Immunology 1978 34 1109
Suppressor T cells which block the induction of cytotoxic T cells in vivo VICTORIA B. TAGART, W. R. THOMAS & G. L. ASHERSON Division of Immunological Medicine, Clinical Research Centre, Watford Road, Harrow HAl 3 UJ, Middlesex
Received 6 October 1977; acceptedfor publication 20 October 1977
Summary. Mice pretreated with cyclophosphamide have an increased ability to produce anti-trinitrophenyl cytotoxic T cells after painting with the contact sensitizing agent picryl chloride. This could be abrogated by injecting normal cells or cells from mice exposed to trinitrophenyl derivatives at the time of painting. If the injection of cells was delayed until 1 day after painting specificity could be demonstrated. Normal cells and cells from mice injected with dinitrobenzene sulphonate were ineffective whereas cells from donors injected with picryl sulphonic acid were inhibitory. Inhibitory cells were also shown in mice painted with picryl chloride, particularly after adult thymectomy. Using this system it was found that cells from picryl chloride but not oxazolone painted mice were inhibitory when injected 1 day after painting the recipients. The suppressor cells from the mice injected with picryl sulphonic acid and from the mice painted with picryl chloride were shown to be cyclophosphamide sensitive T cells and were not affected by adult thymectomy. These properties have helped to classify the suppressor cells induced by trinitrophenyl derivatives. INTRODUCTION Control of the development of cytotoxic lymphocytes is of importance to their activity in graft rejection and tumour immunity (Cerottini &
Brunner, 1974). Evidence for control mechanisms operating in vivo has been indicated by the finding that the frequency of cytotoxic lymphocytes generated in vivo by allogeneic skin grafting or injection of allogeneic cells is considerably less than the frequency of cytotoxic effectors produced in vitro during one way mixed lymphocyte cultures (Cerottini & Brunner, 1974; Feldman, Cohen & Wekerle, 1972). A striking example of this difference is the relative ease with which cytotoxic lymphocytes against trinitrophenyl modified syngeneic lymphocytes can be generated in vitro (Shearer, 1974) and the reported lack of cytotoxic cells after immunization in vivo, either by painting with picryl chloride (Dennert & Hatlen, 1975) or by a local injection of TNP syngeneic spleen cells (Rollinghoff, StarzinskiPowitz, Pfizenmaier & Wagner, 1977). Recently it has been shown that mice pretreated with cyclophosphamide and then either painted with picryl chloride (Tagart, 1977) or immunized in the footpad with TNP modified syngeneic lymphocytes (Rollinghoff et al., 1977) will develop cytotoxic cells in vivo. Here we show that the effect of cyclophosphamide is due to the inactivation of a T lymphocyte which on stimulation with antigen suppresses the generation of cytotoxic lymphocytes.
MATERIALS AND METHODS Animals and tumours Six to 10-week-old female CBA mice bred at the Clinical Research Centre were used. In some ex-
Correspondence: Dr V. B. Tagart, Mergenthaler Laboratory for Biology, The Johns Hopkins University, Baltimore, Maryland 21218, U.S.A.
0019-2805/78/0600-1109502.00 (@1978 Blackwell Scientific Publications
1109
1110
Victoria B. Tagart, W. R. Thomas & G. L. Asherson
periments mice were thymectomized at 3-6 weeks. The Gardner lymphoma (kindly donated by Dr E. Leuchars, Chester Beatty Research Institute, London) was passaged weekly in CBA mice. Immunization Mice were painted on the shaved abdomen and front paws with a total of 0 15 ml of 2 5% picryl chloride (PCI) or 3 % oxazolone (Ox) in ethanol. Mice were given 200 mg/kg cyclophosphamide (CY) (Endoxana, WB Pharmaceuticals Ltd., England) intraperitoneally. Picryl sulphonic acid (PSA) was made up to a 1 % solution in phosphate buffered saline, neutralized and injected intravenously. The sodium salt of 2,4-dinitrobenzene sulphonic acid (DNBS) was made up to a 5 % solution in distilled water and injected intravenously.
General design of experiments Recipient mice were given cyclophosphamide and 2 or 3 days later painted with picryl chloride. After 5 days the draining lymph nodes (axillary, brachial and inguinal) were removed, dissociated by pressing through a fine wire mesh and assayed for cytotoxic activity. Where donor lymphocytes were transferred to the CY treated mice this was done on the day of painting (day 0) and immediately prior to painting, or 1 day later.
for 60 min in guinea-pig complement which had been absorbed with agarose and murine spleen cells, washed twice and injected intravenously into recipient mice. Using a 1 : 1 mixture of spleen and lymph node cells about 50% of lymphocytes were killed by the anti-G serum and complement as compared to the complement controls. 4 x 107 control lymphocytes or an equivalent number of anti-6 treated cells were injected intravenously into each recipient. Preparation of B lymphocyte depleted populations The method of nylon wool filtration described by Cantor & Simpson (1975) was used. Control cells were kept at 370 and washed in parallel with the filtered cells. The cell recovery from the columns was about 30 % and of these 90-93 % were sensitive to anti-O serum and complement. Equal numbers (4 x 107/mouse) of control or filtered cells were injected into groups of mice. Statistics The statistical significance of the data was determined according to Student's t test using the values for percentage 51Cr release. A P value of less than 0 05 was taken as significant.
RESULTS
Cytotoxic assay This was performed as described previously (Tagart, 1977). Essentially 5'Cr labelled TNP modified or unmodified targets were incubated for 16 h with various numbers of attacking lymphocytes in flat bottomed microtitre trays. 0-1 ml aliquots of the supematants were removed for gamma counting and the percentage specific cytotoxicity calculated from the formula: Ci - Cn percentage specific cytoxicity= Cm - Cn x 100 where Ci is the counts released in the presence of immune cells, Cn the counts released in the presence of normal cells and Cm the maximum counts releaseable, obtained by freezing and thawing 4 times 01 ml targets plus 01 ml water. Cn was between 20% and 35% of the total IICr incorporated. Preparation of T lymphocyte depleted populations Lymphocytes were incubated with AKR anti C3H serum for 30 min on ice, washed twice and incubated
Suppression of cytotoxicity by immune cells Mice painted with PCI show low levels of cytotoxicity when their lymph nodes are assayed 5 days later, and this can be augmented by pretreating the mice with cyclophosphamide 2 or 3 days prior to painting (Tagart, 1977). This augmentation can be prevented by lymphocytes given intravenously on the day of painting (Fig. 1). When lymphocytes are given at this time spleen cells from normal mice, mice painted with PCI (Fig. 1) or mice injected intravenously with PSA (data not shown) are effective. These experiments do not necessarily show whether the suppression is specific because normal cells given to the mice at this time would have adequate time to develop into suppressors in the 5 days between painting and assay. In contrast, normal cells given to mice 1 day after painting are ineffective whereas cells from mice painted with PCI (Fig. 2) or injected with PSA (Fig. 3) suppress the cytotoxicity. However normal cells are not a complete specificity control and other immune cells were used. To control for PSA mice
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In vivo suppressor T cells
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Figure 1. The effect of normal or immune spleen cells on the cytotoxic response when given on the day of painting. Mice were given no cells (A), 4 X 107 normal spleen cells (El) or 4 x 107 spleen cells from mice painted 6 days previously with PCI (0).
4U AnA
10
0
I
100 25 50 200 Ratio of attackers : targets Figure 3. Specific suppression of cytotoxicity by cells from mice injected with PSA. Mice were given no cells (U) or 3 x 107 donor spleen cells i.v. 1 day after painting. Donor mice were injected either with PSA, 4 mg i.v. at -7 days (O O); or 3 5 mg i.v. at - 7 days and 3 0 mg i.v. at -4 days (o---O); or with DNBS 15 mg i.v. at -7 days (@ 0) or 11 mg i.v. at - 7 days and - 4 days (- - -). Mice not pre-treated with CY (Ol).
A
30 20 I
10
A N
0 100 2'00 50 25 Ratio of attackers :targets Figure 2. The effect of normal or immune spleen cells on the cytotoxic response when given 1 day after painting. Mice were given no cells (A), normal cells (El) or immune cells (0) as in Fig. 1.
were injected with DNBS, a hapten which differs from PSA by one nitro group, and is known to induce cells which suppress in vitro DNA synthesis and the generation of contact sensitivity at the doses used (Moorhead, 1976). These cells, when given 1 day after painting, are unable to suppress the cytotoxicity (Fig. 3). The usual specificity control for PCI is the contact sensitiser, oxazolone (Ox). However experiments in which cells from Ox painted mice were given to mice the day after painting gave equivocal results, in that some suppression was seen with Ox cells in some experiments. Further experiments using cells from adult thymectomized mice have helped to clarify this point (see below). The suppressor cell is a CY sensitive T lymphocyte The cell responsible for the suppression of cytotoxicity can be shown to be a T lymphocyte since it is removed by treatment with anti-O serum and
Victoria B. Tagart, W. R. Thomas & G. L. Ashersonz
1112
(b)
.Y x
.2 .2 C)
.Y C-) (L) 0-
U)
PCI PCI PCI PCI CY CY CY
PCI PCI PCI PCI CY CY CY ceilscells N.W.
PCI PCI PCI PCI
PCI PCI PCI PCI CY CY CY cells cells C' 6+C'
CY CY CY
cells cells C' O+C'
cells cells -
N.W.
Figure 4. (a) The effect of anti-O treatment on the ability of lymphocytes to suppress cytotoxicity. 4 x 107 complement treated control lymphocytes or an equivalent number of anti-@ plus complement treated cells (about 2 x 107) were injected i.v. on the day of painting. Compared with mice given no cells, control cells (both ratios) P < 0-01; anti-@ treated cells P > 0 05. Attacker: target, 200: 1 (open columns); attacker: target, 100: 1 (hatched columns). (b) The effect of nylon wool filtration on the ability of lymphocytes to suppress cytotoxicity. On the day of painting one group of mice was given 4 x 107 control lymphocytes and another an equal number of nylon wool (NW) passed lymphocytes. Compared with mice given no cells, control cells (200: 1) P
Suppressor T cells which block the induction of cytotoxic T cells in vivo VICTORIA B. TAGART, W. R. THOMAS & G. L. ASHERSON Division of Immunological Medicine, Clinical Research Centre, Watford Road, Harrow HAl 3 UJ, Middlesex
Received 6 October 1977; acceptedfor publication 20 October 1977
Summary. Mice pretreated with cyclophosphamide have an increased ability to produce anti-trinitrophenyl cytotoxic T cells after painting with the contact sensitizing agent picryl chloride. This could be abrogated by injecting normal cells or cells from mice exposed to trinitrophenyl derivatives at the time of painting. If the injection of cells was delayed until 1 day after painting specificity could be demonstrated. Normal cells and cells from mice injected with dinitrobenzene sulphonate were ineffective whereas cells from donors injected with picryl sulphonic acid were inhibitory. Inhibitory cells were also shown in mice painted with picryl chloride, particularly after adult thymectomy. Using this system it was found that cells from picryl chloride but not oxazolone painted mice were inhibitory when injected 1 day after painting the recipients. The suppressor cells from the mice injected with picryl sulphonic acid and from the mice painted with picryl chloride were shown to be cyclophosphamide sensitive T cells and were not affected by adult thymectomy. These properties have helped to classify the suppressor cells induced by trinitrophenyl derivatives. INTRODUCTION Control of the development of cytotoxic lymphocytes is of importance to their activity in graft rejection and tumour immunity (Cerottini &
Brunner, 1974). Evidence for control mechanisms operating in vivo has been indicated by the finding that the frequency of cytotoxic lymphocytes generated in vivo by allogeneic skin grafting or injection of allogeneic cells is considerably less than the frequency of cytotoxic effectors produced in vitro during one way mixed lymphocyte cultures (Cerottini & Brunner, 1974; Feldman, Cohen & Wekerle, 1972). A striking example of this difference is the relative ease with which cytotoxic lymphocytes against trinitrophenyl modified syngeneic lymphocytes can be generated in vitro (Shearer, 1974) and the reported lack of cytotoxic cells after immunization in vivo, either by painting with picryl chloride (Dennert & Hatlen, 1975) or by a local injection of TNP syngeneic spleen cells (Rollinghoff, StarzinskiPowitz, Pfizenmaier & Wagner, 1977). Recently it has been shown that mice pretreated with cyclophosphamide and then either painted with picryl chloride (Tagart, 1977) or immunized in the footpad with TNP modified syngeneic lymphocytes (Rollinghoff et al., 1977) will develop cytotoxic cells in vivo. Here we show that the effect of cyclophosphamide is due to the inactivation of a T lymphocyte which on stimulation with antigen suppresses the generation of cytotoxic lymphocytes.
MATERIALS AND METHODS Animals and tumours Six to 10-week-old female CBA mice bred at the Clinical Research Centre were used. In some ex-
Correspondence: Dr V. B. Tagart, Mergenthaler Laboratory for Biology, The Johns Hopkins University, Baltimore, Maryland 21218, U.S.A.
0019-2805/78/0600-1109502.00 (@1978 Blackwell Scientific Publications
1109
1110
Victoria B. Tagart, W. R. Thomas & G. L. Asherson
periments mice were thymectomized at 3-6 weeks. The Gardner lymphoma (kindly donated by Dr E. Leuchars, Chester Beatty Research Institute, London) was passaged weekly in CBA mice. Immunization Mice were painted on the shaved abdomen and front paws with a total of 0 15 ml of 2 5% picryl chloride (PCI) or 3 % oxazolone (Ox) in ethanol. Mice were given 200 mg/kg cyclophosphamide (CY) (Endoxana, WB Pharmaceuticals Ltd., England) intraperitoneally. Picryl sulphonic acid (PSA) was made up to a 1 % solution in phosphate buffered saline, neutralized and injected intravenously. The sodium salt of 2,4-dinitrobenzene sulphonic acid (DNBS) was made up to a 5 % solution in distilled water and injected intravenously.
General design of experiments Recipient mice were given cyclophosphamide and 2 or 3 days later painted with picryl chloride. After 5 days the draining lymph nodes (axillary, brachial and inguinal) were removed, dissociated by pressing through a fine wire mesh and assayed for cytotoxic activity. Where donor lymphocytes were transferred to the CY treated mice this was done on the day of painting (day 0) and immediately prior to painting, or 1 day later.
for 60 min in guinea-pig complement which had been absorbed with agarose and murine spleen cells, washed twice and injected intravenously into recipient mice. Using a 1 : 1 mixture of spleen and lymph node cells about 50% of lymphocytes were killed by the anti-G serum and complement as compared to the complement controls. 4 x 107 control lymphocytes or an equivalent number of anti-6 treated cells were injected intravenously into each recipient. Preparation of B lymphocyte depleted populations The method of nylon wool filtration described by Cantor & Simpson (1975) was used. Control cells were kept at 370 and washed in parallel with the filtered cells. The cell recovery from the columns was about 30 % and of these 90-93 % were sensitive to anti-O serum and complement. Equal numbers (4 x 107/mouse) of control or filtered cells were injected into groups of mice. Statistics The statistical significance of the data was determined according to Student's t test using the values for percentage 51Cr release. A P value of less than 0 05 was taken as significant.
RESULTS
Cytotoxic assay This was performed as described previously (Tagart, 1977). Essentially 5'Cr labelled TNP modified or unmodified targets were incubated for 16 h with various numbers of attacking lymphocytes in flat bottomed microtitre trays. 0-1 ml aliquots of the supematants were removed for gamma counting and the percentage specific cytotoxicity calculated from the formula: Ci - Cn percentage specific cytoxicity= Cm - Cn x 100 where Ci is the counts released in the presence of immune cells, Cn the counts released in the presence of normal cells and Cm the maximum counts releaseable, obtained by freezing and thawing 4 times 01 ml targets plus 01 ml water. Cn was between 20% and 35% of the total IICr incorporated. Preparation of T lymphocyte depleted populations Lymphocytes were incubated with AKR anti C3H serum for 30 min on ice, washed twice and incubated
Suppression of cytotoxicity by immune cells Mice painted with PCI show low levels of cytotoxicity when their lymph nodes are assayed 5 days later, and this can be augmented by pretreating the mice with cyclophosphamide 2 or 3 days prior to painting (Tagart, 1977). This augmentation can be prevented by lymphocytes given intravenously on the day of painting (Fig. 1). When lymphocytes are given at this time spleen cells from normal mice, mice painted with PCI (Fig. 1) or mice injected intravenously with PSA (data not shown) are effective. These experiments do not necessarily show whether the suppression is specific because normal cells given to the mice at this time would have adequate time to develop into suppressors in the 5 days between painting and assay. In contrast, normal cells given to mice 1 day after painting are ineffective whereas cells from mice painted with PCI (Fig. 2) or injected with PSA (Fig. 3) suppress the cytotoxicity. However normal cells are not a complete specificity control and other immune cells were used. To control for PSA mice
lIltI
In vivo suppressor T cells
40
50
30
40 .) x
0 0
20
30
u
._
10
.)
20
0L
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I 25 50 100 200 Ratio of attackers :targets I
I
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Figure 1. The effect of normal or immune spleen cells on the cytotoxic response when given on the day of painting. Mice were given no cells (A), 4 X 107 normal spleen cells (El) or 4 x 107 spleen cells from mice painted 6 days previously with PCI (0).
4U AnA
10
0
I
100 25 50 200 Ratio of attackers : targets Figure 3. Specific suppression of cytotoxicity by cells from mice injected with PSA. Mice were given no cells (U) or 3 x 107 donor spleen cells i.v. 1 day after painting. Donor mice were injected either with PSA, 4 mg i.v. at -7 days (O O); or 3 5 mg i.v. at - 7 days and 3 0 mg i.v. at -4 days (o---O); or with DNBS 15 mg i.v. at -7 days (@ 0) or 11 mg i.v. at - 7 days and - 4 days (- - -). Mice not pre-treated with CY (Ol).
A
30 20 I
10
A N
0 100 2'00 50 25 Ratio of attackers :targets Figure 2. The effect of normal or immune spleen cells on the cytotoxic response when given 1 day after painting. Mice were given no cells (A), normal cells (El) or immune cells (0) as in Fig. 1.
were injected with DNBS, a hapten which differs from PSA by one nitro group, and is known to induce cells which suppress in vitro DNA synthesis and the generation of contact sensitivity at the doses used (Moorhead, 1976). These cells, when given 1 day after painting, are unable to suppress the cytotoxicity (Fig. 3). The usual specificity control for PCI is the contact sensitiser, oxazolone (Ox). However experiments in which cells from Ox painted mice were given to mice the day after painting gave equivocal results, in that some suppression was seen with Ox cells in some experiments. Further experiments using cells from adult thymectomized mice have helped to clarify this point (see below). The suppressor cell is a CY sensitive T lymphocyte The cell responsible for the suppression of cytotoxicity can be shown to be a T lymphocyte since it is removed by treatment with anti-O serum and
Victoria B. Tagart, W. R. Thomas & G. L. Ashersonz
1112
(b)
.Y x
.2 .2 C)
.Y C-) (L) 0-
U)
PCI PCI PCI PCI CY CY CY
PCI PCI PCI PCI CY CY CY ceilscells N.W.
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PCI PCI PCI PCI CY CY CY cells cells C' 6+C'
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cells cells C' O+C'
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Figure 4. (a) The effect of anti-O treatment on the ability of lymphocytes to suppress cytotoxicity. 4 x 107 complement treated control lymphocytes or an equivalent number of anti-@ plus complement treated cells (about 2 x 107) were injected i.v. on the day of painting. Compared with mice given no cells, control cells (both ratios) P < 0-01; anti-@ treated cells P > 0 05. Attacker: target, 200: 1 (open columns); attacker: target, 100: 1 (hatched columns). (b) The effect of nylon wool filtration on the ability of lymphocytes to suppress cytotoxicity. On the day of painting one group of mice was given 4 x 107 control lymphocytes and another an equal number of nylon wool (NW) passed lymphocytes. Compared with mice given no cells, control cells (200: 1) P
