The lytic action of IEL toward DLD-l target cells was

inhibited by antibody to XES7, whereas the lytic ability of PBLwas inhibited by antibodies to CD2, CD16, MHC class I, andCD45. A higher concentration of antibody (2 5 ug/ml) had thesame effect. Similar results were obtained for IEL cytotoxicityagainst HT-29 target cells and for PBL cytotoxicity against K-562 target cells (not shown). Surprisingly, anti-CD58 enhancedDLD-l killing by PBL. (The increased cytotoxicity by IEL andPBL with antibody to CD3 was not statistically significant.) Todetermine whether DLD-l cells express CD58, immunofluores-cence microscopy was done. DLD- I monolayers were found toexpress CD54, but not CD58 or MHC class 1, indicating thatantibodies to the latter two were probably directed only towardeffector antigens. The surprising lack ofMHC class I expressionwas verified by flow cytometric analysis of DLD-1 cells in

suspension, ruling out the possibility of low density expressionthat might escape detection by microscopic analysis.

We found previously that SC by IEL against DLD-l target

cells is strongly inhibited by the addition of unlabelled DLD-Icells to the assay, but only weakly so by K-562 cells, indicating

CD2CD3CD8CDIla (LFA1)CD16CD54 (ICAM1)CD58 (LFA3)MHC class IMHC class IICD45 (HLel)YE'#7 (HML-1)

Isotype

IgG2aIgG2aIgGIgGIgGIgG 1IgG2aIgG2aIgG2aIgGIgG2a

SC activity (0N control)

IEL or PBL were incubated for 18 h with DLD-l target cells at a25: 1 effector-to-target cell ratio in the presence of various antibodies(0 5 pig/ml). The results are expressed as a percentage of controlcytotoxicity without antibody. The number ofexperiments performed isin parentheses. Values significantly different from control of 100°%(Student's t-test, paired variables): *P<O.0l; tP<005; tP<0 001.

Table 2. Expression of Fc'; receptors on intraepitheliallymphocytes (TEL), lamina propria lymphocytes

(LPL) and peripheral blood lymphocytes (PBL)

Expression of Fc,' receptors

Lymphocyte Per cent Relative intensitytype positive of fluorescence

IEL 4+1 40+40LPL 5+5 3 0+ 1 1PBL 24+4 3 2+0 4

IEL, LPL and PBL were stained with Fc frag-ments of IgG conjugated to biotin followed bystreptavidin conjugated to FITC, and analysed byflow cytometry. The per cent positive cells and relativeintensity of fluorescence were determined in compari-son with a negative control of cells stained with theFITC conjugate alone.

that the cytotoxic IEL preferentially bound to DLD-I cells [1].Similar cold competition experiments showed that PBL, incontrast, bound both target types to an equal degree. Theradiosensitivities of SC activity by IEL and NK activity by PBLwere also tested. Both were equally sensitive to increasingamounts of;-irradiation, with obliteration of cytotoxic activityat 30 Gy. Participation of monocytes in the SC by IEL wasdiscounted, since plastic adherence followed by depletion ofDR+ cells using magnetic beads coated with goat anti-mouseIgG did not affect lytic activity (data not shown).

I.

-ca)

0c-ia)

0~02 0

-, 1 0a-I.-

0

529

0

*00~0I

IEL PBL

81 +20 (6)142+65 (8)85+ 19 (6)94+6 (7)106+8 (3)95+ 10 (5)86+ 16 (4)89X+Q21 (7)98+22 (3)120+28 (3)44+20 (8)+

80 + 9 (5)*144+44 (3)110+28 (3)107+ 12 (3)41+ 14 (3)t101 + 19 (3)145 +37 (4)t63 + 6 (4)t101 +7 (3)62 + 9 (3)t113+ 13 (3)

_-u

I

A. I. Roberts et al.

1000(a) F

IEL

100 _

U.c

eU)cvD

a)

0

u0'0

100

10

Pi

16 32 48 64

Cel I size (forward angle light scatter)

Fig. 3. Flow cytometric analysis of lymphocyte preparations stained byimmunofluorescence for Fcy receptors: cells were labelled with biotin-conjugated Fc fragments of human IgG, then avidin-FITC (left panel).Control cells received avidin-FITC alone (right panel). Contour plotsdepict ungated populations ofintraepithelial lymphocytes (IEL), laminapropria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) (a,b, c). The test and control plots for IEL and LPL are nearlysuperimposable. PBL show a marked increase in fluorescence intensityabove control for both the main lymphocyte population and themonocytes.

The ability of lymphocytes to kill target cells coated withantibody (ADCC) depends on the presence of Fcy receptors onthe effector cell. Fcy receptors on IEL, LPL, and PBL were

identified by immunofluorescence using Fc fragments andanalysed by flow cytometry (Table 2, Fig. 3). IEL fell into twomain clusters: the major lymphocyte population with a sizerange equivalent to that of PBL, and a minor population oflymphocytes that were larger and more granular [8]. In themajor lymphocyte population, 24+ 4% of PBL expressed Fcreceptors, while only 5% or less of the IEL and LPL were

positive (P < 0 05, n = 3). The density of expression as measuredby relative intensity of fluorescence was the same for alllymphocyte types. Of the subset of PBL representing mainlymonocytes, 85+6% of cells expressed Fcy receptors with a

relative intensity of fluorescence of 6-0 + 0-6.The ability of IEL and LPL to carry out ADCC was tested

against DLD-1 target cells using anti-colon antibodies in sera

from patients with UC (Table 3). PBL are capable ofADCC inthis system, and their activity is augmented by pretreating the

Table 3. The ability of intraepithelial lymphocytes (IEL) and laminapropria lymphocytes (LPL) to carry out ulcerative colitis (UC) serum-

induced cytotoxicity

UC serum-induced cytotoxicity(O/( cytotoxicity)

Effector Treatment ofcell type effector cell 4-h assay 18-h assay

PBL None 7-7+2-6 (6)* 18-0+6-3 (4)tIFN-y 20-1 +94 (5)t

IEL None 0-5+0 5 (6)§ 1-2 + 2-0 (6)¶IFN-y 3-2+0-5 (3)**

LPL None 31 +2 8 (4)tt 2-0+2-7 (4)4+IFN-y 0+0 (5)§§

Peripheral blood lymphocytes (PBL), IEL or LPL from normalsubjects were tested for UC serum-induced cytotoxicity either just afterisolation (None) or after an 18-h culture with 100 U/ml IFN-y. In someexperiments the cytotoxicity assay was extended to 18 h. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity is the differencebetween per cent cytotoxicity with and without serum. The number ofexperiments performed is in parentheses. The following values differedsignificantly (Student's (-test, independent variables: * versus tt, t versus

* versus X, tt versus §§: P < 0 05; * versus t, J versus 4, t versus §§,P<0 01; * versus §, t versus ¶, § versus **: P<0 001.

effector cells with IFN-y [13]. The ADCC activities of IEL andLPL with UC sera, however, were no greater than their SCactivities, even when the assay was extended to 18 h or theeffector cells were pretreated with IFN-y.

DISCUSSION

The present study further examines the differences between SCby IEL and NK by PBL using DLD-1 colon cancer cells astargets, as in three previous studies [1,7,13]. These differencesare summarized Table 4. Antibody inhibition experiments werecorroborated with HT-29 target cells. The SC by IEL towardDLD- 1 cells is inhibited by antibody to aEj37, probably a result ofits ability to block the binding of IEL to colon cancer cells [8], anessential first step in cytotoxicity. In contrast, cytotoxic IEL lyseK-562 cells only minimally [1]. This selective SC activity couldbe explained if colon cancer cells but not K-562 cells express theligand to aEf#7, for which a marker has not yet been identified. Ofcirculating lymphocytes, only an exceedingly small percentageexpress aE#7, unless preactivated [16], and antibody to thismarker has no effect on NK activity by fresh PBL. Rather, NKactivity by PBL against both DLD-1 and K-562 target cells isinhibited by antibodies to a different set of surface molecules:CD2, CD 16, CD45, and MHC class I. While the CD2 and CD45molecules are known to be essential for NK activity by PBL[17,18], the role of CD16 and MHC class I on effector cells hasnot been described. The increased lysis by PBL induced by anti-CD58 antibody is also novel. Oddly, anti-CD58 had no effect onSC activity by IEL, even though a larger number of IEL thanPBL express this marker at equivalent surface densities [19].

The present study assayed IgG-Fc fragment binding tolymphocytes, a technique that can detect Fc receptors that areCD16- and have a low affinity for their ligand. The finding that5% of LPL express Fc receptors is consistent with previousresults using IgG-coated sheep erythrocyte rosetting [20], and is

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t1lI I

0 16 32 48 64

Spontaneous cytotoxicity by intestinal lymphocytes 531

Table 4. Comparison of spontaneous cytotoxic (SC) activity by intraepithelial lymphocytes (IEL) to natural killer (NK) activity byperipheral blood lymphocytes (PBL)

Characteristics of SC/NK activity SC activity by IEL NK activity by PBL

Phenotype of effector cell CD2+CD3+CD4- CD8+CD16-CD56-* CD2+CD3-CD4-CD8-CD16+CD56+*Response of effector cell to IFN-, None* Increased cytotoxicity*Antibody inhibits lysis IE#7 CD2, CD 16, CD45, MHC class IAntibody enhances lysis None CD58Irradiation-sensitive Yes YesMitomycin C-sensitive No* No*Targets killed EC tumours* EC tumours and K-562 cells*

Other characteristic'sADCC activity No YesFc receptors for IgG Few Many

* Findings reported previously [1].ADCC, Antibody-dependent cell-mediated cytotoxicity; EC, epithelial cell.

equivalent to the finding in IEL. It differs, however, from thehigh density of Fc receptors for IgM (CD7) on IEL [21].

ADCC activity is carried out by Fc receptor-bearing effectorcells that bind to the Fc portion of an antibody linked to a targetcell. In earlier reports, LPL had variable ADCC activity againstChang or chicken erythrocyte target cells using rabbit antibody[10,20,22,23], while IEL had no activity [10]. Using DLD-1target cells which are known to bind to IgG I from UC sera [24],this study found no evidence of ADCC activity by IEL or LPL,an expected consequence of their low expression of Fc;'receptors.

The many characteristics that distinguish SC activity by IELfrom NK activity by PBL make these cells unique. In view oftheir proximity to EC and their phenotypic similarity tolymphocytes infiltrating colon cancer [6], IEL may be involvedin the host defence against this type of tumour. In order to definefurther the lytic activity by IEL, other questions are beingaddressed. For example, IEL may need to be activated by ECtumours [25] before lytic activity develops. If IEL prove to beimportant in restricting the development of colon cancer as wellas recurrence after treatment, then their sensitivity to irradiationmay have clinical relevance, since radiation therapy is com-monly used to shrink tumour mass.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes ofHealth (DK 42166).

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25 Hoang P. Crotty B, Dalton HR, Jewell DP. Epithelial cells bearingclass II molecules stimulate allogeneic human colonic intraepitheliallymphocytes. Gut 1992; 33:1089-93.