ADONIS
Clin. exp. Immunol. (1991) 84, 383-388
000991049100157T
Inhibition of T suppressor cell function by local administration of an active cyclophosphamide derivative at the sensitization site J. LIMPENS & R. J. SCHEPER Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
(Accepted for
publication
14
January
1991)
SUMMARY In previous work, we have shown that local administration of active cyclophosphamide (CY) derivatives, or other cytostatic drugs, at the sensitization site induced a similar immunopotentiation to that of systemic CY. Since it is well documented that CY can inhibit suppressor cells, it was proposed that immunoenhancement by locally administered drugs might be based on a similar principle. The objective of the present study was to test this hypothesis, using an experimental model of Ts mediated suppression of delayed type hypersensitivity to sheep erythrocytes. In this model, mice are made tolerant to sheep erythrocytes by i.v. injection of a high dose of sheep erythrocytes. Local treatment of sheep erythrocytes-tolerant mice with the active CY derivative Z7557 at the site of attempted sensitization reversed suppression in a dose-dependent manner. Local treatment with the cytostatic drug etoposide (VP-16) and systemic CY treatment were also effective. In transfer experiments, the function of afferently acting suppressor cells was blocked by local treatment with Z7557 or systemic CY. These data support the concept of anti-suppressor cell activity of locally administered cytostatic drugs. As with CY, the pharmacological basis of this effect remains to be determined. Keywords local chemotherapy cyclophosphamide derivatives immunoenhancement tolerance
T suppressor cell inhibition
immunotherapy. In rodent tumour models it has already been shown that doses CY known to inhibit suppressor cell function markedly facilitate the eradication of established murine tumours (North, 1984; Mokyr, 1987). In human cancer patients low-dose CY treatment is currently being used as an adjunct in immunotherapy trials (Berd, Maguire & Mastrangelo, 1986; Sahasrabudhe et al., 1986; Livingston et al., 1987). Previously, we have reported that strong augmentation of delayed-type hypersensitivity (DTH) responses can also be obtained by local administration of active CY derivatives, and various other cytostatic drugs, at the sensitization site during a restricted period after priming. This was demonstrated in several DTH models in guinea pigs (Boerrigter & Scheper, 1984; Scheper et al., 1984) and in mice (Tan et al., 1986; Limpens & Scheper, 1989; Limpens, Garssen & Scheper 1990a; Limpens et al., 1990b). This local protocol might be a good alternative to systemic treatment with CY, since (i) systemic side effects are avoided; (ii) systemic CY treatment can readily abrogate an ongoing immune response, whereas local drug treatment is most effective if applied during antigenic stimulation; and (iii) local treatment is most warranted in the case of vaccination procedures. In the above-mentioned DTH models, it was inferred that immunopotentiation by local administration of cytostatic drugs was due to selective inhibition of Ts cells. To obtain proof that
INTRODUCTION Considerable evidence exists that cyclophosphamide (CY) induced immunopotentiation results from selective inhibition of suppressor cell function. CY can reverse immunological tolerance and inhibit the generation and/or expression of suppressor T cell (Ts) function in adoptive transfer experiments (Polak & Turk, 1974; Mitsuoka, Baba & Morikawa, 1976; Sy, Miller & Claman, 1977; Gill & Liew, 1978; Asherson, Perera & Thomas, 1979; North, 1984; Sahasrabudhe et al., 1987). Studies to define the cellular targets of the drug have been facilitated by the synthesis of analogs of CY with in vitro activity, such as 4HPCY and maphosphamide (ASTA-Z 7557) (Shand & Howard, 1979; Hilgard et al., 1985). 4-HPCY was shown to be selectively toxic for Ts cells in doses not interfering with T effector functions (Kaufmann, Hahn & Diamantstein, 1980; Ozer et al., 1982; Cowens et al., 1984; Smith, Mihich & Ozer, 1987). The selective toxicity of CY for suppressor cells is not only of theoretical, but also of clinical interest, since reduction of suppression may be an essential component of successful Correspondence: Dr Jacqueline Limpens, Department of Human Genetics, Rijksuniversiteit Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
383
384
J. Limpens & R. J. Scheper
locally administered cytostatic drugs can interfere with immune suppression, we studied the effects of a locally administered active CY derivative, Z7557, in a murine model of Ts mediated suppression. We report that local administration of Z7557 and systemic administration of CY abrogate intravenously induced suppression to sheep erythrocytes and inhibit the function of afferently acting Ts. MATERIALS AND METHODS
Mice and antigens BALB/c mice were bred and maintained in our animal facilities. Unless otherwise stated, female mice were used, at 12-18 weeks of age. Sheep erythrocytes in Alsever's solution were washed three times before use and suspended in phosphate-buffered saline (PBS). Keyhole limpet haemocyanin (KLH) (Calbiochem-Behring Corporation, Hoechst Holland, Amsterdam, The Netherlands) was filter-sterilized and diluted in sterile, nonpyrogenic saline for injection (Limpens et al., 1990b). Drug treatment CY (Farmitalia Carlo Erba, Brussels, Belgium) and activated cyclophosphamide Z7557 (a generous gift of ASTA Werke, Brackwede, Germany) were dissolved, and VP-16 (Etoposid VP-16-213) (Bristol-Myers Products, Zug, Switzerland) was diluted in sterile saline immediately before use. CY was injected by the i.p. route as a single dose (150 mg/kg); VP-16 and Z7557 were administered in the indicated doses in a volume of 40 MI subcutaneously at the sensitization site during 3 days after priming.
Sensitization and elicitation of DTH The DTH assay for sheep erythrocytes was performed as previously described (Tan et al., 1986) with slight modifications. Mice were sensitized at day 0 with 101 sheep erythrocytes in one hind foot-pad. DTH was elicited at day 7 by injecting 107 sheep erythrocytes in a volume of 30 p1 into the left-ear pinna. DTH reactions were determined by measuring the difference in thickness between the left- and the right-ear pinnae with an engineer's micrometer (Mitotoyo, MFG, Tokyo, Japan) 24 and 48 h after challenge. A control group consisting of non-immune mice challenged in the same way as the mice to be tested was always included. The specific DTH response was calculated as the relative increase in ear thickness of immune mice minus the relative increase in ear thickness of control mice. In some experiments, mice were also sensitized with 3 ig KLH in the opposite hind foot-pad and challenged with sheep erythrocytes or KLH. Sensitization and challenge with KLH have been described elsewhere (Limpens & Scheper, 1989; Limpens et al., 1990b). Induction of unresponsiveness to sheep erythrocytes and transfer of suppression Unresponsiveness to sheep erythrocytes was induced by injecting one high dose of 109 sheep erythrocytes intravenously into the tail vein (Ramshaw, Bretscher & Parish, 1976; Liew, 1977). Suppressed mice are incapable of responding to a subsequent sensitization with 108 sheep erythrocytes. To assay afferently acting Ts, spleens of suppressed mice were isolated 4 days after i.v. injection of sheep erythrocytes, placed in RPMI 1640 (GIBCO, Paisley, UK) with 5% fetal calf serum (FCS), minced
with scissors and squeezed through nylon gauze filter. Cells were treated with a Tris-buffered ammonium chloride solution for 1 min on ice to lyse erythrocytes, and then washed extensively, first in RPMI 1640 and subsequently in PBS. Viability was determined by trypan blue exclusion. 8 x 107-101 viable spleen cells in PBS (0 5 ml) were injected into the tail vein of syngeneic recipient mice, which were sensitized subcutaneously with 108 sheep erythrocytes within 1 h of cell transfer. To test for the presence of efferently acting Ts, spleen cells of sheep erythrocytes-suppressed mice were injected intravenously into sheep erythrocytes-sensitized mice and DTH was elicited within 1 h of transfer.
Selective cell depletions by antibodies and complement Nucleated spleen cells from sheep erythrocytes-suppressed mice were pooled, divided in equal portions and treated with an excess of anti-L3T4 (clone GK 1.5; ATCC, Rockville, MD) or anti-Lyt-2 (clone AD4; Cedarlane Laboratories, Hornby, Ontario, Canada) monoclonal antibodies for 30 min at 40C, or were left untreated. Cells were washed and incubated with guinea-pig complement (that had been absorbed with normal BALB/c spleen cells) in a final dilution of 1/10 for 30 min at room temperature, washed extensively and resuspended in PBS. RESULTS Induction of suppression to sheep erythrocytes We could confirm the data of others (Ramshaw et al., 1976; Liew, 1977), that i.v. injection of 109 sheep erythrocytes makes mice unresponsive to DTH induction by s.c. injection of 108 sheep erythrocytes, and that this immune suppression can be transferred by L3T4+ (or Lyti +), Lyt2- splenic T cells (Ramshaw et al., 1976; Thomson et al., 1980) (Table 1). Since there is no consensus regarding the mechanisms of action of these Ts cells and the kinetics of their appearance (Ramshaw et al., 1976; Liew, 1977; Whisler & Stobo, 1978; Kaufmann et al., 1980; Tamura et al., 1985), we first examined the characteristics of the Ts cells in our system. As shown in Fig. 1, spleen cells from BALB/c mice, injected with 109 sheep erythrocytes, contained suppressor cells which substantially impaired the induction of DTH to sheep erythrocytes in syngeneic recipients. Maximum Table 1. Phenotype of afferently acting suppressor cells
Group
Cells transferred
a b c d e
No transfer Non-treated spleen cells C-treated spleen cells Anti-L3T4+ C-treated spleen cells Anti-Lyt2+C-treated spleen cells
48 h ear swelling (% specific increase)
12-5+2-8 0 7 + 3-2* 1-9 + 2.9* 8-9 + 3 6f - 15 + 2-5*
Spleen cells from sheep erythrocyte-tolerant mice were isolated, pooled and portions were treated in vitro with either monoclonal antiL3T4 or anti-Lyt2 plus complement (C) (groups d and e), with C alone (c) or remained untreated (b). Spleen cells were injected intravenously into syngeneic recipients (n = 5), which were sensitized with erythrocytes within I h of transfer. Control mice received no splenic cells (a). * Significantly lower versus group a (P< 0-05). t Not
significantly different versus group a.
Suppressor T cell inhibition by local chemotherapy 80
suppression was reached 4 days after induction of suppression. At this time spleen cells also caused a slight, but non-significant, inhibition of DTH, when transferred during the efferent phase. It thus appears that i.v. injection of a high-dose sheep erythrocytes mainly induces splenic afferently acting Ts at days 3-5.
20
60
385
-
~~~~~~t t
Days between tole ra nce induction and transfer
Fig. 1. Kinetics of appearance of suppressor cells in the spleen. Groups of mice were injected intravenously with 109 sheep erythrocytes on various days relative to spleen isolation and transfer (day 0). Pooled spleen cell suspensions were divided in two portions and injected intravenously into different groups of mice to test for the presence of afferently acting Ts (0) and efferently acting Ts (13) as described in Materials and Methods. Results are expressed as the percentage suppression of DTH +s.e.m. compared with appropriately sensitized controls. * Significantly (P < 0005) and tnon-significantly different from sensitized controls, not transferred with spleen cells.
Effects of cytostatic drugs on unresponsiveness ofsheep erythrocyte-tolerized mice Effects of locally administered cytostatic drugs on the unresponsiveness of DTH in sheep erythrocyte-tolerized mice were examined as follows. Mice were made tolerant to sheep erythrocytes by injecting 109 sheep erythrocytes intravenously 7 days before attempted sensitization with 108 sheep erythrocytes (day 0). Sheep erythrocyte-tolerant mice were treated at the sensitization site with graded amounts of the cytostatic drugs Z7557 and VP-16 or with saline at days 1, 2 and 3. This treatment schedule gives the most reproducible strong enhancement of DTH (unpublished data). For comparison, a group of mice treated with an optimal effective dose of 150 mg/kg CY at day -I was also included. All mice were challenged at day 7. Fig. 2 shows the results of a representative experiment. The unresponsiveness to sheep erythrocytes could be reversed in a dose dependent way by local treatment with Z7557 and VP-16, and by systemic pretreatment with CY. Optimal effective doses were 300 pg Z7557 and 100 pg VP-16, doses previously shown to be also most effective in enhancing DTH to 108 sheep erythrocytes (Tan et al., 1986). The level of reversal varied at 50-140% between experiments (for all drugs tested). Notably, local drugtreatment induced higher levels of DTH in non-tolerant mice than in tolerant mice. In addition, it was found that suppression could also be reversed when sensitization was attempted at later time-points (days 10 and 14) after i.v. injection of sheep erythrocytes. Maximal reversal of suppression was observed if Z7557 was administered at days 1-3 or days -I to +1 relative to attempted sensitization. Administration at earlier time-points (-3 to 1) was less effective and administration at later timepoints (3 to 5) not effective at all (data not shown). In the experiment depicted in Table 2, naive and sheep erythrocyte-tolerant mice were sensitized with sheep erythrocytes in the left hind foot-pad and with a suboptimal amount of KLH in the right hind foot-pad. Z7557 treatment was either performed at the site of sensitization with sheep erythrocytes (left hind foot-pad) or KLH (right hind foot-pad). It was found, that suppression to sheep erythrocytes was only broken by giving Z7557 treatment at the site of sensitization with sheep erythrocytes (group f). Drug treatment of mice at the site of sensitization with KLH enhanced the DTH response to KLH in both naive and sheep erythrocyte-tolerant mice (groups d and h versus b), but had no influence on the DTH to sheep erythrocytes in normal (group c) or sheep erythrocytes-tolerant (group g) mice. It can thus be concluded that Z7557 only reverses suppression to sheep erythrocytes, if the drug is given at the site of attempted sensitization with sheep erythrocytes. -
Tolerance induction
Drug treatment
24 h
ear
10
Dose
swelling (%/ spec. increase) % of 20 30 40 50 c control 100 2
+ ~
-
Z7S57
300 pg/day
Z7557 Z7557 Z7557 Z7557
30 pg/doy 100 pg/day 300pg/day 100 pg/day
VP-16 VP-16
30 pg/day
VP-16
300pg/day 600 pg/day 150 mg/kg
VP-16 CY
~
193
A*I
44
31 76
1000 pg/dayIm
VP- 16
~
I i
-P-
136
Clin. exp. Immunol. (1991) 84, 383-388
000991049100157T
Inhibition of T suppressor cell function by local administration of an active cyclophosphamide derivative at the sensitization site J. LIMPENS & R. J. SCHEPER Department of Pathology, Free University Hospital, Amsterdam, The Netherlands
(Accepted for
publication
14
January
1991)
SUMMARY In previous work, we have shown that local administration of active cyclophosphamide (CY) derivatives, or other cytostatic drugs, at the sensitization site induced a similar immunopotentiation to that of systemic CY. Since it is well documented that CY can inhibit suppressor cells, it was proposed that immunoenhancement by locally administered drugs might be based on a similar principle. The objective of the present study was to test this hypothesis, using an experimental model of Ts mediated suppression of delayed type hypersensitivity to sheep erythrocytes. In this model, mice are made tolerant to sheep erythrocytes by i.v. injection of a high dose of sheep erythrocytes. Local treatment of sheep erythrocytes-tolerant mice with the active CY derivative Z7557 at the site of attempted sensitization reversed suppression in a dose-dependent manner. Local treatment with the cytostatic drug etoposide (VP-16) and systemic CY treatment were also effective. In transfer experiments, the function of afferently acting suppressor cells was blocked by local treatment with Z7557 or systemic CY. These data support the concept of anti-suppressor cell activity of locally administered cytostatic drugs. As with CY, the pharmacological basis of this effect remains to be determined. Keywords local chemotherapy cyclophosphamide derivatives immunoenhancement tolerance
T suppressor cell inhibition
immunotherapy. In rodent tumour models it has already been shown that doses CY known to inhibit suppressor cell function markedly facilitate the eradication of established murine tumours (North, 1984; Mokyr, 1987). In human cancer patients low-dose CY treatment is currently being used as an adjunct in immunotherapy trials (Berd, Maguire & Mastrangelo, 1986; Sahasrabudhe et al., 1986; Livingston et al., 1987). Previously, we have reported that strong augmentation of delayed-type hypersensitivity (DTH) responses can also be obtained by local administration of active CY derivatives, and various other cytostatic drugs, at the sensitization site during a restricted period after priming. This was demonstrated in several DTH models in guinea pigs (Boerrigter & Scheper, 1984; Scheper et al., 1984) and in mice (Tan et al., 1986; Limpens & Scheper, 1989; Limpens, Garssen & Scheper 1990a; Limpens et al., 1990b). This local protocol might be a good alternative to systemic treatment with CY, since (i) systemic side effects are avoided; (ii) systemic CY treatment can readily abrogate an ongoing immune response, whereas local drug treatment is most effective if applied during antigenic stimulation; and (iii) local treatment is most warranted in the case of vaccination procedures. In the above-mentioned DTH models, it was inferred that immunopotentiation by local administration of cytostatic drugs was due to selective inhibition of Ts cells. To obtain proof that
INTRODUCTION Considerable evidence exists that cyclophosphamide (CY) induced immunopotentiation results from selective inhibition of suppressor cell function. CY can reverse immunological tolerance and inhibit the generation and/or expression of suppressor T cell (Ts) function in adoptive transfer experiments (Polak & Turk, 1974; Mitsuoka, Baba & Morikawa, 1976; Sy, Miller & Claman, 1977; Gill & Liew, 1978; Asherson, Perera & Thomas, 1979; North, 1984; Sahasrabudhe et al., 1987). Studies to define the cellular targets of the drug have been facilitated by the synthesis of analogs of CY with in vitro activity, such as 4HPCY and maphosphamide (ASTA-Z 7557) (Shand & Howard, 1979; Hilgard et al., 1985). 4-HPCY was shown to be selectively toxic for Ts cells in doses not interfering with T effector functions (Kaufmann, Hahn & Diamantstein, 1980; Ozer et al., 1982; Cowens et al., 1984; Smith, Mihich & Ozer, 1987). The selective toxicity of CY for suppressor cells is not only of theoretical, but also of clinical interest, since reduction of suppression may be an essential component of successful Correspondence: Dr Jacqueline Limpens, Department of Human Genetics, Rijksuniversiteit Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
383
384
J. Limpens & R. J. Scheper
locally administered cytostatic drugs can interfere with immune suppression, we studied the effects of a locally administered active CY derivative, Z7557, in a murine model of Ts mediated suppression. We report that local administration of Z7557 and systemic administration of CY abrogate intravenously induced suppression to sheep erythrocytes and inhibit the function of afferently acting Ts. MATERIALS AND METHODS
Mice and antigens BALB/c mice were bred and maintained in our animal facilities. Unless otherwise stated, female mice were used, at 12-18 weeks of age. Sheep erythrocytes in Alsever's solution were washed three times before use and suspended in phosphate-buffered saline (PBS). Keyhole limpet haemocyanin (KLH) (Calbiochem-Behring Corporation, Hoechst Holland, Amsterdam, The Netherlands) was filter-sterilized and diluted in sterile, nonpyrogenic saline for injection (Limpens et al., 1990b). Drug treatment CY (Farmitalia Carlo Erba, Brussels, Belgium) and activated cyclophosphamide Z7557 (a generous gift of ASTA Werke, Brackwede, Germany) were dissolved, and VP-16 (Etoposid VP-16-213) (Bristol-Myers Products, Zug, Switzerland) was diluted in sterile saline immediately before use. CY was injected by the i.p. route as a single dose (150 mg/kg); VP-16 and Z7557 were administered in the indicated doses in a volume of 40 MI subcutaneously at the sensitization site during 3 days after priming.
Sensitization and elicitation of DTH The DTH assay for sheep erythrocytes was performed as previously described (Tan et al., 1986) with slight modifications. Mice were sensitized at day 0 with 101 sheep erythrocytes in one hind foot-pad. DTH was elicited at day 7 by injecting 107 sheep erythrocytes in a volume of 30 p1 into the left-ear pinna. DTH reactions were determined by measuring the difference in thickness between the left- and the right-ear pinnae with an engineer's micrometer (Mitotoyo, MFG, Tokyo, Japan) 24 and 48 h after challenge. A control group consisting of non-immune mice challenged in the same way as the mice to be tested was always included. The specific DTH response was calculated as the relative increase in ear thickness of immune mice minus the relative increase in ear thickness of control mice. In some experiments, mice were also sensitized with 3 ig KLH in the opposite hind foot-pad and challenged with sheep erythrocytes or KLH. Sensitization and challenge with KLH have been described elsewhere (Limpens & Scheper, 1989; Limpens et al., 1990b). Induction of unresponsiveness to sheep erythrocytes and transfer of suppression Unresponsiveness to sheep erythrocytes was induced by injecting one high dose of 109 sheep erythrocytes intravenously into the tail vein (Ramshaw, Bretscher & Parish, 1976; Liew, 1977). Suppressed mice are incapable of responding to a subsequent sensitization with 108 sheep erythrocytes. To assay afferently acting Ts, spleens of suppressed mice were isolated 4 days after i.v. injection of sheep erythrocytes, placed in RPMI 1640 (GIBCO, Paisley, UK) with 5% fetal calf serum (FCS), minced
with scissors and squeezed through nylon gauze filter. Cells were treated with a Tris-buffered ammonium chloride solution for 1 min on ice to lyse erythrocytes, and then washed extensively, first in RPMI 1640 and subsequently in PBS. Viability was determined by trypan blue exclusion. 8 x 107-101 viable spleen cells in PBS (0 5 ml) were injected into the tail vein of syngeneic recipient mice, which were sensitized subcutaneously with 108 sheep erythrocytes within 1 h of cell transfer. To test for the presence of efferently acting Ts, spleen cells of sheep erythrocytes-suppressed mice were injected intravenously into sheep erythrocytes-sensitized mice and DTH was elicited within 1 h of transfer.
Selective cell depletions by antibodies and complement Nucleated spleen cells from sheep erythrocytes-suppressed mice were pooled, divided in equal portions and treated with an excess of anti-L3T4 (clone GK 1.5; ATCC, Rockville, MD) or anti-Lyt-2 (clone AD4; Cedarlane Laboratories, Hornby, Ontario, Canada) monoclonal antibodies for 30 min at 40C, or were left untreated. Cells were washed and incubated with guinea-pig complement (that had been absorbed with normal BALB/c spleen cells) in a final dilution of 1/10 for 30 min at room temperature, washed extensively and resuspended in PBS. RESULTS Induction of suppression to sheep erythrocytes We could confirm the data of others (Ramshaw et al., 1976; Liew, 1977), that i.v. injection of 109 sheep erythrocytes makes mice unresponsive to DTH induction by s.c. injection of 108 sheep erythrocytes, and that this immune suppression can be transferred by L3T4+ (or Lyti +), Lyt2- splenic T cells (Ramshaw et al., 1976; Thomson et al., 1980) (Table 1). Since there is no consensus regarding the mechanisms of action of these Ts cells and the kinetics of their appearance (Ramshaw et al., 1976; Liew, 1977; Whisler & Stobo, 1978; Kaufmann et al., 1980; Tamura et al., 1985), we first examined the characteristics of the Ts cells in our system. As shown in Fig. 1, spleen cells from BALB/c mice, injected with 109 sheep erythrocytes, contained suppressor cells which substantially impaired the induction of DTH to sheep erythrocytes in syngeneic recipients. Maximum Table 1. Phenotype of afferently acting suppressor cells
Group
Cells transferred
a b c d e
No transfer Non-treated spleen cells C-treated spleen cells Anti-L3T4+ C-treated spleen cells Anti-Lyt2+C-treated spleen cells
48 h ear swelling (% specific increase)
12-5+2-8 0 7 + 3-2* 1-9 + 2.9* 8-9 + 3 6f - 15 + 2-5*
Spleen cells from sheep erythrocyte-tolerant mice were isolated, pooled and portions were treated in vitro with either monoclonal antiL3T4 or anti-Lyt2 plus complement (C) (groups d and e), with C alone (c) or remained untreated (b). Spleen cells were injected intravenously into syngeneic recipients (n = 5), which were sensitized with erythrocytes within I h of transfer. Control mice received no splenic cells (a). * Significantly lower versus group a (P< 0-05). t Not
significantly different versus group a.
Suppressor T cell inhibition by local chemotherapy 80
suppression was reached 4 days after induction of suppression. At this time spleen cells also caused a slight, but non-significant, inhibition of DTH, when transferred during the efferent phase. It thus appears that i.v. injection of a high-dose sheep erythrocytes mainly induces splenic afferently acting Ts at days 3-5.
20
60
385
-
~~~~~~t t
Days between tole ra nce induction and transfer
Fig. 1. Kinetics of appearance of suppressor cells in the spleen. Groups of mice were injected intravenously with 109 sheep erythrocytes on various days relative to spleen isolation and transfer (day 0). Pooled spleen cell suspensions were divided in two portions and injected intravenously into different groups of mice to test for the presence of afferently acting Ts (0) and efferently acting Ts (13) as described in Materials and Methods. Results are expressed as the percentage suppression of DTH +s.e.m. compared with appropriately sensitized controls. * Significantly (P < 0005) and tnon-significantly different from sensitized controls, not transferred with spleen cells.
Effects of cytostatic drugs on unresponsiveness ofsheep erythrocyte-tolerized mice Effects of locally administered cytostatic drugs on the unresponsiveness of DTH in sheep erythrocyte-tolerized mice were examined as follows. Mice were made tolerant to sheep erythrocytes by injecting 109 sheep erythrocytes intravenously 7 days before attempted sensitization with 108 sheep erythrocytes (day 0). Sheep erythrocyte-tolerant mice were treated at the sensitization site with graded amounts of the cytostatic drugs Z7557 and VP-16 or with saline at days 1, 2 and 3. This treatment schedule gives the most reproducible strong enhancement of DTH (unpublished data). For comparison, a group of mice treated with an optimal effective dose of 150 mg/kg CY at day -I was also included. All mice were challenged at day 7. Fig. 2 shows the results of a representative experiment. The unresponsiveness to sheep erythrocytes could be reversed in a dose dependent way by local treatment with Z7557 and VP-16, and by systemic pretreatment with CY. Optimal effective doses were 300 pg Z7557 and 100 pg VP-16, doses previously shown to be also most effective in enhancing DTH to 108 sheep erythrocytes (Tan et al., 1986). The level of reversal varied at 50-140% between experiments (for all drugs tested). Notably, local drugtreatment induced higher levels of DTH in non-tolerant mice than in tolerant mice. In addition, it was found that suppression could also be reversed when sensitization was attempted at later time-points (days 10 and 14) after i.v. injection of sheep erythrocytes. Maximal reversal of suppression was observed if Z7557 was administered at days 1-3 or days -I to +1 relative to attempted sensitization. Administration at earlier time-points (-3 to 1) was less effective and administration at later timepoints (3 to 5) not effective at all (data not shown). In the experiment depicted in Table 2, naive and sheep erythrocyte-tolerant mice were sensitized with sheep erythrocytes in the left hind foot-pad and with a suboptimal amount of KLH in the right hind foot-pad. Z7557 treatment was either performed at the site of sensitization with sheep erythrocytes (left hind foot-pad) or KLH (right hind foot-pad). It was found, that suppression to sheep erythrocytes was only broken by giving Z7557 treatment at the site of sensitization with sheep erythrocytes (group f). Drug treatment of mice at the site of sensitization with KLH enhanced the DTH response to KLH in both naive and sheep erythrocyte-tolerant mice (groups d and h versus b), but had no influence on the DTH to sheep erythrocytes in normal (group c) or sheep erythrocytes-tolerant (group g) mice. It can thus be concluded that Z7557 only reverses suppression to sheep erythrocytes, if the drug is given at the site of attempted sensitization with sheep erythrocytes. -
Tolerance induction
Drug treatment
24 h
ear
10
Dose
swelling (%/ spec. increase) % of 20 30 40 50 c control 100 2
+ ~
-
Z7S57
300 pg/day
Z7557 Z7557 Z7557 Z7557
30 pg/doy 100 pg/day 300pg/day 100 pg/day
VP-16 VP-16
30 pg/day
VP-16
300pg/day 600 pg/day 150 mg/kg
VP-16 CY
~
193
A*I
44
31 76
1000 pg/dayIm
VP- 16
~
I i
-P-
136
