Immunology 1978 35 223
Inhibition of the E-rosette formation of T-lymphocytes by aggregated human IgG and restoration by levamisole
W. DE COCK, J. DE CREE & H. VERHAEGEN St Bartholomeus, Clinical Research Unit, B-2060
Merksem, Belgium
Received 23 June 1977; accepted for publication 24 November 1977
Summary. Aggregated human IgG significantly inhibited the E-rosette formation of T lymphocytes of healthy subjects and cancer patients. The aggregated IgG induced inhibition of rosette forming cells depended on the time of preincubation at 37°. Levamisole, an anti-anergic chemotherapeutic agent, could partly but significantly prevent the aggregated IgG induced inhibition of the E-rosette formation.
We wondered if the binding of aggregated human IgG (aggHIgG) to T lymphocytes would affect their capacity to form rosettes with sheep erythrocytes and indeed, we found that rosette forming cells (RFC) were inhibited by aggHIgG. As levamisole, an anti-anergic chemotherapeutic agent, has been reported to interact with the Erosette formation of T lymphocytes in vivo (Levo, Rotter & Ramot, 1975; Verhaegen, De Cree, De Cock & Verbruggen, 1977) and in vitro (Biniaminov & Ramot, 1975; Verhaegen, De Cock, De Cree, Verbruggen, Verhaegen-Declercq & Brugmans, 1975) we also studied its effect on aggHIgG-inhibited RFC.
INTRODUCTION The presence of easily detectable Fc receptors was regarded as a characteristic of B cells until recently some authors demonstrated Fc receptors on T cells. Indeed, T cells were found to bind aggregated- or immune complexed IgG via the Fc region, as shown by immunofluorescence (Santana & Turk, 1975; Basten, Miller, Warner, Abraham, Chia & Gamble, 1975; Stout & Herzenberg, 1975), radiography (Basten et al., 1975) or EA-rosette formation (Basten et al., 1975; Ferrarini, Moretta, Abrile & Durante, 1975; Gyongyossy, Arnaiz-Villena, Soteriades-Vlachos & Playfair, 1975; Yoshida & Andersson, 1972).
MATERIALS AND METHODS
Venous blood was collected in heparin from 15 apparently healthy adults and from 11 cancer patients with advanced disease.
Separation of lymphocytes A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml heparinized blood and allowing the erythrocytes to sediment at room temperature for about 1 h. The leucocyte-rich plasma was then layered onto a Ficoll/Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1500 rev/min for 30 min. The
Correspondence: Dr W. De Cock, St Bartholomeus, Clinical Research Unit, B-2060 Merksem, Belgium.
0019-2805/78/0800-0223$02.00 ©) 1978 Blackwell Scientific Publications
223
224
W. De Cock, J. De Cree & H. Verhaegen
lymphocytes at the interface were aspirated and washed with Hanks' balanced salt solution (HBSS, pH 7 7) and the concentration adjusted to 2.106 cells/ ml in HBSS. Viability of the lymphocytes was tested with 1 % tryptan blue and purity was evaluated on a smear stained by May-Grunwald-Giemsa's method.
RESULTS Aggregated human IgG at a final concentration of 125 and 250.ug/ml significantly inhibited E-rosette formation as compared to the effects of human 110 r-
Sheep red blood cells (SRBC) SRBC were always obtained under sterile conditions in Alsever's solution (v/v, 1/1) from the same sheep. The cells were stored at 40 and washed three times with HBSS before use. A 1 % solution of SRBC in HBSS was made. E-rosette test SRBC suspension (0 25 ml) was added to 0 25 ml of lymphocyte suspension, incubated for 5 min at 370, centrifuged at 700 rev/min for 5 min and then incubated for 1 h at 00. After gentle resuspension RFC were counted in a haemocytometer. Two hundred cells were counted and lymphocytes binding three or more SRBC were considered positive.
Experimental design. The effect of immunoglobulin IgG and aggregated immunoglobulin IgG on RFC. Dose-response curve Highly purified human IgG (Pentexe, Miles Laboratories) was used. Aggregates were prepared by heating at 670 for 30 min. 0-2 ml lymphocyte suspension was mixed with 0-2 ml HBSS or 62 5, 125, 250, 500, 1000 ug/ml immunoglobulin heated or not heated. After incubation at 370 for 60 min, lymphocytes were washed and the pellet resuspended in 0-4 ml HBSS. The E-rosette test was then performed. The effect of aggHIgG on RFC. Time-response curve Lymphocyte suspension (0i2 ml) was mixed with 0-2 ml HBSS or 0-2 ml 200 pg aggHIgG/ml. The E-rosette test was performed at different periods of incubation at 370, after washing and resuspension of the pellet in 0 4 ml HBSS. The effect of levamisole on aggHIgG inhibited RFC Lymphocyte (0-2 ml) suspension was mixed with (a) 0 4 ml HBSS, (b) 0 2 ml aggHIgG (200 ug/ml) and 0-2 ml HBSS, (c) 0-2 ml aggHIgG (200 pg/ml) and 0-2 ml levamisole solution (10-6 M), (d) 0-2 ml levamisole solution and 0-2 ml HBSS. After incubation at 370 for 60 min, lymphocytes were washed, the pellet resuspended in 0 4 ml HBSS and the E-rosette test performed.
100 H
90 k c
0 0--
80 V
70
K 60
1
31 62-5 125 250 500 Final concentration (pLg/mnl)
Figure 1. The effect of different concentrations of human IgG (0) and aggregated human IgG (0) on the E-rosette formation of T lymphocytes of healthy subjects. Mean of five experiments ± S.E. Mann-Whitney U test (SIEGEL S. (1956) Nonparametric Statistics, p. 116. McGraw-Hill Book Co., New York.) P = NS NS
Inhibition of the E-rosette formation of T-lymphocytes by aggregated human IgG and restoration by levamisole
W. DE COCK, J. DE CREE & H. VERHAEGEN St Bartholomeus, Clinical Research Unit, B-2060
Merksem, Belgium
Received 23 June 1977; accepted for publication 24 November 1977
Summary. Aggregated human IgG significantly inhibited the E-rosette formation of T lymphocytes of healthy subjects and cancer patients. The aggregated IgG induced inhibition of rosette forming cells depended on the time of preincubation at 37°. Levamisole, an anti-anergic chemotherapeutic agent, could partly but significantly prevent the aggregated IgG induced inhibition of the E-rosette formation.
We wondered if the binding of aggregated human IgG (aggHIgG) to T lymphocytes would affect their capacity to form rosettes with sheep erythrocytes and indeed, we found that rosette forming cells (RFC) were inhibited by aggHIgG. As levamisole, an anti-anergic chemotherapeutic agent, has been reported to interact with the Erosette formation of T lymphocytes in vivo (Levo, Rotter & Ramot, 1975; Verhaegen, De Cree, De Cock & Verbruggen, 1977) and in vitro (Biniaminov & Ramot, 1975; Verhaegen, De Cock, De Cree, Verbruggen, Verhaegen-Declercq & Brugmans, 1975) we also studied its effect on aggHIgG-inhibited RFC.
INTRODUCTION The presence of easily detectable Fc receptors was regarded as a characteristic of B cells until recently some authors demonstrated Fc receptors on T cells. Indeed, T cells were found to bind aggregated- or immune complexed IgG via the Fc region, as shown by immunofluorescence (Santana & Turk, 1975; Basten, Miller, Warner, Abraham, Chia & Gamble, 1975; Stout & Herzenberg, 1975), radiography (Basten et al., 1975) or EA-rosette formation (Basten et al., 1975; Ferrarini, Moretta, Abrile & Durante, 1975; Gyongyossy, Arnaiz-Villena, Soteriades-Vlachos & Playfair, 1975; Yoshida & Andersson, 1972).
MATERIALS AND METHODS
Venous blood was collected in heparin from 15 apparently healthy adults and from 11 cancer patients with advanced disease.
Separation of lymphocytes A preliminary separation was achieved by adding 1 ml of a 6% dextran solution to 10 ml heparinized blood and allowing the erythrocytes to sediment at room temperature for about 1 h. The leucocyte-rich plasma was then layered onto a Ficoll/Isopaque mixture (Lymphoprep, Nyegaard, Oslo, Norway) and centrifuged at 1500 rev/min for 30 min. The
Correspondence: Dr W. De Cock, St Bartholomeus, Clinical Research Unit, B-2060 Merksem, Belgium.
0019-2805/78/0800-0223$02.00 ©) 1978 Blackwell Scientific Publications
223
224
W. De Cock, J. De Cree & H. Verhaegen
lymphocytes at the interface were aspirated and washed with Hanks' balanced salt solution (HBSS, pH 7 7) and the concentration adjusted to 2.106 cells/ ml in HBSS. Viability of the lymphocytes was tested with 1 % tryptan blue and purity was evaluated on a smear stained by May-Grunwald-Giemsa's method.
RESULTS Aggregated human IgG at a final concentration of 125 and 250.ug/ml significantly inhibited E-rosette formation as compared to the effects of human 110 r-
Sheep red blood cells (SRBC) SRBC were always obtained under sterile conditions in Alsever's solution (v/v, 1/1) from the same sheep. The cells were stored at 40 and washed three times with HBSS before use. A 1 % solution of SRBC in HBSS was made. E-rosette test SRBC suspension (0 25 ml) was added to 0 25 ml of lymphocyte suspension, incubated for 5 min at 370, centrifuged at 700 rev/min for 5 min and then incubated for 1 h at 00. After gentle resuspension RFC were counted in a haemocytometer. Two hundred cells were counted and lymphocytes binding three or more SRBC were considered positive.
Experimental design. The effect of immunoglobulin IgG and aggregated immunoglobulin IgG on RFC. Dose-response curve Highly purified human IgG (Pentexe, Miles Laboratories) was used. Aggregates were prepared by heating at 670 for 30 min. 0-2 ml lymphocyte suspension was mixed with 0-2 ml HBSS or 62 5, 125, 250, 500, 1000 ug/ml immunoglobulin heated or not heated. After incubation at 370 for 60 min, lymphocytes were washed and the pellet resuspended in 0-4 ml HBSS. The E-rosette test was then performed. The effect of aggHIgG on RFC. Time-response curve Lymphocyte suspension (0i2 ml) was mixed with 0-2 ml HBSS or 0-2 ml 200 pg aggHIgG/ml. The E-rosette test was performed at different periods of incubation at 370, after washing and resuspension of the pellet in 0 4 ml HBSS. The effect of levamisole on aggHIgG inhibited RFC Lymphocyte (0-2 ml) suspension was mixed with (a) 0 4 ml HBSS, (b) 0 2 ml aggHIgG (200 ug/ml) and 0-2 ml HBSS, (c) 0-2 ml aggHIgG (200 pg/ml) and 0-2 ml levamisole solution (10-6 M), (d) 0-2 ml levamisole solution and 0-2 ml HBSS. After incubation at 370 for 60 min, lymphocytes were washed, the pellet resuspended in 0 4 ml HBSS and the E-rosette test performed.
100 H
90 k c
0 0--
80 V
70
K 60
1
31 62-5 125 250 500 Final concentration (pLg/mnl)
Figure 1. The effect of different concentrations of human IgG (0) and aggregated human IgG (0) on the E-rosette formation of T lymphocytes of healthy subjects. Mean of five experiments ± S.E. Mann-Whitney U test (SIEGEL S. (1956) Nonparametric Statistics, p. 116. McGraw-Hill Book Co., New York.) P = NS NS
