Suppression of Immunoglobulin Synthesis by Lymphocyte Subpopulations in Patients With Crohn's Disease

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1 GASTROENTEROLOGY 1984;86:151-8 Suppression of Immunoglobulin Synthesis by Lymphocyte Subpopulations in Patients With Crohn's Disease STEPHEN P. JAMES, LEONARD M. NECKERS, ALAN S. GRAEFF, JEFFRY COSSMAN, CHARLES M. BALCH, and WARREN STROBER Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, and the Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; and Departments of Surgery and Microbiology, University of Alabama, Birmingham, Alabama In previous studies, patients with mild or inactive Grohn's disease were found to have increased suppressor T-cell activity. To further characterize suppressor T cells in Grohn's disease, studies were carried out with the use of monoclonal antibodies. Excessive suppressor activity was eliminated by removal of OKT 8+ lymphocytes by complementmediated lysis. However, the percentage of OKT 8+ (or Leu 2a+) cells and the ratio of OKT 4+ to OKT 8+ (or Leu 3a+ to Leu 2a+) cells were not significantly different from normal. Although the subgroup of patients with increased suppression of immunoglobulin synthesis had a significantly lower mean Leu 3a to Leu 2a ratio than that of normal subjects, in the whole group of Grohn's patients studied, neither the percentage of Leu 2a+ cells nor the ratio of Leu 3a+ to Leu 2a+ cells correlated with excessive suppression of immunoglobulin synthesis. A subpopulation of Leu 2a+ lymphocytes reactive with the monoclonal antibody HNK-l (Leu 2a+HNK-l +) was increased in patients with Grohn's disease. Furthermore, elimination of HNK-l-reactive lymphocytes by complement-mediated lysis diminished the excessive suppressor cell function in patients with Grohn's disease. The percentage of Leu 2a+HNK-l + Received July 8, Accepted January 6, Address requests for reprints to: Stephen P. James, M.D., Mucosal Immunity Section, LCI, NIAID, Building 1, Room IlN- 25, National Institutes of Health, Bethesda, Maryland 225. This work was performed as part of the National Institute of Allergy and Infectious Diseases Clinical Research Project , approved October 18, The authors thank Dr. Gideon Goldstein, Ortho Pharmaceutical Corporation, Raritan, New Jersey, for providing the OKT 4 and OKT 8 used in the complement-mediated lysis studies. lymphocytes correlated significantly with the suppression of pokeweed mitogen-stimulated immunoglobulin synthesis in vitro. Thus, patients with mild Grohn's disease have an increased suppressor cell activity in vitro which correlates with the presence of a subset of lymphocytes that have an HNK-l +Leu- 2a+ phenotype. Considerable evidence supports the view that immune responses are controlled by complex interactions between subsets of T cells or between T cells and B cells (1). Such interactions play an important role in immune responses in the gastrointestinal tract where, for example, it has been shown that oral administration of an antigen frequently results in the induction of suppressor T cells that inhibit immune responses to the antigen (2). This negative feedback mechanism for inhibition of immune responses may playa vital role in preventing excessive stimulation of the immune system by the large number of antigens in the gastrointestinal tract. Regulatory interactions may also be important in the pathogenesis of certain gastrointestinal diseases, such as Crohn's disease (3). In this regard, we have previously found that peripheral blood T cells from patients with mild or inactive Crohn's disease frequently demonstrate increased suppressor T-cell function in vitro (4). In the present study, the properties of suppressor lymphocytes in patients with Crohn's disease were investigated using monoclonal Abbreviations used in this paper: CDAI, Crohn's disease activity index; FITC, fluorescein isothiocyanate; NK, natural killer; PBA, phosphate-buffered saline with 2% albumin; PBM, peripheral blood mononuclear cells; PWM, pokeweed mitogen; T*, allogenic-irradiated (1 R) normal T cells.

2 June 1984 SUPPRESSOR CELLS IN CROHN'S DISEASE 1511 antibodies reactive with T-cell subsets. The results indicate that lymphocytes from patients with Crahn's disease are qualitatively different from those of normal individuals in that there is an increased number of a subset of suppressor cells, those which are reactive with two monoclonal antibodies, Leu Za and HNK-l. These studies provide further evidence that circulating suppressor lymphocytes are altered in Crohn's disease. Methods Patients Studied Twenty-three patients with Crohn's disease were studied. The diagnosis was based on the presence of typical clinical, radiographic, and pathological features of Crohn's disease. Pathological material was available in 2 of 23 patients and was considered diagnostic or compatible with Crohn's disease. The diagnosis of colonic Crohn's disease depended on the presence of typical clinical features such as skip areas or fistulas or diagnostic biopsy findings. The study included 13 patients with disease of the small bowel, 7 with disease of the colon, and 3 with both small bowel and colon involvement. There were 14 men and 9 women with a mean age of 34.3 yr (range 8-78 yr). Most patients had mild or inactive Crohn's disease, using the Crohn's disease activity index (CD AI) as a measure of disease activity (5). The mean CD AI was 74 (range -254). Nine of the patients were not taking any medication at the time of study, 2 were receiving only antidiarrheal agents, 5 were receiving sulfasalazine with or without antidiarrheal agents, and 7 were receiving corticosteroids. Twenty-two normal individuals, including normal laboratory personnel and blood bank donors, were used as control subjects. Lymphocyte Purification Peripheral blood mononuclear cells (PBM) were isolated from fresh heparinized venous blood by centrifugation on Ficoll-Hypaque (lymphocyte separation medium, Bionetics Laboratory Products, Litton Bionetics, Inc., Kensington, Md.). In some studies, mononuclear cells were separated into T-cell-enriched and B-cell-enriched fractions using immunoabsorbent affinity columns as previously described (6). Briefly, mononuclear cells were suspended in culture medium, which consisted of RPMI 164 (Gibco Laboratories, Grand Island Biological Corporation, Grand Island, N.Y.) containing 25 mm HEPES buffer, 4 mm L-glutamine, penicillin (1 U/ml), streptomycin (1 p.g/ml), and 1% heat-inactivated fetal calf serum (Gibco Laboratories). When the PBM were passed over affinity columns, culture medium was supplemented with 2.5 mm ethylenediaminetetraacetic acid. The column matrix consisted of Sephadex G-2 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) to which purified rabbit antihuman (Fab'lz had been covalently bound. Cells that did not adhere to the column were enriched for T cells (mean 7%), whereas cells that were eluted from the column by mechanical agitation were enriched for B cells (mean 45%) and macrophages (mean 4%). In some experiments, mononuclear cells were studied that had been cryopreserved by freezing in culture medium supplemented with 1% dimethylsulfoxide (Sigma Chemical Company, St. Louis, Mo.) using a controlled-rate freezing apparatus (Vari Rate 2, Virtis Co, Gardiner, N.Y.). Monoclonal Antibodies For indirect immunofluorescence studies, anti Leu 2a, anti-leu 3a, and anti-leu 4 (Becton, Dickenson & Company, Sunnyvale, Calif.) and OKT 4 and OKT 8 (Ortho Pharmaceutical Corporation, Raritan, N.J.) were used in conjunction with fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin (Kirkegaard and Perry Laboratories, Gaithersburg, Md.). For direct immunofluorescence studies, the FITC conjugates of anti-leu 2a, anti-leu 3a, anti-leu 4, and anti-leu 7 (HNK-1, Becton, Dickenson) were used. The OKT 4 alld OKT 8 used in complement-mediated lysis studies were the generous gift of Dr. Gideon Goldstein, Ortho Pharmaceutical. The HNK- 1 monoclonal antibody used in the complement-mediated lysis studies was prepared as previously described (7). Complement-Mediated Lysis of Lymphocytes In some studies, lymphocyte subpopulations were depleted by complement-mediated lysis as follows: 6-1 million PBM were suspended in.1 ml of culture medium with monoclonal antibody at a final concentration of 1 p.g/ml for 1 h at 4 C. The cells were washed once in culture medium and then suspended in a 1: 6 dilution of rabbit complement (Low Tox H Complement, Cedarlane Laboratories Limited, Hornby, Ontario, Canada) for 1 h at 37 C. As determined by direct immunofluorescent staining, this procedure removed >9% of OKT 8+ lymphocytes or >85% of HNK-1 + lymphocytes with the use of these two monoclonal antibodies, respectively. Cell Cultures and Determination of Immunoglobulin Synthesis To measure the regulation of immunoglobulin synthesis, varying numbers of purified T cells and B cells were cultured with pokeweed mitogen (PWM) (Gibco Laboratories) at a final dilution of 1: 1 in I-dram glass vials for 1 days in a 5% CO 2 humidified atmosphere at 37 C as previously described (6). In some experiments, T cells were irradiated with 1 R before culture by exposure to a 137CS source (T*) (Gammator M, Isomedix, Parsippany, N.Y.). At the end of the culture period, culture supernatants were harvested and stored at -2 C before assay. Supernatant immunoglobulin M was determined using a liquid phase, double antibody radioimmunoassay as previously described (6). Quantitation of Lymphocyte Subsets Using Monoclonal Antibodies In immunofluorescence studies, the PBM were depleted of macro phages before study by incubating the

3 1512 JAMES ET AL. GASTROENTEROLOGY Vol. 86, No. 6 PBM in culture medium on 6-cm plastic Petri dishes (Costar #36, Data Packaging Corporation, Cambridge, Mass.) for 9 min at 37 C in order to reduce background immunofluorescence. Indirect immunofluorescence studies were carried out by incubating 5 x 1 5 macrophagedepleted PBM in.1 ml of monoclonal antibody at a final dilution of 1: 5 in phosphate-buffered saline containing 2% bovine serum albumin and.3% sodium azide (PBA). The cells were incubated for 3 min at 4 C, washed twice with cold PBA, and incubated for 3 min at 4 C with a 1: 4 dilution of FITC-conjugated goat antimouse immunoglobulin. Background immunofluorescence was determined by treating cells with a 1: 1 dilution of mouse ascites instead of monoclonal antibody. In dual labeling studies, monocyte-depleted PBM were incubated with FITC conjugates of either Leu Za, Leu 3a, or Leu 4 at a dilution of 1: 5, washed, and incubated in a second step with FITC-conjugated HNK-l using the same conditions. The cells were washed three times with cold PBA after the final incubation. Ten thousand cells were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACS II, Becton, Dickenson & Company, Mountain View, Calif.). Cytotoxicity Assay To determine natural killer cell (NK) activity, PBM were tested for cytotoxic function using as target cells 51Cr_ labeled Chang cells as previously described (8). Cytotoxicity assays were performed in 96-well round-bottomed microtiter plates (Linbro, Flow Laboratories, Inc., McLean, Va.) by mixing variable numbers of PBM with 1 4 target cells. Cell mixtures were incubated for 6 h at 37 C in a 5% CO 2 humidified atmosphere. Culture supernatants were harvested using a 96-well supernatant collecting system (Titertek, Flow Laboratories). Maximal 51Cr release was determined by addition of detergent (Triton X-I, Research Products International Corporation, Elk Grove Village, Ill.). Calculations and Statistical Methods To calculate the percentage of cells in PBM reactive with two FITC-labeled monoclonal antibodies in duallabel studies, single-label fluorescence profiles (with Leu Za, Leu 3a, Leu 4, or HNK-l alone) were compared with the fluorescence profiles of the cells stained with either Leu Za, Leu 3a, or Leu 4 followed by staining with HNK-1. Shifts in fluorescence profiles in doubly labeled samples were taken as an indication of dual labeling of cells. Thus, the percentage of cells reactive with both antibodies was calculated as the difference between the sum of the percentage of cells reactive with each of the two labels alone and the percentage of cells reactive with both labels simultaneously. Flow cytometry data were analyzed on a PDP computer interfaced with the flow cytometer and equipped with statistical programs prepared by the National Institutes of Health computer center. The percentage of suppression of immunoglobulin synthesis was calculated as follows: % Suppression = 1 - -Ig+] x 1, [ Igwhere Ig+ is the immunoglobulin synthesis in the presence of suppressor cells and Ig- is the immunoglobulin synthesis in the absence of suppressor cells. The results of cytotoxicity studies were expressed as the percent of specific lysis: BR] BR experimental counts - % Special lysis = x 1, [ maximal release - where BR is the background release. The significance of differences between mean values was determined using the Mann-Whitney test for nonparametric data. The significance of the correlation between two variables was determined with the Spearman rank test. Results Suppression of Immunoglobulin Synthesis by T Cells From Patients With Crohn's Disease Initially, the ability of T cells from patients with Crohn's disease to suppress immunoglobulin synthesis was tested. T cells, which were from normal individuals or Crohn's disease patients, were obtained from anti-(fab')z columns and added to indicator cultures containing mixtures of cells that were optimized for the ability to synthesize immunoglobulin in vitro. These indicator cultures consisted of normal B cells, T* (providing T-cell help). and PWM. As shown in Figure 1, the addition of T cells from allogeneic normal individuals to the indicator cultures suppressed immunoglobulin synthesis by 57% on the average. The inhibition of immunoglobulin synthesis by allogeneic T cells has been described previously and is presumably due to the effects of the allogeneic mixed-lymphocyte reaction induced in these cultures. When T cells from 16 patients with Crohn's disease were tested under the same conditions, immunoglobulin synthesis was significantly lower than that seen in normal individuals (p <.5). As can be seen in Figure 1, the suppression mediated by T cells from Crohn's disease patients was highly variable and similar to previously published data (4). The degree of inhibition of immunoglobulin synthesis did not correlate with the CDAI, duration of illness, location of disease, or corticosteroid treatment. Removal of Suppressor Cells with OKT 8 and Complement Previous studies have indicated that human peripheral blood T cells are composed of two mutually exclusive.subsets defined by the monoclonal

4 June 1984 SUPPRESSOR CELLS IN CROHN'S DISEASE 1513 ~ :. 1,.."... " <> en S (J) en w :r I- z en >- ~!2' 1, Normal B+T* Normal B + T* Normal B + T* + + Normal T Crohn's T Figure 1. Suppression of pokeweed mitogen (PWM)-stimulated immunoglobulin synthesis by T cells from patients with Crohn's disease. Normal B cells (5 x 1 5 ) were cultured with normal irradiated T cells (T*) (5 x 1 5 ) and PWM. Either T (5 x 1 5 ) cells from normal allogeneic donors or T cells from patients with Crohn's disease were added. After 1 days of culture, the supernatants were assayed for immunoglobulin M by radioimmunoassay. Bars represent mean (n = 16) ± 1 SE. by T cells from patients with Crohn's disease was due to an alteration in the number of cells in peripheral blood reactive with OKT 4 or OKT 8, the percentage of these cells was determined by flow cytometry. The mean percentage of OKT 8+ cells in 12 patients with Crohn's disease (26.8% ± 2.9%) was not significantly different from normal (26.7% ± 1.8%); however, the mean percentage of OKT 4+ cells in patients with Crohn's disease (38.4% ± 3.6%) was significantly (p <.5) lower than normal (46.5% ± 4.4%). These findings were confirmed in 2 of the Crohn's disease patients in further studies using the Leu series of antibodies. Leu 2a reacts with a subpopulation of T cells that is similar, if not identical, to OKT 8, and Leu 3a reacts with a subpopulation of T cells that is similar, if not identical, to OKT 4; in addition, Leu 4 reacts with most sheep red blood cell receptor-bearing periphe:ral blood T cells (13). As shown in Figure 3, the percentage of T cells and Leu 2a + T cells was normal in patients with Crohn's disease; however, the percentage of T cells reactive with Leu 3a was significantly diminished in Crohn's disease (p <.5). There was no significant correlation between the percentage of Leu 2a + cells and the degree of suppression of immunoglobulin synthesis. The percentage of Leu 2a + cells was similar in the 9 patients with greatest suppression of 1, antibodies, OKT 4 (helper/inducer) and OKT 8 (suppressor/cytotoxic) (9-12). In order to determine whether or not the excessive suppression of immunoglobulin synthesis mediated by T cells from some Crohn's disease patients was mediated by cells bearing the OKT 4 or the OKT 8 antigen, T cells from 6 patients who exhibited >9% suppression of immunoglobulin synthesis were first treated with OKT 8 and complement and then added to immunoglobulin synthesis cultures as above. As shown in Figure 2, elimination of OKT 8-bearing T cells in Crohn's disease diminished the suppressor effect mediated by these cells. Thus, suppression of immunoglobulin synthesis by T cells from patients with Crohn's disease requires the presence of a cell population reactive with OKT 8. Percentage of Cells Reactive With Monoclonal Antibodies In order to determine whether the increased suppression of immunoglobulin synthesis mediated < 1 L.--'N'-O-N-'E'-N... O~R~M... A-L ~ T ~ OKT 8 DEPLETED CELLS ADDED TO NORMAL B+ T"+PWM Figure 2. Elimination of suppressor cells by treatment of T cells with OKT 8 and complement. T cells were added to PWM-stimulated cultures as in Figure 1. OKT 8+ cells were depleted by complement-mediated cytolysis as described in Methods. Bars represent mean (n = 6) ± 1 SE.

5 151,! JAMES ET AL. GASTROENTEROLOGY Vol. 86. No.6 LEU 2 LEU 3 LEU 4 8 l- Figure 3. P~rcentag\l of peripheral blood mononuclear cells reactive with Leu 2a, Leu 3a, and Leu 4 ~ in patients with Crahn's dis- E 6 - C/) ease. Reactivity was deter- 2 mined by indirect immunoflu- I I =t - ocb _~_ S _8x>-. o~ - _."'- orescence as described in ~ u 4 fa * ~~- Methods. Mean values are ffi represented by the horizontal... lines (p <.5). - - I-- I _. ~ _ -- otc :It o~ I--, Normal Crohn's Normal Crohn's Normal Crohn's immunoglobulin synthesis (33.8% ± 3.8%). the 2 patients studied (28.% ± 2.5%), and the normal controls (27.5% ± 1.7%). Similarly, in the 9 patients with greatest suppression, the mean pe):"centage of Leu 3a+ cells (39.1% ± 5.2%) was not significantly different from the 2 patients studied (41.4% ± 2.8%), but was significantly (p <.5) lower than nonpal (49.7% ± 1.9%). These results indicate that the increased suppressor cell activity observed in T cell preparations from patients with Crohn's disease is not due to an increase in the percentage of cells reactive with OKT 8 or anti-leu Za. It is possible that the abnormal suppressor activity of T cells from patients with Crohn's disease is due to an alteration in the proportion of Leu 3 + and Leu 2+ lymphocytes. In the group of 2 patients examined, however, the mean ratio of Leu 3a to Leu 2a was not significantly different from normal (Figure 4). In addition, there was no significant correlation between the degree of suppression of immunogloblllin synthesis and the Leu 3a to Leu 2a ratio. Also, there was no significant correlation between disease activity, qs measured by the CDAI, and the ratio of Leu 3a to Leu 2a cells. However, when the subgroup of 9 patients with the greatest degree of suppressor activity (Figure 1) was examined, the mean Leu 3a to Leu 2a ratio (1.8 ±.19) was found to be significantly (p <.5) lower than than in normal subjects (1.n ±.42), in the group of Crohn's patients as a whole (1.82 ±,4), and in the patients with Crohn's disease who did not have increased suppressor activity (2.28 ±.23). In summary, the patients with Crohn's disease and increased suppressor T-cell activity were found to have a normal percentage of Leu za + cells, a diminished percentage of Leu 3a + cells, and a decreased Leu 3a to Leu 2a ratio. Reactivity With HNK-l Monoclonal Antibody Recently, a monoclonal antibody was developed that reacts with medium-sized granular lymphocytes having NK- and K-cell function; this antibody has been designated HNK-1 (Leu 7) (7). The mean percentage of HNK-1-reactive PBM was signifiqmtly (p <.5) increased in 19 patients with ,. : 1. Normal Crohn's Figure 4. Ratio of Leu 3a+ to Leu 2a+ cells in peripheral blood of patients with Crahn's disease. The mean values represented by horizontal lines are not significantly statistically different.

6 June 1984 SUPPRESSOR CELLS IN CROHN'S DISEASE 1515 Crohn's disease (2.3% ± 3.3%) compared to that in 22 normal individuals (12.3% ± 1.7%). Because of this finding, the NK function of PBM was measured in patients with Crohn;s disease using Chang target cells in a 51Cr release assay. As shown in Figure 5, although cytotoxicity mediated by PBM from patients with Crohn's disease was higher than that in normal individuals at some effector cell/target cell ratios, these differences were not statistically significant. Furthermore, there was no significant correlation between the percentage of HNK-1 + cells in peripheral blood and lysis of Chang target cells.. Since a proportion of HNK"l + cells also express T cell antigens defined by monoclonal antibodies (14), we next considered the possibility that the increased proportion of HNK-1-bearing cells in patients with Crohn's disease was due to an increase in the population of cells reactive with both Leu 2a and HNK-1. As shown in Table 1, using dual-label studies, the proportion of Leu 2a-reactive cells that also reacted with HNK-1 was increased twofold in patients with Crohn's disease. In addition, there was a significant correlation (p <.5) between suppression of PWMstimulated immunoglobulin synthesis and the percentage of Leu 2a + HNK-1 + cells in peripheral blood (Figure 6). To further test the possibility that the increased suppressor activity mediated by cells from patients with Crohn's disease is mediated by cells bearing HNK-1, T-cell populations were depleted of HNK-1- Table 1. Reactivity of Peripheral Blood Mononuclear Cells With HNK-l and Leu 2a or Leu 3a % % % Subjects HNK-l+ Leu-2+HNK-i + Leu-3+HNK-l T Crohn's disease 24.9 ± ± ± 1.3 tn = 15) Normal 11.5 ± ± ±.6 (n = 15) Peripheral blood mononuclear cells were labeled with fluorescein isothiocyanate conjugates of Leu 2a, Leu 3a, and HNK-l or sequentially with Leu 2a or Leu 3a followed by HNK-l. The percentage of Leu 2a+ or Leu 3a+ cells reactive with HNK-l was calculated as indicated in Methods. Values are mean ± 1 SE. reactive cells by complement-mediated lysis and then added to indicator cultures as described above. As shown in Table 2, lysis of HNK-1-reactive cells from patients with Crohn's disease r'esulted in a loss of the capacity to inhiditpwm-stirimlated immunoglobulin synthesis. In addition, separate T-cell pbpulations from patients with Crohn's disease Were depleted of either OKT 8-reactlve cells or HNK-1~ reactive cells by complement-mediated lysis, and reconstituted mixtures of the two cell populations were added to indicator cultures capable of immunoglobulin synthesis. It was found that the reconstituted T-cell mixtures did not suppress immunoglobulin in synthesis (Table 3], and, therefore, it is likely that suppression is mediated.by bells reactive with both Leu 2a and HNK-1, rather than by one of two separate interacting cell populations. In summary, 5 CJ) ~ 4 -J S:2 u.. 3 U w C;. CJ) f- 2 Z w U a:. w 1 a.. SPONTANEOUS CYTOTOXICITY AGAINST CHANG TARGET CELLS o Crohn's I2l Normal 1 8 z Vi III w 6 a: a. a. => III t- Z w 4 u a: w a EFFECTOR/T ARGET RATIO Figure 5. Natural killer cell activity in patients with Crohn's disease. The cytotoxic activity, indicated as percentage of specific lysis, of peripheral blood mononuclear cells against 5lCr-labeled Chang target cells in 2 normal individuals and 2 patients with Crohn's disease. Bars represent mean values ±1 SE. Mean values are not statistically different at any of the effector to target ratios studied. 5 Figure % LEU 2+. HNK-1 + Suppression of pokeweed mitogen-stimulated immunoglobulin synthesis as a function of Leu 2+HNK-l + cells in peripheral blood of patients with Crohn's disease (n = 15). The percentage of suppression was determined as described in Methods (p <.5 by the Spearman rank test).

7 1516 JAMES ET AL. GASTROENTEROLOGY Vol. 86, No.6 Table 2. Effect of Depletion of HNK-l + Cells on Suppression of Pokeweed Mitogen-Stimulated IgM Synthesis Cells added to normal B + Tx + PWM Normal T Crohn's disease T HNK-l-depleted normal T HNK-l-depleted Crohn's disease T IgM synthesis (ng/culture) 1,66 ± 2,141 1,937 ± ,656 ± 3,16 11,524 ± 3,79 PWM, pokeweed mitogen; IgM, immunoglobulin M. T cells from normal individuals (n = 7) or patients with Crohn's disease (n = 7) were either not treated or treated with HNK-l and complement as described in Methods. T cells were then added to cultures containing allogeneic normal B cells (5 x 1 5 ), allogeneic-irradiated (1 R) normal T cells (T*) (5 x 1 5 ), and PWM for 1 days. Values are mean IgM (nanograms per culture) in culture supernatants. the results reported above are consistent with the conclusion that the increased suppressor cell activity found in some patients with Crohn's disease is mediated by lymphocytes reactive with both Leu 2a and HNK-1 antibodies. Discussion We have previously demonstrated that many patients with Crohn's disease have excessive suppressor T-cell function in vitro (4). To further define the nature of the cells causing suppression, we first depleted populations of T cells by complementmediated lysis using well-characterized monoclonal antibodies. We found that the excessive suppressor cell function could be eliminated with the monoclonal antibody OKT 8. Next, to determine if the excessive suppressor T-cell activity was due to an increase in the proportion of T cells reactive with OKT 8, the percentage of T cells in peripheral blood reactive with OKT 8 (and Leu 2a) was measured. We found that the percentage of cells reactive with OKT 8 or Leu 2a was normal. In addition, the percentage of these cells did not correlate with the degree of suppression of immunoglobulin synthesis in individual patients. These findings indicate that, although suppression could be eliminated by depleting OKT 8-reactive T cells, an abnormality in the percentage of T cells reactive with OKT 8 (or Leu 2a) does not provide an explanation for the excessive suppressor T-cell activity in patients with Crohn's disease. Finally, although the Leu 3a to Leu 2a ratio was diminished in the subgroup of patients with increased suppressor activity, the mean ratio of cells in the "helper: su ppressor" subpopulations was normal in the whole group of 2 patients with Crohn's disease studied, and this ratio did not correlate with suppression of immunoglobulin synthesis. Because the number of OKT 8+ cells was unchanged despite an alteration in functional suppressor activity, it seemed plausible that lymphocytes from patients with Crohn's disease, defined by OKT 8 (or Leu 2a), actually contained two subpopulations of cells with a different capacity to express suppressor function. In normal individuals, a subset of Leu 2a+ HNK-1 + lymphocytes can efficiently suppress immunoglobulin synthesis in vitro, but they require immune complex activation and are distinct from a second subset of Leu 2a + suppressor lymphocytes that lack HNK-1 antigen expression (Leu 2a+HNK- 1-) (15). In the present study, the subpopulation of cells reactive with both Leu 2a and HNK-1 was increased in patients with Crohn's disease. In addition, there was a significant correiation between suppression of PWM-stimulated immunoglobulin synthesis in vitro and the percentage of both HNK- 1 + and HNK-1 +Leu 2a+ lymphocytes in peripheral blood. Furthermore, depletion of HNK-1 + cells from peripheral blood eliminated the excessive suppressor activity of patients. Finally, reconstituted mixtures of T cells from 2 patients with Crohn's disease depleted of both Leu 2a (OKT 8) or HNK-1 + cells did not suppress immunoglobulin synthesis, indicating that a single population of cells each expressing both antigens was necessary for suppression. These findings indicate that Leu 2a + T-cell populations from patients with Crohn's disease differed qualitatively from normal Leu 2a+ T-cell populations because the patients with Crohn's disease had an increased proportion of Leu 2a+HNK-1 + suppressor cells. These data do not prove that suppression by Leu 2+ HNK-1 + lymphocytes is the only subpopulation of cells that mediate suppressor function in Crohn's disease. As shown in Figure 6, 3 patients with excessive suppressor function did not have an increased percentage of Leu 2+HNK-1 + lymphocytes. Table 3. Effect of Coculture of OKT 8-Depleted and HNK-I-Depleted Cells Fram Crahn's Disease Patients on Suppression of Pokeweed Mitogen Stimulated IgM Synthesis Cells added to normal B + T* + PWM Normal T Patient T Patient OKT 8-depleted Patient HNK-l-depleted Patient OKT 8-depleted + HNK-l-depleted IgM synthesis (ng/culture) Experiment 1 Experiment 2 5,982 7, ,798 1,2 19,288 12,859 8,55 7,842 16,5 PWM, pokeweed mitogen; IgM, immunoglobulin M. T cells from 2 patients with Crohn's disease were treated with OKT 8 or HNK-l and complement as described in Methods. The 5 x 1 5 treated cells were added to normal allogeneic B cells, T*, and PWM as in Table 2.

8 June 1984 SUPPRESSOR CELLS IN CROHN'S DISEASE 1517 The results in these patients may be caused by variability in the test system or may indicate that in some patients other cell subpopulations, such as Leu 2+HNK-1- cells or Leu 3+ cells, may contribute to suppressor function. Further studies with isolated subpopulations of lymphocytes will be required to examine these possibilities. The finding that excessive suppressor activity in some patients is due to a particular subpopulation of T cells that are Leu 2a+HNK-1 +, may explain the disparity in results of different assays for suppressor cell function in Crahn's disease. For example, in several studies of patients with Crohn's disease, suppressor T-cell function was tested by activating lymphocytes with the lectin concanavalin A and then testing the capacity of the concanavalin A treated cells to suppress T-cell proliferation. Using this assay, we found that many patients with Crohn's disease have diminished suppressor T-cell activity (16-18); these results were in apparent contrast to the results of the present study, in which increased suppression of immunoglobulin synthesis was demonstrated. A possible explanation for this difference in results is that the two different assay systems measure the activity of different T-cell subsets. This view is supported by the observation that the percentage of Leu 2a +HNK-1 + cells is not increased after concanavalin A activation (James S, unpublished observations). Furthermore, treatment of concanavalin A-activated T cells with HNK-1 and complement did not diminish the ability of the cells to suppress immunoglobulin synthesis. In all, the suppressor T-cell function mediated by concanavalin A activated cells is not mediated by Leu 2a+HNK-1 + cells, and the increased suppressor T-cell function of Leu 2a+HNK-1 + T cells may not be detected in assays using a concanavalin A activation step. One abnormality identified by this study, which has not been addressed so far, is the finding that the mean percentage of OKT 4 + (or Leu 3a +) T cells is diminished in the patients with Crahn's disease studied here. One explanation of this abnormality is that OKT 4 + T cells are sequestered at sites of tissue injury and are, therefore, reduced in percentage in peripheral blood. Further studies of subpopulations of OKT 4 + T cells will be required to determine the cause of the diminished percentage of these T cells in the peripheral blood of patients with Crahn's disease. The finding of an increased percentage of cells in peripheral blood of patients with Crahn's disease reactive with HNK-1 is of interest with regard to studies of NK-cell function. Previous studies in normal individuals have shown that this antibody reacts predominantly with the subpopulation of medium-sized, granular lymphocytes that mediate NK activity (7). In addition, previous studies of NK function in Crohn's disease have indicated normal or diminished NK activity, which may be related to disease activity (19,2). In our patients who had mild or inactive Crahn's disease, NK functional activity was not significantly different from normal, although the percentage of cells reactive with HNK-1 was increased. This disparity between the number of NK cells, as determined by reactivity with HNK-1, and their functional activity may be due to functional heterogeneity of HNK-1 + cells (14). It is possible that a proportion of HNK-1 + cells from patients with Crohn's disease may not contribute to NK activity as measured in the cytotoxicity assays used. To further investigate the mechanism by which Leu 2a + HNK-1 + cells suppress immunoglobulin synthesis. and to determine their capacity to mediate NK activity, it will be necessary to carry' out studies of suppressor cell function and NK function using purified populations of these cells. One unique feature of the excessi ve su ppressor lymphocyte function demonstrated in some patients with Crohn's disease is that the abnormality is revealed only after the procedures to separate lymphocytes into T- and B-cell fractions. Thus. unlike patients with other diseases characterized by circulating suppressor T cells, such as common variable hypogammaglobulinemia (21), unseparated PBM in most patients with Crohn's disease synthesize normal quantities of immunoglobulin in PWM-stimulated cultures, and cocultures of normal PBM and PBM from patients with Crohn's disease do not indicate excessive suppressor T-cell function. On the basis of these observations, it was suggested that the increased suppressor function in patients with Crohn's disease was accompanied by an increase in the function of cells which blocked or opposed the function of suppressor T cells (4). This hypothesis is supported by studies of murine immune responses. in which it has been demonstrated that there exists a subpopulation of T cells (named "contrasuppressor" cells) that have the ability to block the function of suppressor T cells (22-24). Furthermore, there is evidence that such cells are present in the murine gastrointestinal tract (25). The existence of such cells in humans has not been proved to date. partly because of the inability to precisely identify those subpopulations of T cells that provide help and suppression. The demonstration that the increased suppressor activity of lymphocytes from patients with Crohn's disease is mediated by a particular subpopulation of lymphocytes reactive with Leu 2a and HNK-1, may prove useful in attempting to identify contrasuppressor T cells in humans. In summary, the studies reported here indicate that some patients with Crohn's disease have in-

9 1518 JAMES ET AL. GASTROENTEROLOGY Vol. 86, No.6 creased suppressor cell activity in peripheral blood, and that this increased activity correlates with the presence of a subpopulation of lymphocytes reactive with both Leu 2a and HNK-l antibodies. The significance of the alteration of this lymphocyte population to the pathogenesis of Crohn's disease may be revealed by further studies of the tissue lesions in this disease. It is possible that the increased proportion of these cells in peripheral blood is due to the activation or maturation of these cells in the gastrointestinal tract in Crohn's disease. Alternatively, the absence of such suppressor cells in the lesions, but an increased proportion in peripheral blood, may be indicative of a failure of these cells to migrate into the lesions, where they might play a role in suppressing the destructive inflammatory response. 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The Hague: Martinus Nijhoff, 1981: Victorino RMM, Hodgson HJF. Spontaneous suppressor cell function in inflammatory bowel disease. Clin Exp Immunol 1981;26: Auer 1, Ziemer E, Sommer H. Immune status in Crohn's disease. 1. Decreased in vitro natural killer cell activity in peripheral blood. Clin Exp ImmunoI198;42: Ginsburg CH, Dambrauskas JT, Ault KA, Falchuck ZM. Impaired natural killer cell activity in patients with inflammatory bowel disease: evidence for a qualitative defect. Gastroenterology 1983;85: Waldmami. TA, Durm M, Broder S, Blackman M, Blaese RM, Strober W. Role of suppressor T cells in pathogenesis of common variable hypogammaglobulinemia. Lancet 1974; ii: DeKruyff RH, Simonson BG, Siskind GW. Cellular interactions in immune regulation. Hapten specific suppression by non-t cells and T cell mediated reversal of suppression. J Exp Med 1981;154: Gershon RK, Eardley DD, Durum S, et al. Contrasuppression. A novel immunoregulatory activity. 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