214
Eur. J. Immunol. 1979. 9: 214-219
M. A. Aldo-Benson
Marlene A. Aldo-BensonO Indiana University Schooi of Medicine, Division of Rheumatology, Indianapolis
Antigen-specific suppressor cells in hapten-specific carrier-determined tOkl'anCe* Tolerance to the hapten 2,4-dinitrophenyl (DNP) induced by the injection of DNP coupled to isologous IgG (carrier-determined tolerance) is associated with a receptor blockade of antigen-binding lymphocytes. In the present study, hapten-specific suppressor cells were detected in the spleens of mice made tolerant by intravenous injection of 200 vg DNP-IgG. When spleen cells from mice rendered tolerant to DNP were co-cultured with normal spleen cells in Marbrook cultures, the response to DNPFicoll was suppressed, while the response to sheep red blood cells was not altered. Depletion of T cells from these spleens restored the normal anti-DNP response. The suppressor cells were not detectable in the spleen lymphocyte population of mice in the early stages of tolerance but were present on day 7 after injection of tolerogen, and disappeared by day 14. Mice injected with larger doses of 1 mg or four weekly doses of 200 pg DNP-IgG did not have detectable suppressor cells. Thus, it appears that a short-lived suppressor T cell is generated in carrier-determined tolerance. This cell most likely plays a minor role in the mechanism of carrier-determined tolerance and may be associated with the receptor blockade which is seen early in tolerance.
1 Introduction Several methods of tolerance induction have been described which utilize haptens bound to nonimmunogenic carriers as tolerogens. In some models, the tolerance is thought to be confined to bone marrow-derived cells (B cells) while in others, thymus-derived cells (T cells) or both T and B cells are involved [1-61. In carrier-determined tolerance (CDT), mice injected with a hapten, 2,4-dinitrophenyl, bound to isologous gamma globulin (DNP-IgG), are tolerant to that hapten when it is presented in its immunogenic form, i.e. bound to heterologous protein keyhole limpet hemocyanin (KLH) [7]. Although this has been thought by some to be an example of B cell tolerance, Borel et al. have determined that both T and B cells are involved in this form of tolerance [5].
In previous investigations of the mechanisms of CDT, we have found that the hapten DNP persisted on the surface of the antigen-binding lymphocytes in spleens of tolerant animals [8]. This hapten-bearing cell persisted as long as the animal was tolerant and was no longer present when tolerance disappeared [9, 101. We have postulated that the hapten-bearing cell represents receptor blockade of DNP-binding cells and that this is a major mechanism of tolerance induction. However, early in tolerance, normal numbers of antigen-binding cells
with receptors free to bind the determinant were present in addition to hapten-bearing cells [8], and T cell involvement has been demonstrated in this method of tolerance induction [S]. Thus, complete blockage of all antigen-binding cells seemed unlikely, and the possibility of an additional mechanism of tolerance induction must be considered. The present studies were designed to investigate the role of a suppressor cell mechanism in CDT. It was found that spleen cells from animals made tolerant by an intravenous injection of DNP-IgG are able to specifically suppress the in vitro anti-DNP response of normal spleen cells. The possible role of a suppressor cell mechanism CDT is discussed.
2 Materials and methods 2.1 Animals Seven-week-old male (C57BL/6 X DBA/2)Fl hybrid (BDF1) mice were obtained from Jackson Laboratories (Bar Harbor, ME) or Cox Laboratories (Indianapolis, IN). Three-week-old BDF, mice (Cox Laboratories) were used in some experiments.
2.2 Hapten-carrierconjugates and antigens [I 22271
*
This study was supported in part by USPHS grants No. 5507RR05371 and 1 ROI Ca23886-01A1, Arthritis Center Grant No. AM20582 and by an award from the Grace M. Showalter Trust. Fellow of the Arthritis Foundation,
Correspondence: Marlene A. Aldo-Benson, Department of Medicine, Division of Rheumatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Abbreviations: DNP: 2,4-Dinitrophenyl TNP 2,4,6-Trinitrophenyl SRBC: Sheep red blood cells K L H Keyhole limpet hemocyanin DNPS: Dinitrophenyl sulfonic acid PFC. Plaque-forming cells HBSS: Hanks' balanced salt solution MEM. Minimum essential medium FCS: Fetal calf serum HBC: Hapten-bearing cell(s) CDT: Carrier-determined tolerance
0014-2980/79/0303-0214$02.50/0
IgG,, was separated from the serum of BALB/c mice bearing the tumor RPC5 (obtained from Dr. Yves Borel) by starch block electrophoresis, as previously described [ 111. Dinitrophenyl sulfonic acid (DNPS), twice recrystallized (Eastman Kodak, Rochester, NY), was bound to IgGza by the method of Eisen [12]. The number of moles of DNP/mol of carrier are referred to by subscripts. All conjugates were tested for their capacity to induce tolerance in BDF, mice prior to use in these experiments by injecting the DNP-IgG intravenously (i.v.) and immunizing with DNP-KLH. The primary immune response was assayed 5 days later by the Rittenberg modification of the hemolytic plaque-forming cell (PFC) assay [ 131. DNP,,Ficoll was a gift from Dr. E. Goldings. Sheep red blood cells (SRBC) (Colorado Serum Co., Denver, CO) were washed twice in sterile Hanks' balanced salt solution (HBSS), and a 1% suspension in HBSS was used. 0 Verlag Chernie, GmbH, D-6940 Weinheim, 1979
215
Suppressor cells in carrier-determined tolerance
Eur. J. Immunol. 1979. 9: 214-219
2.3 Cell cultures
2.6 Statistics
A modification of the Marbrook method of antibody stimulation in vitro was used to assay suppressor cell activity [14].
The immune response was expressed as PFCkulture. For each group of four or more cultures, the results were expressed as the geometric mean f SE of PFC. All groups were compared using Student's t-test. In some experiments, rewlts were expressed as percentage of the control response, obtained by dividing the geometric mean of the experimental group hy the geometric mean of the control group.
Briefly, sterile spleen cell suspensions were made in Eagle's Minimum essential medium (MEM, Gibco, Grand Island Biological Co., Grand Island, NY), supplemented with 2.2 gA of sodium hydrogen carbonate, 110 mgA of sodium pyruvate (Gibco) and 1 0 % fetal calf serum (FCS, Microbiologic Associates, Bethesda, MD). One ml of this suspension (2 X 10' cells) and antigen were placed in the culture tube, and this tube was placed in a 125 ml wide-mouth erlenmeyer flask which contained 50 ml of Eagle's MEM. After four days of incubation at 37 "C with 5 % COz, cells were harvested and the antibody response assayed by the hemolytic PFC assay using either TNP-coupled SRBC (TNP-SRBC) or SRBC alone as target cells [12].
2.4 T cell depletion Spleen cells of BDFl mice, made tolerant seven days earlier by i.v. injection of 200 pg DNP,,IgG,,, were suspended in Eagle's MEM. They were mixed with rabbit anti-mouse Thy-1 antiserum (Accurate Chemical Scientific Corp., Hicksville, NY) and rabbit complement, incubated at 37°C for 30 min and then passed through a Ficoll-Hypaque gradient. (Pharmacia, Uppsala, Sweden). The recovered cells were 98% viable by trypan blue dye exclusion and contained 96% B cells, as determined by staining with fluorescein labeled anti-mouse IgG.
2.5 Experimental design Seven or eight-week-old BDF, mice were injected i.v. with varying doses of DNP-IgG,,. At intervals ranging from 3 to 14 days, these mice were sacrificed and spleen cell suspensions prepared as noted above. Similar spleen cell suspensions were made from the spleens of normal uninjected BDF, mice of the same age. Groups of cultures were set up with either 1 ml of normal cells, 1 ml of tolerant cells, o r a combination of 1 ml normal and 1 ml tolerant cells. To each culture, either 50 ng/ml of DNP,,Ficoll o r 7 pl/ml of 1%SRBC was added. Thus, cultures containing 2 ml of cell suspension had twice the amount of antigen as those containing 1 ml volumes.
3 Results 3.1 Presence of suppressor cells Initially, seven-week-old BDFl mice were injected with 200 yg of DNP-IgG2,. Seven days after this injection they were sacrificed and their spleen cells used in cultures as described in Sect. 2.5. The results of three separate experiments (Table 1) showed that tolerant spleen cells alone had a decreased antiDNP response when compared to the response of normal spleen cells. A combination of normal and tolerant spleen cells in a 1 : 1 ratio showed a significantly decreased anti-DNP response when compared to normal spleen cells alone (98, 86 and 51% suppression; p < 0.001, < 0.01 and < 0.05, respectively). However, the anti-SRBC response of combined tolerant and normal spleen cells was equal to or greater than the anti-SRBC response of normal spleen cells alone. Cultures of normal spleen cells in 2-ml volumes had the same anti-DNP response as normal spleen cells in I-ml volumes (902 k 82 vs. 1250 f 232, p > 0.2). In these experiments, spleen cells from tolerant animals specifically suppressed the anti-DNP response of normal spleen cells in vitro. To determine what ratio of normal to tolerant cells would give optimal suppression, a similar experiment was designed using a normal cell concentration of 2 X 107/ml and tolerant cells at concentrations varying between 2 X lo7 and 5 X 1O6/ml. Equal volumes of normal and tolerant cell suspensions were used, and DNP,,Ficoll at a concentration of 50 ng/ml was added to each culture. Fig. 1 demonstrates that a 1 : 1 ratio of normal to tolerant cells effected a 5 2 % suppression (p 0.2, p > 0.5).
Table 1. Antigen-specific suppression of normal spleen cells by tolerant spleen cells Antigen
Nomial
DNP-FIc~II SKHC'
7
3
I \p.
I L
Suppreshi
Normal tolerant"' PE'C'/culturc ( * S . F . )
.folcrant"'
32(17 (282) 1710 (400)
64 (0.5) 5010 (422)
5 0 ( I 20)
0.2
< 0.005 >0.1
0.5, p>O.l) even though they were themselves still tolerant (p 0.5).
(%)
3.4 Effect of T cell depletion on suppressor activity
1030 (194) X60 (370) 5 0 (73)
83 5
>o.s
528 (X5) 370 ( 5 ' ) ) 60 (33)
70 1I
> 0.1
< 0.01
Eur. J. Immunol. 1979. 9: 214-219
M. A. Aldo-Benson
Marlene A. Aldo-BensonO Indiana University Schooi of Medicine, Division of Rheumatology, Indianapolis
Antigen-specific suppressor cells in hapten-specific carrier-determined tOkl'anCe* Tolerance to the hapten 2,4-dinitrophenyl (DNP) induced by the injection of DNP coupled to isologous IgG (carrier-determined tolerance) is associated with a receptor blockade of antigen-binding lymphocytes. In the present study, hapten-specific suppressor cells were detected in the spleens of mice made tolerant by intravenous injection of 200 vg DNP-IgG. When spleen cells from mice rendered tolerant to DNP were co-cultured with normal spleen cells in Marbrook cultures, the response to DNPFicoll was suppressed, while the response to sheep red blood cells was not altered. Depletion of T cells from these spleens restored the normal anti-DNP response. The suppressor cells were not detectable in the spleen lymphocyte population of mice in the early stages of tolerance but were present on day 7 after injection of tolerogen, and disappeared by day 14. Mice injected with larger doses of 1 mg or four weekly doses of 200 pg DNP-IgG did not have detectable suppressor cells. Thus, it appears that a short-lived suppressor T cell is generated in carrier-determined tolerance. This cell most likely plays a minor role in the mechanism of carrier-determined tolerance and may be associated with the receptor blockade which is seen early in tolerance.
1 Introduction Several methods of tolerance induction have been described which utilize haptens bound to nonimmunogenic carriers as tolerogens. In some models, the tolerance is thought to be confined to bone marrow-derived cells (B cells) while in others, thymus-derived cells (T cells) or both T and B cells are involved [1-61. In carrier-determined tolerance (CDT), mice injected with a hapten, 2,4-dinitrophenyl, bound to isologous gamma globulin (DNP-IgG), are tolerant to that hapten when it is presented in its immunogenic form, i.e. bound to heterologous protein keyhole limpet hemocyanin (KLH) [7]. Although this has been thought by some to be an example of B cell tolerance, Borel et al. have determined that both T and B cells are involved in this form of tolerance [5].
In previous investigations of the mechanisms of CDT, we have found that the hapten DNP persisted on the surface of the antigen-binding lymphocytes in spleens of tolerant animals [8]. This hapten-bearing cell persisted as long as the animal was tolerant and was no longer present when tolerance disappeared [9, 101. We have postulated that the hapten-bearing cell represents receptor blockade of DNP-binding cells and that this is a major mechanism of tolerance induction. However, early in tolerance, normal numbers of antigen-binding cells
with receptors free to bind the determinant were present in addition to hapten-bearing cells [8], and T cell involvement has been demonstrated in this method of tolerance induction [S]. Thus, complete blockage of all antigen-binding cells seemed unlikely, and the possibility of an additional mechanism of tolerance induction must be considered. The present studies were designed to investigate the role of a suppressor cell mechanism in CDT. It was found that spleen cells from animals made tolerant by an intravenous injection of DNP-IgG are able to specifically suppress the in vitro anti-DNP response of normal spleen cells. The possible role of a suppressor cell mechanism CDT is discussed.
2 Materials and methods 2.1 Animals Seven-week-old male (C57BL/6 X DBA/2)Fl hybrid (BDF1) mice were obtained from Jackson Laboratories (Bar Harbor, ME) or Cox Laboratories (Indianapolis, IN). Three-week-old BDF, mice (Cox Laboratories) were used in some experiments.
2.2 Hapten-carrierconjugates and antigens [I 22271
*
This study was supported in part by USPHS grants No. 5507RR05371 and 1 ROI Ca23886-01A1, Arthritis Center Grant No. AM20582 and by an award from the Grace M. Showalter Trust. Fellow of the Arthritis Foundation,
Correspondence: Marlene A. Aldo-Benson, Department of Medicine, Division of Rheumatology, Indiana University School of Medicine, Indianapolis, IN 46202, USA Abbreviations: DNP: 2,4-Dinitrophenyl TNP 2,4,6-Trinitrophenyl SRBC: Sheep red blood cells K L H Keyhole limpet hemocyanin DNPS: Dinitrophenyl sulfonic acid PFC. Plaque-forming cells HBSS: Hanks' balanced salt solution MEM. Minimum essential medium FCS: Fetal calf serum HBC: Hapten-bearing cell(s) CDT: Carrier-determined tolerance
0014-2980/79/0303-0214$02.50/0
IgG,, was separated from the serum of BALB/c mice bearing the tumor RPC5 (obtained from Dr. Yves Borel) by starch block electrophoresis, as previously described [ 111. Dinitrophenyl sulfonic acid (DNPS), twice recrystallized (Eastman Kodak, Rochester, NY), was bound to IgGza by the method of Eisen [12]. The number of moles of DNP/mol of carrier are referred to by subscripts. All conjugates were tested for their capacity to induce tolerance in BDF, mice prior to use in these experiments by injecting the DNP-IgG intravenously (i.v.) and immunizing with DNP-KLH. The primary immune response was assayed 5 days later by the Rittenberg modification of the hemolytic plaque-forming cell (PFC) assay [ 131. DNP,,Ficoll was a gift from Dr. E. Goldings. Sheep red blood cells (SRBC) (Colorado Serum Co., Denver, CO) were washed twice in sterile Hanks' balanced salt solution (HBSS), and a 1% suspension in HBSS was used. 0 Verlag Chernie, GmbH, D-6940 Weinheim, 1979
215
Suppressor cells in carrier-determined tolerance
Eur. J. Immunol. 1979. 9: 214-219
2.3 Cell cultures
2.6 Statistics
A modification of the Marbrook method of antibody stimulation in vitro was used to assay suppressor cell activity [14].
The immune response was expressed as PFCkulture. For each group of four or more cultures, the results were expressed as the geometric mean f SE of PFC. All groups were compared using Student's t-test. In some experiments, rewlts were expressed as percentage of the control response, obtained by dividing the geometric mean of the experimental group hy the geometric mean of the control group.
Briefly, sterile spleen cell suspensions were made in Eagle's Minimum essential medium (MEM, Gibco, Grand Island Biological Co., Grand Island, NY), supplemented with 2.2 gA of sodium hydrogen carbonate, 110 mgA of sodium pyruvate (Gibco) and 1 0 % fetal calf serum (FCS, Microbiologic Associates, Bethesda, MD). One ml of this suspension (2 X 10' cells) and antigen were placed in the culture tube, and this tube was placed in a 125 ml wide-mouth erlenmeyer flask which contained 50 ml of Eagle's MEM. After four days of incubation at 37 "C with 5 % COz, cells were harvested and the antibody response assayed by the hemolytic PFC assay using either TNP-coupled SRBC (TNP-SRBC) or SRBC alone as target cells [12].
2.4 T cell depletion Spleen cells of BDFl mice, made tolerant seven days earlier by i.v. injection of 200 pg DNP,,IgG,,, were suspended in Eagle's MEM. They were mixed with rabbit anti-mouse Thy-1 antiserum (Accurate Chemical Scientific Corp., Hicksville, NY) and rabbit complement, incubated at 37°C for 30 min and then passed through a Ficoll-Hypaque gradient. (Pharmacia, Uppsala, Sweden). The recovered cells were 98% viable by trypan blue dye exclusion and contained 96% B cells, as determined by staining with fluorescein labeled anti-mouse IgG.
2.5 Experimental design Seven or eight-week-old BDF, mice were injected i.v. with varying doses of DNP-IgG,,. At intervals ranging from 3 to 14 days, these mice were sacrificed and spleen cell suspensions prepared as noted above. Similar spleen cell suspensions were made from the spleens of normal uninjected BDF, mice of the same age. Groups of cultures were set up with either 1 ml of normal cells, 1 ml of tolerant cells, o r a combination of 1 ml normal and 1 ml tolerant cells. To each culture, either 50 ng/ml of DNP,,Ficoll o r 7 pl/ml of 1%SRBC was added. Thus, cultures containing 2 ml of cell suspension had twice the amount of antigen as those containing 1 ml volumes.
3 Results 3.1 Presence of suppressor cells Initially, seven-week-old BDFl mice were injected with 200 yg of DNP-IgG2,. Seven days after this injection they were sacrificed and their spleen cells used in cultures as described in Sect. 2.5. The results of three separate experiments (Table 1) showed that tolerant spleen cells alone had a decreased antiDNP response when compared to the response of normal spleen cells. A combination of normal and tolerant spleen cells in a 1 : 1 ratio showed a significantly decreased anti-DNP response when compared to normal spleen cells alone (98, 86 and 51% suppression; p < 0.001, < 0.01 and < 0.05, respectively). However, the anti-SRBC response of combined tolerant and normal spleen cells was equal to or greater than the anti-SRBC response of normal spleen cells alone. Cultures of normal spleen cells in 2-ml volumes had the same anti-DNP response as normal spleen cells in I-ml volumes (902 k 82 vs. 1250 f 232, p > 0.2). In these experiments, spleen cells from tolerant animals specifically suppressed the anti-DNP response of normal spleen cells in vitro. To determine what ratio of normal to tolerant cells would give optimal suppression, a similar experiment was designed using a normal cell concentration of 2 X 107/ml and tolerant cells at concentrations varying between 2 X lo7 and 5 X 1O6/ml. Equal volumes of normal and tolerant cell suspensions were used, and DNP,,Ficoll at a concentration of 50 ng/ml was added to each culture. Fig. 1 demonstrates that a 1 : 1 ratio of normal to tolerant cells effected a 5 2 % suppression (p 0.2, p > 0.5).
Table 1. Antigen-specific suppression of normal spleen cells by tolerant spleen cells Antigen
Nomial
DNP-FIc~II SKHC'
7
3
I \p.
I L
Suppreshi
Normal tolerant"' PE'C'/culturc ( * S . F . )
.folcrant"'
32(17 (282) 1710 (400)
64 (0.5) 5010 (422)
5 0 ( I 20)
0.2
< 0.005 >0.1
0.5, p>O.l) even though they were themselves still tolerant (p 0.5).
(%)
3.4 Effect of T cell depletion on suppressor activity
1030 (194) X60 (370) 5 0 (73)
83 5
>o.s
528 (X5) 370 ( 5 ' ) ) 60 (33)
70 1I
> 0.1
< 0.01
