Acta haemat. 62: 326-330 (1979)
Role of Serum Factors and Adherent Cells in Cloning of Human T Lymphocytes in Agar Culture1 H. D. Flad, A. J. Ulmer, M. H. Claesson and H. G. Opitz Laboratory of Immunology, Department of Microbiology, University of Ulm, Ulm; Anatomy Department A, University of Copenhagen, Copenhagen, and Institute for Immunology and Oncology, Bayer AG, Wuppertal
Key Words. Colony formation • Human T lymphocytes • 2-Mercaptoethanol • Serum factors Abstract. In the presence of serum, 2-mercaptoethanol-treated bovine serum albumin enhances T cell colony formation as does 2-mercaptoethanol. The factor only partially substitutes for FCS, but neither for the mitogen phytohemagglutinin nor for conditioned medium derived from cultures of adherent cells.
The low molecular weight thiol 2-mercaptoethanol (2-ME) has been shown to enhance antibody and mitogen induced re sponses of murine spleen cells in vitro [1, 5]. In systems measuring clonal proliferation of cells 2-ME enhances T lymphocyte colony formation [10], and is absolutely necessary for murine B lymphocyte colony formation m.
Recently Opitz et al. [8, 9] showed that a 2-ME-activated serum factor (MaSF) gener ated from fetal calf serum or mouse serum was capable of substituting for 2-ME or macrophages and for serum in the primary antibody response of mouse spleen cells in 1 Supported by ‘Deutsche Forschungsgemein schaft, Sonderforschungsbereich 112, Project C4’.
vitro. This factor had a molecular weight which was similar to that of albumin. The purpose of this study was to investi gate the effects of this factor on the growth of human T lymphocytes in three different systems. Material and Methods Cloning of Human T Lymphocytes. For growth of human T lymphocytes in agar, three different methods were employed: (1) A two-step method according to Claesson et al. [2], in which mononuclear cells were preincubated for 18 h with PHA followed by seeding of the cells in agar. (2) A one-step method, in which mononuclear cells or nonadherent cells were incorporated di rectly into the agar together with the mitogen PHA [3], (3) A one-step micromethod recently de veloped by Ulmer and Flad [13] in which blood mononuclear cells are grown in capillary tubes. There is no requirement for factors, erythrocytes
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Introduction
Serum Factors and Cloning of Human T Lymphocytes
% activated or nonactivatad 8 S A
327 (39)
% activated or nonactivated BSA
Fig. 1. The effect of addition of 2-ME, 2-ME-activated and nonactivated BSA to agar on colony growth of human T lymphocytes (2-step method). Cultures contain 5% FCS and 0.6% hu man AB serum. Left panel: © = Control cultures
without BSA and without 2-ME. All cultures without 2-ME. Right panel: • = Control cultures without BSA and with 2-ME (5 X 10~! M). All cultures contain 2-ME (5 X 10'5 M). Data are means of quadruplicates + SD.
or autologous serum for colony growth obtained by this method. Preparation of Serum Factors. 1 g of bovine serum albumin (Behringwerke, Marburg, FRG) was dissolved in 10 ml of RPMI 1640 medium and 2-ME was added at a final concentration of 10_3M. The preparation was incubated and di
alyzed as described [4, 8], A control preparation, not incubated with 2-ME was treated in the same way. Adherent Cell Conditioned Medium (AC-CM). A standard batch of AC-CM was prepared from adherent blood mononuclear cells as described elsewhere [3],
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Fig. 2. The effect of addition of 2-ME, 2-ME-activated and nonactivated BSA to agar on growth of human T lymphocytes (one-step micromcthod). All cul tures contain 10% FCS.
Flad/Ulmer/Claesson/Opitz
328 (40)
As shown in figure 1, activated BSA which is BSA treated with 2-ME - added at the second step of the culture system, en hances the number of colonies. The enhance ment is similar to that obtained by the addi tion of 2-ME. The control preparation nontreated BSA - has no enhancing effect on colony growth but is also enhancing in the presence of 2-ME. We asked the question whether or not the BSA preparations could be a substitute for serum. In table I two experiments are given. Both 2-ME-activated BSA and nonactivated BSA partially substitute for FCS in the preincubation step (table I). They do not substitute for the mitogen PHA (data not shown). On the other hand, 2-ME-acti vated BSA is able to substitute for FCS in the second step ( = agar step) while nonactivated BSA is not. It should be noted, how ever, that no colony growth was obtained in the complete absence of serum, i.e., in the absence of 0.6°/o human AB serum. The influence of activated and nonactivated BSA was tested in a one-step mi cromethod which was recently developed in our laboratory [13]. Activated BSA had again a strong enhancing effect on T cell colony growth which was even more pro nounced than the enhancing effect of 2-ME (fig- 2). Finally, the question was asked whether activated or nonactivated BSA were able to substitute for adherent cells. This was tested in a one-step system which had been shown to depend on the presence of adherent cells or conditioned medium derived from cul tures of adherent cells (AC-CM) [3]. Mononuclear cells were depleted of ad herent cells by incubation in plastic Petri
Tabic I. Two-step method of human T cell colony formation a Replacement of FCS by 2-ME activated and nonactivated BSA during preincubation with PHA1 Addition of
Colonies per 5,000 M N C iSEM 0 2-ME
RPMI 1640 BSA activated (10%) BSA nonactivated (10%) FCS (10%)
4± 28 ± 21 ± 52 ±
1 3 3 3
+ 2-ME 3± 1
83 ± 3
1 106 mononuclear cells in 1 ml were preincubated under various conditions with PHA for 18 h at 37 °C and 5,000 cells were plated per Petri dish.
b Replacement of FCS by 2-ME activated and nonactivated BSA in the second step1 Addition of
RPMI 1640 BSA activated (10%) BSA nonactivated (10%) FCS (10%)
Colonies per 5,000 M N C iSEM 0 2-ME
+ 2-ME
8 35 11 26
51 ± 22 ± 21 ± 56 ±
± ± ± ±
1 3 1 2
4 3 4 4
1 106mononuclear cells in 1 ml were preincubated in 10% FCS with PHA for 18 h at 37°C and 5,000 cells were plated per Petri dish under various conditions. All cultures contain 0.6% human AB serum.
dishes and the nonadherent cells were as sayed for colony formation. The results in figure 3 show that 2-ME-activated BSA can substitute for 2-ME when used in a final concentration of 10%. Nonactivated BSA did not have any effect on colony formation. When AC-CM was not present in the culture medium colony formation was reduced by at least 90%. Neither 2-ME-activated BSA, nor nonactivated BSA nor 2-ME could sub-
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Results
Serum Factors and Cloning oí Human T Lymphocytes
329 (41)
Albumin and transferrin are known to be essential promoters of the growth of human lymphocytes [11, 12], In the growth of ma crophage and granulocyte colonies, these substances partially substitute for serum [6]. When adding BSA or FeCl3 to FCS, Sachs [10] obtained similar numbers of colonies as by the addition of autologous plasma, but the results varied markedly from lectin to lectin and donor to donor [10]. Our results indicate that 2-ME-treated BSA substitutes better for serum than nontreated BSA.
Fig. 3. The effect of activated and nonactivated BSA on colony growth of human T lymphocytes (one-step method). Nonadherent mononuclear cells are used. All cultures contain 5°/o FCS and 0.6°/o AB-serum. A = Plus 2-ME plus adherent cell conditioned medium (AC-CM); O = no 2-ME plus AC-CM; X = plus 2-ME without AC-CM. Data are means of quadruplicates + SD.
stitute for the presence of AC-CM in the cul ture medium. Finally, activated BSA could not substitute for serum in the culture medi um (data not shown in figure 3).
Discussion The results show that 2-ME-treated BSA enhances T cell colony formation similar to 2-ME. The factor was, however, unable to substitute for AC-CM in the one-step method. This is in contrast to our findings in the primary antibody response in vitro, in which 2-ME-treated fetal calf serum was capable of substituting for macrophages as well as for 2-ME [8].
1 Broome, J. D. and Jeng, M. W.: Promotion of replication in lymphoid cells by specific thiols and disulfides in vitro. J. exp. Med. 138: 574-592 (1973). 2 Claesson, M. H.; Rodger, M. B.; Johnson, G. R.; Whittingham, S., and Metcalf, D.: Colony formation by human T-lymphocytes in agar medium. Clin. exp. Immunol. 28: 526-534 (1977). 3 Claesson, M. H.; Whittingham, S.; Rodger, M. B., and Burgess, A. W.: Colony growth of hu man T-lymphocytes in agar. Effect of a soluble factor from adherent cells. Eur. J. Immunol. 7: 608-612 (1977). 4 Claesson, M. H.; Fiad, H.-D., and Opitz, H. G.: B- and T-lymphocyte colony formation in agar. 2-Mercaptoethanol (2-ME) activated al bumin can substitute for 2-ME. Cell Immunol. 46: 398-404 (1979). 5 Click, R. E.; Beck, L., and Alter, B. J.: En hancement of antibody synthesis in vitro by mercaptoethanol. Cell. Immunol. 3: 156-160 (1972). 6 Guilbert, L. J. and Iscove, N. N.: Partial re placement of serum by selenite, transferrin, al bumin and lecithin in haemopoietic cell cul tures. Nature, Lond. 263: 594-595 (1976). 7 Metcalf, D.: Role of mercaptoethanol and en dotoxin in stimulating B-Iymphocyte colony formation in vitro. J. Immun. 116: 635-638 (1976).
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
References
330 ( 42)
Flad/Ulmer/Claesson/Opitz
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
8 Opitz, H. G.; Opitz, U.; Lemke, H.; Hewlett, the growth of activated human lymphocytes. J. G. ; Schreml, W., and Flad, H.-D.: The role of biol. Chem. 251: 987-992 (1976). fetal calf serum in the primary immune re 12 Tomey, D. C.; Imrie, R. C., and Mueller, G. sponse in vitro. J. exp. Med. 145: 1029-1038 C : Identification of transferrin as a lympho (1977). cyte growth promoter in human serum. Expl 9 Opitz, H. G.; Opitz, U.; Lemke, H.; Flad, Cell Res. 74: 163-169 (1972). H. -D.; Hewlett, G., and Schlumberger, H. D.: 13 Ulmer, A. J. and Flad, H. D.: One-stage stimu Humoral primary immune response in vitro in lation of human T lymphocyte colony forming a homologous mouse system: Replacement of units (TL-CFU) in a micro agar culture in glass fetal calf serum by a 2-mercaptoethanol or capillaries. Immunology 38: 393-399 (1979). macrophage activated fraction of mouse se rum. J. Immun. 119: 2089-2094 (1977). 10 Sachs, L.: Control of cloning of normal hu man T-lymphocytes by transferrin, albumin and different lectins. Clin. exp. Immunol. 33: H. D. Flad, Laboratory of Immunology, 495-498 (1978). Department of Microbiology, 11 Spicker-Polet, H. and Polet, H.: Identification University of Ulm, Ulm (FRG) of albumin as the serum factor essential for
Role of Serum Factors and Adherent Cells in Cloning of Human T Lymphocytes in Agar Culture1 H. D. Flad, A. J. Ulmer, M. H. Claesson and H. G. Opitz Laboratory of Immunology, Department of Microbiology, University of Ulm, Ulm; Anatomy Department A, University of Copenhagen, Copenhagen, and Institute for Immunology and Oncology, Bayer AG, Wuppertal
Key Words. Colony formation • Human T lymphocytes • 2-Mercaptoethanol • Serum factors Abstract. In the presence of serum, 2-mercaptoethanol-treated bovine serum albumin enhances T cell colony formation as does 2-mercaptoethanol. The factor only partially substitutes for FCS, but neither for the mitogen phytohemagglutinin nor for conditioned medium derived from cultures of adherent cells.
The low molecular weight thiol 2-mercaptoethanol (2-ME) has been shown to enhance antibody and mitogen induced re sponses of murine spleen cells in vitro [1, 5]. In systems measuring clonal proliferation of cells 2-ME enhances T lymphocyte colony formation [10], and is absolutely necessary for murine B lymphocyte colony formation m.
Recently Opitz et al. [8, 9] showed that a 2-ME-activated serum factor (MaSF) gener ated from fetal calf serum or mouse serum was capable of substituting for 2-ME or macrophages and for serum in the primary antibody response of mouse spleen cells in 1 Supported by ‘Deutsche Forschungsgemein schaft, Sonderforschungsbereich 112, Project C4’.
vitro. This factor had a molecular weight which was similar to that of albumin. The purpose of this study was to investi gate the effects of this factor on the growth of human T lymphocytes in three different systems. Material and Methods Cloning of Human T Lymphocytes. For growth of human T lymphocytes in agar, three different methods were employed: (1) A two-step method according to Claesson et al. [2], in which mononuclear cells were preincubated for 18 h with PHA followed by seeding of the cells in agar. (2) A one-step method, in which mononuclear cells or nonadherent cells were incorporated di rectly into the agar together with the mitogen PHA [3], (3) A one-step micromethod recently de veloped by Ulmer and Flad [13] in which blood mononuclear cells are grown in capillary tubes. There is no requirement for factors, erythrocytes
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Introduction
Serum Factors and Cloning of Human T Lymphocytes
% activated or nonactivatad 8 S A
327 (39)
% activated or nonactivated BSA
Fig. 1. The effect of addition of 2-ME, 2-ME-activated and nonactivated BSA to agar on colony growth of human T lymphocytes (2-step method). Cultures contain 5% FCS and 0.6% hu man AB serum. Left panel: © = Control cultures
without BSA and without 2-ME. All cultures without 2-ME. Right panel: • = Control cultures without BSA and with 2-ME (5 X 10~! M). All cultures contain 2-ME (5 X 10'5 M). Data are means of quadruplicates + SD.
or autologous serum for colony growth obtained by this method. Preparation of Serum Factors. 1 g of bovine serum albumin (Behringwerke, Marburg, FRG) was dissolved in 10 ml of RPMI 1640 medium and 2-ME was added at a final concentration of 10_3M. The preparation was incubated and di
alyzed as described [4, 8], A control preparation, not incubated with 2-ME was treated in the same way. Adherent Cell Conditioned Medium (AC-CM). A standard batch of AC-CM was prepared from adherent blood mononuclear cells as described elsewhere [3],
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Fig. 2. The effect of addition of 2-ME, 2-ME-activated and nonactivated BSA to agar on growth of human T lymphocytes (one-step micromcthod). All cul tures contain 10% FCS.
Flad/Ulmer/Claesson/Opitz
328 (40)
As shown in figure 1, activated BSA which is BSA treated with 2-ME - added at the second step of the culture system, en hances the number of colonies. The enhance ment is similar to that obtained by the addi tion of 2-ME. The control preparation nontreated BSA - has no enhancing effect on colony growth but is also enhancing in the presence of 2-ME. We asked the question whether or not the BSA preparations could be a substitute for serum. In table I two experiments are given. Both 2-ME-activated BSA and nonactivated BSA partially substitute for FCS in the preincubation step (table I). They do not substitute for the mitogen PHA (data not shown). On the other hand, 2-ME-acti vated BSA is able to substitute for FCS in the second step ( = agar step) while nonactivated BSA is not. It should be noted, how ever, that no colony growth was obtained in the complete absence of serum, i.e., in the absence of 0.6°/o human AB serum. The influence of activated and nonactivated BSA was tested in a one-step mi cromethod which was recently developed in our laboratory [13]. Activated BSA had again a strong enhancing effect on T cell colony growth which was even more pro nounced than the enhancing effect of 2-ME (fig- 2). Finally, the question was asked whether activated or nonactivated BSA were able to substitute for adherent cells. This was tested in a one-step system which had been shown to depend on the presence of adherent cells or conditioned medium derived from cul tures of adherent cells (AC-CM) [3]. Mononuclear cells were depleted of ad herent cells by incubation in plastic Petri
Tabic I. Two-step method of human T cell colony formation a Replacement of FCS by 2-ME activated and nonactivated BSA during preincubation with PHA1 Addition of
Colonies per 5,000 M N C iSEM 0 2-ME
RPMI 1640 BSA activated (10%) BSA nonactivated (10%) FCS (10%)
4± 28 ± 21 ± 52 ±
1 3 3 3
+ 2-ME 3± 1
83 ± 3
1 106 mononuclear cells in 1 ml were preincubated under various conditions with PHA for 18 h at 37 °C and 5,000 cells were plated per Petri dish.
b Replacement of FCS by 2-ME activated and nonactivated BSA in the second step1 Addition of
RPMI 1640 BSA activated (10%) BSA nonactivated (10%) FCS (10%)
Colonies per 5,000 M N C iSEM 0 2-ME
+ 2-ME
8 35 11 26
51 ± 22 ± 21 ± 56 ±
± ± ± ±
1 3 1 2
4 3 4 4
1 106mononuclear cells in 1 ml were preincubated in 10% FCS with PHA for 18 h at 37°C and 5,000 cells were plated per Petri dish under various conditions. All cultures contain 0.6% human AB serum.
dishes and the nonadherent cells were as sayed for colony formation. The results in figure 3 show that 2-ME-activated BSA can substitute for 2-ME when used in a final concentration of 10%. Nonactivated BSA did not have any effect on colony formation. When AC-CM was not present in the culture medium colony formation was reduced by at least 90%. Neither 2-ME-activated BSA, nor nonactivated BSA nor 2-ME could sub-
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
Results
Serum Factors and Cloning oí Human T Lymphocytes
329 (41)
Albumin and transferrin are known to be essential promoters of the growth of human lymphocytes [11, 12], In the growth of ma crophage and granulocyte colonies, these substances partially substitute for serum [6]. When adding BSA or FeCl3 to FCS, Sachs [10] obtained similar numbers of colonies as by the addition of autologous plasma, but the results varied markedly from lectin to lectin and donor to donor [10]. Our results indicate that 2-ME-treated BSA substitutes better for serum than nontreated BSA.
Fig. 3. The effect of activated and nonactivated BSA on colony growth of human T lymphocytes (one-step method). Nonadherent mononuclear cells are used. All cultures contain 5°/o FCS and 0.6°/o AB-serum. A = Plus 2-ME plus adherent cell conditioned medium (AC-CM); O = no 2-ME plus AC-CM; X = plus 2-ME without AC-CM. Data are means of quadruplicates + SD.
stitute for the presence of AC-CM in the cul ture medium. Finally, activated BSA could not substitute for serum in the culture medi um (data not shown in figure 3).
Discussion The results show that 2-ME-treated BSA enhances T cell colony formation similar to 2-ME. The factor was, however, unable to substitute for AC-CM in the one-step method. This is in contrast to our findings in the primary antibody response in vitro, in which 2-ME-treated fetal calf serum was capable of substituting for macrophages as well as for 2-ME [8].
1 Broome, J. D. and Jeng, M. W.: Promotion of replication in lymphoid cells by specific thiols and disulfides in vitro. J. exp. Med. 138: 574-592 (1973). 2 Claesson, M. H.; Rodger, M. B.; Johnson, G. R.; Whittingham, S., and Metcalf, D.: Colony formation by human T-lymphocytes in agar medium. Clin. exp. Immunol. 28: 526-534 (1977). 3 Claesson, M. H.; Whittingham, S.; Rodger, M. B., and Burgess, A. W.: Colony growth of hu man T-lymphocytes in agar. Effect of a soluble factor from adherent cells. Eur. J. Immunol. 7: 608-612 (1977). 4 Claesson, M. H.; Fiad, H.-D., and Opitz, H. G.: B- and T-lymphocyte colony formation in agar. 2-Mercaptoethanol (2-ME) activated al bumin can substitute for 2-ME. Cell Immunol. 46: 398-404 (1979). 5 Click, R. E.; Beck, L., and Alter, B. J.: En hancement of antibody synthesis in vitro by mercaptoethanol. Cell. Immunol. 3: 156-160 (1972). 6 Guilbert, L. J. and Iscove, N. N.: Partial re placement of serum by selenite, transferrin, al bumin and lecithin in haemopoietic cell cul tures. Nature, Lond. 263: 594-595 (1976). 7 Metcalf, D.: Role of mercaptoethanol and en dotoxin in stimulating B-Iymphocyte colony formation in vitro. J. Immun. 116: 635-638 (1976).
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
References
330 ( 42)
Flad/Ulmer/Claesson/Opitz
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 7:36:58 PM
8 Opitz, H. G.; Opitz, U.; Lemke, H.; Hewlett, the growth of activated human lymphocytes. J. G. ; Schreml, W., and Flad, H.-D.: The role of biol. Chem. 251: 987-992 (1976). fetal calf serum in the primary immune re 12 Tomey, D. C.; Imrie, R. C., and Mueller, G. sponse in vitro. J. exp. Med. 145: 1029-1038 C : Identification of transferrin as a lympho (1977). cyte growth promoter in human serum. Expl 9 Opitz, H. G.; Opitz, U.; Lemke, H.; Flad, Cell Res. 74: 163-169 (1972). H. -D.; Hewlett, G., and Schlumberger, H. D.: 13 Ulmer, A. J. and Flad, H. D.: One-stage stimu Humoral primary immune response in vitro in lation of human T lymphocyte colony forming a homologous mouse system: Replacement of units (TL-CFU) in a micro agar culture in glass fetal calf serum by a 2-mercaptoethanol or capillaries. Immunology 38: 393-399 (1979). macrophage activated fraction of mouse se rum. J. Immun. 119: 2089-2094 (1977). 10 Sachs, L.: Control of cloning of normal hu man T-lymphocytes by transferrin, albumin and different lectins. Clin. exp. Immunol. 33: H. D. Flad, Laboratory of Immunology, 495-498 (1978). Department of Microbiology, 11 Spicker-Polet, H. and Polet, H.: Identification University of Ulm, Ulm (FRG) of albumin as the serum factor essential for
