- Email: [email protected]
Production of antibodies in vitro in cultures of murine lymphocytes
304
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
The colonies themselves may be fixed in situ and their nonsecreted components analyzed. Finally, the enumeration and characterization of B cell colonies growing on paper disks permit the study of repertoire selection, as LPS-driven colony formation reveals the available repertoire of antigen-binding clones while antigen-driven colonies represent a selected subset of clones expressed during the immune response.
[24] P r o d u c t i o n o f A n t i b o d i e s in V i t r o in C u l t u r e s of Murine Lymphocytes By
TOSHIKI ISHIHARA
and C.
GARRISON FATHMAN
Introduction The elucidation of the mechanism(s) of antibody production is of great importance in understanding the immune system. This mechanism(s) includes interactions among T cells, B cells, antigen, antigen-presenting cells, various factors, and, for certain responses, requires histocompatibility among interacting cells. In an attempt to understand these interactions, several assay systems have been developed to study antibody production in vitro. For detection of antibody production in vitro, two techniques are commonly used as follows: 1. Plaque assay (see this volume [15]) 2. Direct measurement of antibody in culture supernatant The plaque assay is a method for counting antibody producing cells in culture; however, the total amount of antibody produced cannot be detected. The latter method uses ELISA or RIA to quantitate antibody secretion. The methodology described here is a system we have developed in our laboratory to directly measure antibody in culture by ELISA.I,2 This system relies on helper T cell clones as a source of T cells, and antigenspecific antibody is assayed directly from the culture supernatant of T 1 M. Shigeta, S. T a k a h a r a , S. J. K n o x , T. Ishihara, E. S. Vitetta, and C. G. F a t h m a n , J. Immunol. 136, 34 (1986). 2 T. Ishihara, S. Takahara, and C. G. F a t h m a n , J. Immunol. 136, 39 (1986).
METHODS IN ENZYMOLOGY, VOL. 150
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
[24]
in vitro PRODUCTIONOF ANTIBODIES
305
cell-depleted spleen cells reconstituted with cloned T helper cells by an ELISA method. 3 In this system, T cells, B cells, antigen-presenting cells, and antigen are cocultured for 6 days and antigen-specific antibodies are measured on the supernatant using an ELISA assay. To get reliable and reproducible data, highly enriched populations of cells are required. As a source of T cells instead of T cell clones, immunized lymph node cells, 4 or T cell hybridomas, 5 can be used. As a source of B cells, murine spleen cells are depleted of T cells by treatment with anti-Thy-1 antibody or by a cocktail of anti-T cell antibodies and complement (see this series, Vol. 108 [21]). This T cell-depleted fraction contains the accessory cell population. Removal of this population from the B cell source is occasionally necessary (e.g., assessing the major histocompatibility complex (MHC) restriction of interaction between T and accessory cells). This can be achieved by twice passing the B cell source through a Sephadex G-10 column 6 (see this series, Vol. 108 [29]). Methods Materials
Culture plate, 24-well (Flow Laboratories) Pipets (sterile Pasteur pipets; sterile 1-, 5-, and 10-ml volumetric glass or plastic pipets Test tubes (sterile 10 and 50 ml) Syringe (sterile for G-10 column) Glass wool Culture dish (sterile, 5 cm diameter) Hemocytometer for counting cells Microtiter plate (96-well for ELISA) (Flow Laboratories) H202 o-Phenylenediamine (Sigma Chemical Co.) Ficoll-Hypaque Sephadex G-10 (Pharmacia) H2804 Tween 20 ELISA plate reader (Flow Laboratories) 3A. Voller, A. Bartlett, and D. E. Bidwell,J. Clin. Pathol. 1, 507 (1978). 4 R. J. Noelle, E. C. Snow, J. W. Uhr, and E. S. Vitetta, Proc. Natl. Acad. Sci. U.S.A. 80, 6628 0983). s N. W. Roehm, P. Marrack, and J. W. Kappler, J. Exp. Med. 158, 317 (1983). 6 L. Ly and R. Mishell, J. Immunol. Methods 5, 239 (1974).
306
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
Media Hanks' balanced salt solution (HBSS) for washing cells RPMI-1640 medium Complete media for culture consists of RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 12 mM HEPES, 3 x 10-5 M 2-mercaptoethanol, 100 U/ml penicillin, and 100/zg/ml streptomycin
Antibodies Anti-Thy-I antibody (available from New England Nuclear) (we use AT83A anti-Thy-l.2 monoclonal antibody)7 Horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody can be obtained commercially (Miles Laboratories)
Complement Guinea pig or rabbit complement (Cedarlane Laboratories, Ltd., Westbury, NY)
Buffers PBS Citrate buffer, 0.1 M, pH 5.0, for substrate buffer in the ELISA assay
Preparation of B Cells (see also this series, Vol. 108 [21]) Murine spleen cells are treated with anti-Thy-1 antibody and complement to deplete T cells. The optimal dilution of anti-Thy-1 antibody and complement should be determined in preliminary tests. The efficiency of T cell depletion can be assayed by the response of the T-depleted population to T cell mitogens such as concanavalin A by cytofluorography (see this series, Vol. 108 [19]) or by direct visualization by fluorescence microscopy (see this series, Vol. 108 [41]). We usually recover approximately 5 x 10 7 B + accessory cells from one spleen following this procedure: 1. Mice are killed by cervical dislocation, the spleen is removed, and placed in cold HBSS. 2. Single-cell suspensions are prepared by standard techniques (see this series, Vol. 108 [6]). 3. Cell suspensions are transferred to 15-ml test tubes and allowed to stand for 5 min to allow debris to settle. 4. Transfer supernatant containing cell suspension to another tube. 5. Wash the cell twice with HBSS by centrifugation at 1500 rpm. 7 M. Sarmiento, M. R. L o k e n , and F. W. Fitch,
Hybridoma 1,
15 (1981).
[24]
in vitro PRODUCTIONOF ANTIBODIES
307
6. To the pelleted cells, add appropriately diluted anti-Thy-1 antibody (4 ml/2 spleen/tube) and resuspend gently. 7. Incubate on ice for 30 min. 8. Fill the tube with ice-cold HBSS and centrifuge. 9. Remove all supernatant. 10. Add diluted complement (as determined previously) (2 ml/tube). 11. Incubate at 37° in a water bath for 45 min. 12. Wash cells twice with HBSS as above. 13. Repeat steps 6-12. 14. After washing, the cells are counted for viable cells and resuspended in complete media for culture.
Preparation of T Cells T cell clones are maintained by restimulation every 2 weeks with antigen and syngeneic irradiated spleen cells as described 8 (see also this volume [27]). For antibody production assays in vitro, these clones should be harvested 9 to 14days after the last restimulation to assure low background and a good response. 1. Harvest the T cell clones from culture by centrifugation at 1500 rpm. 2. Layer the cells onto 3 ml Ficoll-Hypaque. 3. Centrifuge at 1500 rpm (approximately 400 g) for 20 min at room temperature. 4. Collect the interface using a Pasteur ldipet and place the cells into fresh HBSS. 5. Wash the cells twice with HBSS. 6. Count the number of viable cells. 7. Suspend the cell pellets in complete media.
Removal of Adherent Cell Population (see also this series, Vol. 108 [29]) Adherent cell populations can be removed by twice passage over Sephadex G-10 columns. The cloned T cells can also be passaged over Sephadex G-10 columns to reduce the background. In our laboratory, a sterile disposable 5-ml syringe is used as a column. The Sephadex G-10 gel must be swollen in saline and autoclaved prior to use. The glass wool should also be autoclaved and washed prior to use. The amount of Sephadex G-10 used will vary depending upon the number of cells to be applied a M. Shigeta and C. G. Fathman, lmmunogenetics 14, 415 (1981).
308
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
to the column. We use I ml gel (wet volume) for 3-5 x 10 7 cells and the recovery rate is 40-60% for B cells and 90-100% for T cells. 1. Pack Sephadex G-10 gel into a 5-ml sterile syringe plugged with glass wool. 2. Wash the column with more than a 4-fold excess of gel volume of HBSS containing 5% FCS. 3. Apply the cell suspension (5 × 10 7 cells/m], approximately) and collect the effluent in a 50-ml sterile test tube. 4. Wash the column with 20-25 ml of HBSS/5% FCS and collect the effluent in the same test tube as in step 3. 5. Centrifuge the cells and resuspend the pelleted cells in HBSS/5% FCS. 6. Repeat steps 3-4. 7. Count viable cells. 8. Centrifuge the cells and resuspend the pelleted cells in complete media. Culture
A 24-well culture plate is used for this assay in our laboratory, but 96well microculture plates can also be used. The cells and antigen are mixed and cultured in a humidified 37° 4.5% CO2 incubator for 6 days; 1 x 10 4 cloned T cells and 3 × 106 B cells are cultured in 1 ml total volume. For supplementation of adherent cells following G-10 column passage, T celldepleted irradiated spleen cells, 1,2,9peritoneal macrophages,l° or dendritic cells, ll can be used. We use 1-3 × 106 T cell-depleted irradiated spleen cells per well. The dose of antigen used may vary depending on what antigen is being used and what type of response is being investigated. In the T N P - K L H system, the low-dose IgG response is seen at a concentration of 5 x 10-2 p,g/m] antigen and the high-dose IgM response is seen at I0/,~g/ml. 1.2 ELISA
The optimal dilution of peroxidase conjugated antibody and its specificity for Ig subclasses should be determined with control reagents prior to use.
9 y . Asano, M. Shigeta, C. G. Fathman, A. Singer, and R. J. Hodes, J. Exp. Med. 156, 350 (1982). l0 A. Bandeira, G. Pobor, S. Pettersson, and A. Countinho, J. Exp. Med. 157, 312 (1983). II K. Inaba, A. Granelli-Piperno, and R. M. Steinman, J. Exp. Med. 1 ~ 2040 (1983).
[25]
in vitro ANTIBODYPRODUCTION
309
1. To coat each well of the 96-well microtiter assay plate, 100/~1 of antigen solution (10/xg/ml in PBS) is added to each well and incubated for 1 hr at room temperature. 2. The wells are washed three times by adding 200/~l of PBS/0.05% Tween 20 (PBS-Tween) and decanting. 3. A 50-gl aliquot of culture supernatant obtained from 6-day cultures (see above) is then added to each well and incubated for 2 hr at room temperature. 4. Wash three times with PBS-Tween. 5. Add 50 ml of appropriately diluted peroxidase-conjugated anti-Ig antibody (specific for the subclass of interest) and incubate for 2 hr at room temperature. 6. Wash four to five times with PBS-Tween. 7. Add 50/zl of substrate solution containing 3 mg/ml o-phenylenediamine and 0.012% H202 in citrate buffer, pH 5.0. 8. Incubate for 5-30 min and, when the color change is observed, the reaction can be stopped by the addition of 50/.d of 4 N H2SO4. 9. Measure the optical density at 492 nm of each well with an ELISA plate reader.
[25] I n V i t r o P r o d u c t i o n o f A n t i b o d y in C u l t u r e s of H u m a n P e r i p h e r a l Blood L y m p h o c y t e s By DALE T. UMETSUand RAIF S. GEHA
Introduction The in vitro induction of antibody synthesis in murine systems has been a powerful technique available for many years; it has allowed investigators to dissect the requirements for the activation of B lymphocytes. The induction of antibody synthesis in human B cells has been more difficult, mostly because of the difficulty in immunizing human subjects. Because of this difficulty, most antibody responses examined have been polyclonal, and not antigen specific. We will describe in vitro techniques for the induction of polyclonal antibody synthesis. One of these techniques involves the use of human helper/inducer T cell clones, which is also able to induce IgE antibody synthesis. METHODS IN ENZYMOLOGY, VOL. 150
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
The colonies themselves may be fixed in situ and their nonsecreted components analyzed. Finally, the enumeration and characterization of B cell colonies growing on paper disks permit the study of repertoire selection, as LPS-driven colony formation reveals the available repertoire of antigen-binding clones while antigen-driven colonies represent a selected subset of clones expressed during the immune response.
[24] P r o d u c t i o n o f A n t i b o d i e s in V i t r o in C u l t u r e s of Murine Lymphocytes By
TOSHIKI ISHIHARA
and C.
GARRISON FATHMAN
Introduction The elucidation of the mechanism(s) of antibody production is of great importance in understanding the immune system. This mechanism(s) includes interactions among T cells, B cells, antigen, antigen-presenting cells, various factors, and, for certain responses, requires histocompatibility among interacting cells. In an attempt to understand these interactions, several assay systems have been developed to study antibody production in vitro. For detection of antibody production in vitro, two techniques are commonly used as follows: 1. Plaque assay (see this volume [15]) 2. Direct measurement of antibody in culture supernatant The plaque assay is a method for counting antibody producing cells in culture; however, the total amount of antibody produced cannot be detected. The latter method uses ELISA or RIA to quantitate antibody secretion. The methodology described here is a system we have developed in our laboratory to directly measure antibody in culture by ELISA.I,2 This system relies on helper T cell clones as a source of T cells, and antigenspecific antibody is assayed directly from the culture supernatant of T 1 M. Shigeta, S. T a k a h a r a , S. J. K n o x , T. Ishihara, E. S. Vitetta, and C. G. F a t h m a n , J. Immunol. 136, 34 (1986). 2 T. Ishihara, S. Takahara, and C. G. F a t h m a n , J. Immunol. 136, 39 (1986).
METHODS IN ENZYMOLOGY, VOL. 150
Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.
[24]
in vitro PRODUCTIONOF ANTIBODIES
305
cell-depleted spleen cells reconstituted with cloned T helper cells by an ELISA method. 3 In this system, T cells, B cells, antigen-presenting cells, and antigen are cocultured for 6 days and antigen-specific antibodies are measured on the supernatant using an ELISA assay. To get reliable and reproducible data, highly enriched populations of cells are required. As a source of T cells instead of T cell clones, immunized lymph node cells, 4 or T cell hybridomas, 5 can be used. As a source of B cells, murine spleen cells are depleted of T cells by treatment with anti-Thy-1 antibody or by a cocktail of anti-T cell antibodies and complement (see this series, Vol. 108 [21]). This T cell-depleted fraction contains the accessory cell population. Removal of this population from the B cell source is occasionally necessary (e.g., assessing the major histocompatibility complex (MHC) restriction of interaction between T and accessory cells). This can be achieved by twice passing the B cell source through a Sephadex G-10 column 6 (see this series, Vol. 108 [29]). Methods Materials
Culture plate, 24-well (Flow Laboratories) Pipets (sterile Pasteur pipets; sterile 1-, 5-, and 10-ml volumetric glass or plastic pipets Test tubes (sterile 10 and 50 ml) Syringe (sterile for G-10 column) Glass wool Culture dish (sterile, 5 cm diameter) Hemocytometer for counting cells Microtiter plate (96-well for ELISA) (Flow Laboratories) H202 o-Phenylenediamine (Sigma Chemical Co.) Ficoll-Hypaque Sephadex G-10 (Pharmacia) H2804 Tween 20 ELISA plate reader (Flow Laboratories) 3A. Voller, A. Bartlett, and D. E. Bidwell,J. Clin. Pathol. 1, 507 (1978). 4 R. J. Noelle, E. C. Snow, J. W. Uhr, and E. S. Vitetta, Proc. Natl. Acad. Sci. U.S.A. 80, 6628 0983). s N. W. Roehm, P. Marrack, and J. W. Kappler, J. Exp. Med. 158, 317 (1983). 6 L. Ly and R. Mishell, J. Immunol. Methods 5, 239 (1974).
306
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
Media Hanks' balanced salt solution (HBSS) for washing cells RPMI-1640 medium Complete media for culture consists of RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 12 mM HEPES, 3 x 10-5 M 2-mercaptoethanol, 100 U/ml penicillin, and 100/zg/ml streptomycin
Antibodies Anti-Thy-I antibody (available from New England Nuclear) (we use AT83A anti-Thy-l.2 monoclonal antibody)7 Horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody can be obtained commercially (Miles Laboratories)
Complement Guinea pig or rabbit complement (Cedarlane Laboratories, Ltd., Westbury, NY)
Buffers PBS Citrate buffer, 0.1 M, pH 5.0, for substrate buffer in the ELISA assay
Preparation of B Cells (see also this series, Vol. 108 [21]) Murine spleen cells are treated with anti-Thy-1 antibody and complement to deplete T cells. The optimal dilution of anti-Thy-1 antibody and complement should be determined in preliminary tests. The efficiency of T cell depletion can be assayed by the response of the T-depleted population to T cell mitogens such as concanavalin A by cytofluorography (see this series, Vol. 108 [19]) or by direct visualization by fluorescence microscopy (see this series, Vol. 108 [41]). We usually recover approximately 5 x 10 7 B + accessory cells from one spleen following this procedure: 1. Mice are killed by cervical dislocation, the spleen is removed, and placed in cold HBSS. 2. Single-cell suspensions are prepared by standard techniques (see this series, Vol. 108 [6]). 3. Cell suspensions are transferred to 15-ml test tubes and allowed to stand for 5 min to allow debris to settle. 4. Transfer supernatant containing cell suspension to another tube. 5. Wash the cell twice with HBSS by centrifugation at 1500 rpm. 7 M. Sarmiento, M. R. L o k e n , and F. W. Fitch,
Hybridoma 1,
15 (1981).
[24]
in vitro PRODUCTIONOF ANTIBODIES
307
6. To the pelleted cells, add appropriately diluted anti-Thy-1 antibody (4 ml/2 spleen/tube) and resuspend gently. 7. Incubate on ice for 30 min. 8. Fill the tube with ice-cold HBSS and centrifuge. 9. Remove all supernatant. 10. Add diluted complement (as determined previously) (2 ml/tube). 11. Incubate at 37° in a water bath for 45 min. 12. Wash cells twice with HBSS as above. 13. Repeat steps 6-12. 14. After washing, the cells are counted for viable cells and resuspended in complete media for culture.
Preparation of T Cells T cell clones are maintained by restimulation every 2 weeks with antigen and syngeneic irradiated spleen cells as described 8 (see also this volume [27]). For antibody production assays in vitro, these clones should be harvested 9 to 14days after the last restimulation to assure low background and a good response. 1. Harvest the T cell clones from culture by centrifugation at 1500 rpm. 2. Layer the cells onto 3 ml Ficoll-Hypaque. 3. Centrifuge at 1500 rpm (approximately 400 g) for 20 min at room temperature. 4. Collect the interface using a Pasteur ldipet and place the cells into fresh HBSS. 5. Wash the cells twice with HBSS. 6. Count the number of viable cells. 7. Suspend the cell pellets in complete media.
Removal of Adherent Cell Population (see also this series, Vol. 108 [29]) Adherent cell populations can be removed by twice passage over Sephadex G-10 columns. The cloned T cells can also be passaged over Sephadex G-10 columns to reduce the background. In our laboratory, a sterile disposable 5-ml syringe is used as a column. The Sephadex G-10 gel must be swollen in saline and autoclaved prior to use. The glass wool should also be autoclaved and washed prior to use. The amount of Sephadex G-10 used will vary depending upon the number of cells to be applied a M. Shigeta and C. G. Fathman, lmmunogenetics 14, 415 (1981).
308
MODELS AND ASSAYS OF B AND T LYMPHOCYTES
[24]
to the column. We use I ml gel (wet volume) for 3-5 x 10 7 cells and the recovery rate is 40-60% for B cells and 90-100% for T cells. 1. Pack Sephadex G-10 gel into a 5-ml sterile syringe plugged with glass wool. 2. Wash the column with more than a 4-fold excess of gel volume of HBSS containing 5% FCS. 3. Apply the cell suspension (5 × 10 7 cells/m], approximately) and collect the effluent in a 50-ml sterile test tube. 4. Wash the column with 20-25 ml of HBSS/5% FCS and collect the effluent in the same test tube as in step 3. 5. Centrifuge the cells and resuspend the pelleted cells in HBSS/5% FCS. 6. Repeat steps 3-4. 7. Count viable cells. 8. Centrifuge the cells and resuspend the pelleted cells in complete media. Culture
A 24-well culture plate is used for this assay in our laboratory, but 96well microculture plates can also be used. The cells and antigen are mixed and cultured in a humidified 37° 4.5% CO2 incubator for 6 days; 1 x 10 4 cloned T cells and 3 × 106 B cells are cultured in 1 ml total volume. For supplementation of adherent cells following G-10 column passage, T celldepleted irradiated spleen cells, 1,2,9peritoneal macrophages,l° or dendritic cells, ll can be used. We use 1-3 × 106 T cell-depleted irradiated spleen cells per well. The dose of antigen used may vary depending on what antigen is being used and what type of response is being investigated. In the T N P - K L H system, the low-dose IgG response is seen at a concentration of 5 x 10-2 p,g/m] antigen and the high-dose IgM response is seen at I0/,~g/ml. 1.2 ELISA
The optimal dilution of peroxidase conjugated antibody and its specificity for Ig subclasses should be determined with control reagents prior to use.
9 y . Asano, M. Shigeta, C. G. Fathman, A. Singer, and R. J. Hodes, J. Exp. Med. 156, 350 (1982). l0 A. Bandeira, G. Pobor, S. Pettersson, and A. Countinho, J. Exp. Med. 157, 312 (1983). II K. Inaba, A. Granelli-Piperno, and R. M. Steinman, J. Exp. Med. 1 ~ 2040 (1983).
[25]
in vitro ANTIBODYPRODUCTION
309
1. To coat each well of the 96-well microtiter assay plate, 100/~1 of antigen solution (10/xg/ml in PBS) is added to each well and incubated for 1 hr at room temperature. 2. The wells are washed three times by adding 200/~l of PBS/0.05% Tween 20 (PBS-Tween) and decanting. 3. A 50-gl aliquot of culture supernatant obtained from 6-day cultures (see above) is then added to each well and incubated for 2 hr at room temperature. 4. Wash three times with PBS-Tween. 5. Add 50 ml of appropriately diluted peroxidase-conjugated anti-Ig antibody (specific for the subclass of interest) and incubate for 2 hr at room temperature. 6. Wash four to five times with PBS-Tween. 7. Add 50/zl of substrate solution containing 3 mg/ml o-phenylenediamine and 0.012% H202 in citrate buffer, pH 5.0. 8. Incubate for 5-30 min and, when the color change is observed, the reaction can be stopped by the addition of 50/.d of 4 N H2SO4. 9. Measure the optical density at 492 nm of each well with an ELISA plate reader.
[25] I n V i t r o P r o d u c t i o n o f A n t i b o d y in C u l t u r e s of H u m a n P e r i p h e r a l Blood L y m p h o c y t e s By DALE T. UMETSUand RAIF S. GEHA
Introduction The in vitro induction of antibody synthesis in murine systems has been a powerful technique available for many years; it has allowed investigators to dissect the requirements for the activation of B lymphocytes. The induction of antibody synthesis in human B cells has been more difficult, mostly because of the difficulty in immunizing human subjects. Because of this difficulty, most antibody responses examined have been polyclonal, and not antigen specific. We will describe in vitro techniques for the induction of polyclonal antibody synthesis. One of these techniques involves the use of human helper/inducer T cell clones, which is also able to induce IgE antibody synthesis. METHODS IN ENZYMOLOGY, VOL. 150
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.