Regulation of contact sensitivity reaction: Contrasuppressor T cells and contrasuppressor factor downregulate efferent T suppressor cells

CELLULAR

IMMUNOLOGY

144,95-104 (1992)

Regulation of Contact Sensitivity Reaction: Contrasuppressor T Cells and Contrasuppressor Factor Downregulate Efferent T Suppressor Cells WLODZIMIERZPTAK,KRZYSZTOFBRYNIARSKI,MARIAN SZCZEPANIK, MARIA PTAK, AND AGNIESZKAPOLEWSKA Department ~flmmunology,

Copernicus Medical Scl~ool, CracoM,, Poland

Received February 12, 1992; accepted June 29, 1992

Contact sensitivity (CS) reaction mediated by CD 4+8- Th 1 cells is under the control of several antigen-specific regulatory lymphocytes. Reaction is downregulated at the induction stage by T afferent suppressorT cells (Ts-aff) that prevent immunization and at the effector stageby efferent T suppressor cells (Ts-eff) that made immune Th 1 cells inoperative. Both suppressor cells are CD 4-8’ Th 1 effector cells and are protected against the suppressiveaction of Ts-eff cells by CD 4+8- contrasuppressorT cells (Tcs). As hasbeen already shown there are also regulatory interactions between regulatory cells themselves and Ts-aff cells in addition to their effect on precursorsof Th 1 cells, also preventing the induction of Ts-eff cells. The present experiments extend thesefindings and demonstrate that Ts-eff cells are also under negative control of Tcs lymphocytes. Likewise, antigen-specific factor produced by contrasuppressor T-T cell hybridoma, used in lieu of Tcs cells, impedes the activation of Ts-eff cells. In both casesregulation is aimed at the precursors of Ts-eff cells. Our experiments demonstrate that the outcome of immunization is dependent not only on the balance between immune cells and regulatory cells, but also on interactions between regLIkItOI’y cells themselves. 0 1992 Academic Press, 1~.

INTRODUCTION Contact sensitivity (CS)’ is an important in vivo manifestation of cell-mediated immunity (1). The nature of theseT cell-mediated immune responsesis highly complex and severalpopulations of T cells participate in the expressionand regulation of contact sensitivity reaction. Th 1 CD4+8- effector T cells, which initiate the response, are downregulated by two types of CD4-8+ suppressor cells which control the response at afferent (Ts-aff) (2, 3) or efferent (Ts-eff) level (4-7), respectively. Contrasuppressor T cells (Tcs) render effector cells resistant to the effects of Ts-eff cell action (8, 9). Regulatory lymphocytes form a very complex feedback mechanism in which they can regulate themselves mutually ( lo- 12). We have recently demonstrated that the Ts-aff I Abbreviations used:C’, rabbit complement; CS, contact sensitivity; FCS, fetal calfserum; iv, intravenous; mAb, monoclonal antibody; OX, 4-ethoxymethylene-2-phenyloxazolone; PCl, picryl chloride, TNP chloride; Tcs. contrasuppressor T cells: Tcs-F, contrasuppressor factor; Th, thymocytes; TNP, trinitrophenyl: TNPOX-Th, TNP and OX-haptenated thymocytes; TNP-Th, TNP-haptenated thymocytes; TNP-Th-Ab, TNPhaptenated thymocytes treated with anti-TNP IgG 1 mAb; Ts-eff, efferent suppressorcells. 95 0008-8749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cells which inhibit immunization of Th 1 effector lymphocytes mediating a CS response simultaneously prevented the induction of Ts-eff cells ( 13). We report here that interactions between regulatory cells are rather common and we show that contrasuppressor T cells and antigen-specific factor produced by contrasuppressor T-T hybridoma (14) not only protect effector lymphocytes against suppression by Ts-eff cells, but also inhibit the recruitment of the latter cells from their precursors. However, they do not affect the functional activity of Ts-eff cells once these cells have been induced. Our results strongly suggest that interplay between effector cells and regulatory subsets of T lymphocytes and between regulatory cells themselves can determine the quality and quantity of the resulting immunity. MATERIALS AND METHODS Mice. CBA/J male mice were from the breeding unit, Department of Immunology, Cracow, Poland, and were used between 6 and 8 weeks of age. Each experimental group consisted of four or five mice. Reagents. The following reagents were used: twice-recrystallized picryl chloride (trinitrophenyl, TNP, chloride; PCl) (Chemtronics, Swannanoa, NC); trinitrobenzene sulfonic acid (Eastman Organic Chemicals, Rochester, NY); Sepharose4B (Pharmacia Fine Chemicals, Piscataway, NJ); and 4-ethoxymethylene-2-phenyloxazolone (OX) (British Drug House, Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, NY). Induction of regulatory cells. Thymocytes (Th) were labeled with TNP and/or OX, as described previously (5). When Th were double labeled (i.e., with TNP and OX), TNP substitution was followed by OX substitution. Cells were washed extensively in phosphate-buffered saline supplemented with 1%fetal calf serum (FCS). TNP-Th were incubated for 30 min at 4°C with an affinity-purified anti-TNP monoclonal IgGl antibody (3 pg per 1 X 10’ cells) (TNP-Th-Ab). TNP-Th or TNP-Th-Ab (3 X lo’), unless stated otherwise, were injected iv into recipients to induce Ts-eff or Tcs cells, respectively. Spleen cells of these animals were used as regulatory cells 7 days after injection, unless stated otherwise. Antibodies. Monoclonal anti-L3T4 (CD4) antibody was purchased from BectonDickinson (Mountain View, CA). Monoclonal anti-Lyt 2.1 (CD8) antibody was kindly provided by Dr. F. W. Shen (Sloan-Kettering Institute, New York, NY). Monoclonal anti-TNP IgGl hybridoma cells (H 1-a l- 109.3) were obtained from Dr. F. T. Liew (Molecular Biology Institute, La Jolla, CA). This antibody was affinity purified by absorption onto a TNP-KLH-Sepharose 4B affinity column and was eluted with 3 M guanidine. TNP-specific contrasuppressorfactor (Tcs-F). Cells (2 X 106) of T-T contrasuppressor hybridoma (clone AF5.C6, H-2k) (14) were injected ip into pristane-pretreated CBA mice. Ten days later mice were tapped and peritoneal exudate was cleared by centrifugation. The exudate (0.05 ml) diluted in 5 ml RPM1 1640 medium fully protected 7 X 10’ immune cells against suppression by 5 X lo7 Ts-eff cells (seebelow). In some experiments mice were injected iv with 0.25 ml of Tcs-F on one or two occasions. Peritoneal exudate induced by BW 5147 thymoma (H-2k), fusion partner for AF5 hybridoma, was used as a control. Ascitic fluids contained from 20 to 30 mg of protein per milliliter. Treatment of regulatory cells. Spleen cells from mice immunized with TNP-Th or TNP-Th-Ab were treated with anti-CD4 or anti-CD8 antibody for 30 min at 4°C

CONTRASUPPRESSOR T CELLS IN CS

97

washed,and then incubated for 30 min at 37°C in rabbit complement (C’) (prescreened for low natural cytotoxicity to mouse spleen cells). As shown previously Ts-eff cells are CD4-8+ and Tcs are CD4+8- (8, 9). Active sensitization and adoptive transfer of contact sensitivity. Animals were skin sensitized with PC1or OX by painting the shaved abdomen as well as their four feet with 0.15 ml of 5% PC1or 3% OX (in 3: 1 ethanol:acetone) and were tested for contact sensitivity by application of 0.8% (w/v) PC1 or OX in olive oil to both ears 4 days later ( 15). Ear swelling was assessedby measuring the ears immediately before application and 24 hr later using an engineer’s micrometer (Mitutoyo, Paramus, NJ). Results are expressed in units of lop3 cm f SD and the average ear thickness in nontreated mouse is approximately 20 units. In each experiment, ears of separategroups of nonimmunized controls were similarly challenged and measured.The background swelling response in the control group averaged about 1 to 2 units and was subtracted from the swelling response of the experimental animal group, thus the results are presented as a net increase in ear swelling. In adoptive transfer, 7 X 10’ spleen and lymph node cells (mixed at the ratio 1:1) (6, 16) of animals skin painted 4 days earlier with PC1 or OX were injected iv into recipients which were ear challenged immediately, and ear swelling was measured 24 hr later. In all figures the results are also expressed as percentagesof responses,which are calculated according to the formula: (experimental - negative control)/(positive control - negative control) X 100. Testfor efirent suppression (Ts-ef). Spleen and lymph node cells (7 X 10’) from animals immunized by skin painting were mixed with 5 X 10’ spleen cells (or equivalents) from mice injected 7 days earlier with hapten-substituted Th. This cell mixture was incubated for 30 min at 37°C and then injected into naive recipient mice. Animals were challenged immediately on the ears and were assayedfor contact sensitivity 24 hr later (5, 6). Test .for contrasuppression. Spleen and lymph node cells (7 X 10’) from animals immunized by skin painting were incubated with 5 X 10’ cells (or equivalent) with putative contrasuppressor activity and subsequently with Ts-eff cells (5 X 10’) before being injected into recipients. Both incubations were held at 37°C for 30 min (9, 14). Statistics. Evaluation of the statistical significance of the results was done using double-tailed Student’s t test. RESULTS Contrasuppressor T Cells and Factor Inhibit the Generation qf Ts-efl Cells Since our former results suggestedthe existence of direct correlation between the activation of Tcs cells and the inhibition of Ts-eff cells (8) we examined whether the injection of TNP-Th-Ab that triggers Tcs cells, simultaneously or subsequent to TNPTh, would inhibit Ts-eff cell activity. Mice were injected with 3 X 10’ TNP-Th, and 3 X 10’ TNP-Th-Ab were administered simultaneously or 2 or 4 days later. Seven days from the beginning of experiment spleen cells of these mice were treated with anti-CD 4 mAb plus C’ and were tested in passivetransfer experiment for Ts-eff activity. Our results indicate that the injection of TNP-Th induced good Ts-eff activity and that spleen cells of these animals suppressedalmost completely the passive transfer of CS by immune cells (Fig. 1, compare groups A and B). Spleen cells of animals which received simultaneously TNP-Th and TNP-Th-Ab had no suppressive activity (group C). This inhibitory effect on generation of suppressor cells was also observed when

98

PTAK ET AL. Treatment Group

with TNP-Th

of mice injected (donors

at doy 0

of Ts-eff

Efferent

suppressor

cell assay (CD473+cells)

cells)

No mgulatofy cells added (+ve control)

100

27 95

No additional treatment lNP-lb-Ab

(d. 0)

TNP-lb-Ah

(d. 2)

TNP-Th-Ab

(d. 4)

AF5.M

(d. 0)

K=5.C6 N5.CB

(d. 2) (d. 4)

65 35 97 59 24 0

2

4

6

-

6

Units of swelling x 10m3 cm

FIG. 1. Injection of TNP-Th incubated with IgG 1 anti-TNP monoclonal antibody or injection of monoclonal Tcs-F prevents the induction of Ts-eff cells. CBA mice were injected iv with 3 X lo7 TNP-Th. Some mice received simultaneously or 2 or 4 days later iv injection of 3 X 10’ TNP-Th incubated before injection with IgG 1 anti-TNP monoclonal antibody (3 rg per 1 X IO’ cells).Parallel groupsof mice were given simultaneously or 2 or 4 days after TNP-Th administration iv injection of 0.25 ml of ascitic fluid from mice inoculated with AF5 Tcs cell hybridoma or its fusion partner BW 5 147.Sevendays from the beginning of the experiment spleen cells of mice were tested for efferent suppressor cell activity. The assay for Ts-eff cells consisted of combining 7 X 10’ immune spleen and lymph node cells of mice skin sensitized with PC14 days earlier with 5 X IO’ cells from experimental animals. Cell mixtures were incubated for 30 min at 37°C and then injected iv into naive recipients. Mice were immediately challenged with PC1and the ear swelling contact sensitivity reaction was measured 24 hr later. In this and in the following figures, nonspecific ear swelling of negative control (nontreated) mice that were similarly challenged with antigen was subtracted from experimental value (see Materials and Methods). Results are presented as the net increase in ear swelling in units X 10e3cm f SD. Numbers opposite bars represent percentage of responses(control positive response = 100%).In no casedid the injection of TNP-Th or TNP-Th-Ab induce a significant contact sensitivity by itself. Since injection of ascites from BW 5 147-injected animals had no influence on Ts-eff cells, results are not shown. In Student’s t test group A was significantly different than groups B, E, and H (P < 0.001) and D and G (P < 0.01).

TNP-Th-Ab was injected 2 days later (group D) but injection at Day 4 had little effect (group E). A parallel experiment was done using, in lieu of TNP-Th-Ab, a factor (TcsF) produced by contrasuppressor T-T hybridoma ( 14). As a control we used ascitic fluid induced by BW 5147 (H-2k) cells. The results were almost identical as in the previous experiment, i.e., Tcs-F given simultaneously with TNP-Th abrogated the activity of Ts-eff cells, while injection at Day +4 had little effect (compare groups F and H). Injection at Day +2 had intermediary effect (group G). Ascitic fluid from BW 5 147-injected animals had no effect whatsoever on the generation of Ts cells (results not shown). It was concluded that immunization that activates Tcs cells or administration of Tcs hybridoma cell product (Tcs-F) inhibits the generation of functional Ts-eff cells when performed at the same time or shortly after the procedures that activate Ts-eff cells. Lack of Influence of Ts-eff Cells on the Generation of Tcs Cells The next question we asked was whether downregulation of Ts-eff cells by Tcs is unidirectional or whether these two cells can regulate reciprocally. We simultaneously

99

CONTRASUPPRESSOR T CELLS IN CS

injected a constant number of TNP-Th (3 X 107)and varying numbers TNP-Th-Ab, or vice versa. Spleen cells of these mice were treated 7 days later with either anti-CD4 mAb plus C’ (to test for Ts-eff) or anti-CD8 mAb plus C’ (to test for Tcs) and were examined for appropriate regulatory activity. Figure 2 shows that simultaneous generation of Tcs and Ts-eff cells affected the activity of the latter, even when as little as 3 X lo6 TNP-Th-Ab were injected together with TNP-Th (Fig. 2, groups B and E). In contrast, simultaneous injection of both antigens had no effect on the activity of Tcs cells independent of the number of TNP-Th cells used (Fig. 3). Tcs Cells and Tcs-F Do Not Act by Masking Ts-efActivity To rule out the possibility that Tcs cells or the factor that inhibits Ts-eff generation were functioning by masking or suppressing the activity of functional Ts-eff cells we took advantage of the fact that Ts-eff cells are CD 4-8+ and Tcs cells are CD 4+8-. Thus, 5 X lo7 Ts-eff cells induced by TNP-Th were incubated with 5 X lo7 Tcs cells induced by TNP-Th-Ab or with Tcs-F (0.25 ml in 5 ml RPM1 1640 medium plus 5% FCS) and then were treated with anti-CD4 mAb and C’. We then assayedthe treated cells for Ts-eff suppressor activity. The results in Fig. 4 show that nontreated Ts-eff cells significantly suppressedthe passive transfer of CS (groups A and B) and previous incubation with Tcs cells (group C) or Tcs-F (group D) had no influence on their activity. These results indicate that Tcs cells have no effect on the activity of functional Ts-eff cells. Contrasuppressor T Cells and Factor (Tcs-F) Prevent Induction of Ts-eff Cells in Antigen-Specifc Manner In the next experiment we tested the antigen-specificity of the observed downregulation of Ts-eff cells by Tcs cells. Mice were injected with TNP-Th-Ab together with

Induction TNP-Th (number

Group

of regulotoly cells TNP-Th-Ab of cells injected)

Efferent

suppressor

cell assay

-

regulatory cells (+ve conhl)

A

No

6

3x107

(CD473+)

100 20

C

3x107

3x107

91

II

3x107

1x107

75

E

3x107

3x106

57

F

3x107

lItlO

30

0

i

i Units of swelling

s

e

x 1 Om3cm

FIG. 2. Simultaneous injection of TNP-Th incubated with IgGI anti-TNP monoclonal antibody prevents in a dose-dependentmanner the induction of Ts-eff cells by TNP-Th. CBA mice were injected with 3 X 10’ TNP-Th alone or simultaneously received varying numbers of TNP-Th incubated before injection with IgG 1 anti-TNP mAb (3 pg per 1 X 10’ cells). Seven days later spleen cells of these mice were treated with anti-CD4 mAb plus c’ and tested for Ts-eff activity as described in the legend for Fig. 1. In Student’s t test group A was significantly different from groups B and F (P < 0.001) and group E (P < 0.01).

100 Group

PTAK ET AL. Induction TNP-Th-Ab (number No regulatory

of regulatory

cells TNP-Th of cells injected)

Contrasuppressor

cell assoy

(CD4+8-)

100

cells (+ve cc&ml)

109

3x107 3x107

3X107

3x107

1x107

3x107

3x106

3x10’

1x106

91 93 +

97 88

I 0

2

4 Units of swelling

a

6 x 1 Op3cm

FIG. 3. Injection of TNP-Th does not influence the generation of Tcs cells by TNP-Th-Ab. CBA mice were injected iv with 3 X lo7 TNP-Th incubated before injection with IgGl anti-TNP mAb (3 &I X 10’ cells) only, or received simultaneously varying numbers of TNP-Th. Seven days later spleen cells of these mice were treated with anti-CD8 mAb plus C’ to remove Ts-eff cells and were tested for Tcs cell activity. Equivalent of 5 X 10’ of nontreated cells were incubated for 30 min at 37°C with 7 X IO’ immune spleen and lymph node cells of mice skin sensitized with PCI 4 days before. Subsequently 5 X IO’ Ts-eff cells (induced by TNP-Th) were added and incubation proceeded for another 30 min at 37°C. Mixtures of cells were injected into naive recipient animals that were then immediately challenged with PCI. The ear swelling CS reaction was measured 24 hr later. No statistical differences between groups were found.

TNP-Th or OX-Th. Seven days later animals were sensitized with PC1 or OX, correspondingly, and were tested for CS 4 days later. The results shown in Fig. 5 demonstrate that both TNP-Th and OX-Th caused a significant inhibition of CS reaction

Group

Incubation

of Ts-eff

Effennt

cells with

A

No regulatory cells added (+ve control)

B

No incubation

C

MP-Th-Ab

D

K5.C6

suppressor

cell assoy (CD4-8+

cells

100 22

cells

22 26

i

i

4

Units of swelling x 10T3

s cm

Ftc. 4. Inhibition of Ts-eff cell activity by simultaneous induction of Tcs cells is not due to the “masking” of Ts-eff cells by Tcs cells or Tcs-F. CBA mice were injected iv with 3 X 10’ TNP-Th or 3 X 10’ TNP-Th incubated before the administration of anti-TNP IgG 1 mAb (3 pg per 1 X IO’ cells). TNP-Th-induced Tseff cells (5 X 10’) were incubated for 30 min at 37°C with the same number of TNP-Th-Ab-induced Tcs cells or with 0.25 ml of peritoneal exudate produced by contrasuppressor T-T cell hybridoma (AF5C6 diluted in 5 ml RPMI 1640 medium plus 5% FCS). Incubation was followed by treatment with anti-CD4 mAb plus C to remove Tcs cells. Cells were assayedfor Ts-eff cell activity as described in the legend for Fig. 1. In Student’s f test group A is significantly different from groups B, C, and D (P < 0.001).

101

CONTRASUPPRESSOR T CELLS IN CS Group

Treatment

of mice

Sensitisation

Contact sensitivity

reaction

A

No treatment

PCI

100

B

TNP-Th

PCI

20

C

TNP-Th + TNP-Th-Ab

PCI

93

D

TNP-Th + AF5.C6

PCI

E

No treatment

ox

100

F

ox-Th

ox

26

G

OX-Th + TNP-Th-Ab

OX

21

H

OX-Th + AF5.C6

ox

33

07

0

2

4

6

6

10

Units of swelling x 1Dm3cm FIG. 5. Prevention of induction of Ts-eff cells by Tcs cells and Tcs-F is antigen specific. CBA mice were injected with 3 X IO7 TNP-Th or TNP-OX. Groups of mice were injected simultaneously with 3 X IO’ TNP-Th incubated before injection with anti-TNP-Th IgGl mAb (3 jqg per I X IO’ cells) or alternatively received iv injection of either 0.25 ml of ascitic fluid from mice inoculated with AF5 Tcs cell hybridoma or its fusion partner BW 5147. Seven days later animals were sensitized with homologous hapten and were tested for CS 4 days later. In Student’s t test group A was significantly different from group B (P < 0.001); group E was statistically different from groups F, G, and H (P < 0.001).

in antigen-specific manner (compare groups B vs A and F vs E). Injection of TNPTh-Ab alleviated the suppressive effect only to homologous (i.e., TNP-Th) tolerogen (group C) but had no influence on suppression to unrelated hapten oxazolone (group G). We also tested the antigen specificity of Tcs-F produced by AF5 hybridoma cells. Mice were injected with 3 X lo7 TNP-Th or OX-Th and simultaneously received 0.25 ml ascitic fluid from mice inoculated with either AF 5 cells or thymoma BW 5 147 cells. Seven days later animals were sensitized with PC1or OX, respectively, and after additional 4 days they were tested with appropriate hapten for CS reaction. The results in Fig. 5 show that mice which received only TNP-Th were suppressed,while mice given simultaneously Tcs-F developed a significant CS reaction upon sensitization with PC1(compare groups B and D). This influence was antigen-specific since injection of Tcs-F had no influence on the development of CS to oxazolone (groups F and H). Ascitic fluid induced by BW 5 147 cells had no effect (results not shown). TNP-Specific Contrasuppressor Factor Prevents the Generation of Oxazolone-Specijc Ts-efl Cells If TNP and OX Are Linked to the Same Carrier Additionally we tested the activity of TNP-specific Tcs-F under conditions when both haptens, TNP and OX, were attached to the same carrier. Mice were injected with 3 X lo7 double-labeled Th (TNP-OX-Th), and received iv injection of Tcs-F or no treatment (see previous experiment). Seven days later groups of mice were skin-

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ET AL.

sensitized with PC1or OX and tested for CS 4 days later. The results in Fig. 6 show that TNP-OX-Th generated unresponsiveness (mediated by Ts-eff cells, see (Fig. 5)) to both haptens (compare groups A and B). As expected,the injection of Tcs-F alleviated the induction of suppression not only to TNP, but also, interestingly enough, to the unrelated hapten, OX (group C). These results suggestthat Tcs-F of a given specificity can change a tolerogenic signal into an immunogenic one not only in an antigenspecific manner, but also to unrelated hapten provided that these two determinants are present on the same carrier. DISCUSSION Cell-mediated immune responsesto contact sensitizing agentssuch as picryl chloride or oxazolone mediated by CD4+ effector T cells are under the stringent control of several regulatory cells (3, 5, 8). These latter cells form a very complex mechanism in which the activity of one population of regulatory cells may influence the activity of another population with a distinct function (13, 17). Thus, we have previously found that Ts-aff cells which prevent active immunization, i.e., recruitment of effector cells of CS, simultaneously inhibit the induction of Ts-eff cells (13). Our present experiments extend these findings and demonstrate that CD4+8- Tcs cells or their antigen-specific product not only make immune cells refractory to downregulation by CD4-8+ Ts-eff cells but, additionally, also directly inhibit the activity of Ts-eff cells. These two types of regulatory cells can be distinguished both by functional activity and cell surface markers (9) as well as in terms of distinct conditions that are necessary for their activation. In the TNP system Ts-eff cells are generated by TNP-conjugated syngeneic cells (3, 5) while Tcs cells are generated by immune complexes with IgGl antibody (18) or by certain specialized antigen-presenting cells (e.g., Langerhans cells) (8). In some of our experiments we have also used, a monoclonal Tcs factor produced by TT cell hybridoma in lieu of Tcs cells. Its activity cannot be distinguished from the activity of Tcs cells (14). Diminished Ts-eff cell activity in a situation when Tcs cells are generated simultaneously or shortly thereafter may be due to direct inhibition or masking of functional

Group

Treatment

Contact

of mice PCI

I 0

1

I

I

sensitivity

reaction ox

: :!. : : II IO I2 0 1 4 6 Units of swelling x 1 O-3 cm

: 8

: 10

: ,a

FIG. 6. TNP-specific Tcs-F prevents the development of Ts-eff to unrelated hapten, oxazolone. if TNP and OX are on the same carrier. CBA mice were injected iv with 3 X IO’ double-labeled Th (TNP-OX-Th). Some mice received a simultaneous iv injection of 0.25 ml Tcs-F (see Fig. 4). Seven days later groups of mice were skin-sensitized with PCI or OX and tested with immunizing hapten 4 days later. In Student’s t test, independent of the hapten used for sensitization (PC1 or OX), group A was significantly different from group

B.

CONTRASUPPRESSOR T CELLS IN CS

103

Ts-eff cells or, alternatively, due to decreased recruitment of these cells. The first assumption seemsto us less likely for two reasons: the removal of Tcs cells does not expose latent suppressive activity when both regulatory cells were induced simultaneously moreover, the exposure of Ts-eff cells to Tcs cells or Tcs-F does not change their suppressiveactivity. Therefore, we interpret thesedata to indicate that antagonistic relationship between Tcs and Ts-eff cells occurs at the level of induction of functional Ts-eff cells in vivo. In our experiments we were not able to show a reverse relationship, i.e., influence of Ts-eff cells on Tcs cells. However, it is not necessarily a general phenomenon, since it has been found in a different system that activity of Tcs cells is, under normal conditions, counterbalanced by dominant control of T suppressor cells ( 19). Our results are reminiscent of previous results in which we have shown that Ts-aff cells-in addition to their inhibitory effect on active immunization-simultaneously and undirectionally prevented induction of Ts-eff lymphocytes, but had no inhibitory effect on the suppressive activity of already existing Ts-eff cells (13). The reason why generation of Ts-eff cells is so tightly and unidirectionally controlled by two different types of other regulatory cells is not clear at present. However, it makes teleological sensethat Tcs cells that protect immune lymphocytes against suppression by Ts-eff cells, simultaneously shut off the generation of the latter thus preventing unnecessary biological expenditure. In contrast, generation of Ts-aff cells remains unimpeded in the presence of Tcs cells (unpublished data) and vice versa. Inhibition of Ts-eff cells by Tcs cells was antigen specific, since the TNP-specific Tcs factor has no influence on the generation of OX-specific Ts-eff ceils. However, when OX was present on cells already substituted with TNP, the generation of OXspecific Ts-eff was also downregulated in the presence of TNP-specific Tcs-F. We interpret these findings in terms of two independent antigen-specific recognition events: one in which antigen (TNP or OX) is recognized by precursors of Th 1 effector cells and the other where, as shown previously, contrasuppressor cascade is triggered by antigen-specific Tcs-F and homologous antigen; the final, presumably short-range, product of the cascadeworks in an antigen nonspecific manner thus also preventing the generation of Ts-eff cells to the unrelated antigen present in its vicinity (20). There are several possible mechanisms by which regulatory cells could reciprocally interregulate their activities. There is now an increasing body of evidence that communication between different cells of the immune system is executed through antigenspecific (factors) or nonspecific (interleukins, prostaglandins) mediators in a complex interacting network. The cross-inhibitory nature of Th 1 and Th 2 cell interactions by IFN-7 and IL 10, respectively, is well documented (2 l-24). Regulatory cells, as demonstrated repeatedly, can produce an array of antigen-specific factors, which upor downregulate the activity of other cells participating in the immune response (5, 6,25-28). Although the structure and mode ofaction ofthese factors is largely unknown (29) some of them, including Tcs-F, produced by AF 5 clone (unpublished data) have been shown to react with antibody directed against the a and/or /3 chains of TCR (30-32). Antigen specificity of regulatory events observed in our experiments justifies the assumption that Tcs-F, the product of contrasuppressor cells, plays a significant role in the observed phenomenon. It is not clear at present whether its action is aimed directly at Ts-eff cell precursor or also involves other cells. Indirect action of Tcs-F through CD4-8+ intermediary cell has been found to be responsible for protection of immune CD4+ cells against suppression (20). Thus, although AF5.C6 factor by itself does not exhibit any lymphokine activity when tested for IL 1, IL 2, IL 3, IL 4, IL 5,

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or GM-CSF activity and AF 5 cells do not transcribe any known lymphokine gene ( 14) we cannot entirely exclude that the action of Tcs-F is executed ultimately by any of the known cytokines. In summary, our experiments demonstrate that in contact sensitivity system Tcs cells or their antigen-specific product not only prevent the downregulatory activity of Ts-eff cells presumably by inhibiting their recruitment from precursor cells, but also they confirm the notion that the net effect of the immune response is dependent on the interplay between effector and regulatory cells, as well as between regulatory cells themselves.Their balance may be a critical factor determining T effector cell activation state. When the activation dominates the net functional effect is the induction of immunity. ACKNOWLEDGMENT This work was supported by a grant from the Committee for Scientific Research (K.B.N.).

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