Immunology, 1975, 28, 485.
T Cell-dependent Helper and Suppressive Influences in an Adoptive IgG Antibody Response G. F. MITCHELLt§ * Schweizerisches Forschungsinstitut, Davos, and t Basel Institute for Immunology, Grenzacherstrasse 483, Basel, Switzerland S. ARRENBRECHT*t
AND
(Received 20th May 1974; accepted for publication 20th June 1974) Summary. When immune spleen cells of mice immunized to a hapten carrier preparation 4 days previously were transferred to normal syngeneic hosts, they began to produce 7S antibody (presumably ofthe IgG class), provided that relatively small numbers ofcells (about 1/10 spleen equivalent) were transferred. Increasing the number of transferred cells resulted in less IgG antibody formed. Depletion of the immune spleen cells of T cells by treatment with anti-theta serum and complement prevented IgG antibody formation. IgG antibody production by untreated and anti-theta serum-treated immune spleen cells could be enhanced (reinduced) by addition of small numbers of cells enriched for carrier-activated T cells. These results suggest that T cells are necessary to stimulate antigen-activated B cells into IgG antibody production. Larger numbers of 'carrier-activated T cells' depressed IgG antibody production. Both enhancement and depression could be demonstrated to be antigen-specific. IgG antibody production by high numbers of transferred immune spleen cells could be induced by treating the cells prior to transfer with suboptimal amounts of anti-theta serum and complement. It is argued that this results from the elimination of a T cell-dependent suppressor influence arising during a normal immune response.
INTRODUCTION Antibody production to a number of antigens is increased by collaboration of thymusderived (T) cells and bone marrow-derived (B) cells (Katz and Benacerraf, 1972). In a number of cases, IgM antibody responses have been shown to be less thymus (or T cell) dependent than IgG antibody responses (Mitchell, 1974). It is not known which step in the differentiation of small lymphocytes into antibody-forming (plasma) cells is enhanced or triggered by T cells. The experiments of Roelants and Askonas (1972) suggest that B cells can be triggered into proliferation by antigen without the collaboration of T cells, but will not go on to produce antibodies. T cells might thus be necessary to induce IgG antibody production in antigen triggered B cells (e.g. Cheers and Miller, 1972). This suggestion can be tested by transfer of spleen cells of immunized mice which are not yet making IgG antibody, but will do so after transfer. Upon transfer, the ratio t Present address and correspondence: Dr S. Arrenbrecht, Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. § Present address: The Walter and Eliza Hall Institute, Melbourne, Australia.
485
486 S. Arrenbrecht and G. F. Mitchell of T cells to B cells can be altered artificially by anti-theta treatment and/or supplementation with T cells. In the experiments described below we have studied the anti-DNP antibody response to dinitrophenylated bovine gamma-globulin (DNP-BGG), a response which is thymus-dependent, and for which the kinetics of IgM and IgG formation have been determined (Arrenbrecht, 1974).
MATERIALS AND METHODS Animals For preliminary experiments, Charles River mice (Dr Wander, AG, Berne) were used; for the experiments reported, C3H/HeJ mice were used. Four to 6-week-old mice were used as thymocyte donors, 8-12-week-old mice for immunization and as recipients of spleen cells from immunized mice ('immune spleen cells'-ISC), and 15-20-week-old mice were used as irradiated recipients of thymocytes.
Immunization Mice were immunized with 100 pg of DNP-BGG (substitution rates DNP12- and DNP1 7-BGG). The preparation and immunogenicity of these antigens have been described (Arrenbrecht, 1974). Other antigens were sheep erythrocytes (SRC) (108 per mouse), keyhole limpet haemocyanin (KLH) (Pacific Biomarine Supply Company, Venice, U.S.A.) (100 jg per mouse), and BGG heat-aggregated, at 20 mg/ml and 600 for 30 minutes (100 Mg per mouse). All antigens were given in saline intraperitoneally (i.p.). Preparation of 'antigen (carrier) activated T cells' Mice were irradiated with 750 rads from a Phillips X-ray machine. The dose was lethal after a maximum of 10 days. Irradiated animals received antigen and/or thymocytes intravenously (i.v.) (1/2 thymus equivalent) within 3 hours after irradiation. Seven days later, their spleens were used as a source of 'antigen (carrier) activated T cells' (Mitchell and Miller, 1968). Cell suspensions Spleens and thymus were squashed with a loose fitting pestle in a glass tube at 380 or finely cut with scissors and the lumps repeatedly aspirated with a disposable syringe. All further handling of the cells was at 40. The medium was either Hanks's or Earle's balanced salt solution (BSS). After suspension, the cells were washed once and counted with Turk's solution and BSS with Evans Blue to assess viability. Cell suspensions with less than 80 per cent viable cells were discarded.
Antibody-forming cell (ABFC) assay ABFC were detected by the Jerne plaque test using agarose in minimal Eagle's medium with 5 per cent foetal calf serum. Indicator erythrocytes were SRC or SRC substituted with trinitrophenyl (TNP) groups by the method of Rittenberg and Pratt (1969). Indirect (presumably 7S, IgG) ABFC were detected by incubating the plates, prior to the addition of complement, with a 1:200 dilution of a rabbit anti-Brucella serum raised against bacteria coated with a hyperimmune mouse-anti-Brucella serum. This serum caused a depression of 3rd and 4th day (presumably IgM) ABFC counts by approximately
487 Adoptive IgG Antibody Response 1/4. This was taken into account when indirect ABFC numbers were calculated (see Fig. 1). Bleeding Mice were bled either from the retro-orbital plexus or by heart puncture and the serum separated.
Haemagglutinations Haemagglutination titres (HAT) were determined in U-type Microtiter plates (Cooke Engineering Company, Alexandria, Virginia, U.S.A.). Phosphate-buffered saline (PBS) containing 1 mg/ml of bovine serum albumin was used as diluent. Final red cell concentration was 025. For the detection of antibody (AB) in sera from DNP-BGG immunized mice, TNP-SRC as prepared for the ABFC assay or TNP-HRC (human erythrocytes) were used. For anti-SRC HAT, the first well showing no carpet formation was taken as the endpoint. For anti-TNP HAT, plates were stood upright, until the cells began to slide down and the titre was read as the first well with an even streaming of cells. In some experiments, we tried to inactivate IgM type antibodies in serum by 2-mercaptoethanol (2-ME) (0-1 M final concentration, 370, 30 minutes). However, increased HAT were often observed after 2-mercaptoethanol treatment and the method was therefore considered to be unreliable. Instead, sucrose gradient ultracentrifugation was used to differentiate between 19S (1gM) and 7S (IgG) antibodies.
Ultracentrifugation Linear gradients of sucrose in PBS, pH 7-2, were prepared by layering 1 l-ml amounts of 40, 30, 20 and 10 per cent sucrose solutions on top of each other. The tubes were left undisturbed for 1 hour, then 0X2 ml of serum, diluted with the same volume of water, was overlaid. Such gradients were swung in a Beckman Spinco SW 39 rotor at 35000 rev/min for 9 hours. Concave gradients (10-40 per cent sucrose in PBS) were prepared in a gradient mixer, and serum applied as above. Concave gradients were swung in a SW 50-1 rotor at 50,000 rev/min for 7 hours. At the end of the run, fractions were collected from the bottom of the tubes through a hole punched with a 25 gauge needle. Three drops usually constituted one fraction (about 300 p1). Haemoglobin (5.4S) served as a marker. For an assessment of the amounts of AB present in the IgM and IgG region of the gradients, HAT of the individual fraction were determined. The sum of the HAT of those fractions making up one peak was used as a measure of the relative amount ofAB (RAAB) present in the two regions of each gradient. T cell-depletion of spleen cells An anti-theta serum was prepared in a rabbit by injection of the saline-insoluble fraction of three C3H mouse brains, emulsified in saline, s.c. Three weeks later, a second injection was given i.p. Ten days later, blood was collected and 25 ml of the serum absorbed with AKR mouse brain (saline-insoluble fraction), spleen, lymph node, bone marrow and thymus cells from thirty mice. Cytotoxicity tests were performed by first incubating 2-5 x 106 cells with 100 pl of serial antiserum dilutions in BSS in the cold, followed by one wash and incubation in 100 yl of 10 per cent agarose-absorbed guinea-pig serum in BSS at G
S. Arrenbrecht and G. F. Mitchell 370 for 30 minutes. After one wash, the cells were resuspended in BSS with 0 05 per cent Trypan Blue. While unabsorbed serum had a high cytotoxic titre for C3H, AKR and Charles River lymph node, thymus and bone marrow cells, the absorbed serum had no cytotoxic effect on AKR cells, but still killed 90 per cent of C3H and Charles River thymocytes, 60-70 per cent of lymph node cells and about 10 per cent bone marrow cells (two experiments in triplicate). The highest serum dilution giving this maximal killing of lymph node cells was used to calculate the amount of serum needed to deplete spleen cells of theta-positive cells. Thus, 1 ml of serum was incubated with 3 x 108 cells in 6 ml of BSS, followed after one wash by 6 ml of 10 per cent agarose-absorbed complement. Before transfer, cells so treated were washed twice.
488
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T Cell-dependent Helper and Suppressive Influences in an Adoptive IgG Antibody Response G. F. MITCHELLt§ * Schweizerisches Forschungsinstitut, Davos, and t Basel Institute for Immunology, Grenzacherstrasse 483, Basel, Switzerland S. ARRENBRECHT*t
AND
(Received 20th May 1974; accepted for publication 20th June 1974) Summary. When immune spleen cells of mice immunized to a hapten carrier preparation 4 days previously were transferred to normal syngeneic hosts, they began to produce 7S antibody (presumably ofthe IgG class), provided that relatively small numbers ofcells (about 1/10 spleen equivalent) were transferred. Increasing the number of transferred cells resulted in less IgG antibody formed. Depletion of the immune spleen cells of T cells by treatment with anti-theta serum and complement prevented IgG antibody formation. IgG antibody production by untreated and anti-theta serum-treated immune spleen cells could be enhanced (reinduced) by addition of small numbers of cells enriched for carrier-activated T cells. These results suggest that T cells are necessary to stimulate antigen-activated B cells into IgG antibody production. Larger numbers of 'carrier-activated T cells' depressed IgG antibody production. Both enhancement and depression could be demonstrated to be antigen-specific. IgG antibody production by high numbers of transferred immune spleen cells could be induced by treating the cells prior to transfer with suboptimal amounts of anti-theta serum and complement. It is argued that this results from the elimination of a T cell-dependent suppressor influence arising during a normal immune response.
INTRODUCTION Antibody production to a number of antigens is increased by collaboration of thymusderived (T) cells and bone marrow-derived (B) cells (Katz and Benacerraf, 1972). In a number of cases, IgM antibody responses have been shown to be less thymus (or T cell) dependent than IgG antibody responses (Mitchell, 1974). It is not known which step in the differentiation of small lymphocytes into antibody-forming (plasma) cells is enhanced or triggered by T cells. The experiments of Roelants and Askonas (1972) suggest that B cells can be triggered into proliferation by antigen without the collaboration of T cells, but will not go on to produce antibodies. T cells might thus be necessary to induce IgG antibody production in antigen triggered B cells (e.g. Cheers and Miller, 1972). This suggestion can be tested by transfer of spleen cells of immunized mice which are not yet making IgG antibody, but will do so after transfer. Upon transfer, the ratio t Present address and correspondence: Dr S. Arrenbrecht, Department of Cell Biology, The Weizmann Institute of Science, Rehovot, Israel. § Present address: The Walter and Eliza Hall Institute, Melbourne, Australia.
485
486 S. Arrenbrecht and G. F. Mitchell of T cells to B cells can be altered artificially by anti-theta treatment and/or supplementation with T cells. In the experiments described below we have studied the anti-DNP antibody response to dinitrophenylated bovine gamma-globulin (DNP-BGG), a response which is thymus-dependent, and for which the kinetics of IgM and IgG formation have been determined (Arrenbrecht, 1974).
MATERIALS AND METHODS Animals For preliminary experiments, Charles River mice (Dr Wander, AG, Berne) were used; for the experiments reported, C3H/HeJ mice were used. Four to 6-week-old mice were used as thymocyte donors, 8-12-week-old mice for immunization and as recipients of spleen cells from immunized mice ('immune spleen cells'-ISC), and 15-20-week-old mice were used as irradiated recipients of thymocytes.
Immunization Mice were immunized with 100 pg of DNP-BGG (substitution rates DNP12- and DNP1 7-BGG). The preparation and immunogenicity of these antigens have been described (Arrenbrecht, 1974). Other antigens were sheep erythrocytes (SRC) (108 per mouse), keyhole limpet haemocyanin (KLH) (Pacific Biomarine Supply Company, Venice, U.S.A.) (100 jg per mouse), and BGG heat-aggregated, at 20 mg/ml and 600 for 30 minutes (100 Mg per mouse). All antigens were given in saline intraperitoneally (i.p.). Preparation of 'antigen (carrier) activated T cells' Mice were irradiated with 750 rads from a Phillips X-ray machine. The dose was lethal after a maximum of 10 days. Irradiated animals received antigen and/or thymocytes intravenously (i.v.) (1/2 thymus equivalent) within 3 hours after irradiation. Seven days later, their spleens were used as a source of 'antigen (carrier) activated T cells' (Mitchell and Miller, 1968). Cell suspensions Spleens and thymus were squashed with a loose fitting pestle in a glass tube at 380 or finely cut with scissors and the lumps repeatedly aspirated with a disposable syringe. All further handling of the cells was at 40. The medium was either Hanks's or Earle's balanced salt solution (BSS). After suspension, the cells were washed once and counted with Turk's solution and BSS with Evans Blue to assess viability. Cell suspensions with less than 80 per cent viable cells were discarded.
Antibody-forming cell (ABFC) assay ABFC were detected by the Jerne plaque test using agarose in minimal Eagle's medium with 5 per cent foetal calf serum. Indicator erythrocytes were SRC or SRC substituted with trinitrophenyl (TNP) groups by the method of Rittenberg and Pratt (1969). Indirect (presumably 7S, IgG) ABFC were detected by incubating the plates, prior to the addition of complement, with a 1:200 dilution of a rabbit anti-Brucella serum raised against bacteria coated with a hyperimmune mouse-anti-Brucella serum. This serum caused a depression of 3rd and 4th day (presumably IgM) ABFC counts by approximately
487 Adoptive IgG Antibody Response 1/4. This was taken into account when indirect ABFC numbers were calculated (see Fig. 1). Bleeding Mice were bled either from the retro-orbital plexus or by heart puncture and the serum separated.
Haemagglutinations Haemagglutination titres (HAT) were determined in U-type Microtiter plates (Cooke Engineering Company, Alexandria, Virginia, U.S.A.). Phosphate-buffered saline (PBS) containing 1 mg/ml of bovine serum albumin was used as diluent. Final red cell concentration was 025. For the detection of antibody (AB) in sera from DNP-BGG immunized mice, TNP-SRC as prepared for the ABFC assay or TNP-HRC (human erythrocytes) were used. For anti-SRC HAT, the first well showing no carpet formation was taken as the endpoint. For anti-TNP HAT, plates were stood upright, until the cells began to slide down and the titre was read as the first well with an even streaming of cells. In some experiments, we tried to inactivate IgM type antibodies in serum by 2-mercaptoethanol (2-ME) (0-1 M final concentration, 370, 30 minutes). However, increased HAT were often observed after 2-mercaptoethanol treatment and the method was therefore considered to be unreliable. Instead, sucrose gradient ultracentrifugation was used to differentiate between 19S (1gM) and 7S (IgG) antibodies.
Ultracentrifugation Linear gradients of sucrose in PBS, pH 7-2, were prepared by layering 1 l-ml amounts of 40, 30, 20 and 10 per cent sucrose solutions on top of each other. The tubes were left undisturbed for 1 hour, then 0X2 ml of serum, diluted with the same volume of water, was overlaid. Such gradients were swung in a Beckman Spinco SW 39 rotor at 35000 rev/min for 9 hours. Concave gradients (10-40 per cent sucrose in PBS) were prepared in a gradient mixer, and serum applied as above. Concave gradients were swung in a SW 50-1 rotor at 50,000 rev/min for 7 hours. At the end of the run, fractions were collected from the bottom of the tubes through a hole punched with a 25 gauge needle. Three drops usually constituted one fraction (about 300 p1). Haemoglobin (5.4S) served as a marker. For an assessment of the amounts of AB present in the IgM and IgG region of the gradients, HAT of the individual fraction were determined. The sum of the HAT of those fractions making up one peak was used as a measure of the relative amount ofAB (RAAB) present in the two regions of each gradient. T cell-depletion of spleen cells An anti-theta serum was prepared in a rabbit by injection of the saline-insoluble fraction of three C3H mouse brains, emulsified in saline, s.c. Three weeks later, a second injection was given i.p. Ten days later, blood was collected and 25 ml of the serum absorbed with AKR mouse brain (saline-insoluble fraction), spleen, lymph node, bone marrow and thymus cells from thirty mice. Cytotoxicity tests were performed by first incubating 2-5 x 106 cells with 100 pl of serial antiserum dilutions in BSS in the cold, followed by one wash and incubation in 100 yl of 10 per cent agarose-absorbed guinea-pig serum in BSS at G
S. Arrenbrecht and G. F. Mitchell 370 for 30 minutes. After one wash, the cells were resuspended in BSS with 0 05 per cent Trypan Blue. While unabsorbed serum had a high cytotoxic titre for C3H, AKR and Charles River lymph node, thymus and bone marrow cells, the absorbed serum had no cytotoxic effect on AKR cells, but still killed 90 per cent of C3H and Charles River thymocytes, 60-70 per cent of lymph node cells and about 10 per cent bone marrow cells (two experiments in triplicate). The highest serum dilution giving this maximal killing of lymph node cells was used to calculate the amount of serum needed to deplete spleen cells of theta-positive cells. Thus, 1 ml of serum was incubated with 3 x 108 cells in 6 ml of BSS, followed after one wash by 6 ml of 10 per cent agarose-absorbed complement. Before transfer, cells so treated were washed twice.
488
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