CLINICAL
IMML’NOLOCY
AND
IMMUNOP.XfHOLOGY
11,
1255130 (1978)
Suppressor Activity of Lymphocytes from Patients with Systemic Lupus Erythematosus M. Laboratory
TARDIEU’
AND
of Immunology. Institut National Me’dicale. U 56. HiSpita d’Enfants.
J. M. DUPUY de lu SantP et de la Recherche BicPtre. 94270. France
Received December 28. 1977 Lymphocytes from 16 patients with SLE disease were studied for their ability to proliferate and to induce stimulation of normal control ailogeneic lymphocytes. In 14 patients, the PHA response was normal. A low proliferative response, however, was shown in MLC when patients lymphocytes were cultured with irradiated control cells. The immunological response of two other SLE patients displayed quite a different pattern. These two patients exhibited negative delayed hypersensitivity skin tests and had a low proliferative response of lymphocytes in PHA cultures and in a one-way as well as in a two-way MLC. The absence of reactivity was not due to a lack of responder cells but to the presence of a radio sensitive suppressor activity. In these two patients, however, the relationship between abnormal cell-mediated functions and SLE disease is unclear and may be circumstantial.
INTRODUCTION
Impairment of cell-mediated immunity is thought to play an important role in the pathogenesis of systemic lupus erythematosus (SLE) (1, 2). Previous studies of cell-mediated immunity in patients with SLE displayed, however, conflicting results (3-6). We report herein results obtained in 16 patients with SLE disease in whom immunological studies revealed a deficiency in cell-mediated immunity of a variable intensity. In two of them, the lymphocyte populations exerted a radiosensitive inhibitory activity detected in mixed leukocyte culture. PATIENTS AND METHODS Patient and control populations. Sixteen patients, aged 8 to 26 years, with systemic lupus erythematosus (SLE) were available for study. Patient selection was based upon the fulfillment of criteria of the Rheumatism Association for classification as SLE (7). Activity of the disease was defined by the association of acute clinical manifestations, erythrocyte sedimentation rate (ESR) above 30 mm/hr, serum C4 level below 30 mg/dl, and anti-DNA binding over 25% according to the Farr assay (8). Eight patients had active disease and eight had inactive disease. Five patients of the former group were studied before treatment and the three others just after the onset of therapy. The 11 treated patients received steroid therapy (2 mg/kg/24 hr) at the time of the testing, given alone in six of them and associated with cyclophosphamide (2 to 3 mg/kg/24 hr) in five. I Requests for reprints should be addressed to Marc Tardieu, HBpital d’enfants, INSERM 94270 Bicetre, France.
U 56,
12.5 oo90-1229/78/1)112-0125$01.00/O Copyrishr All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
In two patients who had a low PHA response of lymphocytes, sequential studies were carried out over 2 and I years, respectively. Patient 1, an 1 l-year-old black girl, had a multisystemic disease, including renal involvement. with typical biological pattern (Table I). Steroid therapy (2 mgikg) was started in association with cyclophosphamide (3 mgikg). After 2 years of treatment the disease had stabilized and steroid therapy was progressively discontinued. Patient 2 was an 12-year-old white girl. She was well until 1 year before admission when two successive episodes of hemolytic anaemia led to steroid treatment (2 mg/kg/24 hr) for 6 weeks. She was referred to the pediatric department for SLE without renal involvement. Biological tests are summarized in Table 1. Search for circulating immune complexes as tested by the Clq technique t 12) was positive. The patient was treated with prednisone (2 mg/kg) alone. After 2 months of treatment, all laboratory tests showed a marked improvement and the disease had stabilized I year later. Control lymphocytes originated from 30 normal children or young adults of ages 10 to 25 years with a mean age of 15. Mrthods. Phytohaemagglutinin (PHA) and mixed leukocyte culture (MLC) responses of lymphocytes were performed as previously described (13). Briefly, venous blood was collected in heparinized test tubes and lymphocytes were separated on a Ficoll-Hypaque gradient. Cell suspensions were washed five times at room temperature and counted, and viability was determined by the Trypan blue exclusion test. Cells were resuspended at a concentration of 5 x 10’ in 0.5 ml of culture medium containing 90% RPM1 1640 (Gibco. Glasgow, Scotland) and 10%
BIOLOGICAL
DATA
AT ADMISSION
ESR (mm/hr) C4 (mg/dl) (N = 50 it 30) Double-stranded anti-DNA Cryoglobulinaemia Lymphocytotoxic antibody Lymphocytes Count (per ~1) Surface markers: EAC rosettes” (N = 1220%) Anti-Ig” (N = IO-32%) E rosettes” (N = 50-75%) Anti-T cell” (N = 49-78%) DHR” Tetanus toxoid antibody titers (III/ml) Before secondary immunization After immunization
TABLE 1 1,~Two SLE PATIEWS
WITH
A Low PHA
RESPONSE”
Patient 1
Patient 2
100 7 90% + -
120 30 80% + -
1350 3% NT 58% NT 0.01 2
1624 NT 29% 62% 67%~ 0.04 0.2
” Abbreviations used: NT, not tested; f, present; and -, absent. * E- and EAC-rosette technique (9). ” Cytotoxicity with anti-immunoglobulin antiserum and complement (10). ” Cytotoxicity with anti-T-cell antiserum and complement (11). ” Delayed hypersensitivity (DHR) skin tests to tuberculin, varidase, candidin, and PHA antigens.
SUPPRESSOR
ACTIVITY
IN
SLE
127
normal AB serum (from volunteer blood donors) with 1% glutamine (Eurobio, Paris) and penicillin (100 &ml). The PHA response was studied on lymphocytes (5 x lV/tube cultured for 3 days with or without 80 pg/ml of PHA-M (Difco, Detroit, Michigan). In MLC experiments, stimulator cells were X-irradiated with 3000 rads. Tubes containing an equal number of responder and stimulator cells (2.5 x 10s of each) were kept in culture for 6 days. Each experiment was done in triplicate and cells were cultured at 37°C in a humid atmosphere containing 5%CO,. DNA synthesis of control and of stimulated cells was determined by incorporation of tritiated thymidine added 16 hr before termination of the cultures. Results were expressed as counts per minute (cpm) c standard errors. In each experiment determination of cell count and cell viability was also performed at the end of the culture and showed no difference between control and patient cultures. Experimental design. Each MLC study in patients included several cultures performed simultaneously: cultures between patient and control lymphocytes as well as between two control populations. For each of these two types, three cultures were performed and compared: one MLC in a two-way and two MLC in a one-way culture (e.g., patient lymphocytes cultured with nonirradiated as well as with irradiated control lymphocytes and control cells cultured with irradiated patient cells). RESULTS
PHA stimulation of lymphocytes was performed 30 times in 16 patients. In 14 of them, the response was within the normal range. Two other patients (1 and 2), showed a lower response to PHA (Table 2). Patient 2 was studied before any treatment and patient 1 just after initiation of steroid therapy. When lymphocytes of all patients were studied in a two-way MLC, the pattern of responses was similar to that observed in the PHA study: Patients 1 and 2 displayed responses lower than those of the 14 other patients and of the control group (Table 2). Attention was then focused on the ability of the cells to simulate or to respond in MLC. Lymphocytes from all SLE patients responded poorly to irradiated allogeneic control cells. However, when these patients were studied for their ability to stimulate normal cells, they were able to do it as well as normal controls (Table 2).
When the results obtained in two-way and in one-way MLC (control cells cultured with patient irradiated lymphocytes) were compared, the proliferative responses were similar for all patients, except for Patients 1 and 2. In these two patients, the responses obtained in two-way MLC were significantly lower than those observed in one-way MLC since a normal MLC response of control cells was restored following X-irradiation of patient lymphocytes (Table 2). The proliferative response of lymphocytes from Patient 1 was studied 10 times over 2 years of evolution. The PHA response remained low and did not appear to be influenced by the type of treatment or by the evolution of the disease. A similar depression was observed in MLC when patient and control cells were cultured in two-way systems. In all experiments, the increase of the proliferative response of control cells occurred following in vitro X-irradiation of patient cells. Similar
[jH]Thymidine Cultures
incorporation
-
in lymphocytes from”.“:
-~ Patients 3- I6
Controls
Patient 1
Patient 3
1,196 -t 630 (30)
322 k 80 ( IO)
PHAl‘
51.888 + 5,724 (30)
8,992 t 6,314 (IO)
479 + 96 (4) 16,430 k 8,888 14)
44,387 2 4,322 (16)
Two-way MLC”
17,545 +- 2.800 (24)
7,741 k 2.150 (9)
6,051 + 2.710 14)
14,677 k 2.790 05)
14,000 k 2.512 (24)
1,405 _f 523 (9) 14,811 ” 4,721 (9)
3,482 r 615 (4) 12,472 c 1,620 (4)
5 698 zt 2.158 ( (15)
No stimulation
One-way MLC Responder cells (no irradiation)” Stimulator cells (X-irradiation)’
14,000 -t 2,512
(24)
1.039 t 517 (16)
Il.768 2 1.339 (15)
’ Results are expressed in cpm + SE. ’ The number of experiments is in parentheses. ’ Tested lymphocytes were cultured with PHA (80 &ml) for 3 days. ” Tested lymphocytes were cocultured with control lymphocytes. Results obtained when stimulator and respondor cells were both irradiated and cocultured, or when cultures were performed with one X-irradiated population alone, were within the range of the background. ’ Tested lymphocytes were used as responder cells in a 6-day MLC performed with irradiated control lymphocytes (3000 rads of X-irradiation). ’ Tested lymphocytes were used as stimulator cells (3000 rads of X-irradiation) in a 6-day MLC performed with nonirradiated control lymphocytes.
results were obtained in four separate experiments year of evolution.
performed
in Patient 2 over 1
DISCUSSION
The present work, performed in 16 patients with SLE disease, studied the cellular immune function as tested by lymphocyte proliferation in the presence of PHA as well as in one-way and in two-way mixed leukocyte cultures. In 14 patients, the proliferative response of lymphocytes cultured in the presence of PHA was normal. This contrasted with the impairment of lymphocyte proliferation in MLC. In one-way MLC, irradiated patient lymphocytes behaved as normal stimulator cells. In the reverse manner, however, patient lymphocytes cultured with irradiated control cells displayed in all cases a proliferative response lower than that of controls (Table 2). These data indicate that patients with SLE disease exhibit a T-cell function deficiency of a variable intensity. The discrepancy observed in the lymphocyte proliferative response tested either by PHA or by MLC may explain, in part, the frequent conflicting results obtained in previous studies of cell-mediated immunity in patients with SLE (3-6). Since impairment of cellular immunity in such patients has been related either to cellular or to humoral factors (2), great care was taken to exclude an in vitro effect of humoral factors which has been reported to interfere with responses of SLE
SUPPRESSOR
ACTIVITY
IN
129
SLE
lymphocytes in MLC (14) or in PHA cultures (2). Lymphocytes were washed five times at room temperature and subsequently cultured in normal AB serum, not in sera from patients with SLE. In addition, in Patients 1 and 2, neither lymphocytotoxins nor inhibitory factors were found in serum. The evidence against the hypothetical role of serum factors responsible for the low proliferative responses in Patients 1 and 2 is based on the radiosensitivity of the inhibition (Table 2). It is doubtful also that the therapy itself influenced some of the results since onset or modification of the therapeutic regimen did not alter the results. The two patients who had negative skin tests and who were unable to respond to PHA, gave interesting results in the MLC study. Their cells, cultured with allogeneic control cells in two-way tests, yielded a lower response than that of the 14 other patients and of 24 normal controls. This lower response was not related to the lack of responder cells. Indeed, when the cells of these two patients were used as stimulator cells and were irradiated, the response of control allogeneic lymphocytes was higher than that obtained in a two-way system. This strongly suggested that an active inhibitory process was responsible for the diminution observed in two-way MLC since the inhibitory effect was eradicated following in vitro X-irradiation of the cells. This lymphocyte inhibitory activity, repeatably demonstrated over a period of 1 to 2 years, did not seem influenced by the manifestations of the disease or by the presence of circulating immune complexes. The two SLE patients, who displayed a lymphocyte inhibitory activity, had negative delayed hypersensitivity skin tests possibly related to the same mechanism. Conversely, the antibody response to several antigens appeared to be normal. This, therefore, seems to indicate that the inhibitory activity of lymphocytes is acting on T rather than on B cells. Such an inhibition may be related to suppressor radiosensitive T cells as already described in animals (15) and in man (16). Recently, Horowitz et nl. (17) reported that patients with SLE lacked suppressor T-cell function. Suppressor cell activity was induced in cells from many of these patients by incubation with thymosine. The significance, therefore, of a suppressor activity present in two patients only and absent in 14 others from our study is not clear and its relationship to SLE disease may even be circumstantial. ACKNOWLEDGMENTS This work was supported by INSERM, Contract No. 765-006-4 and by Grant No. 701 from UER Paris-Sud. We gratefully acknowledge the excellent technical assistance of Christiane H&y. We thank Dr. J. J. Ballet and Dr. J. L. Touraine for performing the determination of lymphocyte surface markers. We are indepted to Dr. F. Daguillard and Dr. D. Frommel for stimulating discussions and to Dr. J. P. Revillard, Dr. M. Hermier, Dr. C. Griscelli. Dr. J. Dormont, and Dr. C. Bach for allowing us to study their patients.
REFERENCES I. Hahn, B. H., Bagby, M. K., and Osterland, C. K., Amer. J. Med. 55, 25, 2. Horwitz, D. A., and Cousan, J. B., Amer. .I. Med. 58, 829, 1975. 3. Mellbye, 0. J., Lindstri)m, F. D., Eberle, B. J.. and Williams, Jr., R. C., C/in. 157, 1972. 4. Senyk, G., Hadley, W. K., Attias. M. R., and Talal, N., Arthrifis Rheum. 5. Horwitz, D. A., Arthritis Rheum. 15, 353, 1972.
1973. Exp. 17, 553,
Immunol. 1974.
15,
130
‘I’AKDIEII
AND
DI’PL’l
6. Rosenthal, C. J.. Arthriris Rhrum. 18, 207, 1975. 7. Cohen. A. S., and Canoso. J. J.. Arrlrriris Rheum. 15. 540, 1972. 8. Pincus. T., Schur. P. H., Rose. J. A.. Decker. J. L.. and Talal. N.. .YIJII,CreI. ./. .\I~c/. 281. 701. 1969. 9. Bianco, C., Patrick, R.. and Nussenzweig. V., J. &I. M
IMML’NOLOCY
AND
IMMUNOP.XfHOLOGY
11,
1255130 (1978)
Suppressor Activity of Lymphocytes from Patients with Systemic Lupus Erythematosus M. Laboratory
TARDIEU’
AND
of Immunology. Institut National Me’dicale. U 56. HiSpita d’Enfants.
J. M. DUPUY de lu SantP et de la Recherche BicPtre. 94270. France
Received December 28. 1977 Lymphocytes from 16 patients with SLE disease were studied for their ability to proliferate and to induce stimulation of normal control ailogeneic lymphocytes. In 14 patients, the PHA response was normal. A low proliferative response, however, was shown in MLC when patients lymphocytes were cultured with irradiated control cells. The immunological response of two other SLE patients displayed quite a different pattern. These two patients exhibited negative delayed hypersensitivity skin tests and had a low proliferative response of lymphocytes in PHA cultures and in a one-way as well as in a two-way MLC. The absence of reactivity was not due to a lack of responder cells but to the presence of a radio sensitive suppressor activity. In these two patients, however, the relationship between abnormal cell-mediated functions and SLE disease is unclear and may be circumstantial.
INTRODUCTION
Impairment of cell-mediated immunity is thought to play an important role in the pathogenesis of systemic lupus erythematosus (SLE) (1, 2). Previous studies of cell-mediated immunity in patients with SLE displayed, however, conflicting results (3-6). We report herein results obtained in 16 patients with SLE disease in whom immunological studies revealed a deficiency in cell-mediated immunity of a variable intensity. In two of them, the lymphocyte populations exerted a radiosensitive inhibitory activity detected in mixed leukocyte culture. PATIENTS AND METHODS Patient and control populations. Sixteen patients, aged 8 to 26 years, with systemic lupus erythematosus (SLE) were available for study. Patient selection was based upon the fulfillment of criteria of the Rheumatism Association for classification as SLE (7). Activity of the disease was defined by the association of acute clinical manifestations, erythrocyte sedimentation rate (ESR) above 30 mm/hr, serum C4 level below 30 mg/dl, and anti-DNA binding over 25% according to the Farr assay (8). Eight patients had active disease and eight had inactive disease. Five patients of the former group were studied before treatment and the three others just after the onset of therapy. The 11 treated patients received steroid therapy (2 mg/kg/24 hr) at the time of the testing, given alone in six of them and associated with cyclophosphamide (2 to 3 mg/kg/24 hr) in five. I Requests for reprints should be addressed to Marc Tardieu, HBpital d’enfants, INSERM 94270 Bicetre, France.
U 56,
12.5 oo90-1229/78/1)112-0125$01.00/O Copyrishr All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
In two patients who had a low PHA response of lymphocytes, sequential studies were carried out over 2 and I years, respectively. Patient 1, an 1 l-year-old black girl, had a multisystemic disease, including renal involvement. with typical biological pattern (Table I). Steroid therapy (2 mgikg) was started in association with cyclophosphamide (3 mgikg). After 2 years of treatment the disease had stabilized and steroid therapy was progressively discontinued. Patient 2 was an 12-year-old white girl. She was well until 1 year before admission when two successive episodes of hemolytic anaemia led to steroid treatment (2 mg/kg/24 hr) for 6 weeks. She was referred to the pediatric department for SLE without renal involvement. Biological tests are summarized in Table 1. Search for circulating immune complexes as tested by the Clq technique t 12) was positive. The patient was treated with prednisone (2 mg/kg) alone. After 2 months of treatment, all laboratory tests showed a marked improvement and the disease had stabilized I year later. Control lymphocytes originated from 30 normal children or young adults of ages 10 to 25 years with a mean age of 15. Mrthods. Phytohaemagglutinin (PHA) and mixed leukocyte culture (MLC) responses of lymphocytes were performed as previously described (13). Briefly, venous blood was collected in heparinized test tubes and lymphocytes were separated on a Ficoll-Hypaque gradient. Cell suspensions were washed five times at room temperature and counted, and viability was determined by the Trypan blue exclusion test. Cells were resuspended at a concentration of 5 x 10’ in 0.5 ml of culture medium containing 90% RPM1 1640 (Gibco. Glasgow, Scotland) and 10%
BIOLOGICAL
DATA
AT ADMISSION
ESR (mm/hr) C4 (mg/dl) (N = 50 it 30) Double-stranded anti-DNA Cryoglobulinaemia Lymphocytotoxic antibody Lymphocytes Count (per ~1) Surface markers: EAC rosettes” (N = 1220%) Anti-Ig” (N = IO-32%) E rosettes” (N = 50-75%) Anti-T cell” (N = 49-78%) DHR” Tetanus toxoid antibody titers (III/ml) Before secondary immunization After immunization
TABLE 1 1,~Two SLE PATIEWS
WITH
A Low PHA
RESPONSE”
Patient 1
Patient 2
100 7 90% + -
120 30 80% + -
1350 3% NT 58% NT 0.01 2
1624 NT 29% 62% 67%~ 0.04 0.2
” Abbreviations used: NT, not tested; f, present; and -, absent. * E- and EAC-rosette technique (9). ” Cytotoxicity with anti-immunoglobulin antiserum and complement (10). ” Cytotoxicity with anti-T-cell antiserum and complement (11). ” Delayed hypersensitivity (DHR) skin tests to tuberculin, varidase, candidin, and PHA antigens.
SUPPRESSOR
ACTIVITY
IN
SLE
127
normal AB serum (from volunteer blood donors) with 1% glutamine (Eurobio, Paris) and penicillin (100 &ml). The PHA response was studied on lymphocytes (5 x lV/tube cultured for 3 days with or without 80 pg/ml of PHA-M (Difco, Detroit, Michigan). In MLC experiments, stimulator cells were X-irradiated with 3000 rads. Tubes containing an equal number of responder and stimulator cells (2.5 x 10s of each) were kept in culture for 6 days. Each experiment was done in triplicate and cells were cultured at 37°C in a humid atmosphere containing 5%CO,. DNA synthesis of control and of stimulated cells was determined by incorporation of tritiated thymidine added 16 hr before termination of the cultures. Results were expressed as counts per minute (cpm) c standard errors. In each experiment determination of cell count and cell viability was also performed at the end of the culture and showed no difference between control and patient cultures. Experimental design. Each MLC study in patients included several cultures performed simultaneously: cultures between patient and control lymphocytes as well as between two control populations. For each of these two types, three cultures were performed and compared: one MLC in a two-way and two MLC in a one-way culture (e.g., patient lymphocytes cultured with nonirradiated as well as with irradiated control lymphocytes and control cells cultured with irradiated patient cells). RESULTS
PHA stimulation of lymphocytes was performed 30 times in 16 patients. In 14 of them, the response was within the normal range. Two other patients (1 and 2), showed a lower response to PHA (Table 2). Patient 2 was studied before any treatment and patient 1 just after initiation of steroid therapy. When lymphocytes of all patients were studied in a two-way MLC, the pattern of responses was similar to that observed in the PHA study: Patients 1 and 2 displayed responses lower than those of the 14 other patients and of the control group (Table 2). Attention was then focused on the ability of the cells to simulate or to respond in MLC. Lymphocytes from all SLE patients responded poorly to irradiated allogeneic control cells. However, when these patients were studied for their ability to stimulate normal cells, they were able to do it as well as normal controls (Table 2).
When the results obtained in two-way and in one-way MLC (control cells cultured with patient irradiated lymphocytes) were compared, the proliferative responses were similar for all patients, except for Patients 1 and 2. In these two patients, the responses obtained in two-way MLC were significantly lower than those observed in one-way MLC since a normal MLC response of control cells was restored following X-irradiation of patient lymphocytes (Table 2). The proliferative response of lymphocytes from Patient 1 was studied 10 times over 2 years of evolution. The PHA response remained low and did not appear to be influenced by the type of treatment or by the evolution of the disease. A similar depression was observed in MLC when patient and control cells were cultured in two-way systems. In all experiments, the increase of the proliferative response of control cells occurred following in vitro X-irradiation of patient cells. Similar
[jH]Thymidine Cultures
incorporation
-
in lymphocytes from”.“:
-~ Patients 3- I6
Controls
Patient 1
Patient 3
1,196 -t 630 (30)
322 k 80 ( IO)
PHAl‘
51.888 + 5,724 (30)
8,992 t 6,314 (IO)
479 + 96 (4) 16,430 k 8,888 14)
44,387 2 4,322 (16)
Two-way MLC”
17,545 +- 2.800 (24)
7,741 k 2.150 (9)
6,051 + 2.710 14)
14,677 k 2.790 05)
14,000 k 2.512 (24)
1,405 _f 523 (9) 14,811 ” 4,721 (9)
3,482 r 615 (4) 12,472 c 1,620 (4)
5 698 zt 2.158 ( (15)
No stimulation
One-way MLC Responder cells (no irradiation)” Stimulator cells (X-irradiation)’
14,000 -t 2,512
(24)
1.039 t 517 (16)
Il.768 2 1.339 (15)
’ Results are expressed in cpm + SE. ’ The number of experiments is in parentheses. ’ Tested lymphocytes were cultured with PHA (80 &ml) for 3 days. ” Tested lymphocytes were cocultured with control lymphocytes. Results obtained when stimulator and respondor cells were both irradiated and cocultured, or when cultures were performed with one X-irradiated population alone, were within the range of the background. ’ Tested lymphocytes were used as responder cells in a 6-day MLC performed with irradiated control lymphocytes (3000 rads of X-irradiation). ’ Tested lymphocytes were used as stimulator cells (3000 rads of X-irradiation) in a 6-day MLC performed with nonirradiated control lymphocytes.
results were obtained in four separate experiments year of evolution.
performed
in Patient 2 over 1
DISCUSSION
The present work, performed in 16 patients with SLE disease, studied the cellular immune function as tested by lymphocyte proliferation in the presence of PHA as well as in one-way and in two-way mixed leukocyte cultures. In 14 patients, the proliferative response of lymphocytes cultured in the presence of PHA was normal. This contrasted with the impairment of lymphocyte proliferation in MLC. In one-way MLC, irradiated patient lymphocytes behaved as normal stimulator cells. In the reverse manner, however, patient lymphocytes cultured with irradiated control cells displayed in all cases a proliferative response lower than that of controls (Table 2). These data indicate that patients with SLE disease exhibit a T-cell function deficiency of a variable intensity. The discrepancy observed in the lymphocyte proliferative response tested either by PHA or by MLC may explain, in part, the frequent conflicting results obtained in previous studies of cell-mediated immunity in patients with SLE (3-6). Since impairment of cellular immunity in such patients has been related either to cellular or to humoral factors (2), great care was taken to exclude an in vitro effect of humoral factors which has been reported to interfere with responses of SLE
SUPPRESSOR
ACTIVITY
IN
129
SLE
lymphocytes in MLC (14) or in PHA cultures (2). Lymphocytes were washed five times at room temperature and subsequently cultured in normal AB serum, not in sera from patients with SLE. In addition, in Patients 1 and 2, neither lymphocytotoxins nor inhibitory factors were found in serum. The evidence against the hypothetical role of serum factors responsible for the low proliferative responses in Patients 1 and 2 is based on the radiosensitivity of the inhibition (Table 2). It is doubtful also that the therapy itself influenced some of the results since onset or modification of the therapeutic regimen did not alter the results. The two patients who had negative skin tests and who were unable to respond to PHA, gave interesting results in the MLC study. Their cells, cultured with allogeneic control cells in two-way tests, yielded a lower response than that of the 14 other patients and of 24 normal controls. This lower response was not related to the lack of responder cells. Indeed, when the cells of these two patients were used as stimulator cells and were irradiated, the response of control allogeneic lymphocytes was higher than that obtained in a two-way system. This strongly suggested that an active inhibitory process was responsible for the diminution observed in two-way MLC since the inhibitory effect was eradicated following in vitro X-irradiation of the cells. This lymphocyte inhibitory activity, repeatably demonstrated over a period of 1 to 2 years, did not seem influenced by the manifestations of the disease or by the presence of circulating immune complexes. The two SLE patients, who displayed a lymphocyte inhibitory activity, had negative delayed hypersensitivity skin tests possibly related to the same mechanism. Conversely, the antibody response to several antigens appeared to be normal. This, therefore, seems to indicate that the inhibitory activity of lymphocytes is acting on T rather than on B cells. Such an inhibition may be related to suppressor radiosensitive T cells as already described in animals (15) and in man (16). Recently, Horowitz et nl. (17) reported that patients with SLE lacked suppressor T-cell function. Suppressor cell activity was induced in cells from many of these patients by incubation with thymosine. The significance, therefore, of a suppressor activity present in two patients only and absent in 14 others from our study is not clear and its relationship to SLE disease may even be circumstantial. ACKNOWLEDGMENTS This work was supported by INSERM, Contract No. 765-006-4 and by Grant No. 701 from UER Paris-Sud. We gratefully acknowledge the excellent technical assistance of Christiane H&y. We thank Dr. J. J. Ballet and Dr. J. L. Touraine for performing the determination of lymphocyte surface markers. We are indepted to Dr. F. Daguillard and Dr. D. Frommel for stimulating discussions and to Dr. J. P. Revillard, Dr. M. Hermier, Dr. C. Griscelli. Dr. J. Dormont, and Dr. C. Bach for allowing us to study their patients.
REFERENCES I. Hahn, B. H., Bagby, M. K., and Osterland, C. K., Amer. J. Med. 55, 25, 2. Horwitz, D. A., and Cousan, J. B., Amer. .I. Med. 58, 829, 1975. 3. Mellbye, 0. J., Lindstri)m, F. D., Eberle, B. J.. and Williams, Jr., R. C., C/in. 157, 1972. 4. Senyk, G., Hadley, W. K., Attias. M. R., and Talal, N., Arthrifis Rheum. 5. Horwitz, D. A., Arthritis Rheum. 15, 353, 1972.
1973. Exp. 17, 553,
Immunol. 1974.
15,
130
‘I’AKDIEII
AND
DI’PL’l
6. Rosenthal, C. J.. Arthriris Rhrum. 18, 207, 1975. 7. Cohen. A. S., and Canoso. J. J.. Arrlrriris Rheum. 15. 540, 1972. 8. Pincus. T., Schur. P. H., Rose. J. A.. Decker. J. L.. and Talal. N.. .YIJII,CreI. ./. .\I~c/. 281. 701. 1969. 9. Bianco, C., Patrick, R.. and Nussenzweig. V., J. &I. M
