Clin. exp. Immunol. (1991) 85, 498-503
ADONIS 000991049100267B
Human colonic intra-epithelial lymphocytes are suppressor cells P. HOANG, H. R. DALTON & D. P. JEWELL Gastroenterology Unit, Radcliffe Infirmary, Oxford, England
(Acceptedfor publication 24 April 1991)
SUMMARY Human colonic intra-epithelial lymphocytes (IEL) suppress the proliferation of autologous lamina propria lymphocytes, but not autologous peripheral blood mononuclear cells, when stimulated with phytohaemagglutinin. This suppressor function is mediated by a CD8-dependent soluble factor and is not related to the expression of the yb T cell receptor. These findings may be relevant to the induction of mucosal tolerance. However, there is no defect in suppressor activity of colonic IEL in inflammatory bowel disease. Keywords inflammatory bowel disease lamina propria lymphocytes CD8 lymphocytes lymphocytes
y6
INTRODUCTION Intra-epithelial lymphocytes (TEL) are the first immune cells to come in contact with luminal antigens. However, in humans their role remains to be defined (Brandtzaeg et al., 1989; Mowat, 1990). TEL are predominantly CD8+ cells, as shown in tissue section and in isolated populations (Greenwood, Austin & Dobbins, 1983; Selby et al., 1984; Cerf-Bensussan, Guy-Grand & Griscelli, 1985; Hirata et al., 1986; Ebert et al., 1986 and our
SUBJECTS AND METHODS
Subjects Surgical colonic resection specimens were obtained from 12 control subjects (10 with colonic carcinoma and two with colonic adenoma). These subjects had a median age of 70 5 years (range 37-83 years); eight were men and four women. In addition, colonic resection specimens were obtained from eight patients with irritable bowel disease (IBD) (six with ulcerative colitis and two with Crohn's disease). These patients had a median age of 28 5 years (range 19-61); four were men and four women. At the time of surgery all patients with IBD were receiving corticosteroids. The specimens from the IBD patients were taken from the least inflamed areas of tissue. Specimens from control subjects were taken at least 5 cm from any macroscopic lesion and were subsequently shown to be histologically normal.
unpublished results). In mice, TEL have been reported as having both cytotoxic and suppressor functions (Mowat et al., 1983). However, human functional studies have been hampered by the difficulties in isolating a pure population of TEL. No natural killer (NK) activity has been demonstrated (Cerf-Bensussan et al., 1985). TEL have been shown to possess immunoregulatory activity when added to peripheral blood mononuclear cells (PBMC) in a pokeweed mitogen (PWM)/immunoglobulin system (Greenwood et al., 1983). Low numbers of TEL helped immunoglobulin synthesis. Increasing the numbers of TEL abrogated this effect and caused suppression of immunoglobulin synthesis. However, other investigators (Smart et al., 1988) reported that TEL have no effect on immunoglobulin synthesis. Ebert et al. (1986) showed that small intestinal TEL do not suppress autologous PBMC, and, more recently, that TEL suppress allogeneic PBMC (Ebert, 1990). Both these studies used mitogen-driven proliferation assays. The aim of this study was to examine the putative suppressor function of human colonic TEL on proliferation of autologous lamina propria lymphocytes (LPL) and test the hypothesis that inflammatory bowel disease is associated with a functional defect in TEL.
Lymphocyte isolation Mucosal lymphocytes. To isolate colonic IEL a modification of a method previously described for the small intestine was used (Ebert et al., 1986). Immediately after resection, the specimen was washed with HBSS without calcium and magnesium (Flow, Irvine, UK) containing antibiotics (gentamycin 50 Mg/ml, penicillin 100 U/ml, streptomycin 100 pg/ml). The mucosa was carefully dissected. After further washing, the mucosal strips were immersed in RPMI 1640 (GIBcO, Paisley, UK) supplemented with 10% fetal calf serum (FCS), antibiotics as above, and 1 mmol dithiothreitol (Sigma, St Louis, MO) for 10 min. The specimen was washed again three times with HBSS, supplemented as above. It was then incubated with 0 75 mmol EDTA in a shaking water bath at 37°C and 140 oscillations/min for 30 min and the supernatant recovered. After two incubations with 0-75 mmol EDTA (the supernatant being collected on each occasion), the supernatant was centrifuged at 500 g and washed
Correspondence: Dr D. P. Jewell, Gastroenterology Unit, Radcliffe Infirmary, Woodstock Road, Oxford, OX2 6HE, UK.
498
Colonic IEL are suppressor cells in RPMI 1640 three times. The pellet was resuspended in RPMI 1640 and stored overnight at 40C, in order to achieve an optimum cell separation (Ebert et al., 1986). Further purification was achieved by passing this crude preparation down a glass wool column (Sigma) and performing a two-step Percoll gradient of 44% and 67-5% at 800 g, as previously described (Cerf-Bensussan et al., 1987). The IEL were recovered from the interface. The LPL were isolated from the same resection specimens using a modification of an established technique (Bull & Bookman, 1977). Briefly, the mucosal strips were washed in 5 mmol EDTA in a shaking water bath at 37 C for 40 min. After washing with HBSS, the mucosal strips were minced into fine pieces and then incubated overnight in a shaking water bath at 37 C with 100 ml RPMI 1640 and 10% FCS plus 30 mg Clostridium histolyticum collagenase (Boehringer Mannheim, Mannheim, Germany. Following vigorous agitation the digestate was passed through a 100 pm nylon mesh filter (Lockertex, Warrington, UK) and washed three times in HBSS. The LPL were recovered after centrifugation at 500 g over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient, after which they were washed in RPMI 1640 and 10% FCS three times and resuspended to a concentration of I x 106 cells/ml. PBMC. Heparinized venous blood was obtained from two control patients. PBMC were isolated over a Ficoll-Paque gradient, washed and suspended in RPMI 1640 medium to a concentration of I x 106 cells/ml.
Phenotypic analysis The purity of the IEL population was assessed by the CD4/CD8 ratio (Simultest helper/suppressor (CD4/CD8) monoclonal antibody, Becton Dickinson, Sunnyvale, CA) and flow cytometry (FACScan, Becton Dickinson) (Senju et al., 1991). Functional assays LPL (5 x 104) were cultured with or without differing numbers of IEL (2-5 x 104-1 x 105), together with a range of concentrations of phytohaemagglutinin (PHA) (5-10 Mg/ml) in 96-well Ushaped plates in a final volume of 160 p1 at 37°C with 5% CO2. The cultures were performed for varying lengths of time (3-10 days). All experiments were performed in triplicate. Sixteen hours before the end of the culture the cells were pulsed with 1 pCi 3H-methyl thymidine (Amersham Laboratories, Amersham, UK). At the end of the culture the cells were harvested on to filter paper discs using a semi-automated cell harvester (Skatron, Lier, Norway), suspended in 10 ml scintillation fluid (Optiphase Safe; LKB, Poole, UK) and counted in a scintillation counter (1215 Rackbeta scintillation counter, LKB). In the proliferation assays, the triplicate values varied by < 10% from the median value in > 85% of assays. Two experiments using 5 x 104 autologous PBMC (instead of LPL) were performed. In other experiments, I x 105 irradiated PBMC (25 Gy) and 1 x 105 colonic epithelial cells (depleted of any contaminating CD8 + IEL) were used instead of IEL. The colonic epithelial cells were isolated from control colonic resection specimens using a method described previously (Mayer & Shlein, 1987). These experiments were performed in order to exclude potential effects due to cell crowding and epithelial cell contamination respectively.
499
Lymphocyte subpopulation experiments In three experiments yb+ and yb- subpopulations of IEL were isolated. This was achieved by a magnetic depletion technique. Dynabeads coated with goat anti-mouse IgG (Dynabeads M450, Dynal, Oslo, Norway) were incubated with a murine monoclonal antibody to the 6 chain of human yb T cell receptor (T Cell Sciences, Cambridge, MA). After washing, IEL were incubated with the labelled beads for 45 min at 4°C and yb + cells were removed by applying a magnetic field. The y6 + subpopulation, which comprised a median of 21% (range 16-34%) of the total IEL population, was recovered attached to the magnetic beads, leaving the yb- population in the supernatant. This procedure was repeated and adequate purity of the yb+ and y6IEL was confirmed by staining with the above monoclonal antibody and analysis by flow cytometry (> 90% yb+ and yb-, respectively). Each population of cells was washed in RPMI 1640 and resuspended to a concentration of I x 106 cells/ml. Using magnetic beads pre-coated with human CD8 monoclonal antibody (Dynal) and similar methodology as described for the y6 populations, CD8+ and CD8- IEL populations were isolated. Functional assays were performed with 1 x 105 ybIEL, yb+ IEL, CD8+ or CD8- IEL using LPL and PHA, as described above. Supernatant experiments Several experiments were performed to assess the effect of IEL supernatant on the proliferative response of LPL stimulated with PHA. Aliquots (1 ml) of I x 106 IEL/ml were cultured alone in 24-well plates at 37°C for 3 days. The supernatant was harvested, centrifuged, filtered and stored at -20°C until required. Allogeneic LPL (5 x 104) were cultured with 10 pg/ml PHA with and without 50-pl aliquots of IEL supernatant in a final volume of 160 pl. The proliferative response was then assayed at day 6, as described previously. Ethics Approval for the study was given by the Central Oxford Research and Ethics Committee. Statistical analysis The data were analysed by the Mann-Whitney U-test where appropriate. P< 0-05 was considered significant. RESULTS Mucosal lymphocyte populations The median yield of IEL from control tissue was 5-25 x 105 cells/ g tissue (range 3-33-9-2 x 105) and from IBD patients 4-5 x 105 cells/g (range 2-25-6-0 x 105). The viability was always greater than 95%. The median CD4/CD8 ratio of IEL in controls was 0-22 (range 0 08-0-32) and in IBD patients 0-22 (range 0-030 40). The IEL populations contained > 85% lymphocytes in all experiments. After 6 days of culture the median proliferative response of IEL alone in controls was 159 d/min (range 72-200) and of IEL plus 10 pg/ml PHA, 900 d/min (range 119-3500). In IBD the median rate of proliferation of IEL alone was 187 d/min (range 153-251) and TEL plus 10 pg/ml PHA, 572 d/min (range 34310 550).
500
P. Hoang, H. R. Dalton & D. P. Jewell 100
Table 1. Proliferative response of 5 x 104 LPL stimulated with 10 pg/ml PHA after 6 days culture (d/min)
r
x
80 F0
0
0s
60 FBE Q-
Comparison of the effect of 1 x 105 autologous IEL and irradiated (25 Gy) PBMC
I
x 105
LPL+PHA
LPL+PHA+IEL
LPL+PHA+PBMC
85 747
58 162
121 945 (autologous PBMC) 125 698 (allogeneic PBMC)
*0
40 FeC
>I
c
20 F-
Comparison of the effect of I x 105 autologous IEL and differing numbers (parentheses) of autologous epithelial cells (EC)
0
LPL+ PHA
LPL+PHA +IEL
Fig. 1. A typical experiment which shows the effect of varying cell ratios on the suppressor function of IEL. The incorporation of 3H-thymidine by 5 x 104 LPL stimulated with 10 pg/ml PHA measured after 6 days culture with autologous IEL, using IEL/LPL ratios of 1:2 (0); 1: 1 (A&); and 2:1 (0).
to
140
x
~0
120
C)
100 (n 0
CL C) a)
a)
80
60 40
a-
20 0
LPL PHA
LPL+ PHA+IEL
Fig. 2. Suppressor function ofcontrol IEL (12 experiments). Autologous IEL (I x 105) produced a significant reduction in the proliferative responses of 5 x 104 LPL stimulated with 10 pg/ml PHA after 6 days of culture (P < 0 01).
IEL function in controls
Preliminary experiments were performed to assess the optimum period of culture. The IEL exhibited no suppressor activity before day 3, and after day 9 (although still present) the d/min tailed off, making accurate interpretation difficult. Therefore, the suppressor activity of IEL was assayed at day 6. Further experiments were performed to investigate the effect of differing doses of PHA on the suppressor function of IEL. IEL significantly suppressed the response of LPL to PHA at concentrations of 5, 7 5 (data not shown) and 10 yg/ml. Furthermore, in several experiments (n=6) IEL suppressed the proliferative response of LPL over a range of IEL/LPL cell ratios (2: 1 to 1: 2) in a dose-dependent fashion (Fig. 1). After 6 days of culture, in 11 out of 12 control subjects IEL suppressed the proliferative response to 10 yg/ml PHA by more than 40% using a ratio of IEL/LPL of 2: 1. The median d/min of LPL and PHA alone was 73 942 (range 11 267-134 990). With the addition of IEL this fell to 19 933 (range 2717-85 231). This was significant at the P < 0-01 level (Fig. 2). The median viability of the IEL/LPL cell mixture at day 0 was 95oo (range 94-98%),
LPL+PHA
LPL+PHA+IEL
LPL+PHA+EC
68 606
20601
94261 (I x 105EC) 93447 (3 x 104EC)
and at day 6, 68% (range 54-72%). The viability of LPL and IEL cultured alone for 6 days was similar. To exclude the possibility that the suppressor effect of IEL was due to contamination of the IEL by epithelial cells, two experiments were performed to assess the putative suppressor function of autologous colonic epithelial cells (depleted of contaminating CD8 + IEL) on the proliferative response of LPL. Epithelial cells showed no suppressor activity, and in fact enhanced the proliferative response of LPL (Table 1). Two experiments were performed using a population of PBMC irradiated with 25 Gy (instead of IEL). Irradiated PBMC showed no suppressive effect on the uptake of 3H-methyl thymidine of LPL stimulated with PHA (Table 1), indicating that the suppressor activity of IEL was not due simply to cell crowding. In two other experiments, the effect of IEL on the proliferation of autologous PBMC was compared with their effect on autologous LPL. In both experiments IEL showed significant suppressor activity against LPL, but did not alter the 3Hthymidine uptake of autologous PBMC (Table 2). This is additional evidence that the suppressor effect is unlikely to be due to cell crowding or the toxic effects of cell death. A positively selected CD8+ IEL population ( > 95% CD8+) showed suppressor activity on autologous LPL proliferation. In contrast, a CD8- IEL population (> 95O CD8-) showed no suppressor activity (Table 3). However, both yb+ and ybdepleted IEL exert a suppressive influence on autologous LPL (Table 4). These data indicate that the suppressor effect observed is CD8-dependent. Furthermore, as the y6 + and CD8 + populations were positively selected (and therefore contained no colonic epithelial cells), this effect is not due simply to epithelial cell contamination. The suppressor activity of IEL is mediated by a soluble factor as supernatant from unstimulated IEL suppressed the 3H-thymidine uptake of allogeneic LPL (Table 5). IEL function in IBD The suppressor activity of IEL in patients with IBD was investigated in a similar manner to that described for control patients, using the proliferative response of autologous LPL stimulated with 10 pg/ml PHA after 6 days of culture. No
Colonic IEL are suppressor cells
501
Table 2. Proliferative responses of 5 x 104 LPL to 10 ,g/ml PHA with and without I x 105 autologous IEL, compared with the response of 5 x 104 autologous PBMC to PHA with and without 1 x 105 IEL after 6 days of culture (d/min) LPL + PHA LPL + PHA +IEL PBMC+ PHA PBMC+ PHA + IEL
Experiment 1 Experiment 2
88 122 68606
36827 20601
70281 15378
72023 20311
Table 3. The effect of I x 105 undepleted IEL (IEL), CD8-depleted IEL (CD8- IEL) and positively selected CD8+ IEL (CD8+ IEL) on the proliferation of 5 x 104 autologous LPL after stimulation with 10 g/ml PHA and 6 days of culture (d/min)
LPL+PHA
LPL+PHA+IEL
LPL+PHA+CD8+ IEL
LPL+PHA+CD8- IEL
85747 58 336
58 162 21 667
14739 23 120
98049
Experiment 1 Experiment 2
Table 4. Effect of I x 105 undepleted IEL (IEL), yb-depleted IEL (IELyb-) and positively selected y6+ IEL (IEL yb+) on the proliferation of 5 x 104 autologous LPL after stimulation with 10 jg/ml PHA and 6 days of culture (d/min)
Experiment 1 Experiment 2 Experiment 3
LPL+ PHA
LPL+ PHA + IEL
LPL + PHA+ IEL(y -)
LPL + PHA + IEL(y6+)
78737 98376 14389
43697 19265 4982
59474 20415 7009
6999 5828
Table 5. Effect of 50 pl IEL supernatant on the proliferative response of 5 x 104 LPL stimulated with 10 pg/mT PHA after 6 days of culture (d/min). Supernatant from unstimulated LPL harvested at day 3 was used as a control.
Experiment 1 Experiment 2 Experiment 3
LPL + PHA
LPL+ PHA + IEL supernatant
LPL + PHA + LPL supernatant
58336 14398 141219
44397 3768 50 380
166447
significant difference was found between patients with IBD and controls subjects. In IBD the median d/min of LPL plus 10 yg/ ml PHA was 80034 (range 23 560-199 439). With the addition of TEL this fell significantly (P
ADONIS 000991049100267B
Human colonic intra-epithelial lymphocytes are suppressor cells P. HOANG, H. R. DALTON & D. P. JEWELL Gastroenterology Unit, Radcliffe Infirmary, Oxford, England
(Acceptedfor publication 24 April 1991)
SUMMARY Human colonic intra-epithelial lymphocytes (IEL) suppress the proliferation of autologous lamina propria lymphocytes, but not autologous peripheral blood mononuclear cells, when stimulated with phytohaemagglutinin. This suppressor function is mediated by a CD8-dependent soluble factor and is not related to the expression of the yb T cell receptor. These findings may be relevant to the induction of mucosal tolerance. However, there is no defect in suppressor activity of colonic IEL in inflammatory bowel disease. Keywords inflammatory bowel disease lamina propria lymphocytes CD8 lymphocytes lymphocytes
y6
INTRODUCTION Intra-epithelial lymphocytes (TEL) are the first immune cells to come in contact with luminal antigens. However, in humans their role remains to be defined (Brandtzaeg et al., 1989; Mowat, 1990). TEL are predominantly CD8+ cells, as shown in tissue section and in isolated populations (Greenwood, Austin & Dobbins, 1983; Selby et al., 1984; Cerf-Bensussan, Guy-Grand & Griscelli, 1985; Hirata et al., 1986; Ebert et al., 1986 and our
SUBJECTS AND METHODS
Subjects Surgical colonic resection specimens were obtained from 12 control subjects (10 with colonic carcinoma and two with colonic adenoma). These subjects had a median age of 70 5 years (range 37-83 years); eight were men and four women. In addition, colonic resection specimens were obtained from eight patients with irritable bowel disease (IBD) (six with ulcerative colitis and two with Crohn's disease). These patients had a median age of 28 5 years (range 19-61); four were men and four women. At the time of surgery all patients with IBD were receiving corticosteroids. The specimens from the IBD patients were taken from the least inflamed areas of tissue. Specimens from control subjects were taken at least 5 cm from any macroscopic lesion and were subsequently shown to be histologically normal.
unpublished results). In mice, TEL have been reported as having both cytotoxic and suppressor functions (Mowat et al., 1983). However, human functional studies have been hampered by the difficulties in isolating a pure population of TEL. No natural killer (NK) activity has been demonstrated (Cerf-Bensussan et al., 1985). TEL have been shown to possess immunoregulatory activity when added to peripheral blood mononuclear cells (PBMC) in a pokeweed mitogen (PWM)/immunoglobulin system (Greenwood et al., 1983). Low numbers of TEL helped immunoglobulin synthesis. Increasing the numbers of TEL abrogated this effect and caused suppression of immunoglobulin synthesis. However, other investigators (Smart et al., 1988) reported that TEL have no effect on immunoglobulin synthesis. Ebert et al. (1986) showed that small intestinal TEL do not suppress autologous PBMC, and, more recently, that TEL suppress allogeneic PBMC (Ebert, 1990). Both these studies used mitogen-driven proliferation assays. The aim of this study was to examine the putative suppressor function of human colonic TEL on proliferation of autologous lamina propria lymphocytes (LPL) and test the hypothesis that inflammatory bowel disease is associated with a functional defect in TEL.
Lymphocyte isolation Mucosal lymphocytes. To isolate colonic IEL a modification of a method previously described for the small intestine was used (Ebert et al., 1986). Immediately after resection, the specimen was washed with HBSS without calcium and magnesium (Flow, Irvine, UK) containing antibiotics (gentamycin 50 Mg/ml, penicillin 100 U/ml, streptomycin 100 pg/ml). The mucosa was carefully dissected. After further washing, the mucosal strips were immersed in RPMI 1640 (GIBcO, Paisley, UK) supplemented with 10% fetal calf serum (FCS), antibiotics as above, and 1 mmol dithiothreitol (Sigma, St Louis, MO) for 10 min. The specimen was washed again three times with HBSS, supplemented as above. It was then incubated with 0 75 mmol EDTA in a shaking water bath at 37°C and 140 oscillations/min for 30 min and the supernatant recovered. After two incubations with 0-75 mmol EDTA (the supernatant being collected on each occasion), the supernatant was centrifuged at 500 g and washed
Correspondence: Dr D. P. Jewell, Gastroenterology Unit, Radcliffe Infirmary, Woodstock Road, Oxford, OX2 6HE, UK.
498
Colonic IEL are suppressor cells in RPMI 1640 three times. The pellet was resuspended in RPMI 1640 and stored overnight at 40C, in order to achieve an optimum cell separation (Ebert et al., 1986). Further purification was achieved by passing this crude preparation down a glass wool column (Sigma) and performing a two-step Percoll gradient of 44% and 67-5% at 800 g, as previously described (Cerf-Bensussan et al., 1987). The IEL were recovered from the interface. The LPL were isolated from the same resection specimens using a modification of an established technique (Bull & Bookman, 1977). Briefly, the mucosal strips were washed in 5 mmol EDTA in a shaking water bath at 37 C for 40 min. After washing with HBSS, the mucosal strips were minced into fine pieces and then incubated overnight in a shaking water bath at 37 C with 100 ml RPMI 1640 and 10% FCS plus 30 mg Clostridium histolyticum collagenase (Boehringer Mannheim, Mannheim, Germany. Following vigorous agitation the digestate was passed through a 100 pm nylon mesh filter (Lockertex, Warrington, UK) and washed three times in HBSS. The LPL were recovered after centrifugation at 500 g over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) gradient, after which they were washed in RPMI 1640 and 10% FCS three times and resuspended to a concentration of I x 106 cells/ml. PBMC. Heparinized venous blood was obtained from two control patients. PBMC were isolated over a Ficoll-Paque gradient, washed and suspended in RPMI 1640 medium to a concentration of I x 106 cells/ml.
Phenotypic analysis The purity of the IEL population was assessed by the CD4/CD8 ratio (Simultest helper/suppressor (CD4/CD8) monoclonal antibody, Becton Dickinson, Sunnyvale, CA) and flow cytometry (FACScan, Becton Dickinson) (Senju et al., 1991). Functional assays LPL (5 x 104) were cultured with or without differing numbers of IEL (2-5 x 104-1 x 105), together with a range of concentrations of phytohaemagglutinin (PHA) (5-10 Mg/ml) in 96-well Ushaped plates in a final volume of 160 p1 at 37°C with 5% CO2. The cultures were performed for varying lengths of time (3-10 days). All experiments were performed in triplicate. Sixteen hours before the end of the culture the cells were pulsed with 1 pCi 3H-methyl thymidine (Amersham Laboratories, Amersham, UK). At the end of the culture the cells were harvested on to filter paper discs using a semi-automated cell harvester (Skatron, Lier, Norway), suspended in 10 ml scintillation fluid (Optiphase Safe; LKB, Poole, UK) and counted in a scintillation counter (1215 Rackbeta scintillation counter, LKB). In the proliferation assays, the triplicate values varied by < 10% from the median value in > 85% of assays. Two experiments using 5 x 104 autologous PBMC (instead of LPL) were performed. In other experiments, I x 105 irradiated PBMC (25 Gy) and 1 x 105 colonic epithelial cells (depleted of any contaminating CD8 + IEL) were used instead of IEL. The colonic epithelial cells were isolated from control colonic resection specimens using a method described previously (Mayer & Shlein, 1987). These experiments were performed in order to exclude potential effects due to cell crowding and epithelial cell contamination respectively.
499
Lymphocyte subpopulation experiments In three experiments yb+ and yb- subpopulations of IEL were isolated. This was achieved by a magnetic depletion technique. Dynabeads coated with goat anti-mouse IgG (Dynabeads M450, Dynal, Oslo, Norway) were incubated with a murine monoclonal antibody to the 6 chain of human yb T cell receptor (T Cell Sciences, Cambridge, MA). After washing, IEL were incubated with the labelled beads for 45 min at 4°C and yb + cells were removed by applying a magnetic field. The y6 + subpopulation, which comprised a median of 21% (range 16-34%) of the total IEL population, was recovered attached to the magnetic beads, leaving the yb- population in the supernatant. This procedure was repeated and adequate purity of the yb+ and y6IEL was confirmed by staining with the above monoclonal antibody and analysis by flow cytometry (> 90% yb+ and yb-, respectively). Each population of cells was washed in RPMI 1640 and resuspended to a concentration of I x 106 cells/ml. Using magnetic beads pre-coated with human CD8 monoclonal antibody (Dynal) and similar methodology as described for the y6 populations, CD8+ and CD8- IEL populations were isolated. Functional assays were performed with 1 x 105 ybIEL, yb+ IEL, CD8+ or CD8- IEL using LPL and PHA, as described above. Supernatant experiments Several experiments were performed to assess the effect of IEL supernatant on the proliferative response of LPL stimulated with PHA. Aliquots (1 ml) of I x 106 IEL/ml were cultured alone in 24-well plates at 37°C for 3 days. The supernatant was harvested, centrifuged, filtered and stored at -20°C until required. Allogeneic LPL (5 x 104) were cultured with 10 pg/ml PHA with and without 50-pl aliquots of IEL supernatant in a final volume of 160 pl. The proliferative response was then assayed at day 6, as described previously. Ethics Approval for the study was given by the Central Oxford Research and Ethics Committee. Statistical analysis The data were analysed by the Mann-Whitney U-test where appropriate. P< 0-05 was considered significant. RESULTS Mucosal lymphocyte populations The median yield of IEL from control tissue was 5-25 x 105 cells/ g tissue (range 3-33-9-2 x 105) and from IBD patients 4-5 x 105 cells/g (range 2-25-6-0 x 105). The viability was always greater than 95%. The median CD4/CD8 ratio of IEL in controls was 0-22 (range 0 08-0-32) and in IBD patients 0-22 (range 0-030 40). The IEL populations contained > 85% lymphocytes in all experiments. After 6 days of culture the median proliferative response of IEL alone in controls was 159 d/min (range 72-200) and of IEL plus 10 pg/ml PHA, 900 d/min (range 119-3500). In IBD the median rate of proliferation of IEL alone was 187 d/min (range 153-251) and TEL plus 10 pg/ml PHA, 572 d/min (range 34310 550).
500
P. Hoang, H. R. Dalton & D. P. Jewell 100
Table 1. Proliferative response of 5 x 104 LPL stimulated with 10 pg/ml PHA after 6 days culture (d/min)
r
x
80 F0
0
0s
60 FBE Q-
Comparison of the effect of 1 x 105 autologous IEL and irradiated (25 Gy) PBMC
I
x 105
LPL+PHA
LPL+PHA+IEL
LPL+PHA+PBMC
85 747
58 162
121 945 (autologous PBMC) 125 698 (allogeneic PBMC)
*0
40 FeC
>I
c
20 F-
Comparison of the effect of I x 105 autologous IEL and differing numbers (parentheses) of autologous epithelial cells (EC)
0
LPL+ PHA
LPL+PHA +IEL
Fig. 1. A typical experiment which shows the effect of varying cell ratios on the suppressor function of IEL. The incorporation of 3H-thymidine by 5 x 104 LPL stimulated with 10 pg/ml PHA measured after 6 days culture with autologous IEL, using IEL/LPL ratios of 1:2 (0); 1: 1 (A&); and 2:1 (0).
to
140
x
~0
120
C)
100 (n 0
CL C) a)
a)
80
60 40
a-
20 0
LPL PHA
LPL+ PHA+IEL
Fig. 2. Suppressor function ofcontrol IEL (12 experiments). Autologous IEL (I x 105) produced a significant reduction in the proliferative responses of 5 x 104 LPL stimulated with 10 pg/ml PHA after 6 days of culture (P < 0 01).
IEL function in controls
Preliminary experiments were performed to assess the optimum period of culture. The IEL exhibited no suppressor activity before day 3, and after day 9 (although still present) the d/min tailed off, making accurate interpretation difficult. Therefore, the suppressor activity of IEL was assayed at day 6. Further experiments were performed to investigate the effect of differing doses of PHA on the suppressor function of IEL. IEL significantly suppressed the response of LPL to PHA at concentrations of 5, 7 5 (data not shown) and 10 yg/ml. Furthermore, in several experiments (n=6) IEL suppressed the proliferative response of LPL over a range of IEL/LPL cell ratios (2: 1 to 1: 2) in a dose-dependent fashion (Fig. 1). After 6 days of culture, in 11 out of 12 control subjects IEL suppressed the proliferative response to 10 yg/ml PHA by more than 40% using a ratio of IEL/LPL of 2: 1. The median d/min of LPL and PHA alone was 73 942 (range 11 267-134 990). With the addition of IEL this fell to 19 933 (range 2717-85 231). This was significant at the P < 0-01 level (Fig. 2). The median viability of the IEL/LPL cell mixture at day 0 was 95oo (range 94-98%),
LPL+PHA
LPL+PHA+IEL
LPL+PHA+EC
68 606
20601
94261 (I x 105EC) 93447 (3 x 104EC)
and at day 6, 68% (range 54-72%). The viability of LPL and IEL cultured alone for 6 days was similar. To exclude the possibility that the suppressor effect of IEL was due to contamination of the IEL by epithelial cells, two experiments were performed to assess the putative suppressor function of autologous colonic epithelial cells (depleted of contaminating CD8 + IEL) on the proliferative response of LPL. Epithelial cells showed no suppressor activity, and in fact enhanced the proliferative response of LPL (Table 1). Two experiments were performed using a population of PBMC irradiated with 25 Gy (instead of IEL). Irradiated PBMC showed no suppressive effect on the uptake of 3H-methyl thymidine of LPL stimulated with PHA (Table 1), indicating that the suppressor activity of IEL was not due simply to cell crowding. In two other experiments, the effect of IEL on the proliferation of autologous PBMC was compared with their effect on autologous LPL. In both experiments IEL showed significant suppressor activity against LPL, but did not alter the 3Hthymidine uptake of autologous PBMC (Table 2). This is additional evidence that the suppressor effect is unlikely to be due to cell crowding or the toxic effects of cell death. A positively selected CD8+ IEL population ( > 95% CD8+) showed suppressor activity on autologous LPL proliferation. In contrast, a CD8- IEL population (> 95O CD8-) showed no suppressor activity (Table 3). However, both yb+ and ybdepleted IEL exert a suppressive influence on autologous LPL (Table 4). These data indicate that the suppressor effect observed is CD8-dependent. Furthermore, as the y6 + and CD8 + populations were positively selected (and therefore contained no colonic epithelial cells), this effect is not due simply to epithelial cell contamination. The suppressor activity of IEL is mediated by a soluble factor as supernatant from unstimulated IEL suppressed the 3H-thymidine uptake of allogeneic LPL (Table 5). IEL function in IBD The suppressor activity of IEL in patients with IBD was investigated in a similar manner to that described for control patients, using the proliferative response of autologous LPL stimulated with 10 pg/ml PHA after 6 days of culture. No
Colonic IEL are suppressor cells
501
Table 2. Proliferative responses of 5 x 104 LPL to 10 ,g/ml PHA with and without I x 105 autologous IEL, compared with the response of 5 x 104 autologous PBMC to PHA with and without 1 x 105 IEL after 6 days of culture (d/min) LPL + PHA LPL + PHA +IEL PBMC+ PHA PBMC+ PHA + IEL
Experiment 1 Experiment 2
88 122 68606
36827 20601
70281 15378
72023 20311
Table 3. The effect of I x 105 undepleted IEL (IEL), CD8-depleted IEL (CD8- IEL) and positively selected CD8+ IEL (CD8+ IEL) on the proliferation of 5 x 104 autologous LPL after stimulation with 10 g/ml PHA and 6 days of culture (d/min)
LPL+PHA
LPL+PHA+IEL
LPL+PHA+CD8+ IEL
LPL+PHA+CD8- IEL
85747 58 336
58 162 21 667
14739 23 120
98049
Experiment 1 Experiment 2
Table 4. Effect of I x 105 undepleted IEL (IEL), yb-depleted IEL (IELyb-) and positively selected y6+ IEL (IEL yb+) on the proliferation of 5 x 104 autologous LPL after stimulation with 10 jg/ml PHA and 6 days of culture (d/min)
Experiment 1 Experiment 2 Experiment 3
LPL+ PHA
LPL+ PHA + IEL
LPL + PHA+ IEL(y -)
LPL + PHA + IEL(y6+)
78737 98376 14389
43697 19265 4982
59474 20415 7009
6999 5828
Table 5. Effect of 50 pl IEL supernatant on the proliferative response of 5 x 104 LPL stimulated with 10 pg/mT PHA after 6 days of culture (d/min). Supernatant from unstimulated LPL harvested at day 3 was used as a control.
Experiment 1 Experiment 2 Experiment 3
LPL + PHA
LPL+ PHA + IEL supernatant
LPL + PHA + LPL supernatant
58336 14398 141219
44397 3768 50 380
166447
significant difference was found between patients with IBD and controls subjects. In IBD the median d/min of LPL plus 10 yg/ ml PHA was 80034 (range 23 560-199 439). With the addition of TEL this fell significantly (P
