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Human mucosal T-cell cytotoxicity
GASTROENTEROLOGY
1988;94:960-7
Human Mucosal T-Cell Cytotoxicity FERGUS SHANAHAN, RICHARD DEEM, RAMIN BERNARD LEMAN, and STEPHAN TARGAN Department of Medicine, Los Angeles, California
University
of California
Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CDS-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effecters do not have Fc receptors for immunoglobulin G, and are therefore distinct from T, cells, which mediate antibodydependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7. In contrast, peripheral blood anti-CD3-T effecters are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As antiCD3triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.
urunderstanding of the nature and mechanisms of human lymphocyte-mediated cytotoxicity 0 has advanced considerably in recent years (l-4). It has become clear from studies using peripheral blood lymphocytes (PBLs) that spontaneous cytotoxicity may be a feature of both natural killer (NK) cells and T cells, and that each of these distinct lymphocyte subsets may kill by more than one mechanism (12). However, the extent to which such studies can be extrapolated to the mucosal immune system is not
at Los Angeles,
NAYERSINA,
Center
for the Health
Sciences,
certain. As cytotoxic mechanisms may have a role in mediating the tissue injury that occurs in certain intestinal inflammatory disorders (3-g), a precise delineation of the effector cell populations within the human intestinal mucosa is important. There is general agreement that cultured mucosal lymphocytes exhibit mitogen-induced cellular cytotoxicity (10-12) and lymphokine-activated killer (LAK) cell activity (13-15). Whether freshly isolated mucosal cells exhibit spontaneous NK activity (10-19) or antibody-dependent cellular cytotoxicity (12,16,20-22)has been controversial. We recently found that although unfractionated mucosal lymphocyte preparations have little activity in cytotoxic assays, a small subset of cells with significant NK activity can be demonstrated if specifically enriched for, using monoclonal antibody (NKH-1) panning (151.
Mucosal cytotoxic T cells have been less well studied. Although cells with a surface antigen phenotype compatible with that of cytolytic T cells are present within the intestinal mucosa (23,24),their lytic function has not been clearly demonstrated. Recently, Roche and colleagues (6) showed that intestinal T lymphocytes from patients with inflammatory bowel disease, but not from control patients, could kill red cells coated with epithelial antigens. The significance of this finding is not yet certain because the levels of cytotoxicity were low and lysis occurred only over a restricted range of effector-totarget ratios (6).However, the T-cell killing observed is of interest because it was not restricted by the major histocompatibility complex (MHC) and is, therefore, distinct from classical T-cell cytotoxicity
Abbreviations used in this paper: anti-CD3-T, antXD3triggered T cell; LAK, lymphokine-activated killer; LPL, lamina propria lymphocyte; LU, lytic unit; MHC, major histocompatibility complex; NK, natural killer; PBL, peripheral blood lymphocyte. 0 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
HUMAN MUCOSAL
April 1988
(25). Evidence for non-MHC-restricted cytotoxic T cells in the peripheral blood is now well established (l,2), and a subset of these cells may be conveniently studied in vitro by triggering their activity with antibodies to the CD3 component of the T-cell antigen receptor complex (26). Cytolytic activity, which is triggered by anti-CD3 monoclonal antibodies, is thought to be an indirect measure of in vivo primed cytotoxic T cells when the antigen to which they are reactive is not known (26). Within the human intestinal mucosa, where there is constant and varied antigenic exposure, a high prevalence of in vivo primed T cells would be expected. Therefore, the purpose of this study was (a) to determine whether anti-CD3 triggered lysis could be used to demonstrate cytotoxic T-cell activity in freshly isolated lymphocyte preparations from noninflamed human mucosa, (b) to determine the phenotype of the mucosal anti-CD%triggered T cells (anti-CDS-Ts) in comparison with that of anti-CD3Ts in the peripheral blood, and (c) to examine the target cell restrictions of this form of non-MHCrestricted cytotoxicity within the mucosa.
Materials and Methods Study
Population
Peripheral blood specimens were obtained from healthy volunteers. The intestinal specimens were obtained from patients undergoing surgical resection of the colon at the University of California, Los Angeles, Center for the Health Sciences. The entire study was approved by the hospital’s human subject protection committee. In this series of experiments all tissue specimens were from patients with colonic carcinoma, and were taken from an uninvolved area of the colon. Target
Cells
and
Culture
Medium
The target cell lines K562 (an NK-sensitive human erythroleukemia cell line), HEL 92.1.7 (an NK-sensitive human erythroleukemia line), Jurkat (an NK-sensitive human acute lymphoblastic T-cell leukemia line), IM-9 (an NK-resistant human B-lymphoblast cell line), Daudi (an NK-resistant human B-lymphoblast cell line), and Raji (an NK-resistant human B-lymphoblast cell line) were obtained from American Type Culture Collection, Rockville, Md. Target cell lines U266/AFlO [an NK-sensitive human immunoglobulin (Ig)E-producing myeloma line] and GM-1056 (an NK-resistant human IgA-producing Blymphoblast cell line) were obtained from Dr. Andrew Saxon, University of California, Los Angeles. All target cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 in RPM1 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% fetal calf serum. Monocional
Antibodies
Monoclonal antibodies T8 (anti-CD8), T4 (antiCD4], T3 (anti-CD3), Tll (anti-CD2), NKH-1, NKH-lA, and
CYTOTOXIC
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anti-NKH-l-RDl were purchased from Coulter Immunology, Hialeah, Fla. Monoclonal antibodies Leu llb (antiCD16), Leu lla-FITC (fluorescein isothiocyanate-conjugated anti-CDlG), Leu 4-FITC (anti-CD3), Leu7, Leu 7-FITC, Leu 2b (anti-CD8), and Leu 2a-PE (phycoerythrinconjugated anti-CD8) were purchased from BectonDickinson (Mountain View, Calif.). The monoclonal antibody 13.3, which recognizes an epitope on the human homologue of the murine T-200 (CD45) molecule, was generously provided by Walter Newman, Otsuka Pharmaceuticals, Rockville, Md., after purification by, absorption to, and elution from protein A-Sepharose (27). This antibody is functionally identical to the 13.1 monoclonal antibody and binds to the same epitope of the CD45 molecule (28). Isolation
of Effector
Cells
Peripheral blood mononuclear cells were isolated from healthy volunteers by separation on Ficoll-Hypaque gradients (29). The cells at the interface were recovered, washed three times, and resuspended in RPM1 1640 with 10% fetal calf serum. Lamina propria mononuclear cells were isolated using a technique modified from that originally described by Bull and Bookman (30), which has previously been described in detail (15). The entire procedure was performed in 1 day without interruption. Peripheral blood and lamina propria mononuclear cell preparations were depleted of adherent cells by incubating the cells (5 x 106/ml) on lOO-mm plastic tissue culture dishes (Corning Glass Works, Corning, N.Y.) in a humidified atmosphere (5% COZ in air, 37%) for 2 h. The nonadherent cells, referred to as PBLs and lamina propria lymphocytes (LPLs), were then removed, washed, and resuspended in medium. Enrichment Panning
of Lymphocyte
Subsets
by
In some experiments, a panning technique modified from that originally described by Wysocki and Sato (31) and described in detail elsewhere (15) was used to enrich for LPL and PBL subsets. In brief, nonadherent LPLs or PBLs (lo7 cells/ml) were incubated with subsetspecific monoclonal antibody (0.25 pg/lO” cells) for 60 min at 4°C. The cells were then washed and resuspended in medium at 5 x lo6 cells/ml, and 30 x lo6 cells were added to 100 x 15-mm plastic Petri dishes (Falcon 1029, BectinDickinson Labware, Oxnard, Calif.). These dishes were previously coated with 6 ml of purified goat F(ab’), antimouse IgG Fc or antimouse IgM (p-chain specific) (Jackson Laboratories, Avondale, Pa.) (20 &ml in 0.05 M Tris, pH 9.5) for 120 min at room temperature and then washed three times with medium. After a 90-min incubation of the coated dishes at 4”C, the nonadherent cells were removed by gentle swirling and aspiration. The plates were then gently washed twice and the adherent ceils were removed by vigorous washing. Depletion
of Lymphocyte
Subsets
In some experiments PBLs and LPLs were depleted of lymphocyte subsets by complement lysis. In brief, the
962 SHANAHAN ET AL.
cells were incubated for 1 h fixing monoclonal antibodies. incubated at 22°C with a 1:16 rabbit complement (Pel-Freez, Depletion
GASTROENTEROLOGYVol. 94. No. 4
at 22°C with complementThey were then washed and final dilution of 3-4-wk-old Rogers, Ark.).
of Fc, Rf Lymphocytes
Cells bearing Fc receptors for IgG (Fc,R+) were depleted by immobilization on immune complex monolayers, as described previously (32). In brief, human IgG (Gammimmune, Cutter Laboratories Inc., Berkeley, Calif.) was diluted to 500 pg/ml in phosphate-buffered saline, and 3 ml was added to lOO-mm tissue culture dishes (Falcon) and incubated for 30 min at 22”C, followed by 30 min at 4°C. The dishes were washed three times with phosphatebuffered saline, and then 2 ml of 16 @g/ml rabbitantihuman IgG (y specific) (Miles Scientific, Naperville, Ill.] was added for 30 min at 22°C followed by 30 min at 4’C. The dishes were washed three times and stored at 4°C for up to 2 wk. Up to 30 x lo6 cells were added to each dish and centrifuged at 400 g for 5 min; the plates were then rotated 180” and centrifuged a second time. The dishes were swirled and nonadherent (Fc,R-) cells were removed.
amplitudes of the electrical signals were converted from analog to digital by means of the Analog to Digital Converter. The data were analyzed and histograms displayed by means of the Multiparameter Data Acquisition and Display Systems (MDADS). Dead cells, erythrocytes, platelets, and monocytes were excluded from the analysis by gating on the lymphocyte population. Cytotoxicity
Cytotoxicity tests were performed in 96-well, Vbottom microplates (Cooke, Alexandria, Va.) in a total volume of 200 ~1. Target cells (K562 unless otherwise stated) were labeled with 200 &i NaZ51Cr04 (New England Nuclear Corp., Boston, Mass.] for 1 h at 37°C and washed three times; lo4 target cells were used in each microtiter well along with varying numbers of effector cells. After an incubation period of 12 h (or 4 h where stated), 100 ~1 of supernatant was collected and the p.ercentage of cytotoxicity was estimated from released radioactivity according to the following formula: Cytotoxicity (%] Test release - spontaneous release
= 80%
lmmunofluorescent Cytometry
Staining
and
Flow
Peripheral blood lymphocytes and lamina propria lymphocytes (5 x 105) were suspended in 100 ~1 of phosphate-buffered saline supplemented with fetal calf serum (2%) and sodium azide (O.l%), and phycoerythrinor Red Dye-l- and fluorescein isothiocyanate-conjugated monoclonal antibodies (10 &test for Becton-Dickinson monoclonals and 2.5 pi/test for Coulter monoclonals) were added for 30 min at 4°C. For cells purified by panning or complement-depleted cell populations, staining was performed by indirect immunofluorescence. Monoclonal antibody-treated cells were washed and 0.1 pg of dichlorotriazinyl amino fluorescein conjugated-purified F(ab’)z, goat-antimouse IgG (H & L) (Jackson Immuno Research Laboratories, Avondale, Pa.) was added for 30 min at 4°C. For direct, two-color staining, control cells were stained with fluorochrome-conjugated isotype-matched mouse immunoglobulin. For indirect staining, unconjugated, isotype-matched mouse immunoglobulin monoclonal antibody-treated cells were stained with the secondary, fluorochrome-conjugated antibody. Stained cells were washed twice with phosphate-buffered saline with 2% fetal calf serum, resuspended, and fixed with 1% paraformaldehyde in phosphate-buffered saline. Analysis of lymphocyte subsets was performed using an EPICS 541 Flow Cytometer (Coulter). Red and green fluorochromes were excited using 400 mW of 488-nm light from an argon laser. Fluorescent emission was collected by means of a focusing lens placed at 90” relative to the laser-stream intersection. Forward angle light scatter was collected by a pickup lens (photodiode) in the laser light path. A 488-nm dichroic filter was placed at a 45” angle to the light path to separate the 90” scatter from the red and green fluorescent emissions. A 590-nm-long pass filter was placed in front of the red photomultiplier tube. The
Assay
of total label - spontaneous release
x 100.
A wide range of effector-to-target ratios was used to generate cytotoxicity curves. All assays were performed in triplicate and results were expressed as mean percent cytotoxicity 2 SD. In some experiments results were expressed in terms of lytic units (LU) per lo7 effector cells. Lytic units were calculated from a semilogarithmic curve plotting the natural logarithm of effector cell number versus the percent of cytotoxicity. One lytic unit is the number of effector cells required to induce 30% specific 51Cr release from lo4target cells. Statistics Student’s t-test for paired data was used to determine whether differences in cytotoxicity values under different conditions were significant. Values of p < 0.05 were considered significant.
Results Cytolytic Activity of Freshly Isolated Lamina Propria Lymphocytes in the Presence and Absence of Anti-CD3 Monoclonal Antibody
Freshly isolated, unfractionated LPLs exhibited no significant lytic activity (CO.25 LU) against K562 target cells at effector-to-target ratios up to 1OO:l. However, significant cytolytic activity (164 k 74 LU, mean -+ SD; n = 14) was found when the same cells were pretreated (37°C for 1 h) with 1 pug/ml anti-CD3, but not with an isotype-matched control mouse monoclonal antibody (CO.25 LU). A representative experiment with the data expressed in terms of percent cytotoxicity at different effector-
HUMAN MUCOSAL CYTOTOXIC T CELLS
April 1988
60
?? Untreated LPL ?? Fcu R depleted
AntccD3-LPL Wh)
Control LPL
963
LPL
AntlcD3-LPL (4 h)
Antl-CD3-LPL
I 0
LPL (12 h)
100
200
LYTIC UNITS LPL (4 h) 0
20
40
60
80
100
EFFECTOR TO TARGET RATIO Figure
1. Representative cytotoxicity curves indicating the cytolytic activity (against K562 targets) of freshly isolated LPLs compared with that of LPLs pretreated for 1 h with anti-CD3 monoclonal antibody, in 4- and 12-h assays. Values are the mean f SD of a single experiment performed in triplicate, which is representative of 6 (4 h) to 14 (12 h) similar experiments. Pretreatment with a control isotype-matched monoclonal antibody had no effect on cytotoxic activity (not shown).
to-target ratios is shown in Figure 1. From preliminary titration experiments using LPLs and PBLs, the concentration of anti-CD3 was found to be lo-fold higher than that required to elicit maximal anti-CD3T activity and was used in all subsequent experients. The anti-CD3-triggered LPL cytotoxicity was observed in all of 14 consecutive intestinal specimens. In contrast, in the peripheral blood, we recently found significant anti-CD3-triggered PBL cytotoxicity in only 25% of normal subjects (33, and Deem R, Shanahan F. Niederlehner A, and Targan S, unpublished). Anti-CD3-Triggered Fc, Receptors
Cytotoxic
Figure 2. Anti-CD3-triggered T-cell cytotoxicity against K562 targets by LPLs, before and after depletion of cells bearing Fc receptors for IgG (Fc,R). Values are the mean lytic units 2 SE, n = 4; p = 0.065 for the anti-CD3-T comparison using paired data.
Surface Antigen Phenotype of the Lamina Propria Anti-CD3-Triggered T Cell Is CDl6- Leu 7- CD3+ CD8+ CD4- CDZ+ It has previously been reported that anti-CD3Ts within PBLs are in general CD16-, Leu 7+ (26). In this study (Table l), the anti-CD3-T cytotoxic activity of PBLs from 7 control subjects was mediated by CD16-, Leu 7+ cells, although in 1 subject (No. 7, Table 1) there was also significant lytic activity in the Leu 7- population (which contained only 0.2% residual Leu 7+ cells). This variability is consistent with the findings of others (26), and is not surprising as the surface antigen defined by the Leu 7 antibody has not per se been inextricably linked either functionally or biochemically to the lytic process. Unlike PBLs, mucosal CD3+ T cells are consistently CD16-, Leu 7-. In keeping with other reports (13,15,23,24), we found that these surface antigens are not present within LPL populations (flow cytom-
T Cells Lack
When LPLs were depleted of Fc,R+ cells by adherence to monolayers of immune complexes, the anti-CD3-T activity was either unchanged or enriched (Figure 2). Therefore, the anti-CD3-Ts within LPLs lack Fc receptors for IgG (Fc,JR-) and are distinct from T, cells, which mediate antibodydependent cellular cytotoxicity. This is consistent with our previous results with PBLs (33). Control studies that confirmed the effectiveness of the Fc,R+ cell depletion included (a) its elimination of all NK activity and CD16+ cells and (b) a consistently >75% reduction of antibody-dependent cellular cytotoxicity activity from CD16- cells [data not shown), as determined against K562 target cells that were pretreated with K562 antiserum (provided by Dr. D. Hudig, University of Nevada, Reno, Nev.).
Table 1. Anti-CD3-Triggered T-Cell Cytotoxicity in the Peripheral Blood Is Mediated Predominantly by the Leu 7+(CD16 -) Subpopulation” Lytic units (mean + SD) Subject
Leu 7+
Leu 7-
1
437
2 28
2
642
2 64
3
168
? 24
32 + 5 11 -+ 2
4
215
? 25
5
464
2 63
6
973
2 115
7
158 f
37
67 5 9 3 2 0.6
4 r
0.2
44 2 6 70 2 5
a CD16 cells were first depleted (
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SHANAHAN ET AL.
Figure 3. Anti-CDs-triggered T cell cytotoxicity against K562 targets by LPLs is mediated by CD8+, CD4-, CDZ+ cells. The monoclonal markers were examined using separate intestinal specimens. For each monoclonal marker, LPLs were enriched (284%) and depleted (55%) by panning. The values are the mean f SD of triplicates from experiments using separate intestinal specimens.
GASTROENTEROLOGY Vol. 94, No.
CD8
CD4
4
CD2
1
0
200
400
600
LYTIC UNITS
etry; CD16 = 0%, Leu 7 = O%, n = 11). As expected, anti-CD3-T activity was not affected by treatment of LPLs with Leu 7 and complement, whereas with PBLs from the majority of donors, it was consistently reduced (X0%) by this procedure (33, and data not shown). In contrast to the findings with CD16 and Leu 7, the LPL and PBL anti-CD3-T effecters were phenotypically similar with respect to other surface markers. Positive selection of mucosal lymphocyte subsets using monoclonal antibody panning indicated that the anti-CD3-T effecters within LPLs are CD8+, CD4-, and CDZ+ (Figure 3). Although it has been reported that some CD4+ clones developed from PBLs exhibit anti-CD$triggered lytic activity (34), this has not been found with freshly isolated PBLs. In this study, anti-CD3-Ts within PBLs from 3 consecutively examined subjects were CD4-. Peripheral blood lymphocytes were first depleted of CDl6+ cells (
LYTIC UNITS
LYTIC UNITS
LPLs with NKH-1A and complement, which resulted in a SO%-60% reduction of NKH-l+ cells, did not influence anti-CD3-T activity in three separate experiments, but yielded a 50% reduction in lytic activity in one experiment. The variability may reflect donor-to-donor differences in the coexpression of CD3 and NKH-1 on LPLs. Therefore, doublemarker analysis of LPLs bearing CD3 and NKH-1, from four intestinal specimens, was performed and indicated that the NKH-l+ cells that were also CD3+ ranged from 20% to 60%. These results are similar to our previous findings in the peripheral blood (33, and unpublished). Effect of Anti-CD45 (13.3) on Anti-CD3-Triggered Activity The monoclonal antibody 13.3, which reacts with an epitope of the CD45 (T200) molecule, has been shown to inhibit certain cell-mediated forms of cytotoxicity at the effector cell level (27,28,36,37). It inhibits NK cytotoxicity against some but not all NK-sensitive targets, and has been used to define the trigger phase of the NK lytic sequence (36). In contrast, LAK, antibody-dependent cellular cytotoxicity, and classical T-cell cytotoxicity are not inhibited by anti-CD45 antibodies (27,28,37). Using a panel of monoclonal antibodies to different epitopes of the CD45 molecule, we have recently shown that peripheral blood anti-CDS-T cell killing occurs through the CD45 molecule (33) and therefore differs from classical T-cell cytotoxicity. In this study, 13.3 (antiCD45) was also found to inhibit LPL anti-CDS-Tmediated lysis (Figure 4). The degree of inhibition, although partial, was statistically significant. The concentration of 13.3 (0.1 pg/ml) that yielded maximal inhibition was determined from preliminary titration experiments and was the same as that found previously for PBLs (28,33,37). Increasing the concentration lo-fold (1 pg/ml) in two experiments against K562 targets did not elicit any further inhibitory effect (control = 169 and 200 LU; 13.3, 0.1 pglml, 85 and 47 LU; and 13.3, 1 pglml, 92 and 49
HUMAN MUCOSAL CYTOTOXIC T CELLS
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K562
DAUDI
0
100
200
965
were sensitive to anti-CD3-T cytolysis (Figure 5). Of the four NK-resistant target cells examined (Raji, IM9, GM iO56, and Daudi) only Daudi cells were killed by anti-CD3-Ts, and this killing can be inhibited by the CD45 antibody, 13.3 (Figures 4 and 5). These results suggest that the CD45 molecule may be an important determinant of anti-CD3-T sensitivity. This is consistent with our recent study of peripheral blood anti-CD3-T cytotoxicity (33, and unpublished) using a more extensive range of target cell lines.
LYTIC UNITS
Figure 4. Inhibitory effect of anti-CD45 monaclonal ‘antibody (13.3) on anti-CD3-triggered killing of K562 (p = 0.007, n = 4) and Daudi (p = 0.04, n = 3) targets by CPLs. The values shown are mean ? SD. The LPLs were pretreated with anti-CD3 (1 h, 37’C) and anti-CD45 (0.1 pg/ml) was added to the assay. A control isotypematched monoclonal antibody did not influence the inhibitory effects of anti-CD45 (see text), and increasing the concentration of anti-CD45 lo-fold did not alter the result (see text].
LU, respectively). The addition of a control iqotypematched monoclonal antibody to the assay did not inhibit lysis of K562 targets by anti-CD3-Ts in.*two experiments (85, 161 LU versus 87, 158 LU, respectively). Target Cell Restrictions of Lamina Propria Anti-CD3-Triggered T Cells The target specificity of the lamina propria anti-CD3-triggered effector cells differed from that of NK and LAK cells (Figure 5). Some, but not all, NK-sensitive targets and some, but not all, NKresistant targets were susceptible to anti-CD3-T killing, whereas all of the targets in Figure 5 are LAKsusceptible. The presence of an Fc receptor on the target cell has an important role in anti-CDB-T cytotoxicity (38) and is thought to provide an anchdrage site Whereby CD3 on the surface of the effector cell can be crosslinked by the antibody (3940) However, target cell expression of Fc receptors cannot be the orily determinant of susceptibility to ahti-CD3-T lysis because it does not explainthe pattern of target susceptibility seen in Figure 5. K562, Daudi, and Raji cells have high-affinity surface Fc receptors that bind IgG, (41), yet only K562 and Daudi cells, and not Raji cells, are susceptible to anti-CDB-T lysis (Figure 5). Therefore, other factors must influence the target restrictions of anti-CDB-T lysis. Of the four NK-sensitive target cell lines studied (K562, HEL 92.1.7, AFlO, and Jurkat), only those with NK killing that is mediated through the CD45/T200 trigger (K562 and HEL 92.1.7) (27,333
Discussion
,
The results indicate that freshly isolated lymphocytes from the human intestinal lamina propria can mediate a form of T-cell cytotoxicity that is non-MHC-restricted and can be triggered by antiCD3 antibodies in vitro. In this study the cytotoxic activity was observed in all intestinal specimens examined, whereas in a recent survey of peripheral blood, we found significant anti-CD3-T activity in only 25% of healthy subjects (33, and unpublished). The intestinal mucosa might be an immunologic compartment where these cells predominantly reside, and are perhaps exposed and primed to a variety of environmental antigenic stimuli. It has been proposed that anti-CD3-Ts represent in vivo primed cytotoxic T cells, possibly reactive against viral and other antigens (26). In the presence of already primed T cells, it has been suggested that anti-CD3 antibodies might trigger non-MHCrestricted cytotoxicity by bypassing the requirement for antigen triggering. This technique might therefore be of use in identifying such cells, without knowing the nature of the stimulating antigen (26). The phenotype of the intestinal anti-CD3-triggered effector cell is similar to that of its peripheral blood counterpart in being Fc,R-, CD2+, CD3+, CD4-,
E
4
Raji
f
GM-1056
Y
IM9 U2661AFlO
zu E c $
Julkat HEL 92.1.7 K562 0
100
200
300
400
500
LYTIC UNITS Figure 5. Anti-CD3-triggered lamina propria T cells kill some but not all NK-sensitive targets and some but not all NKresistant targets. Values are mean lytic units ? SE, n = 4.
966
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CDs+, and CDl6-. However, as found by others (261, the anti-CDS-Ts within the peripheral blood are Leu 7+, whereas within the intestine, they lack the Leu 7 antigen. These results extend those of our previous report (15) and further indicate phenotypic differences between human cytotoxic effector cells of the mucosal and systemic immune systems. The anti-CD3-T lytic process is unusual in that it can be inhibited by monoclonal antibodies (13.3) to the CD45 antigen. CD45 is known to be coupled to the trigger process for NK lysis of some but not all target cells (27,28,36). Other types of cytotoxicity, such as classical (MHC-restricted) T-cell cytotoxicity, LAK, and antibody-dependent cellular cytotoxicity by NK or T, cells, are not blocked by anti-CD45 antibodies (28,37). Whether this can be used to distinguish different types of mucosal T-cell cytotoxicity is not yet clear, as the degree of inhibition observed in this study was partial. The lytic process triggered by anti-CD3 may not proceed exclusively through the CD45 molecule. Anti-CDS-triggered T cell cytotoxicity, although non-MHC-restricted, has a considerable degree of target specificity, which differs from that of NK and LAK cytotoxicity. The presence of Fc receptors on the target cell is important (381, but cannot be the only determinant of target specificity, because some targets with receptors for the appropriate anti-CD3 isotype and subclass were not susceptible to the anti-CD3-T lytic process, The pattern of target cell restriction, in addition to the inhibitory effects of the anti-CD45 antibody, suggests that the CD45 molecule may have a role in determining target sensitivity to anti-CDS-T cytotoxicity. Only those NK-sensitive targets with NK killing that is mediated through the CD45 molecule, as defined by the inhibitory effects of anti-CD45 monoclonal antibodies (27,28), were susceptible to lamina propria anti-CD3-T lysis. Of the NK-resistant targets studied, only Daudi cells were susceptible to anti-CD3-T cytotoxicity, and this killing was also inhibited by anti-CD45 antibodies. Whether the CD45 molecule is structurally associated with the CD3/Ti T-cell antigen receptor complex or with another antigen receptor, or simply linked to the lytic process, is not yet known. We are at present attempting to resolve this issue using co-capping immunofluorescence and immunoprecipitation studies. The clinical significance of anti-CDS-T cytotoxicity is not certain. Elevated levels of a subset of PBLs with a phenotype compatible with that of the antiCD%triggered effector cell have been found in the acquired immune deficiency syndrome (42), and we have recently found that the peripheral blood of patients with inflammatory bowel disease exhibits elevated levels of anti-CD3-T cytotoxicity (unpub-
GASTROENTEROLOGY
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lished). At the mucosal level, these and other types of cytotoxic effector cells may have a role in mediating tissue damage in inflammatory and other intestinal diseases (3-9). Therefore, a precise demonstration of the full repertoire of killer cell mechanisms within the human mucosa is clearly important. The results of previous studies using unfractionated LPLs, cultured with mitogens, as a measure of cytolytic T-cell function (lo-12,43) cannot adequately distinguish between T-cell-mediated killing and LAK or culture-activated killing (15,37). That freshly isolated non-MHC-restricted T cells within the mucosa of patients with inflammatory bowel disease can kill erythrocytes coated with epithelial antigens is a provocative finding (6). However, the relationship between this type of killing and the anti-CDS-T cytotoxic reaction is not known. The future development of systems for the long-term culture of epithelial cells from controls and patients with inflammatory bowel disease will permit a more direct examination of the role of cytotoxic T-cell interactions with this potential target cell. Finally, it will be important to determine if mucosal anti-CD3-T activity is also elevated in inflammatory intestinal disorders and to examine the regulation of induction and clonality of this response. A pauciclonal pattern would be particularly interesting, and its antigen specificity, if found, might provide a vital clue to the antigenic factor(s) initiating or perpetuating inflammatory bowel disease. References 1. Lanier
2.
3. 4.
5.
6.
7. 8. 9.
10.
LL, Phillips JH. Evidence of three types of cytotoxic lymphocyte. Immunol Today 1986:7:132-4. Lanier LL. Le AM, Cwirla S, Federspiel N, Phillips JH. Antigenic, functional, and molecular genetic studies of human natural killer cells and cytotoxic T lymphocytes not restricted by the major histocompatibility complex. Fed Proc 1986:45:2823-8. James SP, Strober W. Cytotoxic lymphocytes and intestinal disease. Gastroenterology 1986;90:235-40. Elson CO, Kagnoff MF. Fiocchi C, Befus AD, Targan S. Intestinal immunity and inflammation: recent progress. Gastroenterology 1986;91:746-8. Shorter RG, McGill DB. Bahn RC. Cytotoxicity of mononuclear cells for autologous colonic epithelial cells in colonic diseases. Gastroenterology 1984;86:13-22. Roche JK, Fiocchi C. Youngman K. Sensitization to epithelial antigens in chronic mucosal inflammatory disease. Characterization of human intestinal mucosa-derived mononuclear cells reactive with purified epithelial cell-associated components in vitro. J Clin Invest 1985;75:522-30. Strober W, James SP. The immunologic basis of inflammatory bowel disease. J Clin Immunol 1986;6:415-32. MacDermott RP. Cell mediated immunity in gastrointestinal disease. Hum Path01 1986;17:219-33. Shanahan F. Inflammatory bowel disease. In: Targan S, moderator. Immunology of intestinal disease. Ann Intern Med 1987;106:853-70. Chiba M, Barnik W, ReMine SG, Thayer WR, Shorter RG.
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HUMAN
1988
Human colonic intraepithelial and lamina proprial lymphocytes: cytotoxicity in vitro and the potential effects of the isolation method on their functional properties. Gut 1981;22: 177-86. 11. Falchuk ZM, Barnhard E, Machado I. Human colonic mononuclear cells: studies of cytotoxic function. Gut 1981;22:2904. 12. MacDermott RP. Franklin GO, Jenkins KM. Kodner IJ, Nash GS. Weinrieb IJ. Human intestinal mononuclear cells. I. Investigation of antibody-dependent, lectin-induced. and spontaneous cell-mediated cytotoxic capabilities. Gastroen1980;78:47-56. terology KR. Human intestinal mu13. Fiocchi C, Tubbs RR, Youngman cosal mononuclear cells exhibit lymphokine-activated killer cell activity. Gastroenterology 1985;88:625-37. 14. Hogan PG, Hapel AJ, Doe WF. Lymphokine-activated and natural killer cell activity in human intestinal mucosa. J Immunol 1985;135:1731-8. 15. Shanahan F, Brogan M. Targan S. Human mucosal cytotoxic effector cells. Gastroenterology 1987;92:1951-7. ER, Pledger JV. Peripheral, 16. Bland PW, Britton DC, Richens mucosal, and tumour-infiltrating components of cellular immunity in cancer of the large bowel. Gut 1981;22:744-51. 17. Targan S, Britvan L, Kendall R, Vimadalal S, Sol1 A. Isolation of spontaneous and interferon inducible natural killer like cells from human colonic mucosa: lysis of lymphoid and autologous epithelial target cells. Clin Exp Immunol 1983;54: 14-22. 18. Gibson PR, Dow EL, Selby WS, Strickland RG, Jewel1 DP. Natural killer cells and spontaneous cell-mediated cytotoxicity in the human intestine. Clin Exp Immunol 1984;56:43844. 19. Beeken WL. Gundel M, St. Andre-Ukena S. McAuliffe T. In vitro cellular cytotoxicity for a human colon cancer cell line by mucosal mononuclear cells of patients with colon cancer and other disorders. Cancer 1985;55:1024-9. 20. Clancy R, Pucci A. Absence of K cells in human gut mucosa. Gut 1978;19:273-6. 21. Chiba M, Shorter RG, Thayer WR, Bartnik W, ReMine S. K-cell activity in lamina proprial lymphocytes from the human colon. Dig Dis Sci 1979;24:817-22. 22. Fiocchi C, Battisto JR, Farmer RG. Gut mucosal lymphocytes in inflammatory bowel disease. Isolation and preliminary characterization. Dig Dis Sci 1979;24:705-17. N. Schneeberger EE, Bhan AK. Immunohisto23. Cerf-Bensussan logic and immunoelectron microscopic characterization of the mucosal lymphocytes of human small intestine by the use of monoclonal antibodies. J Immunol 1983;130:2615-22. I. Berrebi G. Austin LL, Keren DF, Dobbins WO. 24. Hirata Immunohistological characterization of intraepithelial and lamina propria lymphocytes in control ileum and colon and in inflammatory bowel disease. Dig Dis Sci 1986;31:593-603. 25. Zinkernagel RM, Doherty PC. H-2 compatibility requirement for T-cell-mediated lysis of target cells infected with lymphocytic choriomeningitis virus. Different cytotoxic Tcell specificities are associated with structures coded in H-2K or H-2D. J Exp Med 1975;141:1427-36. JH, Lanier LL. Lectin-dependent and anti-CD3 in26. Phillips duced cytotoxicity are preferentially mediated by peripheral blood
cytotoxic
Immunol 27. Newman
T lymphocytes
expressing
Leu-7
antigen.
1985;136:1579-85, W. Selective
blockade
of human
natural
killer
cells
J
by 28.
29. 30.
31. 32.
33.
34.
35.
36.
37.
38.
39. 40. 41. 42.
43.
a monoclonal
MUCOSAL
CYTOTOXIC
antibody.
Proc
Nat1
T CELLS
Acad
Sci
967
USA
1982;79:3858-62. Newman W, Fast LD, Rose LM. Blockade of NK lysis is a property of monoclonal antibodies that bind to distinct regions of T-200. J Immunol 1983;131:1742-7. Boyum A. Separation of leukocytes from blood and bone marrow. Stand J Clin Lab Invest 1968;21:31-50. Bull DM, Bookman MA. Isolation and functional characterization of human intestinal mucosal lymphoid cells. J Clin Invest 1977;59:966-74. Wysocki LJ, Sato VL. “Panning” for lymphocytes: a method for cell selection. Proc Nat1 Acad Sci USA 1979;75:2844-8. Targan S, Jondal M. Monolayer immune complex (MIC) fractionation of Fc receptor-bearing human spontaneous killer cells. J Immunol Methods 1978;22:123-9. Deem RL, Shanahan F, Niederlehner A, Targan S. Mechanism and specificity of anti-CD3 triggered T cell killing: relationship to NK lysis (abstr). Fed Proc 1987;46:607. Mentzer SJ. Barbosa JA, Burakoff SJ. T3 monoclonal antibody activation of nonspecific cytolysis: a mechanism of CTL inhibition. J Immunol 1985:135:34-8. Lanier LL, Le AM, Civin CI, Loken MR. Phillips JH. The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic lymphocytes. J Immunol 1986;136:4480-6. Targan S, Newman W. Definition of a “trigger” stage in the NK cytolytic reaction sequence by a monoclonal antibody to the glycoprotein T-200. J Immunol 1983:131:1149-53. Shanahan F, Brogan MD, Newman W. Targan SR. K562 killing by K, and IL-2-responsive NK, and T cells involves different post-binding trigger mechanisms. J Immunol 1986; 137:723-6. Leeuwenberg JEM, Spits H. Tax WJM, Cape1 PJA. Induction of nonspecific cytotoxocity by monoclonal anti-T3 antibodies. J Immunol 1985;134:3770-5. MacDonald HR, Nabholz M. T cell-activation. Annu Rev Cell Biol 1986;2:231-53. Bolhuis RLH, Gravekamp C, Van de Griend R. Cell-cell interactions. Clin Immunol Allergy 1986;6:29-90. Anderson CL, Looney RJ. Human leukocyte IgG Fc receptors. Immunol Today 1986:7:264-6. Lewis DE, Puck JM, Babcock GF. Rich RR. Disproportionate expansion of a minor T cell subset in patients with lymphadenopathy syndrome and acquired immunodeficiency syndrome. J Infect Dis 1985;151:555-9. MacDermott RP, Bragdon MJ, Kodner IJ, Bertovich MJ. Deficient cell-mediated cytotoxicity and hyporesponsiveness to interferon and mitogenic lectin activation by inflammatory bowel disease peripheral blood and intestinal mononuclear cells. Gastroenterology 1986;90:6-11.
Received April 6, 1987. Accepted November 23, 1987. Address requests for reprints to: Fergus Shanahan, M.D., Division of Gastroenterology. UCLA Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, California 90024. This work was supported by the UCLA/Harbor Inflammatory Bowel Disease Center (U.S. Public Health Service grant AM 362001, U.S. Public Health Service grant AM 27806, and funds from the Blinder Foundation for Crohn’s Disease Research. Dr. Shanahan is a recipient of a research career development award from the National Foundation for Ileitis and Colitis.
1988;94:960-7
Human Mucosal T-Cell Cytotoxicity FERGUS SHANAHAN, RICHARD DEEM, RAMIN BERNARD LEMAN, and STEPHAN TARGAN Department of Medicine, Los Angeles, California
University
of California
Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CDS-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effecters do not have Fc receptors for immunoglobulin G, and are therefore distinct from T, cells, which mediate antibodydependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7. In contrast, peripheral blood anti-CD3-T effecters are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As antiCD3triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.
urunderstanding of the nature and mechanisms of human lymphocyte-mediated cytotoxicity 0 has advanced considerably in recent years (l-4). It has become clear from studies using peripheral blood lymphocytes (PBLs) that spontaneous cytotoxicity may be a feature of both natural killer (NK) cells and T cells, and that each of these distinct lymphocyte subsets may kill by more than one mechanism (12). However, the extent to which such studies can be extrapolated to the mucosal immune system is not
at Los Angeles,
NAYERSINA,
Center
for the Health
Sciences,
certain. As cytotoxic mechanisms may have a role in mediating the tissue injury that occurs in certain intestinal inflammatory disorders (3-g), a precise delineation of the effector cell populations within the human intestinal mucosa is important. There is general agreement that cultured mucosal lymphocytes exhibit mitogen-induced cellular cytotoxicity (10-12) and lymphokine-activated killer (LAK) cell activity (13-15). Whether freshly isolated mucosal cells exhibit spontaneous NK activity (10-19) or antibody-dependent cellular cytotoxicity (12,16,20-22)has been controversial. We recently found that although unfractionated mucosal lymphocyte preparations have little activity in cytotoxic assays, a small subset of cells with significant NK activity can be demonstrated if specifically enriched for, using monoclonal antibody (NKH-1) panning (151.
Mucosal cytotoxic T cells have been less well studied. Although cells with a surface antigen phenotype compatible with that of cytolytic T cells are present within the intestinal mucosa (23,24),their lytic function has not been clearly demonstrated. Recently, Roche and colleagues (6) showed that intestinal T lymphocytes from patients with inflammatory bowel disease, but not from control patients, could kill red cells coated with epithelial antigens. The significance of this finding is not yet certain because the levels of cytotoxicity were low and lysis occurred only over a restricted range of effector-totarget ratios (6).However, the T-cell killing observed is of interest because it was not restricted by the major histocompatibility complex (MHC) and is, therefore, distinct from classical T-cell cytotoxicity
Abbreviations used in this paper: anti-CD3-T, antXD3triggered T cell; LAK, lymphokine-activated killer; LPL, lamina propria lymphocyte; LU, lytic unit; MHC, major histocompatibility complex; NK, natural killer; PBL, peripheral blood lymphocyte. 0 1988 by the American Gastroenterological Association 0016-5085/88/$3.50
HUMAN MUCOSAL
April 1988
(25). Evidence for non-MHC-restricted cytotoxic T cells in the peripheral blood is now well established (l,2), and a subset of these cells may be conveniently studied in vitro by triggering their activity with antibodies to the CD3 component of the T-cell antigen receptor complex (26). Cytolytic activity, which is triggered by anti-CD3 monoclonal antibodies, is thought to be an indirect measure of in vivo primed cytotoxic T cells when the antigen to which they are reactive is not known (26). Within the human intestinal mucosa, where there is constant and varied antigenic exposure, a high prevalence of in vivo primed T cells would be expected. Therefore, the purpose of this study was (a) to determine whether anti-CD3 triggered lysis could be used to demonstrate cytotoxic T-cell activity in freshly isolated lymphocyte preparations from noninflamed human mucosa, (b) to determine the phenotype of the mucosal anti-CD%triggered T cells (anti-CDS-Ts) in comparison with that of anti-CD3Ts in the peripheral blood, and (c) to examine the target cell restrictions of this form of non-MHCrestricted cytotoxicity within the mucosa.
Materials and Methods Study
Population
Peripheral blood specimens were obtained from healthy volunteers. The intestinal specimens were obtained from patients undergoing surgical resection of the colon at the University of California, Los Angeles, Center for the Health Sciences. The entire study was approved by the hospital’s human subject protection committee. In this series of experiments all tissue specimens were from patients with colonic carcinoma, and were taken from an uninvolved area of the colon. Target
Cells
and
Culture
Medium
The target cell lines K562 (an NK-sensitive human erythroleukemia cell line), HEL 92.1.7 (an NK-sensitive human erythroleukemia line), Jurkat (an NK-sensitive human acute lymphoblastic T-cell leukemia line), IM-9 (an NK-resistant human B-lymphoblast cell line), Daudi (an NK-resistant human B-lymphoblast cell line), and Raji (an NK-resistant human B-lymphoblast cell line) were obtained from American Type Culture Collection, Rockville, Md. Target cell lines U266/AFlO [an NK-sensitive human immunoglobulin (Ig)E-producing myeloma line] and GM-1056 (an NK-resistant human IgA-producing Blymphoblast cell line) were obtained from Dr. Andrew Saxon, University of California, Los Angeles. All target cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2 in RPM1 1640 (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% fetal calf serum. Monocional
Antibodies
Monoclonal antibodies T8 (anti-CD8), T4 (antiCD4], T3 (anti-CD3), Tll (anti-CD2), NKH-1, NKH-lA, and
CYTOTOXIC
T CELLS
961
anti-NKH-l-RDl were purchased from Coulter Immunology, Hialeah, Fla. Monoclonal antibodies Leu llb (antiCD16), Leu lla-FITC (fluorescein isothiocyanate-conjugated anti-CDlG), Leu 4-FITC (anti-CD3), Leu7, Leu 7-FITC, Leu 2b (anti-CD8), and Leu 2a-PE (phycoerythrinconjugated anti-CD8) were purchased from BectonDickinson (Mountain View, Calif.). The monoclonal antibody 13.3, which recognizes an epitope on the human homologue of the murine T-200 (CD45) molecule, was generously provided by Walter Newman, Otsuka Pharmaceuticals, Rockville, Md., after purification by, absorption to, and elution from protein A-Sepharose (27). This antibody is functionally identical to the 13.1 monoclonal antibody and binds to the same epitope of the CD45 molecule (28). Isolation
of Effector
Cells
Peripheral blood mononuclear cells were isolated from healthy volunteers by separation on Ficoll-Hypaque gradients (29). The cells at the interface were recovered, washed three times, and resuspended in RPM1 1640 with 10% fetal calf serum. Lamina propria mononuclear cells were isolated using a technique modified from that originally described by Bull and Bookman (30), which has previously been described in detail (15). The entire procedure was performed in 1 day without interruption. Peripheral blood and lamina propria mononuclear cell preparations were depleted of adherent cells by incubating the cells (5 x 106/ml) on lOO-mm plastic tissue culture dishes (Corning Glass Works, Corning, N.Y.) in a humidified atmosphere (5% COZ in air, 37%) for 2 h. The nonadherent cells, referred to as PBLs and lamina propria lymphocytes (LPLs), were then removed, washed, and resuspended in medium. Enrichment Panning
of Lymphocyte
Subsets
by
In some experiments, a panning technique modified from that originally described by Wysocki and Sato (31) and described in detail elsewhere (15) was used to enrich for LPL and PBL subsets. In brief, nonadherent LPLs or PBLs (lo7 cells/ml) were incubated with subsetspecific monoclonal antibody (0.25 pg/lO” cells) for 60 min at 4°C. The cells were then washed and resuspended in medium at 5 x lo6 cells/ml, and 30 x lo6 cells were added to 100 x 15-mm plastic Petri dishes (Falcon 1029, BectinDickinson Labware, Oxnard, Calif.). These dishes were previously coated with 6 ml of purified goat F(ab’), antimouse IgG Fc or antimouse IgM (p-chain specific) (Jackson Laboratories, Avondale, Pa.) (20 &ml in 0.05 M Tris, pH 9.5) for 120 min at room temperature and then washed three times with medium. After a 90-min incubation of the coated dishes at 4”C, the nonadherent cells were removed by gentle swirling and aspiration. The plates were then gently washed twice and the adherent ceils were removed by vigorous washing. Depletion
of Lymphocyte
Subsets
In some experiments PBLs and LPLs were depleted of lymphocyte subsets by complement lysis. In brief, the
962 SHANAHAN ET AL.
cells were incubated for 1 h fixing monoclonal antibodies. incubated at 22°C with a 1:16 rabbit complement (Pel-Freez, Depletion
GASTROENTEROLOGYVol. 94. No. 4
at 22°C with complementThey were then washed and final dilution of 3-4-wk-old Rogers, Ark.).
of Fc, Rf Lymphocytes
Cells bearing Fc receptors for IgG (Fc,R+) were depleted by immobilization on immune complex monolayers, as described previously (32). In brief, human IgG (Gammimmune, Cutter Laboratories Inc., Berkeley, Calif.) was diluted to 500 pg/ml in phosphate-buffered saline, and 3 ml was added to lOO-mm tissue culture dishes (Falcon) and incubated for 30 min at 22”C, followed by 30 min at 4°C. The dishes were washed three times with phosphatebuffered saline, and then 2 ml of 16 @g/ml rabbitantihuman IgG (y specific) (Miles Scientific, Naperville, Ill.] was added for 30 min at 22°C followed by 30 min at 4’C. The dishes were washed three times and stored at 4°C for up to 2 wk. Up to 30 x lo6 cells were added to each dish and centrifuged at 400 g for 5 min; the plates were then rotated 180” and centrifuged a second time. The dishes were swirled and nonadherent (Fc,R-) cells were removed.
amplitudes of the electrical signals were converted from analog to digital by means of the Analog to Digital Converter. The data were analyzed and histograms displayed by means of the Multiparameter Data Acquisition and Display Systems (MDADS). Dead cells, erythrocytes, platelets, and monocytes were excluded from the analysis by gating on the lymphocyte population. Cytotoxicity
Cytotoxicity tests were performed in 96-well, Vbottom microplates (Cooke, Alexandria, Va.) in a total volume of 200 ~1. Target cells (K562 unless otherwise stated) were labeled with 200 &i NaZ51Cr04 (New England Nuclear Corp., Boston, Mass.] for 1 h at 37°C and washed three times; lo4 target cells were used in each microtiter well along with varying numbers of effector cells. After an incubation period of 12 h (or 4 h where stated), 100 ~1 of supernatant was collected and the p.ercentage of cytotoxicity was estimated from released radioactivity according to the following formula: Cytotoxicity (%] Test release - spontaneous release
= 80%
lmmunofluorescent Cytometry
Staining
and
Flow
Peripheral blood lymphocytes and lamina propria lymphocytes (5 x 105) were suspended in 100 ~1 of phosphate-buffered saline supplemented with fetal calf serum (2%) and sodium azide (O.l%), and phycoerythrinor Red Dye-l- and fluorescein isothiocyanate-conjugated monoclonal antibodies (10 &test for Becton-Dickinson monoclonals and 2.5 pi/test for Coulter monoclonals) were added for 30 min at 4°C. For cells purified by panning or complement-depleted cell populations, staining was performed by indirect immunofluorescence. Monoclonal antibody-treated cells were washed and 0.1 pg of dichlorotriazinyl amino fluorescein conjugated-purified F(ab’)z, goat-antimouse IgG (H & L) (Jackson Immuno Research Laboratories, Avondale, Pa.) was added for 30 min at 4°C. For direct, two-color staining, control cells were stained with fluorochrome-conjugated isotype-matched mouse immunoglobulin. For indirect staining, unconjugated, isotype-matched mouse immunoglobulin monoclonal antibody-treated cells were stained with the secondary, fluorochrome-conjugated antibody. Stained cells were washed twice with phosphate-buffered saline with 2% fetal calf serum, resuspended, and fixed with 1% paraformaldehyde in phosphate-buffered saline. Analysis of lymphocyte subsets was performed using an EPICS 541 Flow Cytometer (Coulter). Red and green fluorochromes were excited using 400 mW of 488-nm light from an argon laser. Fluorescent emission was collected by means of a focusing lens placed at 90” relative to the laser-stream intersection. Forward angle light scatter was collected by a pickup lens (photodiode) in the laser light path. A 488-nm dichroic filter was placed at a 45” angle to the light path to separate the 90” scatter from the red and green fluorescent emissions. A 590-nm-long pass filter was placed in front of the red photomultiplier tube. The
Assay
of total label - spontaneous release
x 100.
A wide range of effector-to-target ratios was used to generate cytotoxicity curves. All assays were performed in triplicate and results were expressed as mean percent cytotoxicity 2 SD. In some experiments results were expressed in terms of lytic units (LU) per lo7 effector cells. Lytic units were calculated from a semilogarithmic curve plotting the natural logarithm of effector cell number versus the percent of cytotoxicity. One lytic unit is the number of effector cells required to induce 30% specific 51Cr release from lo4target cells. Statistics Student’s t-test for paired data was used to determine whether differences in cytotoxicity values under different conditions were significant. Values of p < 0.05 were considered significant.
Results Cytolytic Activity of Freshly Isolated Lamina Propria Lymphocytes in the Presence and Absence of Anti-CD3 Monoclonal Antibody
Freshly isolated, unfractionated LPLs exhibited no significant lytic activity (CO.25 LU) against K562 target cells at effector-to-target ratios up to 1OO:l. However, significant cytolytic activity (164 k 74 LU, mean -+ SD; n = 14) was found when the same cells were pretreated (37°C for 1 h) with 1 pug/ml anti-CD3, but not with an isotype-matched control mouse monoclonal antibody (CO.25 LU). A representative experiment with the data expressed in terms of percent cytotoxicity at different effector-
HUMAN MUCOSAL CYTOTOXIC T CELLS
April 1988
60
?? Untreated LPL ?? Fcu R depleted
AntccD3-LPL Wh)
Control LPL
963
LPL
AntlcD3-LPL (4 h)
Antl-CD3-LPL
I 0
LPL (12 h)
100
200
LYTIC UNITS LPL (4 h) 0
20
40
60
80
100
EFFECTOR TO TARGET RATIO Figure
1. Representative cytotoxicity curves indicating the cytolytic activity (against K562 targets) of freshly isolated LPLs compared with that of LPLs pretreated for 1 h with anti-CD3 monoclonal antibody, in 4- and 12-h assays. Values are the mean f SD of a single experiment performed in triplicate, which is representative of 6 (4 h) to 14 (12 h) similar experiments. Pretreatment with a control isotype-matched monoclonal antibody had no effect on cytotoxic activity (not shown).
to-target ratios is shown in Figure 1. From preliminary titration experiments using LPLs and PBLs, the concentration of anti-CD3 was found to be lo-fold higher than that required to elicit maximal anti-CD3T activity and was used in all subsequent experients. The anti-CD3-triggered LPL cytotoxicity was observed in all of 14 consecutive intestinal specimens. In contrast, in the peripheral blood, we recently found significant anti-CD3-triggered PBL cytotoxicity in only 25% of normal subjects (33, and Deem R, Shanahan F. Niederlehner A, and Targan S, unpublished). Anti-CD3-Triggered Fc, Receptors
Cytotoxic
Figure 2. Anti-CD3-triggered T-cell cytotoxicity against K562 targets by LPLs, before and after depletion of cells bearing Fc receptors for IgG (Fc,R). Values are the mean lytic units 2 SE, n = 4; p = 0.065 for the anti-CD3-T comparison using paired data.
Surface Antigen Phenotype of the Lamina Propria Anti-CD3-Triggered T Cell Is CDl6- Leu 7- CD3+ CD8+ CD4- CDZ+ It has previously been reported that anti-CD3Ts within PBLs are in general CD16-, Leu 7+ (26). In this study (Table l), the anti-CD3-T cytotoxic activity of PBLs from 7 control subjects was mediated by CD16-, Leu 7+ cells, although in 1 subject (No. 7, Table 1) there was also significant lytic activity in the Leu 7- population (which contained only 0.2% residual Leu 7+ cells). This variability is consistent with the findings of others (26), and is not surprising as the surface antigen defined by the Leu 7 antibody has not per se been inextricably linked either functionally or biochemically to the lytic process. Unlike PBLs, mucosal CD3+ T cells are consistently CD16-, Leu 7-. In keeping with other reports (13,15,23,24), we found that these surface antigens are not present within LPL populations (flow cytom-
T Cells Lack
When LPLs were depleted of Fc,R+ cells by adherence to monolayers of immune complexes, the anti-CD3-T activity was either unchanged or enriched (Figure 2). Therefore, the anti-CD3-Ts within LPLs lack Fc receptors for IgG (Fc,JR-) and are distinct from T, cells, which mediate antibodydependent cellular cytotoxicity. This is consistent with our previous results with PBLs (33). Control studies that confirmed the effectiveness of the Fc,R+ cell depletion included (a) its elimination of all NK activity and CD16+ cells and (b) a consistently >75% reduction of antibody-dependent cellular cytotoxicity activity from CD16- cells [data not shown), as determined against K562 target cells that were pretreated with K562 antiserum (provided by Dr. D. Hudig, University of Nevada, Reno, Nev.).
Table 1. Anti-CD3-Triggered T-Cell Cytotoxicity in the Peripheral Blood Is Mediated Predominantly by the Leu 7+(CD16 -) Subpopulation” Lytic units (mean + SD) Subject
Leu 7+
Leu 7-
1
437
2 28
2
642
2 64
3
168
? 24
32 + 5 11 -+ 2
4
215
? 25
5
464
2 63
6
973
2 115
7
158 f
37
67 5 9 3 2 0.6
4 r
0.2
44 2 6 70 2 5
a CD16 cells were first depleted (
964
SHANAHAN ET AL.
Figure 3. Anti-CDs-triggered T cell cytotoxicity against K562 targets by LPLs is mediated by CD8+, CD4-, CDZ+ cells. The monoclonal markers were examined using separate intestinal specimens. For each monoclonal marker, LPLs were enriched (284%) and depleted (55%) by panning. The values are the mean f SD of triplicates from experiments using separate intestinal specimens.
GASTROENTEROLOGY Vol. 94, No.
CD8
CD4
4
CD2
1
0
200
400
600
LYTIC UNITS
etry; CD16 = 0%, Leu 7 = O%, n = 11). As expected, anti-CD3-T activity was not affected by treatment of LPLs with Leu 7 and complement, whereas with PBLs from the majority of donors, it was consistently reduced (X0%) by this procedure (33, and data not shown). In contrast to the findings with CD16 and Leu 7, the LPL and PBL anti-CD3-T effecters were phenotypically similar with respect to other surface markers. Positive selection of mucosal lymphocyte subsets using monoclonal antibody panning indicated that the anti-CD3-T effecters within LPLs are CD8+, CD4-, and CDZ+ (Figure 3). Although it has been reported that some CD4+ clones developed from PBLs exhibit anti-CD$triggered lytic activity (34), this has not been found with freshly isolated PBLs. In this study, anti-CD3-Ts within PBLs from 3 consecutively examined subjects were CD4-. Peripheral blood lymphocytes were first depleted of CDl6+ cells (
LYTIC UNITS
LYTIC UNITS
LPLs with NKH-1A and complement, which resulted in a SO%-60% reduction of NKH-l+ cells, did not influence anti-CD3-T activity in three separate experiments, but yielded a 50% reduction in lytic activity in one experiment. The variability may reflect donor-to-donor differences in the coexpression of CD3 and NKH-1 on LPLs. Therefore, doublemarker analysis of LPLs bearing CD3 and NKH-1, from four intestinal specimens, was performed and indicated that the NKH-l+ cells that were also CD3+ ranged from 20% to 60%. These results are similar to our previous findings in the peripheral blood (33, and unpublished). Effect of Anti-CD45 (13.3) on Anti-CD3-Triggered Activity The monoclonal antibody 13.3, which reacts with an epitope of the CD45 (T200) molecule, has been shown to inhibit certain cell-mediated forms of cytotoxicity at the effector cell level (27,28,36,37). It inhibits NK cytotoxicity against some but not all NK-sensitive targets, and has been used to define the trigger phase of the NK lytic sequence (36). In contrast, LAK, antibody-dependent cellular cytotoxicity, and classical T-cell cytotoxicity are not inhibited by anti-CD45 antibodies (27,28,37). Using a panel of monoclonal antibodies to different epitopes of the CD45 molecule, we have recently shown that peripheral blood anti-CDS-T cell killing occurs through the CD45 molecule (33) and therefore differs from classical T-cell cytotoxicity. In this study, 13.3 (antiCD45) was also found to inhibit LPL anti-CDS-Tmediated lysis (Figure 4). The degree of inhibition, although partial, was statistically significant. The concentration of 13.3 (0.1 pg/ml) that yielded maximal inhibition was determined from preliminary titration experiments and was the same as that found previously for PBLs (28,33,37). Increasing the concentration lo-fold (1 pg/ml) in two experiments against K562 targets did not elicit any further inhibitory effect (control = 169 and 200 LU; 13.3, 0.1 pglml, 85 and 47 LU; and 13.3, 1 pglml, 92 and 49
HUMAN MUCOSAL CYTOTOXIC T CELLS
April 1988
K562
DAUDI
0
100
200
965
were sensitive to anti-CD3-T cytolysis (Figure 5). Of the four NK-resistant target cells examined (Raji, IM9, GM iO56, and Daudi) only Daudi cells were killed by anti-CD3-Ts, and this killing can be inhibited by the CD45 antibody, 13.3 (Figures 4 and 5). These results suggest that the CD45 molecule may be an important determinant of anti-CD3-T sensitivity. This is consistent with our recent study of peripheral blood anti-CD3-T cytotoxicity (33, and unpublished) using a more extensive range of target cell lines.
LYTIC UNITS
Figure 4. Inhibitory effect of anti-CD45 monaclonal ‘antibody (13.3) on anti-CD3-triggered killing of K562 (p = 0.007, n = 4) and Daudi (p = 0.04, n = 3) targets by CPLs. The values shown are mean ? SD. The LPLs were pretreated with anti-CD3 (1 h, 37’C) and anti-CD45 (0.1 pg/ml) was added to the assay. A control isotypematched monoclonal antibody did not influence the inhibitory effects of anti-CD45 (see text), and increasing the concentration of anti-CD45 lo-fold did not alter the result (see text].
LU, respectively). The addition of a control iqotypematched monoclonal antibody to the assay did not inhibit lysis of K562 targets by anti-CD3-Ts in.*two experiments (85, 161 LU versus 87, 158 LU, respectively). Target Cell Restrictions of Lamina Propria Anti-CD3-Triggered T Cells The target specificity of the lamina propria anti-CD3-triggered effector cells differed from that of NK and LAK cells (Figure 5). Some, but not all, NK-sensitive targets and some, but not all, NKresistant targets were susceptible to anti-CD3-T killing, whereas all of the targets in Figure 5 are LAKsusceptible. The presence of an Fc receptor on the target cell has an important role in anti-CDB-T cytotoxicity (38) and is thought to provide an anchdrage site Whereby CD3 on the surface of the effector cell can be crosslinked by the antibody (3940) However, target cell expression of Fc receptors cannot be the orily determinant of susceptibility to ahti-CD3-T lysis because it does not explainthe pattern of target susceptibility seen in Figure 5. K562, Daudi, and Raji cells have high-affinity surface Fc receptors that bind IgG, (41), yet only K562 and Daudi cells, and not Raji cells, are susceptible to anti-CDB-T lysis (Figure 5). Therefore, other factors must influence the target restrictions of anti-CDB-T lysis. Of the four NK-sensitive target cell lines studied (K562, HEL 92.1.7, AFlO, and Jurkat), only those with NK killing that is mediated through the CD45/T200 trigger (K562 and HEL 92.1.7) (27,333
Discussion
,
The results indicate that freshly isolated lymphocytes from the human intestinal lamina propria can mediate a form of T-cell cytotoxicity that is non-MHC-restricted and can be triggered by antiCD3 antibodies in vitro. In this study the cytotoxic activity was observed in all intestinal specimens examined, whereas in a recent survey of peripheral blood, we found significant anti-CD3-T activity in only 25% of healthy subjects (33, and unpublished). The intestinal mucosa might be an immunologic compartment where these cells predominantly reside, and are perhaps exposed and primed to a variety of environmental antigenic stimuli. It has been proposed that anti-CD3-Ts represent in vivo primed cytotoxic T cells, possibly reactive against viral and other antigens (26). In the presence of already primed T cells, it has been suggested that anti-CD3 antibodies might trigger non-MHCrestricted cytotoxicity by bypassing the requirement for antigen triggering. This technique might therefore be of use in identifying such cells, without knowing the nature of the stimulating antigen (26). The phenotype of the intestinal anti-CD3-triggered effector cell is similar to that of its peripheral blood counterpart in being Fc,R-, CD2+, CD3+, CD4-,
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Julkat HEL 92.1.7 K562 0
100
200
300
400
500
LYTIC UNITS Figure 5. Anti-CD3-triggered lamina propria T cells kill some but not all NK-sensitive targets and some but not all NKresistant targets. Values are mean lytic units ? SE, n = 4.
966
SHANAHAN
ET AL.
CDs+, and CDl6-. However, as found by others (261, the anti-CDS-Ts within the peripheral blood are Leu 7+, whereas within the intestine, they lack the Leu 7 antigen. These results extend those of our previous report (15) and further indicate phenotypic differences between human cytotoxic effector cells of the mucosal and systemic immune systems. The anti-CD3-T lytic process is unusual in that it can be inhibited by monoclonal antibodies (13.3) to the CD45 antigen. CD45 is known to be coupled to the trigger process for NK lysis of some but not all target cells (27,28,36). Other types of cytotoxicity, such as classical (MHC-restricted) T-cell cytotoxicity, LAK, and antibody-dependent cellular cytotoxicity by NK or T, cells, are not blocked by anti-CD45 antibodies (28,37). Whether this can be used to distinguish different types of mucosal T-cell cytotoxicity is not yet clear, as the degree of inhibition observed in this study was partial. The lytic process triggered by anti-CD3 may not proceed exclusively through the CD45 molecule. Anti-CDS-triggered T cell cytotoxicity, although non-MHC-restricted, has a considerable degree of target specificity, which differs from that of NK and LAK cytotoxicity. The presence of Fc receptors on the target cell is important (381, but cannot be the only determinant of target specificity, because some targets with receptors for the appropriate anti-CD3 isotype and subclass were not susceptible to the anti-CD3-T lytic process, The pattern of target cell restriction, in addition to the inhibitory effects of the anti-CD45 antibody, suggests that the CD45 molecule may have a role in determining target sensitivity to anti-CDS-T cytotoxicity. Only those NK-sensitive targets with NK killing that is mediated through the CD45 molecule, as defined by the inhibitory effects of anti-CD45 monoclonal antibodies (27,28), were susceptible to lamina propria anti-CD3-T lysis. Of the NK-resistant targets studied, only Daudi cells were susceptible to anti-CD3-T cytotoxicity, and this killing was also inhibited by anti-CD45 antibodies. Whether the CD45 molecule is structurally associated with the CD3/Ti T-cell antigen receptor complex or with another antigen receptor, or simply linked to the lytic process, is not yet known. We are at present attempting to resolve this issue using co-capping immunofluorescence and immunoprecipitation studies. The clinical significance of anti-CDS-T cytotoxicity is not certain. Elevated levels of a subset of PBLs with a phenotype compatible with that of the antiCD%triggered effector cell have been found in the acquired immune deficiency syndrome (42), and we have recently found that the peripheral blood of patients with inflammatory bowel disease exhibits elevated levels of anti-CD3-T cytotoxicity (unpub-
GASTROENTEROLOGY
Vol. 94. No. 4
lished). At the mucosal level, these and other types of cytotoxic effector cells may have a role in mediating tissue damage in inflammatory and other intestinal diseases (3-9). Therefore, a precise demonstration of the full repertoire of killer cell mechanisms within the human mucosa is clearly important. The results of previous studies using unfractionated LPLs, cultured with mitogens, as a measure of cytolytic T-cell function (lo-12,43) cannot adequately distinguish between T-cell-mediated killing and LAK or culture-activated killing (15,37). That freshly isolated non-MHC-restricted T cells within the mucosa of patients with inflammatory bowel disease can kill erythrocytes coated with epithelial antigens is a provocative finding (6). However, the relationship between this type of killing and the anti-CDS-T cytotoxic reaction is not known. The future development of systems for the long-term culture of epithelial cells from controls and patients with inflammatory bowel disease will permit a more direct examination of the role of cytotoxic T-cell interactions with this potential target cell. Finally, it will be important to determine if mucosal anti-CD3-T activity is also elevated in inflammatory intestinal disorders and to examine the regulation of induction and clonality of this response. A pauciclonal pattern would be particularly interesting, and its antigen specificity, if found, might provide a vital clue to the antigenic factor(s) initiating or perpetuating inflammatory bowel disease. References 1. Lanier
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Received April 6, 1987. Accepted November 23, 1987. Address requests for reprints to: Fergus Shanahan, M.D., Division of Gastroenterology. UCLA Center for the Health Sciences, 10833 Le Conte Avenue, Los Angeles, California 90024. This work was supported by the UCLA/Harbor Inflammatory Bowel Disease Center (U.S. Public Health Service grant AM 362001, U.S. Public Health Service grant AM 27806, and funds from the Blinder Foundation for Crohn’s Disease Research. Dr. Shanahan is a recipient of a research career development award from the National Foundation for Ileitis and Colitis.