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Suppression of allograft immunity by 3,4,5,3′,4′',5′-hexachlorobiphennyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro
lrnmunopharmacology, 16 (1988) 13-23 Elsevier IMO 00410
13
Suppression of allograft immunity by 3,4,5,3',4',5'hexachlorobiphenyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro N.I. Kerkvliet and L. Baecher-Steppan College o[" Veterinary Medicine and Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, U.S.A. (Received 10 December 1987; accepted 18 March 1988)
Abstract: Previous studies in our laboratory have established the sensitivity of the in vivo allogeneic cytotoxic T lymphocyte (CTL) response to suppression by 3,4,5,Y,4',5'-hexachlorobiphenyl [(345)2-HxCB], a toxic, Ah receptor-binding polychlorinated biphenyl isomer. The present studies have examined possible cellular mechanisms for this suppression. A modest dose-dependent suppression of the proliferative response to alloantigen in mixed lymphocyte culture (MLC) was observed with lymphocytes from B6 mice exposed to 10 or 100 mg/kg (345)2-HxCB while the CTL response generated in MLC was significantly suppressed only following exposure to 100 mg/kg (345)2-HxCB. The amount of time between treatment with (345)2-HxCB and sacrifice, which ranged from 2 to 23 days, did not appear to influence the degree of immunosuppression produced by (345) 2-HxCB exposure. Mitomycin C-treated lymphocytes from B6 mice treated with (345)2-HxCB were not suppressive when added as third party cells to an independent MLC. However, if the mice were alloimmune, lymphocyte-mediated suppression of the MLC response was observed and directly correlated with the magnitude of the CTL response present in the same population. Thus, (345)2-HxCB-treated mice which had less CTL activity as compared to vehicle-treated mice also had less suppressor activity. Further analysis indicated that stimulator cell lysis by the CTL was likely to be responsible for the inhibitory activity of the alloimmune lymphocytes rather than suppressor cells per se. Avoidance of stimulator cell lysis by using H-2-incompatible MLC stimulator cells revealed the existence of antigen-nonspecific suppressor activity that was greater with lymphocytes from vehicle-treated than from (345)2-HxCB-treated mice, suggesting that both CTL and suppressor cell activities were suppressed by (345)2-HxCB exposure. Direct addition of (345)2-HxCB to lymphocyte cultures in vitro indicated a lack of direct toxicity of (345)2-HxCB on lymphoproliferative responses to mitogen or alloantigen at concentrations equal to or less than 1 x 10 .6 M. Thus, the in-vitro functional integrity of lymphocytes obtained from (345)2-HxCB-treated mice coupled with the lack of a direct lymphotoxic effect of (345)2-HxCB in vitro suggest an indirect mechanism of action for (345)2-HxCB-mediated suppression of CTL activity in vivo. Previous reports implicating suppressor cell induction and/or activation by Ah-receptor-binding halogenated aromatic hydrocarbons that mediate the inhibition of CTL generation were not confirmed in these studies. Key words: Polychlorinated biphenyls; 3,4,5,3',4',5'-Hexachlorobiphenyl; lmmunotoxicity; Suppressor cells; Mixed lymphocyte reactivity; Cytotoxic T cells; Mouse
Correspondence: Dr N.I. Kerkvliet, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, U.S.A. Abbreviations: (345)Z-HxCB, 3,4,5,Y,4',5'-hexachlorobiphenyl; CTL, cytotoxic T lymphocyte; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; M LC, mixed lymphocyte culture; B6, C57B 1/6; D2, DBA/2; C3, C3H; B6M, D2M, C3M, mitomycin-C treated B6, D2, or C3 spleen cells; FBS, fetal bovine serum; Con A, concanavalin A; CTX, cytotoxicity; HAH, halogenated aromatic hydrocarbons.
Introduction
In the preceding report we demonstrated that invivo exposure of C57B1/6 (B6) mice to the toxic, Ah-receptor-binding 3,4,5,3',4',5'-hexachlorobiphenyl [(345)2-HxCB] suppressed the in-vivo generation of cytotoxic T lymphocytes (CTL) to allogeneic P815 tumor cell challenge (Kerkvliet and
0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
14 Baecher-Steppan, 1988). Dose-dependent suppression of the peak CTL response on day 10 12 after tumor challenge was observed in both male and female mice. This suppression was not due to a shift in kinetics of the response nor to a cytotoxic effect of (345)2-HxCB on the growth of the P815 cells. Significant suppression of CTL activity was observed when (345)2-HxCB was given prior to or during the first three days after P815 sensitization, whereas exposure later than 3 days after antigen was not suppressive. These results suggested that an early event in the activation/maturation of CTL was sensitive to (345)2-HxCB exposure whereas mature CTL appeared to be resistant to (345) zHxCB-mediated suppression. Clark et al. (1981, 1983) have reported that exposure of mice to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and related Ah-receptor-binding halogenated aromatic hydrocarbons (HAH) significantly impaired the generation of alloantigenstimulated CTL in vitro in mixed lymphocyte cultures (MLC). Whereas the frequency of CTL precursors in the spleen or lymph nodes was not altered by T C D D exposure (Clark et al., 1981), the demonstration of cells in the thymus of T C D D treated mice capable of inhibiting the in-vitro CTL response led to the proposal that the cellular basis for the suppression of CTL activity by T C D D was due to suppressor T cell activation (Clark et al., 1981). On the basis of these reports, the studies described in this paper have examined the sensitivity of the in-vitro MLC response to (345)2-HxCB and the possible role for suppressor cells in mediating (345)2-HxCB-induced suppression of the CTL response to alloantigen.
Materials and Methods Anima&
B6 (H-2b), DBA/2 (D2; H-2 a) and C3H (C3; H-2 k) mice were purchased from Jackson Laboratory (Bar Harbor, ME). They were maintained as previously described (Kerkvliet and Baecher-Steppan, 1988) and used when they were 8 10 weeks of age.
PCB e.vposure
(345)2-HxCB (Ultrascientific, Hope, RI) was dissolved in acetone and diluted in peanut oil as previously described (Kerkvliet and Baecher-Steppan, 1988). Mice were g'iven a single dose orally by stomach tube at various times relative to P815 sensitization and/or sacrifice. In some experiments, (345)2-HxCB was dissolved in different vehicles, as indicated, and added to the culture medium. A lloimmuniza tion
B6 mice were injected i.p. with 1-10 × 10~' viable P815 (H-2 a) mastocytoma cells obtained from the ascites fluid of D2 mice as previously described (Kerkvliet and Baecher-Steppan, 1988). Lymphocyte cultures
MLC, CTL, and suppressor cell activity was measured following the basic protocols described by Argyris (1977) and Argyris and DeLustro (1977). Single-cell suspensions of splenic lymphocytes from B6, D2, or C3 mice were prepared as previously described (Kerkvliet and Baecher-Steppan, 1988). One-way MLCs were established in triplicate wells of flat-bottom microtiter plates using 3 × l0 s B6 responder spleen cells and 3 x l0 s mitomycin Ctreated D2 (D2m) or B6 [B6m (syngeneic control)] as stimulators in a total volume of 200/A RPMI 1640 containing 5% fetal bovine serum (FBS) and 5 x 10 5 M 2-mercaptoethanol. In one experiment, 6 x l0 s mitomycin C-treated C3 (C3m) splenocytes were used as stimulator cells. Cultures were incubated for 4 or 5 days at 37°C in a humidified atmosphere of 5% CO2. The proliferative response in MLC was assayed by the addition of tritiated thymidine ([3H]TdR; 0.5 l~Ci/well) 24 hours prior to culture termination. The cells were harvested from cultures and processed for liquid scintillation counting as previously described (Kerkvliet et al., 1985). CTL activity present in day 5 MLCs was measured using a s~Cr-release assay as previously described using s~Cr-labeled P815 tumor cells
15 (Kerkvliet et al., 1985) or, in one experiment, concanavalin A (Con A)-stimulated C3 blasts as targets. C3 blasts were prepared by culturing C3 spleen cells for 48 hours at a density of 1 x 106 cells/ml in 75 cm 2 flasks in RPM! 1640 containing 2/~g/ml Con A. After 48 hours, the cells were harvested and labeled with 51Cr as described for the P815 target cells. Effector: target cell ratios of 100:1, 30:1, 10:1, and 3:1 were tested. Percentage of cytotoxicity (% CTX) was calculated as described in the preceding paper (Kerkvliet and Baecher-Steppan, 1988). Cells to be tested for suppressor activity were pretreated with mitomycin C (50 #g/2 x l0 T cells/2 ml for 60 min), washed three times, and added as thirdparty cells to normal M L C / C T L cultures. In all experiments, third-party cell concentrations ranging from 0.6 to 5 × 105/well were tested.
sisted of 6 mice tested individually or in pools of 3 for MLC responses and/or suppressor cell activity. The results of each experiment were independently confirmed at least one time. The mean response of each animal or pool was calculated from triplicate cultures; the means were then used for calculating treatment means and carrying out statistical analysis as decribed in the preceding paper (Kerkvliet and Baecher-Steppan, 1988). In cases where culture replicates were the only measure of variation, the standard deviation of the replicates is presented.
Results
Influence of in-vivo (345)Z-HxCB exposure on the proliferative and differentiative response of splenic lymphocytes in allogeneic MLC
Statistical analysis In most experiments, each treatment group con-
B6 mice were given a single oral dose of 0, t0, or 100 mg/kg (345)2-HxCB. At various times after
TABLE I Influence of exposure to (345)2-HxCB on the proliferative and differentiative response of lymphocytes in MLC a (345)Z-HxCB treatment b (mg/kg)
Time of treatment c (days)
[3H]TdR incorporation (cpm; mean 4, SE) B6 x B6~
B6 × D2M~
% CTX a (10:I)
0 10
-2 -2
2,658 4, 122 1,940 4, 194"
24,779 4- 1,557 18,006 + 1,467" (73)
34.1 4, 2.2 29.7 + 2.2
0 10
-11 -11
1,967 4- 191 1,931 4, 109
12,626 411,221 4-
36.5 4- 1.9 36.6 4- 2.6
0 10 100
-18 -18 -18
2,921 4, 163 2,841 4- 251 2,344 4- 137"
36,046 4- 2,782 30,172 4- 3,074 (84) 25,076 4- 2,473* (70)
56.7 4. 1.0 54.8 4- 1.3 51.1 4, 1.9"
0 10 100
-23 -23 -23
801 + 70 1,022 4- 172 826 + 54
17,947 + 619 13,054 + 1,184" (73) 10,835 ± 604* (60)
26.5 4- 2.9 23.5 4- 2.2 17.5 + 2.5*
618 721 (89)
a Data represent mean + S.E. of 5-6 mice/treatment tested individually. Female mice were used in all experiments except for the day - 2 3 study where data from male mice are shown. b.c (345)2_HxCB was administered orally to B6 mice on the indicated day relative to sacrifice on day 0. d Anti-P815 cytotoxicity generated in day 5 MLC cultures. Cells were harvested and tested at several E:T ratios. Data for an E:T ratio of 10:1 is shown. Values in parenthesis represent the MLC response of cells from (345)
16 dosing, the mice were sacrificed and their splenic lymphocytes were cultured in vitro in M LC using D2M spleen cells as stimulators. Data from several representative experiments are shown in Table I. The ability of lymphocytes to proliferate in response to alloantigen stimulation was dose-dependently suppressed by prior in-vivo exposure to (345)2-HxCB. The 10 mg/kg exposure level appeared to be the minimum effective dose, producing a modest degree of suppression (11 to 27%) of p H ] T d R incorporation which was statistically significant in 2/4 experiments. The MLC-generated CTL response was less sensitive to suppression with a dose of 100 mg/kg required to produce significant suppression. No clear relationship between time of exposure to (345)2-HxCB and time of sacrifice on
the degree of inhibition of MLC or CTL responses was apparent.
Suppression o/" the MLC/CTL response by lymphocytes.from animals exposed to (345)2-HxCB The possibility that suppression of the CTL response in vivo was due to the induction of suppressor cells following (345p-HxCB exposure was examined in several experiments. Initially, mice were treated with 0 or 10 mg/kg (345)2-HxCB and sacrificed two days later for assessment of suppressor cell activity in the spleen since this dose and timing of exposure relative to P815 injection produced significant suppression of the in vivo CTL response. Later experiments were expanded to in-
TABLE lI Ability of mitomycin-treated spleen cells from mice treated with (345)2-HxCB to suppress the MLC,,CTL response" Source of B6Mh HxCB (mg,'kg)
Number of B6M added to MLC Day of treatment
p H]TdR incorporation (cpm) 0
0 10
2 2
0 1 10 100
--7 -7 -7 -7
0 I0 100
-17 --17 17
%CTX (10:1) c 5.0 x 105
5.0 x 105
21,178 ± 1,397 28,294 ± 30,429 ±
581 997
42.6 ± 2.3 42.1 ± 1.6
24,479 ± 2,069 38,372 41,643 38,605 38,674 15,248 ±
± 1,515 ± 1,034 ± 705 ± 237
NT '~ NT NT NT
615 14,522 ± 1,406 16,292 ± 645 13,027 ± 1,235
24.0 ± 0.8 25.4 ± 0.9 24.7 ± 1.9
a Data for day --2 study represent two pools of 3 mice/treatment; mean values ± SE for [3H]TdR cpm is based on 3 replicate wells per pool. Data for % CTX on day - 2 represents the average response of 2 pools + SD. Data for day - 7 represent 1 pool of 3 mice/treatment and mean ± SD is based on 3 replicate wells. Data for day - 17 study represents the mean :t_ SE of 5 mice/treatment tested individually for [3H]TdR and % CTX. The baseline MLC response with no added B6M cells is the mean response of a pool of 3 mice: the SE is based on 3 6 replicate wells. b B6 mice were given the indicated dose of (345)2-HxCB orally on the day of treatment relative to sacrifice on day 0 for utilization of their splenic lymphocytes (B6M) as suppressor cells in MLC. % CTX generated in MLC to which 5 x l0 s B6M cells were added, data shown for E:T ratio of 30:i. d NT - not tested.
17 clude a 100 mg/kg exposure level and various times of sacrifice ranging from 2 to 23 days after (345) 2HxCB exposure. Comparative studies using male and female mice were also conducted. Representative results from these studies are summarized in Table II. The addition of mitomycin C-treated spleen cells from control or (345)2-HxCB-treated B6 mice as third-party cells to a normal B6 x D2M MLC resulted in no change or enhanced [3H]TdR incorporation with no evidence for suppression of the proliferative response due to (345)2-HxCB treatment. The stimulatory effect was not due to proliferation by the mitomycin C-treated third-party cells as these cells did not proliferate in B6M x D2M MLC (data not shown). The generation of CTL in MLC was also not suppressed by the addition of third-party cells from (345)2-HxCB-treat ed mice as compared to the addition of cells from vehicle-treated mice (Table II). This apparent lack of suppressor cell activity in the spleen of (345)2HxCB-treated mice was not related to time of sacrifice after dosing or the sex of the donor animals.
creased from day 6 to day 10, which correlated with the development of cytotoxic activity in the spleen. At all time points examined, the degree of inhibition of MLC proliferation was greater with spleen cells from vehicle-treated mice as compared to spleen cells from (345)2-HxCB-treated mice, which also reflected the amount of CTL activity present in the spleen. Subsequent analysis of all available data from both control and (345)2-HxCB-treated mice showed a high degree of correlation between the amount of cytotoxic activity present in a given spleen cell suspension and the degree of inhibition of the MLC response, which was independent of (345)2-HxCB treatment (Fig. 1). Since these results are in direct contrast to what one would expect biologically (e.g., greater CTL activity should imply less suppressor cell activity), it suggested that the inhibitory effect of the spleen cells in MLC was due not to suppressor cell activity but rather to a loss
80
Suppression of the MLC/CTL response by PS15sensitized lymphocytes from animals exposed to (345) 2_HxCB: The lack of detectable suppressor cell activity in the spleen of mice treated with (345)2-HxCB suggested that (345)2-HxCB treatment alone did not promote the development of nonspecific suppressor cell function. However, these studies did not address the possibility that (345)2-HxCB interacted with antigen challenge to modulate antigen-induced suppressor cell function. In order to test this possibility, suppressor activity associated with spleen cells taken from control and (345)2-HxCB-treated mice at various times after P815 challenge was examined. As summarized in Table III, spleen cells from P815-sensitized mice, when added as mitomycin C-treated third-party cells to a normal B6 x D2M MLC, produced significant suppression of [3H]TdR incorporation as compared to the addition of spleen cells from non-P815-sensitized mice. For vehicletreated mice, the suppressive activity of the spleen cells increased as the time after P815 injection in-
6O
c) 40 •
X 0 ~ 2 0 -
0
0
'
•
i
i
I
2o
40
60
80
1oo
%
Inhibition
of
MLC
Fig. 1. Linear correlation between the cytotoxic and MLC-suppressive activities of spleen cells from P815-sensitized B6 mice. Spleen cells were obtained from B6 mice at various times after sensitization with P815 (H-2 d) mastocytoma cells. The suppressive activity of the spleen cells was measured by their ability to inhibit the proliferative response of a B6 anti-D2 MLC when added as rnitomycin C-treated third-party cells. Cytotoxic activity in the same spleen cell suspension was determined by their ability to lyse 51 Cr-labeled P815 tumor cells in vitro. Data shown represent percent suppression of [3H]TdR incorporation in MLC with 5 × l0 s B6M spleen cells added; % CTX is shown for a 3021 effector to target cell ratio.
18 o f M L C s t i m u l a t o r cells c a u s e d by t h e i r lysis by the
q u e n t l y f o c u s e d on the p o s s i b i l i t y t h a t s u p p r e s s o r
C T L p r e s e n t in the s a m e splenic p o p u l a t i o n . T h i s
cell a c t i v i t y m i g h t
be p r e s e n t
in the spleen
of
i n t e r p r e t a t i o n was s u p p o r t e d by the results o f a d -
P815-sensitized m i c e d u r i n g t h e early stages o f sen-
d i t i o n a l studies in w h i c h we d e m o n s t r a t e d t h a t mi-
s i t i z a t i o n p r i o r to the d e v e l o p m e n t o f m a t u r e C T L
t o m y c i n C - t r e a t e d C T L w e r e still f u n c t i o n a l effec-
in the spleen (i.e., p r i o r to d a y 6). B a s e d o n pre-
t o t cells c a p a b l e o f l y s i n g P815 t a r g e t cells ( d a t a n o t
v i o u s results w h i c h s h o w e d t h a t s u p p r e s s i o n o f the
s h o w n ) . L i k e w i s e , w h e n M L C w e r e set up w i t h a
C T L r e s p o n s e by ( 3 4 5 ) 2 - H x C B was effective o n l y
c o n s t a n t n u m b e r o f B6 r e s p o n d e r cells a n d d e c r e a s -
w h e n a d m i n i s t e r e d w i t h i n 3 d a y s a f t e r P815 chal-
ing n u m b e r s
lenge ( K e r k v l i e t a n d B a e c h e r - S t e p p a n , 1988), this was also the m o s t logical t i m e f r a m e for (345) 2-
o f D2M spleen cells as s t i m u l a t o r s ,
[ 3 H ] T d R i n c o r p o r a t i o n d e c r e a s e d a c c o r d i n g l y (da-
H x C B t r e a t m e n t to affect s u p p r e s s o r cell d e v e l o p -
ta n o t s h o w n ) . In o r d e r to a v o i d s t i m u l a t o r cell lysis by the C T L p r e s e n t in the spleen cell p o p u l a t i o n ,
we subse-
m e n t . H o w e v e r , as s u m m a r i z e d
in T a b l e IV, n o
s u p p r e s s i o n o f e i t h e r the p r o l i f e r a t i v e o r c y t o t o x i c
TABLE Ill Suppressor and cytotoxic activity in the spleen of P815-sensitized mice treated with (345)2-HxCB" HxCB treatment (mg/kg)
Days after P815 sensitization
Suppressor activityb [3H]TdRdR incorporation (cpm) (5 x 105 B6M)
Cytotoxic activity~ % CTX (100:1)
0 10
NI 6 6
9,367 + 2,114 8,307 ± 1,038(11) 9,532 ± 699 (0)
10.2 4- 3.1 6.2 ± 2.8
0 I0
N1 7 7
13,681 ± 3,766 10,146 ± 1,311 (26) 10,921 ± 1,543 (20)
31.8 ± 3.3 II.2 4- 1.8"
0 10
NI 8 8
8,612 ± 1,239 1,108 4- 175 (87) 3,579 4- 614 (58)
40.9 + 1.4 I5.8 ± 2.1"
0 10
NI 9 9
11,065 ± 1,876 1,384 4- 306 (87) 3,207 4- 666 (71)
58.6 4- 4,3 30.3 ± 6.0*
0 10
NI 10 l0
6,356 ± 1,346 1,064 4- 185 (83) 2,603 ± 284 (59)
72.3 4- 3.0 44.3 4- 5.2*
a B6 mice were treated orally with vehicle (0) or 10 mg/kg (345)
19 T A B L E IV Influence of (345)2-HxCB exposure on splenic suppressor activity of B6 mice during the early stages of the P815 allograft response" N u m b e r of B6M added ( x 10 s)
Source of B6M HxCB (mg/kg)
P815 (dpi) b
[3H]TdR incorporation (cpm)
% C T X (10:1) c
0
5.0
5.0
6,997 ± 969
11,912 ± 985 ll,130 ± 615 11,788 ± 616
52.3 56.9 ± 1.3 57.4 ± 1.2
7,086 + 692
-
66.0 58.8 64.0 59.0 54.9
Experiment 1 0 0 10
N1 2 2
Experiment 2 0 0 10 0 l0
N! 3 3 4 4
8,183 10,385 11,108 9,892 8,566
± + ± ± ±
848 518 234 665 678
+ ± ± 4-
0.9 1.2 1.2 2.7
B6 mice were treated orally with 0 to I0 mg/kg (345)2-HxCB one day prior to the i.p. injection of 1 x 107 P815 tumor cells. Spleen cells were obtained 2, 3, or 4 days after P815 injection (dpi) and tested for their ability to suppress the proliferative and differentiative response when added as mitomycin C-treated (B6M) third-party cells to a normal B6 x D2M MLC. Data reflect mean + SE of 5 to 6 mice/group tested individually except n o n i m m u n e (NI) data where spleen cells from a pool of 3 normal untreated mice were used as B6M and SE represents replicate culture (n = 6) variation. b Day post P815 injection. Anti-P815 cytotoxic activity (% CTX) generated in M L C to which 5 x l0 s B6M were added; data shown for a 10:l effector to target cell ratio.
response in MLC was observed with spleen cells taken from vehicle- or (345)2-HxCB-treated mice on day 2, 3, or 4 after P815 challenge. As an alternate approach to avoiding stimulator cell lysis by the CTL present in the spleen cell population, suppressor cell activity was measured in MLC cultures consisting of B6 responder cells and C3M stimulator cells. Because the anti-P815 CTL activity is directed toward the H-2 d haplotype, the C3 (H-2 k) stimulators would not be lysed. Results of this study using spleen cells from mice injected with P815 10 days previously are shown in Table V. Statistically significant suppression of the proliferative response as measured by [3H]TdR incorporation was observed when 5 x 105 or 2.5 x 105 spleen cells from vehicle-treated mice were added to MLC. Spleen cells from (345)2-HxCB-treated
mice exhibited suppressor activity only when 5 x 105 cells were added and the degree of suppression was significantly less as compared to cells from vehicle-treated animals. Spleen cells from neither vehicle- nor (345) 2 -HxCB-treated mice suppressed the in-vitro generation of the CTL response.
Effect of direct addition of (345)2-HxCB in vitro on the lymphocyte proliferation response to mitogen or alloantigen stimulation Since the induction/enhancement of suppressor cell activity did not appear to be a mechanism responsible for suppression of the CTL response following (345)2-HxCB exposure, the ability of (345)2-HxCB to directly alter the T lymphocyte response to alloantigen in vitro in MLC was examined. At the
20 TABLE V Effect of adding mitomycin C-treated P815 (H-2d)-sensitized spleen cells from control or (345)e-HxCB-treated B6 mice on proliferation and CTL activity generated in B6 × C3M (H-2 k) M L ( ? Sourcc of B6 M
Number of B6M added ( x l0 s) to MLC,'CTL
HxCB (mg/kg)
[3H]TdR incorporation (cpm; mean ± SE)
P815 (dpi)
0
% CTX (10:1) h
1.25
2.5
5.0
0
12,409 ± 622 ( 12.9)c 13,632 ± 285 (4.3)
11,484 ± 559 d (19.4) 13,124 ± 457 c (7.8)
8,652 ± 509 a (39.2) 11,263 ± 659 d,~ (20.9)
14,241 ± 521 0
l0
I0
10
5.0
34.1 m 0.5 27.8 ± 1.7 (18.5) 30.4 ± 1.6 (10.9)
Data are presented as mean ± SE of 6 mice/treatment tested individually. Spleen cells were obtained from B6 mice treated orally with 0 or 10 mg/kg (345)2-HxCB one day prior to the i.p. injection of 1 x 10; P815 tumor cells and sacrificed 10 days after P815 challenge. The cells were treated with mitomycin C and added as third-party cells to MLC established with 3 x l0 s 86 responder spleen cells and 6 x l0 s C3M stimulator spleen cells. The MLC proliferative response was measured by the addition of p H ] T d R to cultures 72 hours after initiation, 24 hours prior to harvest. Anti-H-U-CTL activity was quantitated by the lysis of Con A-stimulated, s ~Cr_labeled C3 blasts. b Percent cytotoxicity at a 10:1 effector to target cell ratio. c Percent suppression relative to MLC/CTL responses without third-party B6M cells present shown in parenthesis. a Significantly different from response with no addition of third-party cells, p < 0.05. Significantly different from 0 mg/kg, p < 0.05.
TABLE VI Direct effect of (345)2-HxCB on the lymphoproliferative response to alloantigen in MLC or concanavalin A ~ Treatment HxCB (M)
[3H]TdR incorporation (cpm; mean ± SD) Vehicle (%)
MLC
Con A
Vehicle
+ HxCB
Vehicle
+ HxCB
Experiment l: p-Dioxane 0 0 10 5 0.25 10 ~' 0.025 10 7 0.0025 l0 s 0.00025
3.086 17.996 26,087 21,584
± 517 ± 1,977 ± 1,848 + 4,044
21,088 ± 4,505 2,240 15,277 19,921 24,510
± [79 ± 2,860 ± 2,043 ± 5,843
I35,420 139,295 138,170 146,728
139,400 ± 4,630 ± 2,467 I08,245 ± 3,212" ± 671 141,207 ± 4,474 ± I2,988 136,253 ± 3,894 ± 1,782 148,185 ± 2,771
Expcriment 2: Acetone 0 0 l0 5 0.25 10 ~' 0.025 10 7 0.0025 10 -~ 0.00025
8,425 21,621 22,284 23,179
3_ 1,535 ± 635 ± 893 ± 1,805
18,869 + 3,750 12,288 I9,981 20,534 20,444
± 2,766 ± 4,499 + 940 ± 2,915
114,981 121,562 119,031 125,515
± ± ± ±
Data represent mean ± SD of 3-6 replicate cultures. * Significantly different from vehicle control, p < 0.05.
115,333 ± 7,625 2,357 123,284 ± 940 321 I21,896 ± 5,583 6,616 116.804 ± 6,705 3,027 122,470 ± 4,440
21 same time, the direct effect of (345)2-HxCB on the in-vitro lymphoproliferative response to the T cell mitogen Con A was examined. The results of two experiments are summarized in Table VI using two different vehicles, p-dioxane or acetone. Due to the limited solubility of (345) 2HxCB in either solvent, the highest concentration that could be tested was 1 x 10 -5 M at a vehicle concentration of 0.25%. At this concentration, significant vehicle effects were produced on the lymphoproliferative response to alloantigen in MLC but not to Con A. Effects of (345)2-HxCB on the MLC response were produced only at the 10 -5 M concentration and thus could not be differentiated from effects due to vehicle only. Significant suppression of the Con A response was observed at 10-5 M (345)2-HxCB using p-dioxane as vehicle but this effect was not confirmed in the second experiment using acetone as vehicle. Studies using dimethylsulfoxide as vehicle also failed to separate vehicle effects from specific effects of (345)2-HxCB (data not shown). Based on these results, the direct effect of (345)2-HxCB on the proliferative response of T cells to mitogen or alloantigen remains in question.
Discussion In the preceding paper, we described the sensitivity of the in-vivo allogeneic CTL response to suppression by the toxic, Ah receptor-binding (345) 2HxCB isomer. The studies reported in this paper have examined the sensitivity of the in-vitro M L C / C T L response to suppression by this isomer and the possible role of suppressor cells in mediating its immunosuppressive effect. These latter studies were precipitated in part on the basis of the results of Clark et al. (1981, 1983), which suggested that the cellular basis for the suppression of CTL activity by T C D D and related Ah receptor-binding PCB isomers was due to suppressor T cell activation via altered lymphocyte processing in the thymus (Nagarkatti et al., 1984). In the current studies, B6 mice were exposed to a single oral dose of (345)2-HxCB and sacrificed at various times thereafter for assessment of the func-
tional activity of their splenocytes as responders in M L C / C T L or as third-party suppressors of an independent MLC/CTL. The effect of direct in vitro addition of (345)2-HxCB on the lymphoproliferation response to mitogen or alloantigen was also assessed. Results of the present studies indicated that there was modest dose-dependent suppression of the invitro proliferative response to alloantigen of lymphocytes obtained from mice exposed to 10 or 100 mg/kg (345)2-HxCB whereas the MLC-generated CTL response was significantly suppressed only following exposure to the 100 mg/kg dose. Even at the relatively high dose of 100 mg/kg (345)2-HxCB, the in-vitro CTL response was only modestly suppressed. Furthermore, the magnitude of suppression of the M L C / C T L response did not appear to be influenced by the amount of time that elapsed between dosing and sacrifice, which was assessed over a 3-week period. Thus, in comparison to the in-vivo generation of CTL which was highly suppressed following exposure to a single dose of 10 mg/kg (345)2-HxCB (Kerkvliet and Baecher-Steppan, 1988), the in-vitro M L C / C T L response was considerably more resistant to (345)2-HxCB-in duced suppression. The direct addition of (345)2-HxCB to lymphocyte cultures at concentrations of 1 × 10 6 M or less did not suppress the lymphoproliferative response to either alloantigen in MLC or to the T cell mitogen Con A. Determination of effects at higher concentrations of (345)2-HxCB in vitro were compromised by significant vehicle effects on lymphocyte proliferation. This lack of effect following in-vitro exposure to (345)2-HxCB as well as the functional integrity of lymphocytes taken from animals exposed to doses of (345)Z-HxCB that are highly functionally immunosuppressed when challenged with antigen in vivo may suggest an indirect effect of (345)2-HxCB on T cell responses that is mediated via nonlymphoid target tissue. The possible role of suppressor cells in mediating (345)2-HxCB-induced suppression of CTL development was addressed in the present studies by several different approaches. When naive spleen cells were taken from vehicle- or (345)2-HxCB-treated
22 mice at various times after exposure, treated with mitomycin C and added as third-party cells to a normal MLC, no evidence for suppression of the development of the proliferative or cytotoxic responses was found. This apparent lack of suppresactivity in (345)2-HxCB-treated mice was not related to the time of sacrifice relative to exposure or the sex of the donor animals. The lack of detectable suppressor activity in the spleen of mice treated with (345)2-HxCB suggested that chemical exposure alone did not promote the development of nonspecific suppressor cell function. However, the experimental conditions employed did not address the possibility that (345) 2HxCB interacted with antigen to promote antigeninduced suppressor cell function. This possibility was addressed in several experiments using mitomycin C-treated spleen cells obtained from vehicleor (345)2-HxCB-treated mice at various times after P815 injection as putative suppressor cells. The results of these studies indicated that spleen cells from P815-sensitized mice were indeed suppressive when added as third-party cells to a normal M L C but only when measurable C T L activity was also present in the same population of cells. In fact, a direct correlation was observed between the level of C T L activity present in the spleen cell population and the level of 'suppressor' activity. Thus, (345)2-HxCB treated mice which showed suppressed C T L function also exhibited reduced 'suppressor' function. Assuming that biologically relevant suppressor activity would correlate inversely with C T L activity, these results suggested that the assay was actually measuring CTL-mediated stimulator cell lysis rather than suppressor cell activity. This conclusion was supported by the demonstration that mitomycin C-treated P815-immune spleen cells were still capable of lysing SlCr-labeled P815 target cells. Likewise, when M LCs were established using decreasing numbers of stimulator cells, the proliferative response that was generated decreased in magnitude accordingly. This concept of suppressor responses resulting from the cytotoxic properties of T cells mixed with responder and stimulator cells in M L C has been previously addressed in detail (Fitch et al., 1976; Sinclair et al., 1976; Sugarbaker and Matthews, 1981).
Alternatively, these results suggest that both CTL and suppressor cell activities develop in parallel following P815 injection and that both activities are inhibited by exposure to (345)2-HxCB. This interpretation is supported to some extent by the results of experiments using C3M spleen cells as stimulator cells in MLC. Because the CTL activity generated in PS15-sensitized B6 mice is primarily anti-H-2 d, the H-2 k C3M stimulators were not targets for the C T L present in the B6~ cell suspension used as putative suppressor cells. Nonspecific suppressor activity in these cultures was significantly greater when cells from vehicle-treated as compared to (345)2-HxCB-treated mice were added, suggesting that treatment with (345)2-HxCB inhibited suppressor cell activity. Thus, based on the results reported here, we conclude that the in-vivo suppression of allogeneic CTL activity following acute exposure to (345) 2HxCB is not mediated via altered suppressor cell activity. The relative functional integrity of lymphocytes taken from (345)2-HxCB-treated mice to respond in vitro in M L C / C T L and the inability to directly suppress the M L C response by the addition of (345)2-HxCB to culture suggest an indirect mechanism for (345)2-HxCB-induced suppression of allogeneic T cell responses. As discussed in the preceding paper, the role of elevated corticosteroid levels in (345)2-HxCB-treated mice is under current investigation.
Acknowledgements This work was supported by N I E H S grants ES00040 and ES00210. The authors thank Ms. Lynne Rogers, Word Processing Specialist, for her assistance in preparation of the manuscript.
References Argyris BF. Suppressor cells in the spleen of tumor-allosensitized mice. Cancer Res 1977:37:3390. Argyris B, DeLustro F. Immunologic unresponsiveness of mouse spleen sensitized to allogeneic tumors. Cell Immunol 1977:28:390. Clark DA, (}auldie J, Sweeney G. Dose response, time-course
23 and mechanism for suppression of cytotoxic T cell generation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. In: Poland A, Kimbrough RD, eds. Biological Mechanisms of Dioxin Action. Banbury Report 18. Cold Spring Harbor Laboratory pp. 421-434. Clark DA, Gauldie J, Szewczuk MR, Sweeney G. Enhanced suppressor cell activity as a mechanism of immunosuppression by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Proc Soc Exp Biol Med 1981;168:290. Clark DA, Sweeney G, Safe S, Hancock E, Kilburn DG, Gauldie J. Cellular and genetic basis for suppression of cytotoxic T cell generation by haloaromatic hydrocarbons. Immunopharmacology 1983;6:143. Fitch FW, Engers HD, Cerottini JC, Brunner KT. Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response. J lmmunol 1976;116:716. Kerkvliet NI, Baecher-Steppan L. Suppression of allograft immunity by 3,4,5,3',4',5'-hexachlorobiphenyl. I. Effects of ex-
posure on tumor rejection and cytotoxic T cell activity in vivo. Immunopharmacology 1988;16:1-12. Kerkvliet NI, Brauner JA, Baecher-Steppan L. Effects of dietary technical pentachlorophenol exposure on T cell, macrophage, and natural killer cell activity in C57B1/6 mice. Int J Immunopharmacol 1985;7:239. Nagarkatti PS, Sweeney GD, Gauldie J, Clark DA. Sensitivity to suppression of cytotoxic T cell generation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on the Ah genotype of the murine host. Toxicol Appl Pharmacol 1984;72:169. Sinclair NRStC, Lees MK, Wheeler ME, Vichos EE, Fung FY. Regulation of the immune response. XI. Cell-mediated feedback of an in vitro cell-mediated immune response. Cell lmmunol 1976;27:153. Sugarbaker PH, Matthews W. Cytotoxic cells suppress in vitro generation of cellular immunity by stimulator cell lysis. Cell lmmunol 1981;57:124.
13
Suppression of allograft immunity by 3,4,5,3',4',5'hexachlorobiphenyl. II. Effects of exposure on mixed lymphocyte reactivity and induction of suppressor cell activity in vitro N.I. Kerkvliet and L. Baecher-Steppan College o[" Veterinary Medicine and Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, U.S.A. (Received 10 December 1987; accepted 18 March 1988)
Abstract: Previous studies in our laboratory have established the sensitivity of the in vivo allogeneic cytotoxic T lymphocyte (CTL) response to suppression by 3,4,5,Y,4',5'-hexachlorobiphenyl [(345)2-HxCB], a toxic, Ah receptor-binding polychlorinated biphenyl isomer. The present studies have examined possible cellular mechanisms for this suppression. A modest dose-dependent suppression of the proliferative response to alloantigen in mixed lymphocyte culture (MLC) was observed with lymphocytes from B6 mice exposed to 10 or 100 mg/kg (345)2-HxCB while the CTL response generated in MLC was significantly suppressed only following exposure to 100 mg/kg (345)2-HxCB. The amount of time between treatment with (345)2-HxCB and sacrifice, which ranged from 2 to 23 days, did not appear to influence the degree of immunosuppression produced by (345) 2-HxCB exposure. Mitomycin C-treated lymphocytes from B6 mice treated with (345)2-HxCB were not suppressive when added as third party cells to an independent MLC. However, if the mice were alloimmune, lymphocyte-mediated suppression of the MLC response was observed and directly correlated with the magnitude of the CTL response present in the same population. Thus, (345)2-HxCB-treated mice which had less CTL activity as compared to vehicle-treated mice also had less suppressor activity. Further analysis indicated that stimulator cell lysis by the CTL was likely to be responsible for the inhibitory activity of the alloimmune lymphocytes rather than suppressor cells per se. Avoidance of stimulator cell lysis by using H-2-incompatible MLC stimulator cells revealed the existence of antigen-nonspecific suppressor activity that was greater with lymphocytes from vehicle-treated than from (345)2-HxCB-treated mice, suggesting that both CTL and suppressor cell activities were suppressed by (345)2-HxCB exposure. Direct addition of (345)2-HxCB to lymphocyte cultures in vitro indicated a lack of direct toxicity of (345)2-HxCB on lymphoproliferative responses to mitogen or alloantigen at concentrations equal to or less than 1 x 10 .6 M. Thus, the in-vitro functional integrity of lymphocytes obtained from (345)2-HxCB-treated mice coupled with the lack of a direct lymphotoxic effect of (345)2-HxCB in vitro suggest an indirect mechanism of action for (345)2-HxCB-mediated suppression of CTL activity in vivo. Previous reports implicating suppressor cell induction and/or activation by Ah-receptor-binding halogenated aromatic hydrocarbons that mediate the inhibition of CTL generation were not confirmed in these studies. Key words: Polychlorinated biphenyls; 3,4,5,3',4',5'-Hexachlorobiphenyl; lmmunotoxicity; Suppressor cells; Mixed lymphocyte reactivity; Cytotoxic T cells; Mouse
Correspondence: Dr N.I. Kerkvliet, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, U.S.A. Abbreviations: (345)Z-HxCB, 3,4,5,Y,4',5'-hexachlorobiphenyl; CTL, cytotoxic T lymphocyte; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; M LC, mixed lymphocyte culture; B6, C57B 1/6; D2, DBA/2; C3, C3H; B6M, D2M, C3M, mitomycin-C treated B6, D2, or C3 spleen cells; FBS, fetal bovine serum; Con A, concanavalin A; CTX, cytotoxicity; HAH, halogenated aromatic hydrocarbons.
Introduction
In the preceding report we demonstrated that invivo exposure of C57B1/6 (B6) mice to the toxic, Ah-receptor-binding 3,4,5,3',4',5'-hexachlorobiphenyl [(345)2-HxCB] suppressed the in-vivo generation of cytotoxic T lymphocytes (CTL) to allogeneic P815 tumor cell challenge (Kerkvliet and
0162-3109/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
14 Baecher-Steppan, 1988). Dose-dependent suppression of the peak CTL response on day 10 12 after tumor challenge was observed in both male and female mice. This suppression was not due to a shift in kinetics of the response nor to a cytotoxic effect of (345)2-HxCB on the growth of the P815 cells. Significant suppression of CTL activity was observed when (345)2-HxCB was given prior to or during the first three days after P815 sensitization, whereas exposure later than 3 days after antigen was not suppressive. These results suggested that an early event in the activation/maturation of CTL was sensitive to (345)2-HxCB exposure whereas mature CTL appeared to be resistant to (345) zHxCB-mediated suppression. Clark et al. (1981, 1983) have reported that exposure of mice to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) and related Ah-receptor-binding halogenated aromatic hydrocarbons (HAH) significantly impaired the generation of alloantigenstimulated CTL in vitro in mixed lymphocyte cultures (MLC). Whereas the frequency of CTL precursors in the spleen or lymph nodes was not altered by T C D D exposure (Clark et al., 1981), the demonstration of cells in the thymus of T C D D treated mice capable of inhibiting the in-vitro CTL response led to the proposal that the cellular basis for the suppression of CTL activity by T C D D was due to suppressor T cell activation (Clark et al., 1981). On the basis of these reports, the studies described in this paper have examined the sensitivity of the in-vitro MLC response to (345)2-HxCB and the possible role for suppressor cells in mediating (345)2-HxCB-induced suppression of the CTL response to alloantigen.
Materials and Methods Anima&
B6 (H-2b), DBA/2 (D2; H-2 a) and C3H (C3; H-2 k) mice were purchased from Jackson Laboratory (Bar Harbor, ME). They were maintained as previously described (Kerkvliet and Baecher-Steppan, 1988) and used when they were 8 10 weeks of age.
PCB e.vposure
(345)2-HxCB (Ultrascientific, Hope, RI) was dissolved in acetone and diluted in peanut oil as previously described (Kerkvliet and Baecher-Steppan, 1988). Mice were g'iven a single dose orally by stomach tube at various times relative to P815 sensitization and/or sacrifice. In some experiments, (345)2-HxCB was dissolved in different vehicles, as indicated, and added to the culture medium. A lloimmuniza tion
B6 mice were injected i.p. with 1-10 × 10~' viable P815 (H-2 a) mastocytoma cells obtained from the ascites fluid of D2 mice as previously described (Kerkvliet and Baecher-Steppan, 1988). Lymphocyte cultures
MLC, CTL, and suppressor cell activity was measured following the basic protocols described by Argyris (1977) and Argyris and DeLustro (1977). Single-cell suspensions of splenic lymphocytes from B6, D2, or C3 mice were prepared as previously described (Kerkvliet and Baecher-Steppan, 1988). One-way MLCs were established in triplicate wells of flat-bottom microtiter plates using 3 × l0 s B6 responder spleen cells and 3 x l0 s mitomycin Ctreated D2 (D2m) or B6 [B6m (syngeneic control)] as stimulators in a total volume of 200/A RPMI 1640 containing 5% fetal bovine serum (FBS) and 5 x 10 5 M 2-mercaptoethanol. In one experiment, 6 x l0 s mitomycin C-treated C3 (C3m) splenocytes were used as stimulator cells. Cultures were incubated for 4 or 5 days at 37°C in a humidified atmosphere of 5% CO2. The proliferative response in MLC was assayed by the addition of tritiated thymidine ([3H]TdR; 0.5 l~Ci/well) 24 hours prior to culture termination. The cells were harvested from cultures and processed for liquid scintillation counting as previously described (Kerkvliet et al., 1985). CTL activity present in day 5 MLCs was measured using a s~Cr-release assay as previously described using s~Cr-labeled P815 tumor cells
15 (Kerkvliet et al., 1985) or, in one experiment, concanavalin A (Con A)-stimulated C3 blasts as targets. C3 blasts were prepared by culturing C3 spleen cells for 48 hours at a density of 1 x 106 cells/ml in 75 cm 2 flasks in RPM! 1640 containing 2/~g/ml Con A. After 48 hours, the cells were harvested and labeled with 51Cr as described for the P815 target cells. Effector: target cell ratios of 100:1, 30:1, 10:1, and 3:1 were tested. Percentage of cytotoxicity (% CTX) was calculated as described in the preceding paper (Kerkvliet and Baecher-Steppan, 1988). Cells to be tested for suppressor activity were pretreated with mitomycin C (50 #g/2 x l0 T cells/2 ml for 60 min), washed three times, and added as thirdparty cells to normal M L C / C T L cultures. In all experiments, third-party cell concentrations ranging from 0.6 to 5 × 105/well were tested.
sisted of 6 mice tested individually or in pools of 3 for MLC responses and/or suppressor cell activity. The results of each experiment were independently confirmed at least one time. The mean response of each animal or pool was calculated from triplicate cultures; the means were then used for calculating treatment means and carrying out statistical analysis as decribed in the preceding paper (Kerkvliet and Baecher-Steppan, 1988). In cases where culture replicates were the only measure of variation, the standard deviation of the replicates is presented.
Results
Influence of in-vivo (345)Z-HxCB exposure on the proliferative and differentiative response of splenic lymphocytes in allogeneic MLC
Statistical analysis In most experiments, each treatment group con-
B6 mice were given a single oral dose of 0, t0, or 100 mg/kg (345)2-HxCB. At various times after
TABLE I Influence of exposure to (345)2-HxCB on the proliferative and differentiative response of lymphocytes in MLC a (345)Z-HxCB treatment b (mg/kg)
Time of treatment c (days)
[3H]TdR incorporation (cpm; mean 4, SE) B6 x B6~
B6 × D2M~
% CTX a (10:I)
0 10
-2 -2
2,658 4, 122 1,940 4, 194"
24,779 4- 1,557 18,006 + 1,467" (73)
34.1 4, 2.2 29.7 + 2.2
0 10
-11 -11
1,967 4- 191 1,931 4, 109
12,626 411,221 4-
36.5 4- 1.9 36.6 4- 2.6
0 10 100
-18 -18 -18
2,921 4, 163 2,841 4- 251 2,344 4- 137"
36,046 4- 2,782 30,172 4- 3,074 (84) 25,076 4- 2,473* (70)
56.7 4. 1.0 54.8 4- 1.3 51.1 4, 1.9"
0 10 100
-23 -23 -23
801 + 70 1,022 4- 172 826 + 54
17,947 + 619 13,054 + 1,184" (73) 10,835 ± 604* (60)
26.5 4- 2.9 23.5 4- 2.2 17.5 + 2.5*
618 721 (89)
a Data represent mean + S.E. of 5-6 mice/treatment tested individually. Female mice were used in all experiments except for the day - 2 3 study where data from male mice are shown. b.c (345)2_HxCB was administered orally to B6 mice on the indicated day relative to sacrifice on day 0. d Anti-P815 cytotoxicity generated in day 5 MLC cultures. Cells were harvested and tested at several E:T ratios. Data for an E:T ratio of 10:1 is shown. Values in parenthesis represent the MLC response of cells from (345)
16 dosing, the mice were sacrificed and their splenic lymphocytes were cultured in vitro in M LC using D2M spleen cells as stimulators. Data from several representative experiments are shown in Table I. The ability of lymphocytes to proliferate in response to alloantigen stimulation was dose-dependently suppressed by prior in-vivo exposure to (345)2-HxCB. The 10 mg/kg exposure level appeared to be the minimum effective dose, producing a modest degree of suppression (11 to 27%) of p H ] T d R incorporation which was statistically significant in 2/4 experiments. The MLC-generated CTL response was less sensitive to suppression with a dose of 100 mg/kg required to produce significant suppression. No clear relationship between time of exposure to (345)2-HxCB and time of sacrifice on
the degree of inhibition of MLC or CTL responses was apparent.
Suppression o/" the MLC/CTL response by lymphocytes.from animals exposed to (345)2-HxCB The possibility that suppression of the CTL response in vivo was due to the induction of suppressor cells following (345p-HxCB exposure was examined in several experiments. Initially, mice were treated with 0 or 10 mg/kg (345)2-HxCB and sacrificed two days later for assessment of suppressor cell activity in the spleen since this dose and timing of exposure relative to P815 injection produced significant suppression of the in vivo CTL response. Later experiments were expanded to in-
TABLE lI Ability of mitomycin-treated spleen cells from mice treated with (345)2-HxCB to suppress the MLC,,CTL response" Source of B6Mh HxCB (mg,'kg)
Number of B6M added to MLC Day of treatment
p H]TdR incorporation (cpm) 0
0 10
2 2
0 1 10 100
--7 -7 -7 -7
0 I0 100
-17 --17 17
%CTX (10:1) c 5.0 x 105
5.0 x 105
21,178 ± 1,397 28,294 ± 30,429 ±
581 997
42.6 ± 2.3 42.1 ± 1.6
24,479 ± 2,069 38,372 41,643 38,605 38,674 15,248 ±
± 1,515 ± 1,034 ± 705 ± 237
NT '~ NT NT NT
615 14,522 ± 1,406 16,292 ± 645 13,027 ± 1,235
24.0 ± 0.8 25.4 ± 0.9 24.7 ± 1.9
a Data for day --2 study represent two pools of 3 mice/treatment; mean values ± SE for [3H]TdR cpm is based on 3 replicate wells per pool. Data for % CTX on day - 2 represents the average response of 2 pools + SD. Data for day - 7 represent 1 pool of 3 mice/treatment and mean ± SD is based on 3 replicate wells. Data for day - 17 study represents the mean :t_ SE of 5 mice/treatment tested individually for [3H]TdR and % CTX. The baseline MLC response with no added B6M cells is the mean response of a pool of 3 mice: the SE is based on 3 6 replicate wells. b B6 mice were given the indicated dose of (345)2-HxCB orally on the day of treatment relative to sacrifice on day 0 for utilization of their splenic lymphocytes (B6M) as suppressor cells in MLC. % CTX generated in MLC to which 5 x l0 s B6M cells were added, data shown for E:T ratio of 30:i. d NT - not tested.
17 clude a 100 mg/kg exposure level and various times of sacrifice ranging from 2 to 23 days after (345) 2HxCB exposure. Comparative studies using male and female mice were also conducted. Representative results from these studies are summarized in Table II. The addition of mitomycin C-treated spleen cells from control or (345)2-HxCB-treated B6 mice as third-party cells to a normal B6 x D2M MLC resulted in no change or enhanced [3H]TdR incorporation with no evidence for suppression of the proliferative response due to (345)2-HxCB treatment. The stimulatory effect was not due to proliferation by the mitomycin C-treated third-party cells as these cells did not proliferate in B6M x D2M MLC (data not shown). The generation of CTL in MLC was also not suppressed by the addition of third-party cells from (345)2-HxCB-treat ed mice as compared to the addition of cells from vehicle-treated mice (Table II). This apparent lack of suppressor cell activity in the spleen of (345)2HxCB-treated mice was not related to time of sacrifice after dosing or the sex of the donor animals.
creased from day 6 to day 10, which correlated with the development of cytotoxic activity in the spleen. At all time points examined, the degree of inhibition of MLC proliferation was greater with spleen cells from vehicle-treated mice as compared to spleen cells from (345)2-HxCB-treated mice, which also reflected the amount of CTL activity present in the spleen. Subsequent analysis of all available data from both control and (345)2-HxCB-treated mice showed a high degree of correlation between the amount of cytotoxic activity present in a given spleen cell suspension and the degree of inhibition of the MLC response, which was independent of (345)2-HxCB treatment (Fig. 1). Since these results are in direct contrast to what one would expect biologically (e.g., greater CTL activity should imply less suppressor cell activity), it suggested that the inhibitory effect of the spleen cells in MLC was due not to suppressor cell activity but rather to a loss
80
Suppression of the MLC/CTL response by PS15sensitized lymphocytes from animals exposed to (345) 2_HxCB: The lack of detectable suppressor cell activity in the spleen of mice treated with (345)2-HxCB suggested that (345)2-HxCB treatment alone did not promote the development of nonspecific suppressor cell function. However, these studies did not address the possibility that (345)2-HxCB interacted with antigen challenge to modulate antigen-induced suppressor cell function. In order to test this possibility, suppressor activity associated with spleen cells taken from control and (345)2-HxCB-treated mice at various times after P815 challenge was examined. As summarized in Table III, spleen cells from P815-sensitized mice, when added as mitomycin C-treated third-party cells to a normal B6 x D2M MLC, produced significant suppression of [3H]TdR incorporation as compared to the addition of spleen cells from non-P815-sensitized mice. For vehicletreated mice, the suppressive activity of the spleen cells increased as the time after P815 injection in-
6O
c) 40 •
X 0 ~ 2 0 -
0
0
'
•
i
i
I
2o
40
60
80
1oo
%
Inhibition
of
MLC
Fig. 1. Linear correlation between the cytotoxic and MLC-suppressive activities of spleen cells from P815-sensitized B6 mice. Spleen cells were obtained from B6 mice at various times after sensitization with P815 (H-2 d) mastocytoma cells. The suppressive activity of the spleen cells was measured by their ability to inhibit the proliferative response of a B6 anti-D2 MLC when added as rnitomycin C-treated third-party cells. Cytotoxic activity in the same spleen cell suspension was determined by their ability to lyse 51 Cr-labeled P815 tumor cells in vitro. Data shown represent percent suppression of [3H]TdR incorporation in MLC with 5 × l0 s B6M spleen cells added; % CTX is shown for a 3021 effector to target cell ratio.
18 o f M L C s t i m u l a t o r cells c a u s e d by t h e i r lysis by the
q u e n t l y f o c u s e d on the p o s s i b i l i t y t h a t s u p p r e s s o r
C T L p r e s e n t in the s a m e splenic p o p u l a t i o n . T h i s
cell a c t i v i t y m i g h t
be p r e s e n t
in the spleen
of
i n t e r p r e t a t i o n was s u p p o r t e d by the results o f a d -
P815-sensitized m i c e d u r i n g t h e early stages o f sen-
d i t i o n a l studies in w h i c h we d e m o n s t r a t e d t h a t mi-
s i t i z a t i o n p r i o r to the d e v e l o p m e n t o f m a t u r e C T L
t o m y c i n C - t r e a t e d C T L w e r e still f u n c t i o n a l effec-
in the spleen (i.e., p r i o r to d a y 6). B a s e d o n pre-
t o t cells c a p a b l e o f l y s i n g P815 t a r g e t cells ( d a t a n o t
v i o u s results w h i c h s h o w e d t h a t s u p p r e s s i o n o f the
s h o w n ) . L i k e w i s e , w h e n M L C w e r e set up w i t h a
C T L r e s p o n s e by ( 3 4 5 ) 2 - H x C B was effective o n l y
c o n s t a n t n u m b e r o f B6 r e s p o n d e r cells a n d d e c r e a s -
w h e n a d m i n i s t e r e d w i t h i n 3 d a y s a f t e r P815 chal-
ing n u m b e r s
lenge ( K e r k v l i e t a n d B a e c h e r - S t e p p a n , 1988), this was also the m o s t logical t i m e f r a m e for (345) 2-
o f D2M spleen cells as s t i m u l a t o r s ,
[ 3 H ] T d R i n c o r p o r a t i o n d e c r e a s e d a c c o r d i n g l y (da-
H x C B t r e a t m e n t to affect s u p p r e s s o r cell d e v e l o p -
ta n o t s h o w n ) . In o r d e r to a v o i d s t i m u l a t o r cell lysis by the C T L p r e s e n t in the spleen cell p o p u l a t i o n ,
we subse-
m e n t . H o w e v e r , as s u m m a r i z e d
in T a b l e IV, n o
s u p p r e s s i o n o f e i t h e r the p r o l i f e r a t i v e o r c y t o t o x i c
TABLE Ill Suppressor and cytotoxic activity in the spleen of P815-sensitized mice treated with (345)2-HxCB" HxCB treatment (mg/kg)
Days after P815 sensitization
Suppressor activityb [3H]TdRdR incorporation (cpm) (5 x 105 B6M)
Cytotoxic activity~ % CTX (100:1)
0 10
NI 6 6
9,367 + 2,114 8,307 ± 1,038(11) 9,532 ± 699 (0)
10.2 4- 3.1 6.2 ± 2.8
0 I0
N1 7 7
13,681 ± 3,766 10,146 ± 1,311 (26) 10,921 ± 1,543 (20)
31.8 ± 3.3 II.2 4- 1.8"
0 10
NI 8 8
8,612 ± 1,239 1,108 4- 175 (87) 3,579 4- 614 (58)
40.9 + 1.4 I5.8 ± 2.1"
0 10
NI 9 9
11,065 ± 1,876 1,384 4- 306 (87) 3,207 4- 666 (71)
58.6 4- 4,3 30.3 ± 6.0*
0 10
NI 10 l0
6,356 ± 1,346 1,064 4- 185 (83) 2,603 ± 284 (59)
72.3 4- 3.0 44.3 4- 5.2*
a B6 mice were treated orally with vehicle (0) or 10 mg/kg (345)
19 T A B L E IV Influence of (345)2-HxCB exposure on splenic suppressor activity of B6 mice during the early stages of the P815 allograft response" N u m b e r of B6M added ( x 10 s)
Source of B6M HxCB (mg/kg)
P815 (dpi) b
[3H]TdR incorporation (cpm)
% C T X (10:1) c
0
5.0
5.0
6,997 ± 969
11,912 ± 985 ll,130 ± 615 11,788 ± 616
52.3 56.9 ± 1.3 57.4 ± 1.2
7,086 + 692
-
66.0 58.8 64.0 59.0 54.9
Experiment 1 0 0 10
N1 2 2
Experiment 2 0 0 10 0 l0
N! 3 3 4 4
8,183 10,385 11,108 9,892 8,566
± + ± ± ±
848 518 234 665 678
+ ± ± 4-
0.9 1.2 1.2 2.7
B6 mice were treated orally with 0 to I0 mg/kg (345)2-HxCB one day prior to the i.p. injection of 1 x 107 P815 tumor cells. Spleen cells were obtained 2, 3, or 4 days after P815 injection (dpi) and tested for their ability to suppress the proliferative and differentiative response when added as mitomycin C-treated (B6M) third-party cells to a normal B6 x D2M MLC. Data reflect mean + SE of 5 to 6 mice/group tested individually except n o n i m m u n e (NI) data where spleen cells from a pool of 3 normal untreated mice were used as B6M and SE represents replicate culture (n = 6) variation. b Day post P815 injection. Anti-P815 cytotoxic activity (% CTX) generated in M L C to which 5 x l0 s B6M were added; data shown for a 10:l effector to target cell ratio.
response in MLC was observed with spleen cells taken from vehicle- or (345)2-HxCB-treated mice on day 2, 3, or 4 after P815 challenge. As an alternate approach to avoiding stimulator cell lysis by the CTL present in the spleen cell population, suppressor cell activity was measured in MLC cultures consisting of B6 responder cells and C3M stimulator cells. Because the anti-P815 CTL activity is directed toward the H-2 d haplotype, the C3 (H-2 k) stimulators would not be lysed. Results of this study using spleen cells from mice injected with P815 10 days previously are shown in Table V. Statistically significant suppression of the proliferative response as measured by [3H]TdR incorporation was observed when 5 x 105 or 2.5 x 105 spleen cells from vehicle-treated mice were added to MLC. Spleen cells from (345)2-HxCB-treated
mice exhibited suppressor activity only when 5 x 105 cells were added and the degree of suppression was significantly less as compared to cells from vehicle-treated animals. Spleen cells from neither vehicle- nor (345) 2 -HxCB-treated mice suppressed the in-vitro generation of the CTL response.
Effect of direct addition of (345)2-HxCB in vitro on the lymphocyte proliferation response to mitogen or alloantigen stimulation Since the induction/enhancement of suppressor cell activity did not appear to be a mechanism responsible for suppression of the CTL response following (345)2-HxCB exposure, the ability of (345)2-HxCB to directly alter the T lymphocyte response to alloantigen in vitro in MLC was examined. At the
20 TABLE V Effect of adding mitomycin C-treated P815 (H-2d)-sensitized spleen cells from control or (345)e-HxCB-treated B6 mice on proliferation and CTL activity generated in B6 × C3M (H-2 k) M L ( ? Sourcc of B6 M
Number of B6M added ( x l0 s) to MLC,'CTL
HxCB (mg/kg)
[3H]TdR incorporation (cpm; mean ± SE)
P815 (dpi)
0
% CTX (10:1) h
1.25
2.5
5.0
0
12,409 ± 622 ( 12.9)c 13,632 ± 285 (4.3)
11,484 ± 559 d (19.4) 13,124 ± 457 c (7.8)
8,652 ± 509 a (39.2) 11,263 ± 659 d,~ (20.9)
14,241 ± 521 0
l0
I0
10
5.0
34.1 m 0.5 27.8 ± 1.7 (18.5) 30.4 ± 1.6 (10.9)
Data are presented as mean ± SE of 6 mice/treatment tested individually. Spleen cells were obtained from B6 mice treated orally with 0 or 10 mg/kg (345)2-HxCB one day prior to the i.p. injection of 1 x 10; P815 tumor cells and sacrificed 10 days after P815 challenge. The cells were treated with mitomycin C and added as third-party cells to MLC established with 3 x l0 s 86 responder spleen cells and 6 x l0 s C3M stimulator spleen cells. The MLC proliferative response was measured by the addition of p H ] T d R to cultures 72 hours after initiation, 24 hours prior to harvest. Anti-H-U-CTL activity was quantitated by the lysis of Con A-stimulated, s ~Cr_labeled C3 blasts. b Percent cytotoxicity at a 10:1 effector to target cell ratio. c Percent suppression relative to MLC/CTL responses without third-party B6M cells present shown in parenthesis. a Significantly different from response with no addition of third-party cells, p < 0.05. Significantly different from 0 mg/kg, p < 0.05.
TABLE VI Direct effect of (345)2-HxCB on the lymphoproliferative response to alloantigen in MLC or concanavalin A ~ Treatment HxCB (M)
[3H]TdR incorporation (cpm; mean ± SD) Vehicle (%)
MLC
Con A
Vehicle
+ HxCB
Vehicle
+ HxCB
Experiment l: p-Dioxane 0 0 10 5 0.25 10 ~' 0.025 10 7 0.0025 l0 s 0.00025
3.086 17.996 26,087 21,584
± 517 ± 1,977 ± 1,848 + 4,044
21,088 ± 4,505 2,240 15,277 19,921 24,510
± [79 ± 2,860 ± 2,043 ± 5,843
I35,420 139,295 138,170 146,728
139,400 ± 4,630 ± 2,467 I08,245 ± 3,212" ± 671 141,207 ± 4,474 ± I2,988 136,253 ± 3,894 ± 1,782 148,185 ± 2,771
Expcriment 2: Acetone 0 0 l0 5 0.25 10 ~' 0.025 10 7 0.0025 10 -~ 0.00025
8,425 21,621 22,284 23,179
3_ 1,535 ± 635 ± 893 ± 1,805
18,869 + 3,750 12,288 I9,981 20,534 20,444
± 2,766 ± 4,499 + 940 ± 2,915
114,981 121,562 119,031 125,515
± ± ± ±
Data represent mean ± SD of 3-6 replicate cultures. * Significantly different from vehicle control, p < 0.05.
115,333 ± 7,625 2,357 123,284 ± 940 321 I21,896 ± 5,583 6,616 116.804 ± 6,705 3,027 122,470 ± 4,440
21 same time, the direct effect of (345)2-HxCB on the in-vitro lymphoproliferative response to the T cell mitogen Con A was examined. The results of two experiments are summarized in Table VI using two different vehicles, p-dioxane or acetone. Due to the limited solubility of (345) 2HxCB in either solvent, the highest concentration that could be tested was 1 x 10 -5 M at a vehicle concentration of 0.25%. At this concentration, significant vehicle effects were produced on the lymphoproliferative response to alloantigen in MLC but not to Con A. Effects of (345)2-HxCB on the MLC response were produced only at the 10 -5 M concentration and thus could not be differentiated from effects due to vehicle only. Significant suppression of the Con A response was observed at 10-5 M (345)2-HxCB using p-dioxane as vehicle but this effect was not confirmed in the second experiment using acetone as vehicle. Studies using dimethylsulfoxide as vehicle also failed to separate vehicle effects from specific effects of (345)2-HxCB (data not shown). Based on these results, the direct effect of (345)2-HxCB on the proliferative response of T cells to mitogen or alloantigen remains in question.
Discussion In the preceding paper, we described the sensitivity of the in-vivo allogeneic CTL response to suppression by the toxic, Ah receptor-binding (345) 2HxCB isomer. The studies reported in this paper have examined the sensitivity of the in-vitro M L C / C T L response to suppression by this isomer and the possible role of suppressor cells in mediating its immunosuppressive effect. These latter studies were precipitated in part on the basis of the results of Clark et al. (1981, 1983), which suggested that the cellular basis for the suppression of CTL activity by T C D D and related Ah receptor-binding PCB isomers was due to suppressor T cell activation via altered lymphocyte processing in the thymus (Nagarkatti et al., 1984). In the current studies, B6 mice were exposed to a single oral dose of (345)2-HxCB and sacrificed at various times thereafter for assessment of the func-
tional activity of their splenocytes as responders in M L C / C T L or as third-party suppressors of an independent MLC/CTL. The effect of direct in vitro addition of (345)2-HxCB on the lymphoproliferation response to mitogen or alloantigen was also assessed. Results of the present studies indicated that there was modest dose-dependent suppression of the invitro proliferative response to alloantigen of lymphocytes obtained from mice exposed to 10 or 100 mg/kg (345)2-HxCB whereas the MLC-generated CTL response was significantly suppressed only following exposure to the 100 mg/kg dose. Even at the relatively high dose of 100 mg/kg (345)2-HxCB, the in-vitro CTL response was only modestly suppressed. Furthermore, the magnitude of suppression of the M L C / C T L response did not appear to be influenced by the amount of time that elapsed between dosing and sacrifice, which was assessed over a 3-week period. Thus, in comparison to the in-vivo generation of CTL which was highly suppressed following exposure to a single dose of 10 mg/kg (345)2-HxCB (Kerkvliet and Baecher-Steppan, 1988), the in-vitro M L C / C T L response was considerably more resistant to (345)2-HxCB-in duced suppression. The direct addition of (345)2-HxCB to lymphocyte cultures at concentrations of 1 × 10 6 M or less did not suppress the lymphoproliferative response to either alloantigen in MLC or to the T cell mitogen Con A. Determination of effects at higher concentrations of (345)2-HxCB in vitro were compromised by significant vehicle effects on lymphocyte proliferation. This lack of effect following in-vitro exposure to (345)2-HxCB as well as the functional integrity of lymphocytes taken from animals exposed to doses of (345)Z-HxCB that are highly functionally immunosuppressed when challenged with antigen in vivo may suggest an indirect effect of (345)2-HxCB on T cell responses that is mediated via nonlymphoid target tissue. The possible role of suppressor cells in mediating (345)2-HxCB-induced suppression of CTL development was addressed in the present studies by several different approaches. When naive spleen cells were taken from vehicle- or (345)2-HxCB-treated
22 mice at various times after exposure, treated with mitomycin C and added as third-party cells to a normal MLC, no evidence for suppression of the development of the proliferative or cytotoxic responses was found. This apparent lack of suppresactivity in (345)2-HxCB-treated mice was not related to the time of sacrifice relative to exposure or the sex of the donor animals. The lack of detectable suppressor activity in the spleen of mice treated with (345)2-HxCB suggested that chemical exposure alone did not promote the development of nonspecific suppressor cell function. However, the experimental conditions employed did not address the possibility that (345) 2HxCB interacted with antigen to promote antigeninduced suppressor cell function. This possibility was addressed in several experiments using mitomycin C-treated spleen cells obtained from vehicleor (345)2-HxCB-treated mice at various times after P815 injection as putative suppressor cells. The results of these studies indicated that spleen cells from P815-sensitized mice were indeed suppressive when added as third-party cells to a normal M L C but only when measurable C T L activity was also present in the same population of cells. In fact, a direct correlation was observed between the level of C T L activity present in the spleen cell population and the level of 'suppressor' activity. Thus, (345)2-HxCB treated mice which showed suppressed C T L function also exhibited reduced 'suppressor' function. Assuming that biologically relevant suppressor activity would correlate inversely with C T L activity, these results suggested that the assay was actually measuring CTL-mediated stimulator cell lysis rather than suppressor cell activity. This conclusion was supported by the demonstration that mitomycin C-treated P815-immune spleen cells were still capable of lysing SlCr-labeled P815 target cells. Likewise, when M LCs were established using decreasing numbers of stimulator cells, the proliferative response that was generated decreased in magnitude accordingly. This concept of suppressor responses resulting from the cytotoxic properties of T cells mixed with responder and stimulator cells in M L C has been previously addressed in detail (Fitch et al., 1976; Sinclair et al., 1976; Sugarbaker and Matthews, 1981).
Alternatively, these results suggest that both CTL and suppressor cell activities develop in parallel following P815 injection and that both activities are inhibited by exposure to (345)2-HxCB. This interpretation is supported to some extent by the results of experiments using C3M spleen cells as stimulator cells in MLC. Because the CTL activity generated in PS15-sensitized B6 mice is primarily anti-H-2 d, the H-2 k C3M stimulators were not targets for the C T L present in the B6~ cell suspension used as putative suppressor cells. Nonspecific suppressor activity in these cultures was significantly greater when cells from vehicle-treated as compared to (345)2-HxCB-treated mice were added, suggesting that treatment with (345)2-HxCB inhibited suppressor cell activity. Thus, based on the results reported here, we conclude that the in-vivo suppression of allogeneic CTL activity following acute exposure to (345) 2HxCB is not mediated via altered suppressor cell activity. The relative functional integrity of lymphocytes taken from (345)2-HxCB-treated mice to respond in vitro in M L C / C T L and the inability to directly suppress the M L C response by the addition of (345)2-HxCB to culture suggest an indirect mechanism for (345)2-HxCB-induced suppression of allogeneic T cell responses. As discussed in the preceding paper, the role of elevated corticosteroid levels in (345)2-HxCB-treated mice is under current investigation.
Acknowledgements This work was supported by N I E H S grants ES00040 and ES00210. The authors thank Ms. Lynne Rogers, Word Processing Specialist, for her assistance in preparation of the manuscript.
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