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Induction and persistence of suppression of contact hypersensitivity against bystander haptens and alloantigens in rats
CELLULAR
IMMUNOLOGY
99, 85-94 (1986)
Induction and Persistence of Suppression of Contact Hypersensitivity against Bystander Haptens and Alloantigens in Rats’ JOCHUMPROP,’ IAN V. HUTCHINSON,~AND PETERJ. MORRIS NuJield Department of Surgery, University of Oxford, John Radcli$e Hospital, Oxford OX3 9DV, England Received September 19, 1982; acceptedNovember 2, 1985 The shift of suppressionfrom a tolerizing hapten to a so-calledbystander antigen was investigated in this study using contact hypersensitivity to trinitrochlorobenzene (TNCB) and dinitrofluorobenzene(DNFB) and delayed type hypersensitivity (DTH) to alloantigensin the tat asexperimental models. Primary suppression of contact hypersensitivity was induced by intravenous injection of the water-soluble forms of TNCB and DNFB. A shift of the suppression to the bystander hapten was found if the tolerizing and bystander hapten were mixed and applied to the same area of skin during the sensitization procedure, but not if they were applied to separate areas of skin. With alloantigens, bystander suppression developed only when the sensitizing allogeneic cells were mixed with hapten-modified syngeneic cells. It was not induced by hapten-modified allogeneic cells. Once induced, such bystander suppressionof the responseto haptens persistedindependently of the primarily tolerizing hapten, and it could be adoptively transferred with spleen cells. These results favour the concept that the bystander suppression is mediated by the non-specific action of suppressorcells generated specifically during the mixed sensitization rather than by an antigen bridge. Q 1986 Academic Press,Inc.
INTRODUCTION Suppressor T cells (Ts) are somehow involved in the long-term acceptance of allogeneic grafts. They have been found in spleens of recipient animals with long-surviving heart (l-3), kidney (4, 5), lung (6), and bone marrow (7) grafts, although their activity apparently wanes with time to become undetectable, at least in some models, after 1 to 2 years (6, 7). The maintenance phase of graft acceptance, during which Ts activity was investigated in the above studies, was generated by a variety of treatment protocols, such as active (1) and passive (4, 5) enhancement, cyclophosphamide (7) and cyclosporine (2, 3, 6) immunosuppression. However, there is evidence that this suppression in the maintenance phase of graft acceptancediffers essentially from that found early after transplantation (8, 9), where it is questionable whether in any of ’ This work was supported by grants from the Medical Research Council, United Kingdom, and the National Kidney ResearchFund, United Kingdom. * J. Prop was in receipt of a European Science Exchange Programme Award from The Royal Society. Present address:Department of Experimental Surgery, University Hospital, Groningen, The Netherlands. 3To whom correspondence should be addressed. 85 000%8749186$3.00 Copyright 0 1986 by Academic Press,Inc. All rights of repmdwtion in any form reserved.
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PROP, HUTCHINSON,
AND MORRIS
these treatments graft-specific Ts are responsible for the actual induction of graft acceptance. Recently we described an animal model (10) which provides a method of studying the role of Ts in the induction rather than the maintenance phase of graft acceptance. The key feature of this model is that suppression is induced against the haptenic trinitrophenyl (TNP) determinant. This suppression is then used to suppress the response to alloantigens such that a subsequent renal allograft bearing the same alloantigens will not be rejected (10). The crucial step in the treatment mentioned above is the shift of suppression from one antigen (TNP) to another (alloantigen). This was achieved by injecting the rats with the tolerizing antigen (TNP) linked with the nonsuppressed alloantigen on the same structure: TNP-modified cell membranes. The concept was that the linked antigens, either covalently coupled or associatedon the surface of a presenting cell, form an “antigen bridge” between the Ts and alloreactive cells binding to the alloantigen. In this way, the alloreactive cells could becomethe target of suppression.The hypothesis that an antigen bridge plays a role in the induction of suppression is not proven. The concept is supported by the observation that cytotoxic responsesto minor antigens were suppressedby Ts against allogeneic MHC antigens only if the two setsof antigens were presented on the same Fl cell (11). On the other hand delayed-type hypersensitivity (DTH) reactions to major histocompatibility (MHC) (sub)region antigens could be suppressed by Ts directed against simultaneously presented, but not linked, alloantigens (12). The requirements for a shift of suppression to so-called bystander antigens were investigated in this study using DTH reactions in the rat as an experimental model instead of the immunologically more complex allograft rejection. First, we studied suppression of contact hypersensitivity against trinitrochlorobenzene (TNCB) and dinitrofluorobenzene (DNFB), two haptens extensively used in mice. In a previous study (13), we used these haptens successfully to produce contact hypersensitivity in rats. The present experiments show that, in a rat tolerized to one hapten, a population of Ts is generated against a second, bystander, hapten if that hapten is mixed with the tolerizing hapten and applied to the same area of skin during the sensitization procedure. These Ts then act independently of the primarily suppressedhapten. In further experiments we investigated bystander suppression of DTH against alloantigens and found in this system, too, that allospecific suppression could be induced when the alloantigens were presented simultaneously but not linked with the tolerizing hapten. These results favour the concept that suppressor factors (TsF) rather than an antigen bridge per se cause the shift of suppression, although the hapten and alloantigenic determinants may be simultaneously copresented on the surface of an antigen-presenting cell. MATERIALS
AND METHODS
Rats. Inbred DA rats were used for contact sensitization experiments and DA (RT 1“), WAG (RT l”), BN (RT l”), and (LEW X BN)FI (RT 1”“) rats for DTH to alloantigens. Rats were generally 10 weeks old when they were entered into the experiments and were sex matched in case of cell transfer or allo-DTH experiments. They were bred in the Animal Unit of the John Radcliffe Hospital, Oxford, or obtained from the Animal House of the Charing Cross Hospital, London, Haptens and suppressants. 2,4,6-Trinitro- 1-chlorobenzene (or picryl chloride) (BDH
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
87
Chemicals, Poole, England) and 2,4-dinitro- 1-fluorobenzene (Sigma, Poole, England) were used as haptens, and their water-soluble derivatives trinitrobenzenesulphonic acid sodium salt (TNBS) (Aldrich, Gillingham, England) and dinitrobenzenesulphonic acid sodium salt (DNBS) (Eastman Kodak, Rochester, N.Y.) as tolerizing antigens. Preparation of cell suspensions. Single-cell suspensionswere made by teasing spleens, pushing the cells gently through a steel sieve, and washing them three times in Hanks’ balanced salt solution (HBSS) (Flow Laboratories, Irvine, Scotland). TNP-modljication and mitomycin C treatment of spleen cells. For modification of cells with trinitrophenyl, whole spleen cell suspensions (5 X 10’ nucleated cells/ml) were incubated with 1 mM TNBS at 37°C for 10 min, and then washed three times in HBSS. These haptenated cells were used to produce sensitization to alloantigens. To reduce any nonspecific swelling in the foot-swelling assay (14) the challenging spleen cells were treated with mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan). Suspensionsof 1 to 5 X lo8 cells/ml were incubated with 100 pg mitomycin C/ml at 37C for 30 min, and then washed three times in HBSS. The viability of cells in these suspensions, determined by trypan blue exclusion, was always greater than 90%. Contact sensitization and challenge. Contact sensitivity was induced with 5 mg TNCB or 0.25 mg DNFB dissolved at 10 and 0.5%, respectively, in 4: 1 acetone:olive oil. Fifty microliters of these solutions was painted onto the hair-plucked skin of the belly, unless otherwise mentioned. In experiments with simultaneous but separate sensitization, TNCB and DNFB were applied on opposite sites of the belly. In experiments with mixed sensitization of bystander and tolerizing hapten, TNCB and DNFB were dissolved at the above mentioned concentrations in the mixture and applied to one site. The sites were covered with plaster and surgical tape for 2 days. Rats were challenged on the ears 5 days after sensitization with 0.2 mg TNCB or 0.04 mg DNFB in 20 ~14: 1 acetone:olive oil (concentrations of 1 and 0.2%, respectively) applied to both sides of the ear. The thickness of the ear along the outer margin of the pinna was measured immediately before, and 24,48,72, and 96 hr after challenge, using a Mitutoya engineer’s micrometer with an accuracy of lo-’ mm. The maximum swelling in TNCB-sensitized rats was found 24 to 48 hr after challenge, and in DNFBsensitized animals after 48 to 72 hr. DTH to alloantigens. Rats were sensitized by subcutaneous injection of various doses(1O6to 2 X 10’) of WAG or BN nucleated spleen cells in 0.1 ml HBSS. In the relevant experiments these cells were TNP-modified. DTH reactions were determined 6 days later by injecting 10’ allogeneic or 2 X 10’ semiallogeneic mitomycin C-treated spleen cells in a volume of 50 ~1HBSS into the dorsum of the hind feet, and measuring the swelling of the feet after 24 hr with a Mitutoya engineer’s micrometer. Induction of suppression. Suppression of the DTH response was induced by an intravenous injection of 25 mg TNBS or 20 mg DNBS 7 days before sensitization. The percentage of suppression both in the DTH responseto the hapten in the ear and in the DTH response to alloantigens in the foot was calculated as positive control - experimental x 100%. positive control - negative control Adoptive transfer of suppression. Whole spleen cells ( 108) were injected into sexmatched syngeneic recipient rats which had been irradiated with 2.5 Gy (250-R) Xrays 1 day before cell transfer. Rats were contact sensitized immediately after transfer.
88
PROP, HUTCHINSON,
AND MORRIS
This experimental design allowed the transferred cells to act upon the whole process of sensitization and challenge. Statistical analysis. Differences between groups were considered to be statistically significant if the P values determined with a paired or unpaired Student’s t test were ~0.05. Data given in tables are means + standard errors of the mean. RESULTS Suppression against bystander haptens. The data in Table 1 demonstrate that sensitization with haptens as bystanders of responses to tolerizing haptens results in suppressionof the contact hypersensitivity. Rats were sensitizedwith TNCB and DNFB either on separatesites on the belly (groups.2-4 and 9- 11) or mixed together (groups 5-7 and 12- 14). The ears of positive control rats swelled strongly upon challenge with TNCB (groups 2 and 5) or DNFB (groups 9 and 12). No ear swelling was found in nonsensitized negative controls (groups 1 and 8). Hapten-specific suppressionof contact hypersensitivity against TNCB or DNFB could be induced by prior intravenous inTABLE 1 Bystander Suppression of Contact Hypersensitivity to Haptens Induced by Mixed Sensitization with a Primarily SuppressedAntigen Maximal ear swelling’ Group’
Suppressionb (Day - 12)
Sensitization’ (Day -5)
Challenged (Day 0)
mm X low2 + SEM
% Suppression
I
None
None
TNCB
2.0 f 0.5
2 3 4
None TNBS DNBS
TNCB/DNFB TNCB/DNFB TNCB/DNFB
TNCB TNCB TNCB
14.8 ? 1.6 5.8 f 1.3 11.6 f 2.0
-
5 6 I
None TNBS DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
TNCB TNCB TNCB
18.0 f 1.5 4.6 + 0.7 9.6 r~:1.9
-
8
None
None
DNFB
1.2 + 0.2
9 10 11
None TNBS DNBS
TNCB/DNFB TNCB/DNFB TNCB/DNFB
DNFB DNFB DNFB
16.3 f 2.8 15.0 + 2.8 2.4 + 0.4
-
12 13 14
None TNBS DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
DNFB DNFB DNFB
10.8 f 1.8 4.0 zk0.7 2.3 + 0.4
-
70 25 84 53
9 92 71 89
P
NS >O.ool
’ There were 6 or 7 rats in each group. bSuppressionwas induced by iv injection of either 25 mg TNBS or 20 mg DNBS 7 days before sensitization. cAnimals were sensitized by painting with 5 mg TNCB or 0.25 mg DNFB either on separatesites:TNCB/ DNFB-groups 2-4 and 9-I I -or as a mixture on one site: TNCB + DNFB-groups 5-7 and 12- 14. d Rats were challenged by painting the ears with 0.4 mg TNCB (groups l-7) or 0.08 mg DNFB (groups 8-14) 5 days after sensitization. ’ Maximal increasein ear thicknesswas measuredin the period 24-96 hr after challenge.The % suppression of the responseinduced by pretreatment with TNBS or DNBS was calculated and the statistical significance of the difference with the positive control was determined. NS = not significant.
89
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
jection of TNBS (groups 3 and 6) or DNBS (groups 11 and 14), respectively. This specific suppression did not affect the other hapten if the sensitization had taken place separately (groups 4 and 10). However, if the TNCB- and DNFB-sensitizing haptens were mixed, then the contact hypersensitivity against the bystander hapten was strongly suppressedin rats tolerized with the nonrelated suppressant, TNBS in group 13 and DNBS in group 7. Mechanism of bystander suppression. The suppression of the response against bystander antigens may be mediated during the primary sensitization by nonspecific action of Ts activated by the tolerizing hapten possibly through an antigen bridge. Persistenceof such a nonspecific block of the development of contact hypersensitivity would depend on the presence of the hapten to which the animal was tolerized. Alternatively, bystander suppression may be mediated by Ts specific for the bystander hapten generated during the mixed sensitization. In that case, a shift of suppression towards the bystander antigen might be found persisting independently of the primarily suppressedantigen. We tried to distinguish these possibilities, which are not necessarily mutually exclusive, with two experiments. First, 10 days after mixed sensitization with suppressedand bystander hapten, rats were sensitized with the bystander hapten alone on a site of the belly far from the site of primary sensitization (seeTable 2, groups 5, 6, 11, and 12). Hypersensitivity against TNCB and DNFB presented as bystanders in the primary sensitization appeared to be suppressedpersistently without reexposure TABLE 2 Persistenceof Bystander Suppression in the Absence of the Primarily SuppressedAntigen Maximal ear swelling’
SUP pressionb Group’
(Day -12)
First sensitizationc (Day -5)
Second sensitizationd WY +5)
Challenge’ (Day 0 or IO)
mm x 10-Z f SEM
90 SUP pression
P
None
-
TNCB
2.7 + 0.3
2 3 4
None DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
-
TNCB TNCB TNCB
15.7 kO.8 3.7 It 1.0 4.3 * 1.0
92 87
<0.002 <0.002
5 6
None DNBS
TNCB + DNFB TNCB + DNFB
TNCB TNCB
TNCB TNCB
15.3 * 2.5 6.3 + 1.4
71
<0.02
-
DNFB
1.3 + 0.3
-
DNFB DNFB DNFB
11.0~0.9 2.7 f 0.6 3.3 + 0.6
86 80
<0.005 to.005
DNFB DNFB
DNFB DNFB
18.0 f0.5 4.7 f 0.5
80
I
7 8 9 10
DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
11 12
None TNBS
TNCB + DNFB TNCB + DNFB
a There-were-4-6 rats in each group. b Suppressionwas induced by iv injection of either 25 mg TNBS or 20 mg DNBS 7 days before sensitization. ‘Animals were sensitized by painting with a mixture of TNCB and DNFB. d Animals in groups 5 and 6 or I I and 12 were painted with TNCB or DNFB at a site distant to the first sensitization (seetext). *Rats were challengedby painting the earnwith TNCB or DNFB 5 days after their first (groups 2-4 and 8-10) or second (groups 5-8) sensitization. ‘See footnote e of Table I.
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PROP, HUTCHINSON,
AND MORRIS
to the primarily tolerizing hapten during the second sensitization (Table 2, groups 6 and 12). Hence the bystander suppression of TNCB sensitization was not dependent on reexposure of DNFB in group 6, and vice versa in group 12. As expected, responses in negative control rats (groups 1 and 7) were weak, as were primary responsesto the primarily tolerizing hapten (groups 3 and 10) and its bystander hapten (groups 4 and 9) whereaspositive control animals had strong primary (groups 2 and 8) and secondary (groups 5 and 11) responses.Thus the shift from specific suppression to a persistent bystander suppression is unlikely to be caused by nonspecific action of Ts specific for the tolerized haptens. The second possibility was therefore tested, whether a new population of Ts had been generated during bystander suppression. Spleen cells from animals that had been sensitized with DNFB as a bystander hapten of a suppressedresponseto TNCB were adoptively transferred into rats that subsequently were sensitized with DNFB alone (Table 3). These transferred cells suppressedDNFB sensitization by 59% (group 4). Cells from control rats sensitized with DNFB without the suppressedTNCB amplified the response(group 3). Thus, we conclude that bystander suppression does not depend on reexposure of the primary antigen stimulating nonspecific Ts activity, e.g., through an antigen bridge but, rather, appears to be mediated by newly generated suppressor cells which may possibly be specific for the bystander antigen. Suppression of DTH against bystander alloantigens. In a series of preliminary experiments (data not shown) we determined that optimal DTH to alloantigens in a foot-swelling assaywas produced when rats were sensitized subcutaneously with lo7 spleen cells and challenged 6 days later in the foot pad with 2 X lo7 mitomycin Cinactivated cells. Maximal swelling was observed at 24 hr after challenge. TNP modification of the sensitizing spleen cells using 1 mit4 TNBS did not reduce the response while modification with higher concentrations of TNP did so. The cells used for challenge in these experiments were not modified with TNP. Under these conditions there is a good DTH response to WAG alloantigens in DA rats (Table 4, Experiment A). TABLE 3 Adoptive Transfer of Bystander Suppression with Spleen Cells Cell donorb
Group” 1
2 3 4
Suppression (Day - 17) -
TNBS TNBS
Sensitization (Day -10)
Cell recipient’
Maximal ear swellingd
Sensitization Challenge mm X lo-’ f SEM Pay -5) (Day 0)
% Suppression
P
DNFB DNFB DNFB DNFB
(+70) 59
-
-
DNFB (TNCB + DNFB)
DNFB DNFB DNFB
0.5 f 0.3 12.0 * 1.9 20.1 +- 0.6 5.2 + 1.0
’ There were 6-10 rats in each group. bCell donors were either normal syngeneicrats or were given 25 mg TNBS iv on Day - 17, 12days before adoptive transfer, and a sensitizing dose of DNFB or TNCB + DNFB mixture on Day 10, 5 days before transfer. ’ Recipient rats were irradiated with 250-R X-rays 1 day before transfer of IO* spleen cells from donor rats. The rats were sensitized with DNFB immediately after cell transfer (Day 5) and challenged 5 days later (Day 0) by ear painting with DNFB. d See footnote e of Table 1.
91
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS TABLE 4 Bystander Suppression of Delayed-Type Hypersensitivity to Alloantigens Induced by Mixed Sensitization with a SuppressedHapten Foot swelling’
Expt
Recipient st*na
Suppressionb (Day -13)
Sensitization’ (Day -6)
Challenge” (Day 0)
mm x 10-Z + SEM
% Suppression
P
A
DA
None None
None WAG
WAG WAG
19.5 + 8.0 60.0 + 3.4
B
DA
None None TNBS
None TNP - WAG TNP - WAG
WAG WAG WAG
31.5 + 2.2 65.5 + 3.3 59.0 + 3. I
19
NS
53
<0.05
100
to.005
24
NS
C
DA
None None TNBS
None TNP - DA + WAG TNP - DA + WAG
WAG WAG WAG
26.8 + 1.7 49.2 f 4.3 37.4 f 3. I
D
LEW
None None TNBS None TNBS
None TNP TNP TNP TNP -
BN BN BN BN BN
25.9 f 47.4 f 25.4 k 47.8 + 42.5 k
LEW + BN LEW + BN (LEW X BN)F, (LEW X BN)F,
2.8 4.4 4.0 3.0 2.8
’ The experiments were performed in either DA or LEW inbred rats. There were 6-8 rats in each group. ’ Suppressionto TNP was induced in some groups by iv administration of 25 mg TNBS 7 days before sensitization. ’ Rats were sensitizedby subcutaneousinjection of 10’ allogeneic or 2 X 10’ semiallogeneic spleencells. Where indicated thesecells were TNP modified (seetext). In somegroups mixtures of 10’ TNP-modified syngeneiccells and 10’ unhaptenated allogeneic cells were injected. d Animals were challengedby injecting 2 X 10’ mitomycin C-treated spleencells into the hind foot 6 days after sensitization. ’ Swelling of the foot was measured24 hr after challenge. The percentageof suppressionin TNBS-pretreated groups were calculated and the statistical significance of this suppression compared with the positive controls in each experiment was determined.
To present alloantigens as bystanders of a suppressedresponseto TNP in animals treated with an intravenous injection of TNBS, the sensitizing allogeneic cells were modified with TNP. Rats were tested for DTH 6 days after sensitization by challenging with unmodified cells in the hind feet. However, TNBS suppression did not affect the induction of hypersensitivity with these TNP-modified allogeneic cells (Table 4, Experiment B). To avoid the need for possible MHC restriction of the Ts action which might be held responsible for the absence of suppression in the previous experiment, TNPhaptenated syngeneic cells were mixed with allogeneic cells for sensitization. In this model we observed significant suppression of anti-WAG DTH (Table 4, Experiment C). In a similar experiment, LEW rats were sensitized either with mixtures of TNPhaptenated syngeneic cells plus BN strain cells or with TNP-modified cells from Fl hybrid (LEW X BN)Fi rats (Table 4, Experiment D). Again, the hypersensitivity against bystander antigens in rats previously injected with TNBS was suppressedwhen the sensitizing allogeneic cells were not themselves modified with TNP but were mixed with syngeneic,TNP-modified cells. By contrast, TNP-modified Fl cells, which should have presented TNP-haptenated self-antigens to the LEW rats and thereby avoided genetic restrictions, did not suppressthe anti-BN response.
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PROP, HUTCHINSON,
AND MORRIS
DISCUSSION The immune response to one antigen may be influenced by the simultaneously activated response to another antigen. In this study, rats were made unresponsive to one antigen and then were reexposed to the same antigen together with a second (bystander) antigen. It was shown that contact hypersensitivity against bystander haptens and DTH against bystander alloantigens is suppressedwhen these bystander antigens are mixed with the tolerizing antigen during the sensitization procedure. There is no need for physical linkage of the two, primarily tolerizing antigen and bystander antigen for bystander suppression (Tables 1 and 4). Such bystander suppression of the responseto haptens, once induced, persisted independently of the primarily tolerizing antigen so that, after combined sensitization with bystander and tolerizing antigens, the hypersensitivity to the bystander antigen was suppressedfor a further challenge in the absence of reexposure to the primarily tolerizing antigen. Even subsequent sensitization with the bystander antigen alone could not overcome this suppression (Table 2). This indicates that an active and persistent suppressive mechanism was induced by the combined sensitization. Spleen cells from bystander-suppressedanimals were capable of adoptively transferring the persistent bystander suppression(Table 3). The phenotype of this suppressor cell has not been studied in these experiments, but in a previous study, antigen-specific OX&positive Ts were found to transfer the primarily induced suppression against TNP or dinitrophenyl (DNP) (13). It therefore seemslikely that Ts also mediate the transfer of bystander suppression. It is at least clear that a suppressive cell population has been generated by the combined sensitization. The persistent suppressionin these animals sensitized with bystander and suppressed antigens, challenged, and resensitized with the bystander antigen alone could also be due to local suppression at the challenge site. Local suppression has been found (13) after challenge with actively suppressedantigens, but not after negative responsesin nonsensitized animals. Subsequent challenges within a certain time period are then suppressedin an antigen-nonspecific way. If local suppression plays any role in the bystander suppression, it must have been activated during the first challenge. That again would hint at active suppression developed against the bystander antigen. Cross-reaction of Ts between the tolerizing antigen with the bystander antigen could suggestwrongly that there was an active bystander suppression. However, even though the haptens used in this study are chemically similar, the suppression of their contact hypersensitivity was found to be specific in various experiments: (i) Rats suppressed for one of the haptens developed normal hypersensitivity against the other hapten ( 13). (ii) Such suppressedrats also developed hypersensitivity against the nonsuppressed hapten when simultaneously sensitized with the suppressedhapten at a separate site (Table 1). (iii) Spleen cells adoptively transferred from TNBS-suppressed, DNFBsensitized rats did not suppressDNFB sensitization (Table 3). Thus, we conclude that combined sensitization with the tolerizing haptenic antigen and the bystander antigen shifts suppression towards the bystander hapten, possibly by generating Ts specific for the bystander antigen. Working in a very different immunological system, Herzenberg and Tokuhisa ( 15) have observed that specific Ts against large carrier molecules, such as keyhole limpet hemocyanin, suppressedantibody response to epitopes, e.g., DNP, presented subse-
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
93
quently on the carrier. Suppression to this epitope persisted independently of the carrier; i.e., antibody responsesremained specifically suppressedwhen the DNP was again presented, but on another carrier molecule. These observations are very much like the persistent bystander suppression described in this paper. With alloantigens, the shift of suppression is complicated by the likelihood that there is MHC restriction in the action of Ts ( 16) and their TsF ( 17, 18). Recently it has been shown that in mice the I-J subregion of H2 is involved in this restriction ( 19, 20). The failure to induce bystander suppressionin our experiments with TNP-modified allogeneic cells (Table 4) may be a result of this MHC restriction since suppression was not activated by the TNP on the allogeneic cells. However, even when the requirement for matching MHC was met by sensitization with TNP-modified semiallogeneic cells, no significant bystander suppression developed against the alloantigens (Table 4, Experiment D). Possibly, the TNP-modified BN antigens on the Fl cells are recognised differently from native BN molecules. Hence the responseto BN challenge was not suppressedbut the responseto TNP-BN challenge might have been. Arguing against this view is the observation that the cells responsible for the DTH responsedo not discriminate between normal and modified cells (Table 4, Experiment B, line 2, and Experiment D, line 4). In general, then, to shift suppression to bystander alloantigens, the primarily tolerizing antigen has to be presented during sensitization with the appropriate MHC, and to expressthe bystander suppression the bystander antigen has to be presented during sensitization and challenge in an identical form. In apparent conflict with this MHC restriction of bystander suppression, no restriction was found in the kidney transplantation experiments ( 10) which led to the present study. In those experiments, however, the bystander suppression was not induced with TNP-modified live cells but with modified cell membranes. Unlike the cells, these membranes will be processedby the recipient’s antigen-presenting cells. In that way, the TNP is presented in context with the recipient’s histocompatibility products, so that the MHC-restricted Ts will be activated. In the mechanism of bystander suppression, the supposed role of an antigen bridge is still a matter of debate. Contrasting results were found in studies similar to those reported here; i.e., after combined sensitization with bystander and tolerizing antigens the bystander antigen alone was used for the challenge. For example, DTH responses against horse red blood cells were suppressed,but only if the horse red blood cells had been coated with the tolerizing haemocyanin (21). Similarly, cytotoxic lymphocyte reactions against alloantigens were suppressedby Ts against third-party antigens only alter sensitization with cells of Fl hybrids with the bystander and suppressedhaplotypes (11). In these experiments a mixture without physical linkage of the antigens was ineffective in inducing bystander suppression. These experiments support the concept that linked bystander and tolerizing antigens form an antigen bridge between the antigen-reactive cell and the tolerogen-reactive Ts. In contrast, the work of Bianchi et al. (12) and our own experiments show strong suppression against bystander alloantigens mixed but not linked with suppressedantigens. When the antigens were not mixed but presented simultaneously at different sites, no such suppression developed in our study (Table 1) and in Bianchi’s study (12). The bystander suppression in these experiments could be explained by the action of nonspecific suppressive products releasedafter specific activation of Ts (22). These factors could suppress the response against the bystander antigen which is going on
94
PROP, HUTCHINSON,
AND MORRIS
at the same place or cause the shift of suppression by triggering the generation of specific bystander Ts. The latter possibility is supported by the persistence of the bystander suppression (Table 2) and its adoptive transfer by spleen cells (Table 3). In a preceding study (lo), kidney allograft rejection was suppressedby spleen cells from TNBS-treated rats adoptively transferred 1 day before transplantation together with the TNP-modified cell membranes of the donor type. The present results suggest that instead of modified allogeneic membranes a mixture of modified syngeneic and normal allogeneic membranes might be equally effective in inducing suppression. Of greater importance may be our finding that the bystander suppression once induced develops independently of the primarily suppressedantigen. This explains how permanent acceptance of allografts could develop in the animals experimentally treated only the day before transplantation. We realise that the DTH response against alloantigens as used in this study does often (23) but not always (24), correlate with allograft rejection. Nevertheless, the idea of inducing graft acceptance by generating specific Ts immediately before transplantation is a challenge for further exploration of the complex mechanism of immunological suppression. REFERENCES 1. Marquet, R. L., and Heystek, G. A., Transplantation 31,272, 1981. 2. Bordes-Aznar, J., Kupiec-We&ski, J. W., Duarte, A. J. S., Milford, E. L., Strom, T. B., and Tilney, N. L., Transplantation 35, 185, 1983. 3. Hall, B. M., Jelbart, M. E., and Dorsch, S. E., Transplantation 37, 595, 1984. 4. Batchelor, J. R., Phillips, B. E., and Grennan, D., Transplantation 37, 43, 1984. 5. Barber, W. H., Hutchinson, I. V., and Morris, P. J., Transplantation 38, 548, 1984. 6. Prop, J., Idenburg, V. J. S., and de Jong, B., Transplant. Proc. 17, 248, 1985. 7. Tutschka, P. J., Ki, P. F., Beschomer, W., Hess, A. D., and Santos, G. W., Transplantation 32, 321, 1981. 8. Nagao, T., White, D. J. G., and Came, R. Y., Transplantation 33, 31, 1982. 9. Bordes-Aznar, J., Lear, P. A., Strom, T. B., Tilney, N. L., and Kupiec-Weglinski, J. W., Transplant. Proc. 15, 500, 1983. 10. Hutchinson, 1. V., Barber, W. H., and Morris, P. J., J. Exp. Med. 162, 1409, 1985. 11. Gascoigne, N. R. J., and Crispe, N., Eur. J. Irnmunol. 14, 210, 1984. 12. Bianchi, A. T. J., Hussaarts-Odijk, L. M., and Benner, R., Cell. Immunol. 81,33, 1983. 13. Prop, J., Griffiths, A. J., Hutchinson, I. V., and Morris, P. J., Cell. Immunol. 99, 73, 1986. 14. Wolters, E. A. J., and Benner, R., Transplantation 26, 40, 1978. 15. Herzenberg, L. A., and Tokuhisa, T., J. Exp. Med. 1.55,1730, 1982. 16. Miller, S. D., Sy, M. S., and Claman, H. N., J. Exp. Med. 145, 1071, 1977. 17. Takemori, T., and Tada, T., J. Exp. Med. 142, 1241, 1975. 18. Tsurufuji, M., Benacerraf, B., and Sy, M. S., J. Exp. Med. 158,932, 1983. 19. Asherson, G. L., Colizzi, V., Zembala, M., James, B. B. M., and Watkins, M. C., Cell. Immunol. 83, 389, 1984. 20. Aoki, I., Usui, M., Minami, M., and Dorf, M. E., .I. Immunol. 132, 1735, 1984. 21. Ranshaw, I. A., Bretscher, P. A., and Parish, C. R., Eur. J. Immunol. 6, 674, 1976. 22. Zembala, M. A., Asherson, Cl. L., James, B. B. M., Stein, V. E., and Watkins, M. C., J. Immunol. 129, 1823, 1982. 23. Loveland, B. E., and Mackenzie, I. F. C., Immunology 46, 313, 1981. 24. Kloke, O., and Kolsch, E., Transplantation 38, 526, 1984.
IMMUNOLOGY
99, 85-94 (1986)
Induction and Persistence of Suppression of Contact Hypersensitivity against Bystander Haptens and Alloantigens in Rats’ JOCHUMPROP,’ IAN V. HUTCHINSON,~AND PETERJ. MORRIS NuJield Department of Surgery, University of Oxford, John Radcli$e Hospital, Oxford OX3 9DV, England Received September 19, 1982; acceptedNovember 2, 1985 The shift of suppressionfrom a tolerizing hapten to a so-calledbystander antigen was investigated in this study using contact hypersensitivity to trinitrochlorobenzene (TNCB) and dinitrofluorobenzene(DNFB) and delayed type hypersensitivity (DTH) to alloantigensin the tat asexperimental models. Primary suppression of contact hypersensitivity was induced by intravenous injection of the water-soluble forms of TNCB and DNFB. A shift of the suppression to the bystander hapten was found if the tolerizing and bystander hapten were mixed and applied to the same area of skin during the sensitization procedure, but not if they were applied to separate areas of skin. With alloantigens, bystander suppression developed only when the sensitizing allogeneic cells were mixed with hapten-modified syngeneic cells. It was not induced by hapten-modified allogeneic cells. Once induced, such bystander suppressionof the responseto haptens persistedindependently of the primarily tolerizing hapten, and it could be adoptively transferred with spleen cells. These results favour the concept that the bystander suppression is mediated by the non-specific action of suppressorcells generated specifically during the mixed sensitization rather than by an antigen bridge. Q 1986 Academic Press,Inc.
INTRODUCTION Suppressor T cells (Ts) are somehow involved in the long-term acceptance of allogeneic grafts. They have been found in spleens of recipient animals with long-surviving heart (l-3), kidney (4, 5), lung (6), and bone marrow (7) grafts, although their activity apparently wanes with time to become undetectable, at least in some models, after 1 to 2 years (6, 7). The maintenance phase of graft acceptance, during which Ts activity was investigated in the above studies, was generated by a variety of treatment protocols, such as active (1) and passive (4, 5) enhancement, cyclophosphamide (7) and cyclosporine (2, 3, 6) immunosuppression. However, there is evidence that this suppression in the maintenance phase of graft acceptancediffers essentially from that found early after transplantation (8, 9), where it is questionable whether in any of ’ This work was supported by grants from the Medical Research Council, United Kingdom, and the National Kidney ResearchFund, United Kingdom. * J. Prop was in receipt of a European Science Exchange Programme Award from The Royal Society. Present address:Department of Experimental Surgery, University Hospital, Groningen, The Netherlands. 3To whom correspondence should be addressed. 85 000%8749186$3.00 Copyright 0 1986 by Academic Press,Inc. All rights of repmdwtion in any form reserved.
86
PROP, HUTCHINSON,
AND MORRIS
these treatments graft-specific Ts are responsible for the actual induction of graft acceptance. Recently we described an animal model (10) which provides a method of studying the role of Ts in the induction rather than the maintenance phase of graft acceptance. The key feature of this model is that suppression is induced against the haptenic trinitrophenyl (TNP) determinant. This suppression is then used to suppress the response to alloantigens such that a subsequent renal allograft bearing the same alloantigens will not be rejected (10). The crucial step in the treatment mentioned above is the shift of suppression from one antigen (TNP) to another (alloantigen). This was achieved by injecting the rats with the tolerizing antigen (TNP) linked with the nonsuppressed alloantigen on the same structure: TNP-modified cell membranes. The concept was that the linked antigens, either covalently coupled or associatedon the surface of a presenting cell, form an “antigen bridge” between the Ts and alloreactive cells binding to the alloantigen. In this way, the alloreactive cells could becomethe target of suppression.The hypothesis that an antigen bridge plays a role in the induction of suppression is not proven. The concept is supported by the observation that cytotoxic responsesto minor antigens were suppressedby Ts against allogeneic MHC antigens only if the two setsof antigens were presented on the same Fl cell (11). On the other hand delayed-type hypersensitivity (DTH) reactions to major histocompatibility (MHC) (sub)region antigens could be suppressed by Ts directed against simultaneously presented, but not linked, alloantigens (12). The requirements for a shift of suppression to so-called bystander antigens were investigated in this study using DTH reactions in the rat as an experimental model instead of the immunologically more complex allograft rejection. First, we studied suppression of contact hypersensitivity against trinitrochlorobenzene (TNCB) and dinitrofluorobenzene (DNFB), two haptens extensively used in mice. In a previous study (13), we used these haptens successfully to produce contact hypersensitivity in rats. The present experiments show that, in a rat tolerized to one hapten, a population of Ts is generated against a second, bystander, hapten if that hapten is mixed with the tolerizing hapten and applied to the same area of skin during the sensitization procedure. These Ts then act independently of the primarily suppressedhapten. In further experiments we investigated bystander suppression of DTH against alloantigens and found in this system, too, that allospecific suppression could be induced when the alloantigens were presented simultaneously but not linked with the tolerizing hapten. These results favour the concept that suppressor factors (TsF) rather than an antigen bridge per se cause the shift of suppression, although the hapten and alloantigenic determinants may be simultaneously copresented on the surface of an antigen-presenting cell. MATERIALS
AND METHODS
Rats. Inbred DA rats were used for contact sensitization experiments and DA (RT 1“), WAG (RT l”), BN (RT l”), and (LEW X BN)FI (RT 1”“) rats for DTH to alloantigens. Rats were generally 10 weeks old when they were entered into the experiments and were sex matched in case of cell transfer or allo-DTH experiments. They were bred in the Animal Unit of the John Radcliffe Hospital, Oxford, or obtained from the Animal House of the Charing Cross Hospital, London, Haptens and suppressants. 2,4,6-Trinitro- 1-chlorobenzene (or picryl chloride) (BDH
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
87
Chemicals, Poole, England) and 2,4-dinitro- 1-fluorobenzene (Sigma, Poole, England) were used as haptens, and their water-soluble derivatives trinitrobenzenesulphonic acid sodium salt (TNBS) (Aldrich, Gillingham, England) and dinitrobenzenesulphonic acid sodium salt (DNBS) (Eastman Kodak, Rochester, N.Y.) as tolerizing antigens. Preparation of cell suspensions. Single-cell suspensionswere made by teasing spleens, pushing the cells gently through a steel sieve, and washing them three times in Hanks’ balanced salt solution (HBSS) (Flow Laboratories, Irvine, Scotland). TNP-modljication and mitomycin C treatment of spleen cells. For modification of cells with trinitrophenyl, whole spleen cell suspensions (5 X 10’ nucleated cells/ml) were incubated with 1 mM TNBS at 37°C for 10 min, and then washed three times in HBSS. These haptenated cells were used to produce sensitization to alloantigens. To reduce any nonspecific swelling in the foot-swelling assay (14) the challenging spleen cells were treated with mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan). Suspensionsof 1 to 5 X lo8 cells/ml were incubated with 100 pg mitomycin C/ml at 37C for 30 min, and then washed three times in HBSS. The viability of cells in these suspensions, determined by trypan blue exclusion, was always greater than 90%. Contact sensitization and challenge. Contact sensitivity was induced with 5 mg TNCB or 0.25 mg DNFB dissolved at 10 and 0.5%, respectively, in 4: 1 acetone:olive oil. Fifty microliters of these solutions was painted onto the hair-plucked skin of the belly, unless otherwise mentioned. In experiments with simultaneous but separate sensitization, TNCB and DNFB were applied on opposite sites of the belly. In experiments with mixed sensitization of bystander and tolerizing hapten, TNCB and DNFB were dissolved at the above mentioned concentrations in the mixture and applied to one site. The sites were covered with plaster and surgical tape for 2 days. Rats were challenged on the ears 5 days after sensitization with 0.2 mg TNCB or 0.04 mg DNFB in 20 ~14: 1 acetone:olive oil (concentrations of 1 and 0.2%, respectively) applied to both sides of the ear. The thickness of the ear along the outer margin of the pinna was measured immediately before, and 24,48,72, and 96 hr after challenge, using a Mitutoya engineer’s micrometer with an accuracy of lo-’ mm. The maximum swelling in TNCB-sensitized rats was found 24 to 48 hr after challenge, and in DNFBsensitized animals after 48 to 72 hr. DTH to alloantigens. Rats were sensitized by subcutaneous injection of various doses(1O6to 2 X 10’) of WAG or BN nucleated spleen cells in 0.1 ml HBSS. In the relevant experiments these cells were TNP-modified. DTH reactions were determined 6 days later by injecting 10’ allogeneic or 2 X 10’ semiallogeneic mitomycin C-treated spleen cells in a volume of 50 ~1HBSS into the dorsum of the hind feet, and measuring the swelling of the feet after 24 hr with a Mitutoya engineer’s micrometer. Induction of suppression. Suppression of the DTH response was induced by an intravenous injection of 25 mg TNBS or 20 mg DNBS 7 days before sensitization. The percentage of suppression both in the DTH responseto the hapten in the ear and in the DTH response to alloantigens in the foot was calculated as positive control - experimental x 100%. positive control - negative control Adoptive transfer of suppression. Whole spleen cells ( 108) were injected into sexmatched syngeneic recipient rats which had been irradiated with 2.5 Gy (250-R) Xrays 1 day before cell transfer. Rats were contact sensitized immediately after transfer.
88
PROP, HUTCHINSON,
AND MORRIS
This experimental design allowed the transferred cells to act upon the whole process of sensitization and challenge. Statistical analysis. Differences between groups were considered to be statistically significant if the P values determined with a paired or unpaired Student’s t test were ~0.05. Data given in tables are means + standard errors of the mean. RESULTS Suppression against bystander haptens. The data in Table 1 demonstrate that sensitization with haptens as bystanders of responses to tolerizing haptens results in suppressionof the contact hypersensitivity. Rats were sensitizedwith TNCB and DNFB either on separatesites on the belly (groups.2-4 and 9- 11) or mixed together (groups 5-7 and 12- 14). The ears of positive control rats swelled strongly upon challenge with TNCB (groups 2 and 5) or DNFB (groups 9 and 12). No ear swelling was found in nonsensitized negative controls (groups 1 and 8). Hapten-specific suppressionof contact hypersensitivity against TNCB or DNFB could be induced by prior intravenous inTABLE 1 Bystander Suppression of Contact Hypersensitivity to Haptens Induced by Mixed Sensitization with a Primarily SuppressedAntigen Maximal ear swelling’ Group’
Suppressionb (Day - 12)
Sensitization’ (Day -5)
Challenged (Day 0)
mm X low2 + SEM
% Suppression
I
None
None
TNCB
2.0 f 0.5
2 3 4
None TNBS DNBS
TNCB/DNFB TNCB/DNFB TNCB/DNFB
TNCB TNCB TNCB
14.8 ? 1.6 5.8 f 1.3 11.6 f 2.0
-
5 6 I
None TNBS DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
TNCB TNCB TNCB
18.0 f 1.5 4.6 + 0.7 9.6 r~:1.9
-
8
None
None
DNFB
1.2 + 0.2
9 10 11
None TNBS DNBS
TNCB/DNFB TNCB/DNFB TNCB/DNFB
DNFB DNFB DNFB
16.3 f 2.8 15.0 + 2.8 2.4 + 0.4
-
12 13 14
None TNBS DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
DNFB DNFB DNFB
10.8 f 1.8 4.0 zk0.7 2.3 + 0.4
-
70 25 84 53
9 92 71 89
P
NS >O.ool
’ There were 6 or 7 rats in each group. bSuppressionwas induced by iv injection of either 25 mg TNBS or 20 mg DNBS 7 days before sensitization. cAnimals were sensitized by painting with 5 mg TNCB or 0.25 mg DNFB either on separatesites:TNCB/ DNFB-groups 2-4 and 9-I I -or as a mixture on one site: TNCB + DNFB-groups 5-7 and 12- 14. d Rats were challenged by painting the ears with 0.4 mg TNCB (groups l-7) or 0.08 mg DNFB (groups 8-14) 5 days after sensitization. ’ Maximal increasein ear thicknesswas measuredin the period 24-96 hr after challenge.The % suppression of the responseinduced by pretreatment with TNBS or DNBS was calculated and the statistical significance of the difference with the positive control was determined. NS = not significant.
89
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
jection of TNBS (groups 3 and 6) or DNBS (groups 11 and 14), respectively. This specific suppression did not affect the other hapten if the sensitization had taken place separately (groups 4 and 10). However, if the TNCB- and DNFB-sensitizing haptens were mixed, then the contact hypersensitivity against the bystander hapten was strongly suppressedin rats tolerized with the nonrelated suppressant, TNBS in group 13 and DNBS in group 7. Mechanism of bystander suppression. The suppression of the response against bystander antigens may be mediated during the primary sensitization by nonspecific action of Ts activated by the tolerizing hapten possibly through an antigen bridge. Persistenceof such a nonspecific block of the development of contact hypersensitivity would depend on the presence of the hapten to which the animal was tolerized. Alternatively, bystander suppression may be mediated by Ts specific for the bystander hapten generated during the mixed sensitization. In that case, a shift of suppression towards the bystander antigen might be found persisting independently of the primarily suppressedantigen. We tried to distinguish these possibilities, which are not necessarily mutually exclusive, with two experiments. First, 10 days after mixed sensitization with suppressedand bystander hapten, rats were sensitized with the bystander hapten alone on a site of the belly far from the site of primary sensitization (seeTable 2, groups 5, 6, 11, and 12). Hypersensitivity against TNCB and DNFB presented as bystanders in the primary sensitization appeared to be suppressedpersistently without reexposure TABLE 2 Persistenceof Bystander Suppression in the Absence of the Primarily SuppressedAntigen Maximal ear swelling’
SUP pressionb Group’
(Day -12)
First sensitizationc (Day -5)
Second sensitizationd WY +5)
Challenge’ (Day 0 or IO)
mm x 10-Z f SEM
90 SUP pression
P
None
-
TNCB
2.7 + 0.3
2 3 4
None DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
-
TNCB TNCB TNCB
15.7 kO.8 3.7 It 1.0 4.3 * 1.0
92 87
<0.002 <0.002
5 6
None DNBS
TNCB + DNFB TNCB + DNFB
TNCB TNCB
TNCB TNCB
15.3 * 2.5 6.3 + 1.4
71
<0.02
-
DNFB
1.3 + 0.3
-
DNFB DNFB DNFB
11.0~0.9 2.7 f 0.6 3.3 + 0.6
86 80
<0.005 to.005
DNFB DNFB
DNFB DNFB
18.0 f0.5 4.7 f 0.5
80
I
7 8 9 10
DNBS
TNCB + DNFB TNCB + DNFB TNCB + DNFB
11 12
None TNBS
TNCB + DNFB TNCB + DNFB
a There-were-4-6 rats in each group. b Suppressionwas induced by iv injection of either 25 mg TNBS or 20 mg DNBS 7 days before sensitization. ‘Animals were sensitized by painting with a mixture of TNCB and DNFB. d Animals in groups 5 and 6 or I I and 12 were painted with TNCB or DNFB at a site distant to the first sensitization (seetext). *Rats were challengedby painting the earnwith TNCB or DNFB 5 days after their first (groups 2-4 and 8-10) or second (groups 5-8) sensitization. ‘See footnote e of Table I.
90
PROP, HUTCHINSON,
AND MORRIS
to the primarily tolerizing hapten during the second sensitization (Table 2, groups 6 and 12). Hence the bystander suppression of TNCB sensitization was not dependent on reexposure of DNFB in group 6, and vice versa in group 12. As expected, responses in negative control rats (groups 1 and 7) were weak, as were primary responsesto the primarily tolerizing hapten (groups 3 and 10) and its bystander hapten (groups 4 and 9) whereaspositive control animals had strong primary (groups 2 and 8) and secondary (groups 5 and 11) responses.Thus the shift from specific suppression to a persistent bystander suppression is unlikely to be caused by nonspecific action of Ts specific for the tolerized haptens. The second possibility was therefore tested, whether a new population of Ts had been generated during bystander suppression. Spleen cells from animals that had been sensitized with DNFB as a bystander hapten of a suppressedresponseto TNCB were adoptively transferred into rats that subsequently were sensitized with DNFB alone (Table 3). These transferred cells suppressedDNFB sensitization by 59% (group 4). Cells from control rats sensitized with DNFB without the suppressedTNCB amplified the response(group 3). Thus, we conclude that bystander suppression does not depend on reexposure of the primary antigen stimulating nonspecific Ts activity, e.g., through an antigen bridge but, rather, appears to be mediated by newly generated suppressor cells which may possibly be specific for the bystander antigen. Suppression of DTH against bystander alloantigens. In a series of preliminary experiments (data not shown) we determined that optimal DTH to alloantigens in a foot-swelling assaywas produced when rats were sensitized subcutaneously with lo7 spleen cells and challenged 6 days later in the foot pad with 2 X lo7 mitomycin Cinactivated cells. Maximal swelling was observed at 24 hr after challenge. TNP modification of the sensitizing spleen cells using 1 mit4 TNBS did not reduce the response while modification with higher concentrations of TNP did so. The cells used for challenge in these experiments were not modified with TNP. Under these conditions there is a good DTH response to WAG alloantigens in DA rats (Table 4, Experiment A). TABLE 3 Adoptive Transfer of Bystander Suppression with Spleen Cells Cell donorb
Group” 1
2 3 4
Suppression (Day - 17) -
TNBS TNBS
Sensitization (Day -10)
Cell recipient’
Maximal ear swellingd
Sensitization Challenge mm X lo-’ f SEM Pay -5) (Day 0)
% Suppression
P
DNFB DNFB DNFB DNFB
(+70) 59
-
-
DNFB (TNCB + DNFB)
DNFB DNFB DNFB
0.5 f 0.3 12.0 * 1.9 20.1 +- 0.6 5.2 + 1.0
’ There were 6-10 rats in each group. bCell donors were either normal syngeneicrats or were given 25 mg TNBS iv on Day - 17, 12days before adoptive transfer, and a sensitizing dose of DNFB or TNCB + DNFB mixture on Day 10, 5 days before transfer. ’ Recipient rats were irradiated with 250-R X-rays 1 day before transfer of IO* spleen cells from donor rats. The rats were sensitized with DNFB immediately after cell transfer (Day 5) and challenged 5 days later (Day 0) by ear painting with DNFB. d See footnote e of Table 1.
91
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS TABLE 4 Bystander Suppression of Delayed-Type Hypersensitivity to Alloantigens Induced by Mixed Sensitization with a SuppressedHapten Foot swelling’
Expt
Recipient st*na
Suppressionb (Day -13)
Sensitization’ (Day -6)
Challenge” (Day 0)
mm x 10-Z + SEM
% Suppression
P
A
DA
None None
None WAG
WAG WAG
19.5 + 8.0 60.0 + 3.4
B
DA
None None TNBS
None TNP - WAG TNP - WAG
WAG WAG WAG
31.5 + 2.2 65.5 + 3.3 59.0 + 3. I
19
NS
53
<0.05
100
to.005
24
NS
C
DA
None None TNBS
None TNP - DA + WAG TNP - DA + WAG
WAG WAG WAG
26.8 + 1.7 49.2 f 4.3 37.4 f 3. I
D
LEW
None None TNBS None TNBS
None TNP TNP TNP TNP -
BN BN BN BN BN
25.9 f 47.4 f 25.4 k 47.8 + 42.5 k
LEW + BN LEW + BN (LEW X BN)F, (LEW X BN)F,
2.8 4.4 4.0 3.0 2.8
’ The experiments were performed in either DA or LEW inbred rats. There were 6-8 rats in each group. ’ Suppressionto TNP was induced in some groups by iv administration of 25 mg TNBS 7 days before sensitization. ’ Rats were sensitizedby subcutaneousinjection of 10’ allogeneic or 2 X 10’ semiallogeneic spleencells. Where indicated thesecells were TNP modified (seetext). In somegroups mixtures of 10’ TNP-modified syngeneiccells and 10’ unhaptenated allogeneic cells were injected. d Animals were challengedby injecting 2 X 10’ mitomycin C-treated spleencells into the hind foot 6 days after sensitization. ’ Swelling of the foot was measured24 hr after challenge. The percentageof suppressionin TNBS-pretreated groups were calculated and the statistical significance of this suppression compared with the positive controls in each experiment was determined.
To present alloantigens as bystanders of a suppressedresponseto TNP in animals treated with an intravenous injection of TNBS, the sensitizing allogeneic cells were modified with TNP. Rats were tested for DTH 6 days after sensitization by challenging with unmodified cells in the hind feet. However, TNBS suppression did not affect the induction of hypersensitivity with these TNP-modified allogeneic cells (Table 4, Experiment B). To avoid the need for possible MHC restriction of the Ts action which might be held responsible for the absence of suppression in the previous experiment, TNPhaptenated syngeneic cells were mixed with allogeneic cells for sensitization. In this model we observed significant suppression of anti-WAG DTH (Table 4, Experiment C). In a similar experiment, LEW rats were sensitized either with mixtures of TNPhaptenated syngeneic cells plus BN strain cells or with TNP-modified cells from Fl hybrid (LEW X BN)Fi rats (Table 4, Experiment D). Again, the hypersensitivity against bystander antigens in rats previously injected with TNBS was suppressedwhen the sensitizing allogeneic cells were not themselves modified with TNP but were mixed with syngeneic,TNP-modified cells. By contrast, TNP-modified Fl cells, which should have presented TNP-haptenated self-antigens to the LEW rats and thereby avoided genetic restrictions, did not suppressthe anti-BN response.
92
PROP, HUTCHINSON,
AND MORRIS
DISCUSSION The immune response to one antigen may be influenced by the simultaneously activated response to another antigen. In this study, rats were made unresponsive to one antigen and then were reexposed to the same antigen together with a second (bystander) antigen. It was shown that contact hypersensitivity against bystander haptens and DTH against bystander alloantigens is suppressedwhen these bystander antigens are mixed with the tolerizing antigen during the sensitization procedure. There is no need for physical linkage of the two, primarily tolerizing antigen and bystander antigen for bystander suppression (Tables 1 and 4). Such bystander suppression of the responseto haptens, once induced, persisted independently of the primarily tolerizing antigen so that, after combined sensitization with bystander and tolerizing antigens, the hypersensitivity to the bystander antigen was suppressedfor a further challenge in the absence of reexposure to the primarily tolerizing antigen. Even subsequent sensitization with the bystander antigen alone could not overcome this suppression (Table 2). This indicates that an active and persistent suppressive mechanism was induced by the combined sensitization. Spleen cells from bystander-suppressedanimals were capable of adoptively transferring the persistent bystander suppression(Table 3). The phenotype of this suppressor cell has not been studied in these experiments, but in a previous study, antigen-specific OX&positive Ts were found to transfer the primarily induced suppression against TNP or dinitrophenyl (DNP) (13). It therefore seemslikely that Ts also mediate the transfer of bystander suppression. It is at least clear that a suppressive cell population has been generated by the combined sensitization. The persistent suppressionin these animals sensitized with bystander and suppressed antigens, challenged, and resensitized with the bystander antigen alone could also be due to local suppression at the challenge site. Local suppression has been found (13) after challenge with actively suppressedantigens, but not after negative responsesin nonsensitized animals. Subsequent challenges within a certain time period are then suppressedin an antigen-nonspecific way. If local suppression plays any role in the bystander suppression, it must have been activated during the first challenge. That again would hint at active suppression developed against the bystander antigen. Cross-reaction of Ts between the tolerizing antigen with the bystander antigen could suggestwrongly that there was an active bystander suppression. However, even though the haptens used in this study are chemically similar, the suppression of their contact hypersensitivity was found to be specific in various experiments: (i) Rats suppressed for one of the haptens developed normal hypersensitivity against the other hapten ( 13). (ii) Such suppressedrats also developed hypersensitivity against the nonsuppressed hapten when simultaneously sensitized with the suppressedhapten at a separate site (Table 1). (iii) Spleen cells adoptively transferred from TNBS-suppressed, DNFBsensitized rats did not suppressDNFB sensitization (Table 3). Thus, we conclude that combined sensitization with the tolerizing haptenic antigen and the bystander antigen shifts suppression towards the bystander hapten, possibly by generating Ts specific for the bystander antigen. Working in a very different immunological system, Herzenberg and Tokuhisa ( 15) have observed that specific Ts against large carrier molecules, such as keyhole limpet hemocyanin, suppressedantibody response to epitopes, e.g., DNP, presented subse-
SUPPRESSION OF DTH TO BYSTANDER ANTIGENS
93
quently on the carrier. Suppression to this epitope persisted independently of the carrier; i.e., antibody responsesremained specifically suppressedwhen the DNP was again presented, but on another carrier molecule. These observations are very much like the persistent bystander suppression described in this paper. With alloantigens, the shift of suppression is complicated by the likelihood that there is MHC restriction in the action of Ts ( 16) and their TsF ( 17, 18). Recently it has been shown that in mice the I-J subregion of H2 is involved in this restriction ( 19, 20). The failure to induce bystander suppressionin our experiments with TNP-modified allogeneic cells (Table 4) may be a result of this MHC restriction since suppression was not activated by the TNP on the allogeneic cells. However, even when the requirement for matching MHC was met by sensitization with TNP-modified semiallogeneic cells, no significant bystander suppression developed against the alloantigens (Table 4, Experiment D). Possibly, the TNP-modified BN antigens on the Fl cells are recognised differently from native BN molecules. Hence the responseto BN challenge was not suppressedbut the responseto TNP-BN challenge might have been. Arguing against this view is the observation that the cells responsible for the DTH responsedo not discriminate between normal and modified cells (Table 4, Experiment B, line 2, and Experiment D, line 4). In general, then, to shift suppression to bystander alloantigens, the primarily tolerizing antigen has to be presented during sensitization with the appropriate MHC, and to expressthe bystander suppression the bystander antigen has to be presented during sensitization and challenge in an identical form. In apparent conflict with this MHC restriction of bystander suppression, no restriction was found in the kidney transplantation experiments ( 10) which led to the present study. In those experiments, however, the bystander suppression was not induced with TNP-modified live cells but with modified cell membranes. Unlike the cells, these membranes will be processedby the recipient’s antigen-presenting cells. In that way, the TNP is presented in context with the recipient’s histocompatibility products, so that the MHC-restricted Ts will be activated. In the mechanism of bystander suppression, the supposed role of an antigen bridge is still a matter of debate. Contrasting results were found in studies similar to those reported here; i.e., after combined sensitization with bystander and tolerizing antigens the bystander antigen alone was used for the challenge. For example, DTH responses against horse red blood cells were suppressed,but only if the horse red blood cells had been coated with the tolerizing haemocyanin (21). Similarly, cytotoxic lymphocyte reactions against alloantigens were suppressedby Ts against third-party antigens only alter sensitization with cells of Fl hybrids with the bystander and suppressedhaplotypes (11). In these experiments a mixture without physical linkage of the antigens was ineffective in inducing bystander suppression. These experiments support the concept that linked bystander and tolerizing antigens form an antigen bridge between the antigen-reactive cell and the tolerogen-reactive Ts. In contrast, the work of Bianchi et al. (12) and our own experiments show strong suppression against bystander alloantigens mixed but not linked with suppressedantigens. When the antigens were not mixed but presented simultaneously at different sites, no such suppression developed in our study (Table 1) and in Bianchi’s study (12). The bystander suppression in these experiments could be explained by the action of nonspecific suppressive products releasedafter specific activation of Ts (22). These factors could suppress the response against the bystander antigen which is going on
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AND MORRIS
at the same place or cause the shift of suppression by triggering the generation of specific bystander Ts. The latter possibility is supported by the persistence of the bystander suppression (Table 2) and its adoptive transfer by spleen cells (Table 3). In a preceding study (lo), kidney allograft rejection was suppressedby spleen cells from TNBS-treated rats adoptively transferred 1 day before transplantation together with the TNP-modified cell membranes of the donor type. The present results suggest that instead of modified allogeneic membranes a mixture of modified syngeneic and normal allogeneic membranes might be equally effective in inducing suppression. Of greater importance may be our finding that the bystander suppression once induced develops independently of the primarily suppressedantigen. This explains how permanent acceptance of allografts could develop in the animals experimentally treated only the day before transplantation. We realise that the DTH response against alloantigens as used in this study does often (23) but not always (24), correlate with allograft rejection. Nevertheless, the idea of inducing graft acceptance by generating specific Ts immediately before transplantation is a challenge for further exploration of the complex mechanism of immunological suppression. REFERENCES 1. Marquet, R. L., and Heystek, G. A., Transplantation 31,272, 1981. 2. Bordes-Aznar, J., Kupiec-We&ski, J. W., Duarte, A. J. S., Milford, E. L., Strom, T. B., and Tilney, N. L., Transplantation 35, 185, 1983. 3. Hall, B. M., Jelbart, M. E., and Dorsch, S. E., Transplantation 37, 595, 1984. 4. Batchelor, J. R., Phillips, B. E., and Grennan, D., Transplantation 37, 43, 1984. 5. Barber, W. H., Hutchinson, I. V., and Morris, P. J., Transplantation 38, 548, 1984. 6. Prop, J., Idenburg, V. J. S., and de Jong, B., Transplant. Proc. 17, 248, 1985. 7. Tutschka, P. J., Ki, P. F., Beschomer, W., Hess, A. D., and Santos, G. W., Transplantation 32, 321, 1981. 8. Nagao, T., White, D. J. G., and Came, R. Y., Transplantation 33, 31, 1982. 9. Bordes-Aznar, J., Lear, P. A., Strom, T. B., Tilney, N. L., and Kupiec-Weglinski, J. W., Transplant. Proc. 15, 500, 1983. 10. Hutchinson, 1. V., Barber, W. H., and Morris, P. J., J. Exp. Med. 162, 1409, 1985. 11. Gascoigne, N. R. J., and Crispe, N., Eur. J. Irnmunol. 14, 210, 1984. 12. Bianchi, A. T. J., Hussaarts-Odijk, L. M., and Benner, R., Cell. Immunol. 81,33, 1983. 13. Prop, J., Griffiths, A. J., Hutchinson, I. V., and Morris, P. J., Cell. Immunol. 99, 73, 1986. 14. Wolters, E. A. J., and Benner, R., Transplantation 26, 40, 1978. 15. Herzenberg, L. A., and Tokuhisa, T., J. Exp. Med. 1.55,1730, 1982. 16. Miller, S. D., Sy, M. S., and Claman, H. N., J. Exp. Med. 145, 1071, 1977. 17. Takemori, T., and Tada, T., J. Exp. Med. 142, 1241, 1975. 18. Tsurufuji, M., Benacerraf, B., and Sy, M. S., J. Exp. Med. 158,932, 1983. 19. Asherson, G. L., Colizzi, V., Zembala, M., James, B. B. M., and Watkins, M. C., Cell. Immunol. 83, 389, 1984. 20. Aoki, I., Usui, M., Minami, M., and Dorf, M. E., .I. Immunol. 132, 1735, 1984. 21. Ranshaw, I. A., Bretscher, P. A., and Parish, C. R., Eur. J. Immunol. 6, 674, 1976. 22. Zembala, M. A., Asherson, Cl. L., James, B. B. M., Stein, V. E., and Watkins, M. C., J. Immunol. 129, 1823, 1982. 23. Loveland, B. E., and Mackenzie, I. F. C., Immunology 46, 313, 1981. 24. Kloke, O., and Kolsch, E., Transplantation 38, 526, 1984.