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Rosette formation by lymphoid cells of mice immunized to HSA using SRBC sensitized with an HSA-anti-SRBC conjugate
Journal of ImmunologicalMethods 2 (1972) 11-15. © North-Holland Publishing Company
R O S E T T E F O R M A T I O N BY L Y M P H O I D C E L L S O F MICE I M M U N I Z E D TO H S A U S I N G SRBC S E N S I T I Z E D W I T H A N HSA-ANTI-SRBC CONJUGATE D. S U L I T Z E A N U
* and J.S. HASKILL
Department of Pathology, Queen's University, Kingston. Ontario, Canada
Received 20 June 1972
Accepted 1 July 1972
A sensitive method has been devised to detect serum antibodies and rosette forming cells to a soluble antigen.
The rosette technique (Zaalberg, 1964) has been widely employed to study the interaction of lymphoid cells with the sheep red blood cell (SRBC) antigen. More recently, the method has been adapted to work with soluble antigens. A variety of procedures have been devised to couple the soluble antigens to red blood cells. Thus, Wilson et al. (1969) have used guinea pig RBC sensitized with the Fab' fragment of rabbit anti-guinea pig serum, to prepare rosettes with lymphoid cells of guinea pigs immunized to rabbit Fab'. Perrudet-Badoux and Frei have prepared rosettes with formalinized, tanned SRBC sensitized with thyroglobulin (PerrudetBadoux and Frei, 1969) and with a variety of drugs (Perrudet-Badoux and Frei, 1971). McConnell (1971) has conjugated BSA to sheep and fowl RBC, by means of carbodiimide. In the present communication we describe our experience with an antigen prepared by a modification of the technique of Strausbach et al. (1970): HSA was coupled with glutaraldehyde (Avrameas, 1969) to mouse anti SRBC-IgG and the coupled antibody was used to sensitize SRBC. This antigen was very useful not only for preparing rosettes but, as will be described in a subsequent publication, it was also highly efficient in stimulating a secondary response to HSA without adjuvant, in an adoptive transfer system. Rabbit antiserum to human serum albumin (HSA) was prepared by injecting HSA in complete Freund's adjuvant (three injections of 5 mg HSA each, at weekly intervals, intramuscularly). Mouse antiserum to sheep red blood cells (SRBC) was a gift from Dr. M. Axelrad. A crude IgG fraction was isolated from this antiserum by precipitation with ammonium sulfate at 33% saturation (ASRBC-IgG). Coupling of HSA to ASRBC-IgG (ASRBC-HSA) was done following the technique described by * On Sabbatical leave from the Department of Immunology Hebrew University, Hadassah Medical School, Jerusalem, Israel. 11
12
D. SULITZEANU and J.S. HASKILL
Avrameas (1969). All reagents were diluted in 0.1 M phosphate buffer (PB), pH 6.8. To a mixture of 5 mg ASRBC-IgG and 50 mg HSA in 0.6 ml, 0.05 ml of 1% aqueous solution of glutaraldehyde were added dropwise (approx. 0.01 ml per drop). The mixture was kept at room temperature for 2 hr and then dialyzed against phosphate buffered saline, pH 7.0 (PBS). The coupled antiserum lost very little, if any, hemagglutinating activity against SRBC. SRBC to be sensitized with ASRBC-HSA were filtered through gauze, washed 2 times in cold saline and brought to a concentration of 50% in PBS. 0.3 ml of the SRBC suspension was added to 0.1 ml of 1/10 dilution of ASRBC-HSA. The mixture was kept at 37°C for 2 hr, with occasional mixing. The sensitized SRBC were washed 3 times in PBS and stored as a 10% suspension in PBS containing 0.1% sodium azide. The effectiveness of sensitization was checked in hemagglutination tests with anti-HSA serum. Normal SRBC were not affected by anti-HSA at a dilution of 1/10 or higher, whereas the sensitized RBC were agglutinated in high titer (1/4500). The agglutination was specific as it could be blocked by 10/~g HSA. The sensitized SRBC could be used for one week. Spleens or lymph nodes (popliteal and mesenteric) were teased with needles in Tris-buffered Eagle's minimum essential medium, pH 7.4 (MEM). The cell suspensions were left to stand for 5 rain for the coarse particles to settle. The cells were washed 3 times in MEM and resuspended in MEM containing 7.5% fetal calf serum. They were then passed through a syringe with a 26 gauge needle, counted and adjusted to 5 × 106 cells/ml. 0.1 ml of the lymphoid cell suspension were mixed with 0.1 ml of a 0.5% sensitized SRBC suspension in MEM. When testing for specific inhibition of rosette formation, 0.1 ml of the test protein solution was also added. Cells and solutions were kept cold (0°C) throughout. After mixing, the suspension was transferred into a 0.4 ml plastic centrifuge tube, centrifuged for 5 rain at 1000 rpm in a refrigerated International centrifuge and left in the refrigerator for at least 2 hr, mostly overnight. Finally, the pellet was resuspended, an aliquot was mixed with an equal volume of 1% methyl violet containing 2% rabbit serum and the rosettes were counted in a hemacytometer, in a total area of 9 mm 2. The methyl violet stained the central cell in the rosette so that rosettes could be identified with certainty. Rosettes could be preserved for long intervals by fixing with glutaraldehyde. To a suspension of rosettes in 5 ml MEM, 0.5 ml of 2.5% glutaraldehyde in distilled water was added dropwise. The mixture was left overnight in the refrigerator, washed oncewith, and resuspended in, PBS. The identity of the RFC was best determined in stained preparations of non fixed rosettes. Cell suspensions containing rosettes were centrifuged and the pellet was suspended in a drop of rabbit or mouse serum. A small droplet of the suspension was smeared on a clean glass slide, dried quickly under a fan, fixed for l0 min and stained with Giemsa. CBA mice were immunized with HSA in complete Freund's adjuvant, at concentrations ranging from 2 5 0 - 2 5 0 0 ~tg/ml. Approx. 0.03 ml was given into each of the hind foot pads and 0.1 ml was injected intraperitoneally. Preliminary experiments were carried out to determine the optimal amounts of
Rosette formation in lymphoid cells
13
Table 1 Mice (3 per group) were immunized with HSA in Freund's adjuvant (250 ,g/ml). The lymph node cells were derived from a mixture of popliteal and mesenteric lymph nodes. Data for time 0 represent the frequency of RFC in nodes and spleens of non immunized mice. RFC/106 cells, at days after immunization Cell source Spleen Lymph node
0
3
7
14
21
28
520 220
600 800
150 3000
420 4200
360 4000
1650 1950
ASRBC-HSA needed to sensitize SRBC. Too much of the reagent caused hemagglutination of the SRBC through the free valencies of ASRBC in the complex, resulting in false rosette formation. If too little reagent was used for sensitization, the number of rosettes obtained was small. The correct amount was adjusted by trial and error, to give a maximal number of rosettes with cells o f immune mice, but hardly any rosettes (0.05% or less) with cells of normal mice. Table 1 shows the recovery o f rosette forming cells (RFC) from spleen and lymph nodes of mice immunized to HSA. RFC in amounts exceeding the background could be detected already at 3 days after immunization, but their number reached a maximum between 1 4 - 2 1 days after immunization. Elevated numbers o f RFC could be found in spleens and lymph nodes of specifically immunized mice over a period o f several months. Fig. 1 shows some o f the different cell types seen among the RFC. Rosettes could be fixed by treating with glutaraldehyde and stored subsequently for several weeks in the refrigerator (fig. 2). The specificity of RFC for HSA was checked by inhibition experiments. Addition of HSA (4 mg/ml) to the reaction mixture reduced the number o f rosettes by 95%, while egg albumin in the same concentration had no effect. Lower concentrations o f HSA blocked rosette formation only partially. Non-sensitized SRBC did not form a significant number of rosettes with cells of HSA immune mice (4% of the number formed with sensitized RFC). An important advantage of the technique described here was its reproducibility. Once the coupling reagent is prepared and standardized, it can be divided into equal aliquots and stored frozen for prolonged periods. We have used a single batch for over six months, with no apparent loss in potency. When sensitized red cells are needed, all one has to do is to add the required amount of red cells to the tube containing the coupling reagent and place the mixture at 37°C. Attempts were also made to prepare rosettes with SRBC coupled to HSA directly, by means of glutaraldehyde, but the number of rosettes thus obtained was much smaller. As will be shown in a subsequent communication, enriched suspensions of RFC obtained by fractionation on discontinuous, Ficoll gradients have been used to transfer adoptively memory to HSA. The HSA-sensitized SRBC transferred with the
14
D. SULITZEANU and J.S. HASKILL
n~
t
II
t
A
C
O O
O
Fig. 1. Various types of RFC: (a) medium lymphocyte, (b and c) undifferentiated blast forms, (d) macrophage probably forming rosette through cytophilic antibody.
Rosette j~)rmatMn in h,mphoid cells
15
4" Fig. 2. Rosette fixed with glutaraldehyde. RFCs p r o v e d to be highly efficient in s t i m u l a t i n g the p r o d u c t i o n o f a n t i b o d y to HSA in the r e c i p i e n t mice.
ACKNOWLEDGEMENTS We are indebted to Mrs. Lauretta Eaton for technical assistance. One of us (DS) was a recipient of Canadian MRC fellowship. This work was supported by the Medical Research Council and National Cancer Institute of Canada. REFERENCES Avrameas, S., 1969, lmmunochemistry 6, 43. McConnell, I., 1971, Nature New Biol. 233, 177. Perrudet-Badoux, A. and P.C. Frei, 1969, Clin. Exptl. Immunol. 5, 117. Perrudet-Badoux, A. and P.C. Frei, 1971, Intern. Arch. Allergy 41, 149. Strausbauch, P., A. Sulica and D. Givol, 1970, Nature 227, 68. Wilson, A.B., A. Munro and R.R.A. Coombs, 1969, Intern. Arch. Allergy 35,228. Zaalberg, O.B., 1964, Nature 202, 1231.
R O S E T T E F O R M A T I O N BY L Y M P H O I D C E L L S O F MICE I M M U N I Z E D TO H S A U S I N G SRBC S E N S I T I Z E D W I T H A N HSA-ANTI-SRBC CONJUGATE D. S U L I T Z E A N U
* and J.S. HASKILL
Department of Pathology, Queen's University, Kingston. Ontario, Canada
Received 20 June 1972
Accepted 1 July 1972
A sensitive method has been devised to detect serum antibodies and rosette forming cells to a soluble antigen.
The rosette technique (Zaalberg, 1964) has been widely employed to study the interaction of lymphoid cells with the sheep red blood cell (SRBC) antigen. More recently, the method has been adapted to work with soluble antigens. A variety of procedures have been devised to couple the soluble antigens to red blood cells. Thus, Wilson et al. (1969) have used guinea pig RBC sensitized with the Fab' fragment of rabbit anti-guinea pig serum, to prepare rosettes with lymphoid cells of guinea pigs immunized to rabbit Fab'. Perrudet-Badoux and Frei have prepared rosettes with formalinized, tanned SRBC sensitized with thyroglobulin (PerrudetBadoux and Frei, 1969) and with a variety of drugs (Perrudet-Badoux and Frei, 1971). McConnell (1971) has conjugated BSA to sheep and fowl RBC, by means of carbodiimide. In the present communication we describe our experience with an antigen prepared by a modification of the technique of Strausbach et al. (1970): HSA was coupled with glutaraldehyde (Avrameas, 1969) to mouse anti SRBC-IgG and the coupled antibody was used to sensitize SRBC. This antigen was very useful not only for preparing rosettes but, as will be described in a subsequent publication, it was also highly efficient in stimulating a secondary response to HSA without adjuvant, in an adoptive transfer system. Rabbit antiserum to human serum albumin (HSA) was prepared by injecting HSA in complete Freund's adjuvant (three injections of 5 mg HSA each, at weekly intervals, intramuscularly). Mouse antiserum to sheep red blood cells (SRBC) was a gift from Dr. M. Axelrad. A crude IgG fraction was isolated from this antiserum by precipitation with ammonium sulfate at 33% saturation (ASRBC-IgG). Coupling of HSA to ASRBC-IgG (ASRBC-HSA) was done following the technique described by * On Sabbatical leave from the Department of Immunology Hebrew University, Hadassah Medical School, Jerusalem, Israel. 11
12
D. SULITZEANU and J.S. HASKILL
Avrameas (1969). All reagents were diluted in 0.1 M phosphate buffer (PB), pH 6.8. To a mixture of 5 mg ASRBC-IgG and 50 mg HSA in 0.6 ml, 0.05 ml of 1% aqueous solution of glutaraldehyde were added dropwise (approx. 0.01 ml per drop). The mixture was kept at room temperature for 2 hr and then dialyzed against phosphate buffered saline, pH 7.0 (PBS). The coupled antiserum lost very little, if any, hemagglutinating activity against SRBC. SRBC to be sensitized with ASRBC-HSA were filtered through gauze, washed 2 times in cold saline and brought to a concentration of 50% in PBS. 0.3 ml of the SRBC suspension was added to 0.1 ml of 1/10 dilution of ASRBC-HSA. The mixture was kept at 37°C for 2 hr, with occasional mixing. The sensitized SRBC were washed 3 times in PBS and stored as a 10% suspension in PBS containing 0.1% sodium azide. The effectiveness of sensitization was checked in hemagglutination tests with anti-HSA serum. Normal SRBC were not affected by anti-HSA at a dilution of 1/10 or higher, whereas the sensitized RBC were agglutinated in high titer (1/4500). The agglutination was specific as it could be blocked by 10/~g HSA. The sensitized SRBC could be used for one week. Spleens or lymph nodes (popliteal and mesenteric) were teased with needles in Tris-buffered Eagle's minimum essential medium, pH 7.4 (MEM). The cell suspensions were left to stand for 5 rain for the coarse particles to settle. The cells were washed 3 times in MEM and resuspended in MEM containing 7.5% fetal calf serum. They were then passed through a syringe with a 26 gauge needle, counted and adjusted to 5 × 106 cells/ml. 0.1 ml of the lymphoid cell suspension were mixed with 0.1 ml of a 0.5% sensitized SRBC suspension in MEM. When testing for specific inhibition of rosette formation, 0.1 ml of the test protein solution was also added. Cells and solutions were kept cold (0°C) throughout. After mixing, the suspension was transferred into a 0.4 ml plastic centrifuge tube, centrifuged for 5 rain at 1000 rpm in a refrigerated International centrifuge and left in the refrigerator for at least 2 hr, mostly overnight. Finally, the pellet was resuspended, an aliquot was mixed with an equal volume of 1% methyl violet containing 2% rabbit serum and the rosettes were counted in a hemacytometer, in a total area of 9 mm 2. The methyl violet stained the central cell in the rosette so that rosettes could be identified with certainty. Rosettes could be preserved for long intervals by fixing with glutaraldehyde. To a suspension of rosettes in 5 ml MEM, 0.5 ml of 2.5% glutaraldehyde in distilled water was added dropwise. The mixture was left overnight in the refrigerator, washed oncewith, and resuspended in, PBS. The identity of the RFC was best determined in stained preparations of non fixed rosettes. Cell suspensions containing rosettes were centrifuged and the pellet was suspended in a drop of rabbit or mouse serum. A small droplet of the suspension was smeared on a clean glass slide, dried quickly under a fan, fixed for l0 min and stained with Giemsa. CBA mice were immunized with HSA in complete Freund's adjuvant, at concentrations ranging from 2 5 0 - 2 5 0 0 ~tg/ml. Approx. 0.03 ml was given into each of the hind foot pads and 0.1 ml was injected intraperitoneally. Preliminary experiments were carried out to determine the optimal amounts of
Rosette formation in lymphoid cells
13
Table 1 Mice (3 per group) were immunized with HSA in Freund's adjuvant (250 ,g/ml). The lymph node cells were derived from a mixture of popliteal and mesenteric lymph nodes. Data for time 0 represent the frequency of RFC in nodes and spleens of non immunized mice. RFC/106 cells, at days after immunization Cell source Spleen Lymph node
0
3
7
14
21
28
520 220
600 800
150 3000
420 4200
360 4000
1650 1950
ASRBC-HSA needed to sensitize SRBC. Too much of the reagent caused hemagglutination of the SRBC through the free valencies of ASRBC in the complex, resulting in false rosette formation. If too little reagent was used for sensitization, the number of rosettes obtained was small. The correct amount was adjusted by trial and error, to give a maximal number of rosettes with cells o f immune mice, but hardly any rosettes (0.05% or less) with cells of normal mice. Table 1 shows the recovery o f rosette forming cells (RFC) from spleen and lymph nodes of mice immunized to HSA. RFC in amounts exceeding the background could be detected already at 3 days after immunization, but their number reached a maximum between 1 4 - 2 1 days after immunization. Elevated numbers o f RFC could be found in spleens and lymph nodes of specifically immunized mice over a period o f several months. Fig. 1 shows some o f the different cell types seen among the RFC. Rosettes could be fixed by treating with glutaraldehyde and stored subsequently for several weeks in the refrigerator (fig. 2). The specificity of RFC for HSA was checked by inhibition experiments. Addition of HSA (4 mg/ml) to the reaction mixture reduced the number o f rosettes by 95%, while egg albumin in the same concentration had no effect. Lower concentrations o f HSA blocked rosette formation only partially. Non-sensitized SRBC did not form a significant number of rosettes with cells of HSA immune mice (4% of the number formed with sensitized RFC). An important advantage of the technique described here was its reproducibility. Once the coupling reagent is prepared and standardized, it can be divided into equal aliquots and stored frozen for prolonged periods. We have used a single batch for over six months, with no apparent loss in potency. When sensitized red cells are needed, all one has to do is to add the required amount of red cells to the tube containing the coupling reagent and place the mixture at 37°C. Attempts were also made to prepare rosettes with SRBC coupled to HSA directly, by means of glutaraldehyde, but the number of rosettes thus obtained was much smaller. As will be shown in a subsequent communication, enriched suspensions of RFC obtained by fractionation on discontinuous, Ficoll gradients have been used to transfer adoptively memory to HSA. The HSA-sensitized SRBC transferred with the
14
D. SULITZEANU and J.S. HASKILL
n~
t
II
t
A
C
O O
O
Fig. 1. Various types of RFC: (a) medium lymphocyte, (b and c) undifferentiated blast forms, (d) macrophage probably forming rosette through cytophilic antibody.
Rosette j~)rmatMn in h,mphoid cells
15
4" Fig. 2. Rosette fixed with glutaraldehyde. RFCs p r o v e d to be highly efficient in s t i m u l a t i n g the p r o d u c t i o n o f a n t i b o d y to HSA in the r e c i p i e n t mice.
ACKNOWLEDGEMENTS We are indebted to Mrs. Lauretta Eaton for technical assistance. One of us (DS) was a recipient of Canadian MRC fellowship. This work was supported by the Medical Research Council and National Cancer Institute of Canada. REFERENCES Avrameas, S., 1969, lmmunochemistry 6, 43. McConnell, I., 1971, Nature New Biol. 233, 177. Perrudet-Badoux, A. and P.C. Frei, 1969, Clin. Exptl. Immunol. 5, 117. Perrudet-Badoux, A. and P.C. Frei, 1971, Intern. Arch. Allergy 41, 149. Strausbauch, P., A. Sulica and D. Givol, 1970, Nature 227, 68. Wilson, A.B., A. Munro and R.R.A. Coombs, 1969, Intern. Arch. Allergy 35,228. Zaalberg, O.B., 1964, Nature 202, 1231.