Cell, Vol. 10, 27-34,
January
1977,
Copyright
0 1977 by MIT
Clonal Growth and Maturation to lmmunoglobulin Secretion in Vitro of Every Growth-Inducible B Lymphocyte Jan Andersson University of Uppsala Biomedicum Uppsala, Sweden Antonio Coutinho, Waldemar Melchers Base1 Institute for Immunology Basel, Switzerland
The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately lo4 fold by the addition of 2 x lo6 normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG6 myeloma tumor cells secreting IgG,. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth*upporfing factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cells abolishes their growth-enhancing properties. Thymus cells can be syngeneic or allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-6 week old C,H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of X63-AG6 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shows that these B cells divide every 16 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immunoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.
suspension cultures (Melchers et al., 1975b). These conditions allow us, for the first time, to measure growth of normal murine lymphocytes by an increase in the number of cells in culture. This can be considered a significant advance in our aims to establish continuous cultures of growing normal lymphocytes, to study growth and differentiation control of these lymphocytes, and to evaluate more accurately, while quantitatively, in vitro responses of lymphocytes to stimulation by antigens or mitogens. It became evident, however, that these in vitro growth conditions did not allow every lymphocyte to grow. This, on the other hand, appears mandatory, in particular, for quantitative evaluations of in vitro lymphocyte responses to stimulation, which may be defined as the number of responding cells, the number of divisions which the responding cells undergo, and the efficiency of maturation of the stimulated cells into effector functions. The effector function of B lymphocytes, immunoglobulin secretion, can be measured on the singlecell level by the hemolytic plaque assay (Jerne, Nordin, and Henry, 1963), which has recently been modified so that every immunoglobulin-secreting cell can now be detected (Gronowicz, Coutinho, and Melchers, 1976). Such effector functions of T lymphocytes still cannot be measured on the single-cell level. Thus in the present paper, we have concentrated on clonal growth and maturation of murine B lymphocytes only. Our aim was to find culture conditions in which every B lymphocyte which has the capacity to initiate growth will grow. Normal B cells which can be stimulated to growth by mitogens such as lipopolysaccharide (LPS) (Andersson, Sjdberg, and Mdller, 1972; Andersson et al., 1973) and myeloma tumor cells which have the capacity for growth inherent in their transformed state are used to study influences of alterations in the culture conditions on the frequency of growing cells. Thymus cells prove to be inert “feeder” cells which increase the frequency of normal, LPS-stimulated, growing B cells and of growing myeloma tumor cells to near one. Analysis of the growth kinetics of single clones of immunoglobulin M (IgM)-secreting B cells suggests that every dividing normal B cell secretes immunoglobulin.
Introduction
Results
We have previously described conditions for prolonged growth of normal murine T and B lymphocytes and their maturation into effector functions in
The Influence of Inert Cells, Such as Sheep Red Cells or Purified Thymus Cells, on the Frequency of LPS-Induced B Lymphocytes Growing in Vitro in Suspension Cultures Inert cells are frequently used as “feeder” cells to improve conditions for cellular growth in vitro. For
Lernhardt,
and Fritz
Summary
All correspondence should be addressed to Dr. Melchers at the Base1 institute for Immunology, Grenzacherstrasse 487, Postfach 4005 Easel 5. Switzerland.
Cell 28
in vitro lymphocyte cultures, irradiated syngeneic lymphocytes are often added (Quintans and Lefkovits, 1973). In our initial experiments, two types of inert cells-sheep red cells and purified thymus ceils-were added to suspension cultures of splenic lymphocytes in the presence of LPS to test for their possible influence on the frequencies of growing cells. Since a large excess of inert cells was added to the cultures (see below), growth could no longer be monitored by cell numbers. Growth of B lymphocytes can, however, also be monitored by the number of IgM-secreting, plaque-forming cells (PFC) in culture (Melchers et al., 1975b; Andersson et al., 1976). A modified hemolytic plaque assay, using protein A-coated sheep red cells and immunoglobulin-specific antibodies (Gronowicz et al., 1976), allows us to detect every B cell secreting a given type or class of immunoglobulin. Sheep red cells added to the cultures of limiting numbers of B cells improved the frequency of growing B cells slightly. Growth and maturation into immunoglobulin secretion of B cells could be detected at 10 fold lower B cell concentrations (Figure 1). Addition of nylon-wool-purified (Julius, Simpson, and Herzenberg, 1973) thymus cells obtained from 4-6 week old syngeneic mice, on the other hand, dramatically improved the growth conditions for B cells in suspension cultures (Figure 1). Concentrations of 2 x lo6 thymus cells per ml or above increased the frequency of splenic B cells growing and producing plaques in the presence of LPS by approximately lo4 fold. In fact, it appeared that almost every splenic B cell could initiate growth
and development into plaque-forming cells with LPS in the presence of purified thymus cells. (A more accurate determination of the frequency of LPS-reactive B cells in spleen is given below). Concentrations of thymus cells below lo6 cells per ml became insufficient for this frequency-enhancing effect on B cell growth (Figure 1).
Influence of Thymus Cells on the Frequency of Growing IgG-Secreting Myeloma Cells Myeloma tumor cells of the IgG,-secreting tissue culture line X63-AG8 (Kohler and Milstein, 1975) of the MOPC 21 mouse myeloma (Potter, 1967) initiated growth in suspension cultures at cell concentrations of 3 x 10Vml and upwards when no other cells were added (Figure 2). Addition of 2 x lo6 syngeneic Balb/c thymus cells per ml to suspension cultures of logarithmically growing X63-AG8 tumor cells increased the frequency of growing cells to near 1 (Figure 2; see also below for a more detailed analysis of this frequency). This suggests that every X63-AG8 tumor cell can grow in suspension culture into a single clone of IgG-secreting cells when thymus cells are present in the culture. Since tumor B cells, such as the X63-AG8-cells, could grow more frequently in suspension culture in the presence of thymus cells, it is improbable that thymus cells are involved in the stimulatory phase of B cell growth, since tumor cells do not require addition of external mitogen, such as LPS,
NO OF CELLS/CULTURE
Figure 1. The Effect of Addition of Sheep Red Cells and Mouse Thymus Cells on the Frequency of LPS-Reactive Spleen Cells Which Grow and Secrete IgM-Forming Hemolytic Plaques Graded numbers of spleen cells of C$bTlf mice (abscissa) were cultured in the presence of 50 pg/ml LPS and 2 x 10’ sheep red cells (SK) per ml (+. .+), 1 x 105syngeneic thymus cells per ml (A-A), 5 x lo5 thymus cells per ml (U+), 2 x lo6 thymus cells per ml (O+), or no added cells (O-O). Cultures were assayed for Igkkecreting, plaque-forming cells at day 5. The data in the figure represent the average of five cultures.
Figure 2. The Effect of Addition of Mouse Frequency of Growing X63-AGE Myeloma Secrete lgG,-Forming Hemolytic Plaques Graded numbers the absence (-) thymus cells per assayed at day 5. four cultures.
Thymus Tumor
Cells Cells
on the Which
of X63-AG6 myeloma tumor cells were grown in or presence (+) of 2 x IO8 syngeneic (Balbk) ml. IgG,-secreting, plaque-forming cells were The data in the figure represent the average of
Clonal 29
B Cell Growth
in Vitro
to grow. It is more probable growth-supporting factors auxotrophic in suspension
that thymus cell supply for which B cells are cultures.
Conditioning of Culture Medium and Plastic Dishes Used for Culture by Preincubation with Thymus Cells Myeloma tumor cells of the IgG,-secreting tissue culture line X63-AG8 of MOPC 21 mouse myeloma were grown in culture medium or in plastic dishes which had been conditioned by a 24 hr preincubation with 2 x lo6 thymus cells per ml. The thymus cells were removed after the preincubation period from the dishes by repeated pipetting of the medium, followed by one wash with medium, and from the medium by centrifugation and filtration. It is evident from the data in Table 1 that both the conditioning of the culture dishes and the conditioning of the medium were sufficient to achieve the same enhancing effect on the frequency of growing B cells as did the addition of 2 x lo6 thymus cells per ml. These results indicate that the growth-supporting factors are released from the thymus cells into the medium and, in part, stick to plastic surfaces. Influence of Nonirradiated and of Irradiated Thymus Cells on the Frequency of Growing B Cells Irradiation of syngeneic thymus cells resulted in a total loss of the capacity of these cells to increase the frequencies of growing B cells. Thus when nylon-purified thymus cells of CSH/Tir mice were irradiated with 1500 rad and then added to syngeneic splenic lymphocytes in the presence of LPS, the frequency of growing B cells decreased to levels measured in the absence of any thymus cells Table
1. Effect
of Conditioning
the Culture
IgG,-Secreting
PFC per Culture
(Average
X63-AG8 Culture
per
Cells
6000
Medium
and the Plastic
of Five Cultures)
Emerging
(Table 2). We conclude that the thymus cells cannot be irradiated. The release of growth-supporting factors into the medium and onto the plastic dishes must therefore involve radiation-sensitive release of thus far unknown material which can stick to plastic surfaces. Syngeneic, Semiallogeneic, and Allogeneic Thymus Cells as Growth-Supporting Cells for B Cells Splenic lymphocytes from C57BV6J mice were cultured in the presence of LPS and 2 x lo6 thymus cells per ml from either C57Bl/GJ-mice, C57BV6J x DBA/2 Fl mice, or DBA/2-mice. The number of all IgM-secreting, plaque-forming cells emerging at Table 2. The Effect of Irradiation of Thymus to Enhance the Frequency of LPS-Stimulated. IgM-Secreting ing in Cultures”
PFC per Culture Containing:
(Average
Cells on Their Ability Growing B Cells
of Four Cultures)
Emerg-
2 x lo8 plus Live Thymus Cells per ml
2 x 106 plus Irradiated’ Thymus Cells per ml
60,000
~10,000
>10,000
20,000
>10,000
350
6,000
>10,000
10,000
>10,000
600
2.000
>10.000
210.000
210,000
200
200
6,000
4,000
7,000
80
January
1977,
Copyright
0 1977 by MIT
Clonal Growth and Maturation to lmmunoglobulin Secretion in Vitro of Every Growth-Inducible B Lymphocyte Jan Andersson University of Uppsala Biomedicum Uppsala, Sweden Antonio Coutinho, Waldemar Melchers Base1 Institute for Immunology Basel, Switzerland
The frequency of normal murine B lymphocytes initiating growth in diluted suspension cultures in the presence of a B cell mitogen, such as lipopolysaccharide, can be increased approximately lo4 fold by the addition of 2 x lo6 normal thymus cells per ml. This increase in the frequency of growing cells by thymus cells can also be observed with X63-AG6 myeloma tumor cells secreting IgG,. Thus thymus cells may not contribute growth-stimulating factors, but may supply growth*upporfing factors. Culture medium and plastic dishes can be conditioned by preincubation with thymus cells for a day after which the thymus cells may be omitted from further culture for maximal B cell growth. Irradiation of thymus cells abolishes their growth-enhancing properties. Thymus cells can be syngeneic or allogeneic with the growing B cells. The frequency of growing LPS-reactive, normal B cells in spleen of 6-6 week old C,H/Tif mice was determined by limiting dilution analysis to be one of three splenic B cells. With this limiting dilution analysis, it was also shown that the cloning efficiency of X63-AG6 myeloma tumor cells in suspension culture in the presence of thymus cells is practically 100%. Analysis of the growth kinetics of single clones of LPS-reactive, normal B cells shows that these B cells divide every 16 hr. Within the first 126 hr of growth, every B cell in the clone divides, and every dividing B cell in this clone secretes sufficient immunoglobulin to form a hemolytic plaque. The conditions of in vitro suspension cultures of murine B lymphocytes are therefore perfect to the extent that every B cell capable of growth will grow as a single clone.
suspension cultures (Melchers et al., 1975b). These conditions allow us, for the first time, to measure growth of normal murine lymphocytes by an increase in the number of cells in culture. This can be considered a significant advance in our aims to establish continuous cultures of growing normal lymphocytes, to study growth and differentiation control of these lymphocytes, and to evaluate more accurately, while quantitatively, in vitro responses of lymphocytes to stimulation by antigens or mitogens. It became evident, however, that these in vitro growth conditions did not allow every lymphocyte to grow. This, on the other hand, appears mandatory, in particular, for quantitative evaluations of in vitro lymphocyte responses to stimulation, which may be defined as the number of responding cells, the number of divisions which the responding cells undergo, and the efficiency of maturation of the stimulated cells into effector functions. The effector function of B lymphocytes, immunoglobulin secretion, can be measured on the singlecell level by the hemolytic plaque assay (Jerne, Nordin, and Henry, 1963), which has recently been modified so that every immunoglobulin-secreting cell can now be detected (Gronowicz, Coutinho, and Melchers, 1976). Such effector functions of T lymphocytes still cannot be measured on the single-cell level. Thus in the present paper, we have concentrated on clonal growth and maturation of murine B lymphocytes only. Our aim was to find culture conditions in which every B lymphocyte which has the capacity to initiate growth will grow. Normal B cells which can be stimulated to growth by mitogens such as lipopolysaccharide (LPS) (Andersson, Sjdberg, and Mdller, 1972; Andersson et al., 1973) and myeloma tumor cells which have the capacity for growth inherent in their transformed state are used to study influences of alterations in the culture conditions on the frequency of growing cells. Thymus cells prove to be inert “feeder” cells which increase the frequency of normal, LPS-stimulated, growing B cells and of growing myeloma tumor cells to near one. Analysis of the growth kinetics of single clones of immunoglobulin M (IgM)-secreting B cells suggests that every dividing normal B cell secretes immunoglobulin.
Introduction
Results
We have previously described conditions for prolonged growth of normal murine T and B lymphocytes and their maturation into effector functions in
The Influence of Inert Cells, Such as Sheep Red Cells or Purified Thymus Cells, on the Frequency of LPS-Induced B Lymphocytes Growing in Vitro in Suspension Cultures Inert cells are frequently used as “feeder” cells to improve conditions for cellular growth in vitro. For
Lernhardt,
and Fritz
Summary
All correspondence should be addressed to Dr. Melchers at the Base1 institute for Immunology, Grenzacherstrasse 487, Postfach 4005 Easel 5. Switzerland.
Cell 28
in vitro lymphocyte cultures, irradiated syngeneic lymphocytes are often added (Quintans and Lefkovits, 1973). In our initial experiments, two types of inert cells-sheep red cells and purified thymus ceils-were added to suspension cultures of splenic lymphocytes in the presence of LPS to test for their possible influence on the frequencies of growing cells. Since a large excess of inert cells was added to the cultures (see below), growth could no longer be monitored by cell numbers. Growth of B lymphocytes can, however, also be monitored by the number of IgM-secreting, plaque-forming cells (PFC) in culture (Melchers et al., 1975b; Andersson et al., 1976). A modified hemolytic plaque assay, using protein A-coated sheep red cells and immunoglobulin-specific antibodies (Gronowicz et al., 1976), allows us to detect every B cell secreting a given type or class of immunoglobulin. Sheep red cells added to the cultures of limiting numbers of B cells improved the frequency of growing B cells slightly. Growth and maturation into immunoglobulin secretion of B cells could be detected at 10 fold lower B cell concentrations (Figure 1). Addition of nylon-wool-purified (Julius, Simpson, and Herzenberg, 1973) thymus cells obtained from 4-6 week old syngeneic mice, on the other hand, dramatically improved the growth conditions for B cells in suspension cultures (Figure 1). Concentrations of 2 x lo6 thymus cells per ml or above increased the frequency of splenic B cells growing and producing plaques in the presence of LPS by approximately lo4 fold. In fact, it appeared that almost every splenic B cell could initiate growth
and development into plaque-forming cells with LPS in the presence of purified thymus cells. (A more accurate determination of the frequency of LPS-reactive B cells in spleen is given below). Concentrations of thymus cells below lo6 cells per ml became insufficient for this frequency-enhancing effect on B cell growth (Figure 1).
Influence of Thymus Cells on the Frequency of Growing IgG-Secreting Myeloma Cells Myeloma tumor cells of the IgG,-secreting tissue culture line X63-AG8 (Kohler and Milstein, 1975) of the MOPC 21 mouse myeloma (Potter, 1967) initiated growth in suspension cultures at cell concentrations of 3 x 10Vml and upwards when no other cells were added (Figure 2). Addition of 2 x lo6 syngeneic Balb/c thymus cells per ml to suspension cultures of logarithmically growing X63-AG8 tumor cells increased the frequency of growing cells to near 1 (Figure 2; see also below for a more detailed analysis of this frequency). This suggests that every X63-AG8 tumor cell can grow in suspension culture into a single clone of IgG-secreting cells when thymus cells are present in the culture. Since tumor B cells, such as the X63-AG8-cells, could grow more frequently in suspension culture in the presence of thymus cells, it is improbable that thymus cells are involved in the stimulatory phase of B cell growth, since tumor cells do not require addition of external mitogen, such as LPS,
NO OF CELLS/CULTURE
Figure 1. The Effect of Addition of Sheep Red Cells and Mouse Thymus Cells on the Frequency of LPS-Reactive Spleen Cells Which Grow and Secrete IgM-Forming Hemolytic Plaques Graded numbers of spleen cells of C$bTlf mice (abscissa) were cultured in the presence of 50 pg/ml LPS and 2 x 10’ sheep red cells (SK) per ml (+. .+), 1 x 105syngeneic thymus cells per ml (A-A), 5 x lo5 thymus cells per ml (U+), 2 x lo6 thymus cells per ml (O+), or no added cells (O-O). Cultures were assayed for Igkkecreting, plaque-forming cells at day 5. The data in the figure represent the average of five cultures.
Figure 2. The Effect of Addition of Mouse Frequency of Growing X63-AGE Myeloma Secrete lgG,-Forming Hemolytic Plaques Graded numbers the absence (-) thymus cells per assayed at day 5. four cultures.
Thymus Tumor
Cells Cells
on the Which
of X63-AG6 myeloma tumor cells were grown in or presence (+) of 2 x IO8 syngeneic (Balbk) ml. IgG,-secreting, plaque-forming cells were The data in the figure represent the average of
Clonal 29
B Cell Growth
in Vitro
to grow. It is more probable growth-supporting factors auxotrophic in suspension
that thymus cell supply for which B cells are cultures.
Conditioning of Culture Medium and Plastic Dishes Used for Culture by Preincubation with Thymus Cells Myeloma tumor cells of the IgG,-secreting tissue culture line X63-AG8 of MOPC 21 mouse myeloma were grown in culture medium or in plastic dishes which had been conditioned by a 24 hr preincubation with 2 x lo6 thymus cells per ml. The thymus cells were removed after the preincubation period from the dishes by repeated pipetting of the medium, followed by one wash with medium, and from the medium by centrifugation and filtration. It is evident from the data in Table 1 that both the conditioning of the culture dishes and the conditioning of the medium were sufficient to achieve the same enhancing effect on the frequency of growing B cells as did the addition of 2 x lo6 thymus cells per ml. These results indicate that the growth-supporting factors are released from the thymus cells into the medium and, in part, stick to plastic surfaces. Influence of Nonirradiated and of Irradiated Thymus Cells on the Frequency of Growing B Cells Irradiation of syngeneic thymus cells resulted in a total loss of the capacity of these cells to increase the frequencies of growing B cells. Thus when nylon-purified thymus cells of CSH/Tir mice were irradiated with 1500 rad and then added to syngeneic splenic lymphocytes in the presence of LPS, the frequency of growing B cells decreased to levels measured in the absence of any thymus cells Table
1. Effect
of Conditioning
the Culture
IgG,-Secreting
PFC per Culture
(Average
X63-AG8 Culture
per
Cells
6000
Medium
and the Plastic
of Five Cultures)
Emerging
(Table 2). We conclude that the thymus cells cannot be irradiated. The release of growth-supporting factors into the medium and onto the plastic dishes must therefore involve radiation-sensitive release of thus far unknown material which can stick to plastic surfaces. Syngeneic, Semiallogeneic, and Allogeneic Thymus Cells as Growth-Supporting Cells for B Cells Splenic lymphocytes from C57BV6J mice were cultured in the presence of LPS and 2 x lo6 thymus cells per ml from either C57Bl/GJ-mice, C57BV6J x DBA/2 Fl mice, or DBA/2-mice. The number of all IgM-secreting, plaque-forming cells emerging at Table 2. The Effect of Irradiation of Thymus to Enhance the Frequency of LPS-Stimulated. IgM-Secreting ing in Cultures”
PFC per Culture Containing:
(Average
Cells on Their Ability Growing B Cells
of Four Cultures)
Emerg-
2 x lo8 plus Live Thymus Cells per ml
2 x 106 plus Irradiated’ Thymus Cells per ml
60,000
~10,000
>10,000
20,000
>10,000
350
6,000
>10,000
10,000
>10,000
600
2.000
>10.000
210.000
210,000
200
200
6,000
4,000
7,000
80
