A novel suppressive activity: Complementation between a T cell induced with first-order T-suppressor factor and an I-J-restricted antigen-nonspecific T cell

CELLULAR

IMMUNOLOGY

101,35 l-362 (1986)

A Novel Suppressive Activity: Complementation between a T Cell Induced with First-Order T-Suppressor Factor and an l-J-Restricted Antigen-Nonspecific T Cell’ SUNDARARAJAN JAYARAMAN AND CLIFFORD J. BELLONE Department ofMicrobiology, St. Louis University School ofMedicine, 1402 South Grand Boulevard, St. Louis. Missouri 63104 Received February 24, 1986; accepted April 13, I986 Previous studies demonstrated that the first-order T-suppressor factor (TsF,) requires the presence of antigen to induce idiotype-specific Ts cells which readily suppress phenyltrimethylamino (TMA) hapten-specific delayed-type hypersensitivity (DTH) responses when transferred into already immune recipients. In this study we show that TsF, in the absence of antigen induces a splenic population which limits DTH in recipient mice only when an additional accessory lymphoid population was also cotransferred. Neither of these populations alone was sufficient to mediate suppression and depletion of T cells in either population’s abrogated suppression, indicating the T-cell dependency of the complementing celltypes.Moreover,suppression wasseen only when TMA-TsF,-induced and not normal spleen cell lysate-induced cells were cotransferred with theantigen-induced population, suggesting the requirement for a specific signal to induce the factor-induced population. Further experiments showed that the antigen-induced lymphoid population could be replaced by either heterologous antigen-induced or adjuvant alone-induced splenic populations, indicating the lack of specificity of this secondary population. Further analysis showed that the cell complementation between TMA-TsF,-induced and the nonspecific accessory lymphoid population resulted in antigen-specific and genetically restricted immune suppression. The TsFi-induced lymphoid population was not responsible for the genetic restriction, and furthermore, there was no restriction observed between the two complementing populations. However, matching of the nonspecific accessory cell with the recipient host at the I-J subregion of the H-2 complex was essential for immune suppression. Finally, the activity of complementing cells was found to be independent of cyclophosphamide-sensitive Ts populations of the recipient mice. The ramifications of these findings with reference to the existing suppressor pathways are discussed. 0 1986 Academic press, IIK.

INTRODUCTION

Suppressor T (Ts)* cells and soluble factors (TsF) derived from them have been shown to play an important role in immune regulation (1). Immune suppression is normally produced by a series of interactions between defined Ts subsets. Complex ’ Supported by Grant AI 13 115 from the U.S. Public Health Service. ’ Abbreviations used: ABA, azobenzenearsonate; ATS, anti-thymocyte serum; BSS, balanced-salt solution; c’, complement; CY, cyclophosphamide; DTH, delayed-type hypersensitivity; FCA, Freund’s complete adjuvant; FIA, Freund’s incomplete adjuvant; NSC, normal spleen cell lysate; SC, spleen cells; Tact, T acceptor cell; TMA, phenyltrimethylammonium; Ts, , Ts2, Tsr-first-order, second-order, third-order T suppressor cells; TsF, , TsF*-suppressor factors derived from Ts, , Tsr; tyr(ABA), L-tyrosine-pazobenzenearsonate; tyr(TMA), L-tyrosine-pazophenyltrimethylammonium. 351

352

JAYARAMAN

AND BELLONE

suppressor pathways are usually initiated by either the first-order Ts (Tsr) subset or the Tsr-derived factor(s), called the TsFr (2-13). The antigen systems which display dominant cross-reactive idiotype(s) (Id) produce Id-binding second-order Ts (Tsz) cells in response to Tsr/TsFr (4-6, 9) whereas those which do not express dominant cross-reactive Id produce Tsz cells with antigen specificity (7,10). All the TsF, studied so far, with the exception of one (4,6), require antigen as an accessory signal to induce TsZ cells irrespective of their source, varied biochemical nature, and the presence or absence of genetic restrictions imposed for their biological functions (3, 5,7, 10-12). In the 4-hydroxy-3-nitrophenyl acetyl system, the Ts, cells require antigen (5) whereas the monoclonal hybridomaderived TsFr does not depend on antigen to cause immune suppression ( 13). Despite the fact that antigen is required as a second signal in a majority of systems for the production of TsZ cells, the influence and nature of such secondary signals required for the TsFr-mediated immune suppression have not been thoroughly studied. We have analyzed the regulation of phenyltrimethylamino (TMA) hapten-specific delayed-type hypersensitivity (DTH) responses by Ts cells and their factors induced with the monovalent antigen, L-tyrosine-pazophenyltrimethylammonium [tyr(TMA)] (8, 9, 12, 14-15). The monovalent antigen given in a nontolerogenic fashion first induces the Tsr population by 2 weeks. The Tsl subset, upon disruption, yields TsF, whose biologic properties are distinct from other TsF, reported in Iddefined suppressor systems. The TsF, appears to be a single polypeptide chain containing antigen-binding site(s), an I-J subregion-encoded determinant, and immunoglobulin (Ig)-associated Id determinants. While TsF, induces an efferent-acting Idspecific TsZ subset when given in the presence of the monovalent antigen tyr(TMA) in FCA, neither the TsF, nor antigen alone could independently induce detectable suppressor activity. However, a mixture of splenic cells from these separately inoculated mice reconstituted suppression in recipients previously primed for DTH. This preliminary data suggested to us the possibility that in the hosts receiving TsFr and antigen together, two complementing cell types were triggered: one by antigen and the other by TsFl resulting in an Id-specific efferent-phase Ts population. In this communication, we have begun studies on the induced complementing cell types and the results demonstrate that the separately induced cells, to our surprise, do not resemble the Id-specific Ts2 which is normally induced by TsF, plus antigen, but rather these cell types appear to be cell populations heretofore undescribed. This novel suppression is discussed in the light of existing suppressor pathways. MATERIALS

AND METHODS

Mice. Male A/J, BlO.A, BlO.A(3R), BlO.A(SR), and SWR/J mice were obtained from the Jackson Laboratory (Bar Harbour, Maine) and were 6 to 8 weeks of age at the time of the experiment. Immunization and challenge. One-molar solutions of diazotized trimethylaminoaniline (TMA) (Bachem, Inc., Torrance, Calif.) and parsanilic acid (ABA) (Sigma Chemical Company, St. Louis, MO.) were prepared according to the method of Thomas et al. (16). Syngeneic spleen cells (SC) were coupled with the diazotized TMA or ABA as described earlier (12, 17). Freshly coupled 3 X 10’ TMA-SC or ABA-SC were injected subcutaneously (SC) into two separate sites on the dorsal flank of the animal. Five days after immunization, mice were challenged in the right footpad with 30 ~1 of 10 mM diazotized TMA or ABA in saline. The footpad thickness was mea-

COMPLEMENTING

CELLS

OF

353

SUPPRESSION

sured with a Mitutoyo micrometer (Schlesingers for Tools, Ltd., Brooklyn, N.Y.) 24 hr after challenge. Nonimmunized mice challenged similarly served as the negative control. Cyclophosphamide treatment. Mice were injected intraperitoneally (ip) with 20 mg cyclophosphamide (CY) (Cytoxan, Mead Johnson and Co., Evansville, Ind.) per kilogram body weight 1 day after immunization ( 14). Induction of suppressor cells and factors. To prepare TsFl , naive A/J mice were inoculated ip with 0.2 ml containing 100 pg of tyrosinated TMA [tyr(TMA)] (Biosearch, San Rafael, Calif.) and Freund’s complete adjuvant (FCA) containing Hj7Ra (Difco Laboratories, Detroit, Mich.). The cell-free cytosol factor was extracted from the SC 14 days later as detailed before (8). Cytosol was prepared from normal A/J spleen cells (NSC) in a similar fashion. The induction of Id-specific TsZ has been described earlier ( 12). Naive A/J mice were injected intravenously (iv) with a total of 6 X 10’ cell equivalent of TsFi through a lateral tail vein on Days 0, 1, and 2. Additionally, these mice were injected ip with 50 pg of tyr(TMA) emulsified in 0.1 ml of FCA on Day 0. Seven days later, SC were harvested from these mice and previously shown to contain Id-binding Ts2 ( 12). TsF,-induced T cells were generated by injecting naive A/J or SWR/J mice iv with a total of 6 X 10’ cell equivalent of TsFl on Days 0, 1, and 2. SC were harvested 1 week after the initial injection. The nonspecific accessory T cells were induced by injecting appropriate strains of mice ip with 0.1 ml containing either 50 pg of tyr(TMA) or tyr(ABA) emulsified in FCA, or 0.1 ml containing balanced-salt solution (BSS) emulsified either in FCA or incomplete adjuvant (FIA). SC were obtained 1 week later. Treatment of spleen cells. Spleen cell suspensions (33 X 106/ml) made in sterile BSS were treated for 1 hr at 4°C with anti-Thy- 1 monoclonal ascitic fluid ( 1: 10 final dilution) derived from the hybridoma T24/40.7 (kindly provided by Dr. J. Kappler and Dr. P. Marrack, The National Jewish Hospital, Denver, Colo.) and anti-thymocyte serum (ATS; M.A. Bioproducts; 1: 10 final dilution) followed by treatment with rabbit c’ ( 1:20 final dilution) for 30 min at 37°C. Viability was assessed using trypan blue dye exclusion method. Sixty-two percent of TsF,-induced SC and 75% of tyr(TMA) + FCA-induced SC were recovered as viable after treatment with ATS + anti-Thy-l and c’ treatment. In contrast, more than 95% cells were recovered as viable when SC were treated with c’ alone. Assay ofsuppression. Prospective suppressor cells were transferred iv into mice that had been appropriately immunized 4 days earlier. The mice were then challenged on Day 5 and their footpad swelling responses assessed 24 hr later. All data were represented as arithmetic means f standard error. Percentage suppression was calculated using the formula: % suppression =

positive control - experimental group x loo positive control - negative control .

More than 50% suppression was always significant. RESULTS Idiotype-Spec$c

Ts2 Cells Are Replaced by Complementing

T Cells

The first set of experiments addressed whether TsF,-induced spleen cells could express efferent-acting suppressor function when mixed with SC induced independently

354

JAYARAMAN

AND BELLONE

TABLE 1 Suppression is Mediated by Complementing Cells induced withb Positive controls TsF, + tyr(TMA) in FCA together TsF, alone tyr(TMA) in FCA alone 1:1 mixture of cells induced with TsF, alone and tyr(TMA) in FCA alone Negative controlsd

Cells’

Response (mm + 1 SEM)

Percentage suppression’

0.95 + 0.06 0.35 f 0.06 0.8820.13 0.85+0.11

85 10 14

0.43 + 0.04 0.24 + 0.04

73 -

’ A/J mice were immunized sc with freshly coupled 3 X 10’ syngeneic TMA-SC. Five days after immunization, the mice were challenged in the footpads with 30 pl of 10 mM diazotized TMA in saline. Footpad swelling response was measured 24 hr after challenge. Four mice per group were tested. b One day before challenge, mice were transferred iv with the prospective suppressor cell populations obtained from A/J mice injected 1 week previously with TsF, alone or tyr(TMA) in FCA alone. Some mice were injected with TsF, and tyr(TMA) in FCA together. All mice received 6 X IO’ viable SC. The last group received 3 X 10’ TsF, alone induced SC mixed with 3 X 10’ tyr(TMA) in FCA alone induced SC. ’ Percentage suppression was calculated as described under Materials and Methods. Significant suppression is in bold face. d Negative controls indicate mice challenged only.

with antigen. Hence, groups of mice were injected with either TsF, or antigen alone. As controls, some mice were injected both with TsFl and antigen in order to induce efferent-acting Id-specific Tsz (12). One week later, SC were harvested from these mice and adoptively transferred into mice immunized for DTH responses with TMASC 4 days earlier. The recipient mice were challenged the following day and the footpad swelling responses were measured 24 hr later. As observed earlier (12), SC obtained from mice injected with both TsF, and tyr(TMA) in FCA contained potent efferent-phase suppressive activity (Table 1). Also in accordance with our previous report, neither TsF, nor antigen by itself induced detectable suppressor activity by 1 week. However, a mixture of TsF,- and antigen-induced SC readily suppressed the DTH responses. Thus, the data demonstrate that the efferent suppressive activity normally mediated by Id-specific TsZ ( 12) can also be demonstrated by complementing SC which independently do not express suppressor function. Therefore, we performed a series of experiments to evaluate whether the independently induced cells resemble the Id-specific Ts2 generated by the administration of TsF, and antigen together in the same experimental mice. To determine whether the observed cell complementation were T-cell-dependent, SC obtained from mice injected with TsF, alone or antigen alone were separately treated with ATS and anti-Thy- 1 antibodies plus c’ prior to cell transfer. Control cells were treated with c’ alone. The treated populations were mix-matched as shown in Table 2 and tested for efferent-acting suppressive activity as detailed earlier. Treatment of complementing cell populations with c’ alone did not abolish their suppressive potential. On the other hand, suppression was not seen when both the complementing populations were devoid of T cells. Furthermore, removal of T cells from either one of the complementing populations abrogated suppression, indicating that the T cells from both populations are necessary to bring about immune suppression.

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CELLS OF SUPPRESSION TABLE 2

Cell Complementation

is T-Cell Dependent”

Treatment of cells induced withb TsF,

Response (mm + 1 SEM)

tyr(TMA) in FCA

Positive controls C’

ATS, anti-Thy. 1 + c’ ATS, anti-Thy. 1 + c’ C’

Negative controls

d ATS, anti-Thy- 1 + c’ C’

ATS, anti-Thy- I + c’

0.93 0.45 0.94 0.88 0.90 0.33

+ + + f f f

0.04 0.05 0.03 0.06 0.06 0.02

Percentage suppression’ 80 0 8 5 -

u Immunization and challenge for TMA-specific DTH were performed in A/J mice as described earlier (see Table 1). Four mice per group were tested. * SC were obtained from A/J mice injected with TsF, alone or tyr(TMA) in FCA alone 1 week before. Aliquots were either treated with c’ alone or with a mixture of ATS and anti-Thy- 1 antibodies followed by c’ treatment. Each mouse received 3 X 10’ viable cells of one population mixed with 3 X 10’ of the other population, as indicated, just prior to injection. Cells were given on Day 4 and the mice were challenged on Day 5. ’ Significant suppression is in bold face.

Specificity Requirements for the Induction

of Complementing

Cells

To analyze whether specific signals are required for the induction of complementing populations, the following experiments were performed. Complementing populations were variously induced and transferred into TMA-SC-primed mice to monitor their function. In the first set of experiments either SC from TsF, or normal spleen cell lysate (NSC)-injected mice were cotransferred along with tyr (TMA)-induced populations and, as seen in Table 3, suppression occurred only when the TsFi-induced population was utilized. Thus, the data indicate that in order to observe suppression antigen-induced cells require the presence of a TMA-specific TsFi-induced complementing cell population. Since a specific signal was required for the induction of the factor-induced Ts, we next examined the induction specificity of the complementing antigen-induced population. Hence, TMA-TsFi-induced SC were cotransferred with SC induced by a variety of agents. As can be seen in Table 3, the TsFi-induced population readily complemented to produce suppression with all of the populations tested: these include SC induced with tyr (ABA) in FCA, FCA alone, or FIA alone. Since none of these second populations produced suppression when transferred alone (Table 3), we conclude that the TMA-specific TsFi-induced population can efficiently complement with what appears to be a nonspecifically induced accessory population to produce immune suppression. Suppression Mediated

by Complementing

Cells Is Antigen Specific

We then tested the antigen specificity of suppression produced by complementing cells. To this end, TMA-TsF,-induced SC were cotransferred with the tyr(TMA) in FCA-induced population into mice which had been previously immunized for TMA or ABA-directed DTH responses. Mice were appropriately challenged the following day and their footpad responses measured 24 hr later. As seen in Table 4, the comple-

356

JAYARAMAN

AND BELLONE

TABLE 3 Specificity Requirements for the Induction of Complementing Cells” Response (mm ? 1 SEM)

Percentage suppression’

Positive controls

0.77 + 0.04 (19)d

-

TsF,-induced cells alone TsFi-induced + tyr(TMA) in FCA-induced NSC-induced + tyr(TMA) in FCA-induced

0.70 +- 0.04 (4) 0.40 + 0.04 (12) 0.80 + 0.04 (4)

18 77 0

TsF,-induced + tyr(ABA) in FCA-induced TsFi-induced + BSS in FCA-induced TsFi-induced + BSS in FIA-induced

0.44 + 0.06 (4) 0.45 + 0.05 (4) 0.30 + 0.04 (4)

69 67 96

tyr(TMA) in FCA-induced cells alone tyr(ABA) in FCA-induced cells alone BSS in FCA-induced cells alone BSS in FIA-induced cells alone

0.66 0.70 0.66 0.65

23 15 23 25

Negative controls

0.29 + 0.01 (20)

Cells transferredb

+ 0.06 * 0.04 + 0.13 +- 0.03

(4) (4) (4) (4)

-

’ A/J mice were immunized for TMA-specific DTH responses as described earlier. ‘SC were otained from A/J mice 1 week after stimulation as indicated (see Materials and Methods). Four days alter immunization, each mouse received either 6 X IO’ SC from a single source or a 1: 1 mixture of two different populations (6 X 10’ SC total). Mice were challenged on Day 5 and their footpad responses measured the following day. ‘Significant suppression is in bold face. d Number of mice tested is given in parentheses.

menting cells were only able to suppress the TMA-specific and not the ABA-specific DTH responses. Furthermore, in data not shown here, we have observed that the TMA-TsF,-induced SC also failed to suppress ABA-specific cutaneous hypersensitivity responses when cotransfen-ed with either tyr(TMA) in FCA-induced cells,

TABLE 4 Complementing Cells Produce Antigen-Specific Suppression Cells transferred”

Immunization”

None Complementing cells -

TMA-SC TMA-SC Negative controls

None Complementing -

ABA-SC ABA-SC Negative controls

cells

Response (mm + 1 SEM) 0.68 0.40 0.30 0.53 0.53 0.28

+ f f * + f

0.07 0.04 0.04 0.02 0.04 0.02

Percentage suppressionC 74 -

0

“Each mouse received 3 X 10’ TMA-TsF,-induced and 3 X 10’ tyr(TMA) in FCA-induced SC iv on Day 4. The complementing cells were induced in A/J mice a week before harvest. b Immunization and challenge were performed in A/J mice as described under Materials and Methods. Four mice per group were tested. ’ Significant suppression is in bold face.

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CELLS OF SUPPRESSION TABLE 5

Genetic Restriction is Determined by Nonspecific Accessory Cells” Source of transferred cellsb Recipients

TMA-TsF,-induced

Experiment A A/J(H-2”; I&l’)

Positive controls

A/J

NJ A/J

NJ A/J

NJ

SWR/J Negative controls

NJ Experiment B SWR/J(H-2q; &h-l’) SWR/J SWR/J SWR/J SWR/J

Accessory cells

SWR/J

NJ

Positive controls SWR/J SWR/J

A/J

A/J

SWR/J

Negative controls

SWRJJ

Response (mm ? 1 SEM)

Percentage suppressionC

1.03 + 0.03 0.58 + 0.02 0.85 + 0.03 0.59 + 0.01 0.31 -co.01

74 25 61 -

0.68 0.48 0.68 0.49 0.30

53 0 50 -

f f f f f

0.04 0.04 0.06 0.01 0.05

’ Recipient mice were immunized and elicited for TMA-specific DTH responses as described earlier (see Table 1). Four mice per group were tested. b One week after injections, 3 X 10’ TMA-TsF,-induced SC were mixed either with 3 X 10’ tyr(TMA)induced SC (Experiment A) or with 3 X 10’ SC induced with BSS in FCA (Experiment B) and iv transferred into recipient mice on Day 4. These cells were either induced in A/J or SWR/J mice and mix-matched as indicated. ’ Significant suppression is in bold face.

tyr(ABA) in FCA-induced cells, or FIA-induced complementing cells while these cell mixtures readily suppressed TMA-specific DTH responses. Hence, we conclude that the suppression mediated by these cell populations is antigen specific and probably controlled by the TsF,-induced population. Genetic Restriction Is Imparted

by Nonspecijic Accessory Populations

In many systems, it has been observed that the TsF,-induced Ts cells or their factors are genetically restricted for their function (6,7,9, 13, 18, 19). Therefore, we investigated whether suppression mediated by the complementing cells is also genetically restricted. To this end, complementing cells were induced in either A/J (H-2”; Ighle) or SWR/J (H-2q; &h-l’) strains of mice. They were then mixed in several combinations and adoptively transferred into TMA-SC-primed A/J mice. The recipient mice were then challenged the following day and footpad responses measured 24 hr later. The results obtained are shown in Table 5. As observed earlier, when both cell populations were obtained from A/J mice and transferred into syngeneic A/J recipients, significant suppression was observed. In contrast, when a nonspecific allogeneic accessory population was cotransferred with A/J-derived TsFr-induced cells, suppression was not seen in A/J mice. This suggests that suppression requires a genetic match either between the two donor populations, or between the accessory cell and the recipient mice. The latter requirement appears to be the case since allogeneic TsF,-induced cells along with A/J-derived accessory cells were able to promote suppression in the A/J recipients. In the reciprocal experiments using SWR/J mice as recipients, again the genetic restriction was observed only between the nonspecific

358

JAYARAMAN

AND BELLONE

TABLE 6 Nonspecific Accessory Cell Function Is Restricted bv I-J Subregion” Mice providing tyr(TMA)-induced

cell$ H-21

Strain

NJ

Allotype Igh-1’

Bl0.A Igh-lb BlO.A(SR) &b-lb BlO.A(3R) Igh-lb Positive controlse Negative controls’

H-2K K

K b b

A

B

J

E

C

KKKKJ KKKKd

b b bbbkd

H-2D d

d k

k

d

d

d

Response (mm f 1 SEM) 0.38 + 0.3 1 + 0.38 + 0.83 + 0.69 + 0.26 +

0.07 0.06 0.05 0.04 0.06 0.02

(1 I)d (8) (12) (4) ( 17) (12)

Percentage suppressionc 72 88 73 0 -

’ A/J mice were immunized with TMA-SC as described earlier and received test SC populations on Day 4. Challenge was performed the next day and the footpad swellings were measured 24 hr later. ’ Spleen cells were obtained after tbe injection of tyr(TMA) + FCA in indicated strains of mice. One week later, 3 X 10’ TsFi-induced cells obtained from SWR/J (H-2q; Igh-1’) mice were mixed with 3 X 10’ tyr(TMA) in FCA-primed SC just prior to iv transfer into A/J recipients. Regions of homology of accessory T-cell population with recipient A/J mice are in bold face. ’ Significant suppression is in bold face. d Number of mice tested is given in parentheses. ’ Positive controls indicate primed A/J mice which did not receive any cell. /Negative controls indicate nonimmune A/J mice challenged only.

accessory cells and the recipients, and not between the two complementing cell populations producing suppression. Further experiments were performed to map the subregion specificity of the restricting element between the nonspecific accessory population and recipient mice. Hence, TsF1-induced SC were obtained from SWR/J mice and cotransferred with nonspecific complementing SC obtained from several strains of mice. The cell mixtures were then tested for suppressor function by adoptive transfer into TMA-SC inoculated A/J mice. Results obtained are shown in Table 6. As expected, suppression was observed in A/J recipients when the SWR/J-derived TsF,-induced cells were cotransferred with the A/J-derived nonspecific population. Furthermore, suppression was seen when the nonspecific cells were derived from Bl0.A mice, suggesting that neither the non-H-2 background nor the allotype-linked gene products are restricting elements in this system but rather the major histocompatibility complex (MHC) appears to be important. In data not shown, several strain combinations ruled out participation of the K and D gene products and suggested that the I region played a critical role. Thus, nonspecific accessory cells were obtained from either BIO.A (5R) or B 1O.A (3R) mice since these strains are considered I-J congeneic and used extensively to evaluate the role of I-J subregion in imparting genetic restriction between interacting Ts subsets. The data obtained using these mice demonstrate that suppression was manifested in the A/J strain of mice (I-Jk) only when Bl0.A (5R) (1-J’) cells and not B 10.A (3R) (I-Jb) cells were cotransferred with the TsF,-induced population. In data not shown here, we have observed that neither the TsF1-induced cells alone nor the nonspecific populations by themselves from any of these strains produced suppression. Thus, the data collectively demonstrate the requirement for I-J match-

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CELLS OF SUPPRESSION TABLE I

Complementing Cell Function Does Not Depend on CY-Sensitive Tsg Cell’ Treatment*

Cells transferred’

Response (mm + 1 SEM)

Percentage suppressiorrj

None None None

Positive controls Complementing cells Id-specific Tsz cells

0.84 + 0.07 (8) 0.40 + 0.08 (8) 0.55 + 0.03 (4)

76 50

CY CY CY

Positive controls Complementing cells Id-specific Tsz cells

0.68 k 0.07 (8) 0.39 f 0.06 (8) 0.70 f 0.04 (4)

69 0

-

Negative controls

0.26 f 0.05 (8)

-

0 Immunization and challenge of A/J mice for TMA-specific DTH responses were performed as described in Table 1. * Some mice received 20 mg CY/kg body weight 1 day after immunization. ’ Indicated SC were generated (see Table 1) 1 week before and transferred (6 X 10’ per mouse) iv 4 days after immunization. Mice were challenged on Day 5 and their footpad responses measured the following &Y. d Percentage suppression was calculated by comparing responses of experimental group with similarly treated positive controls. Significant suppression is in bold face. eNumber of mice tested is given in parentheses.

ing of the nonspecific accessory population with the recipient A/J mice to manifest suppression in concert with TsF,-induced cells. Complementing

Cells Do Not Require CY-Sensitive Ts3for Function

We have previously shown that Id-specific Tsz cells function by interacting with a CY-sensitive third-order Ts subset (Ts3) which is coinduced along with DTH-mediating T cells after normal antigen stimulation (14). Hence, we then examined whether complementing cells also require interaction with CY-sensitive Ts3 to inhibit DTH responses. To this end, syngeneic complementing SC were transferred into CYtreated as well as untreated mice which were previously primed for TMA-specific DTH responses. As controls, Id-specific Ts2 known to depend on CY-sensitive Ts3 subset (14) were also tested for their ability to cause suppression in control and CYtreated mice. Data in Table 7 show that complementing cells readily suppressed the DTH responses in both CY-treated and untreated mice. In sharp contrast, the Idspecific TsZ failed to suppress the immune responses of CY-treated mice confirming our earlier observations that these cells depend on a CY-sensitive Ts3 population for function (14). Thus, the data indicate that the complementing cells, unlike the Idspecific TsZ, do not depend on CY-sensitive cells of the recipient mice to produce suppression. DISCUSSION The data presented in this paper document that the inoculation of TsF, in the absence of antigen results in the induction of a unique efferent-acting T-cell population heretofore unidentified. The TsF,-induced T cell does not exhibit suppressive activity and to become functional requires the presence of a nonspecifically induced

360

JAYARAMAN I.

TMA-TsF,

+ tyr(TMA)

AND BELLONE

together

m

H-2and TMA-SC

II.

immunization

Igh

Id-specific restrIcted

Tsp y

-

Id+T%

0

DTH

TMA-Ts?

Nonspecific stimulation

-

Accessory

T Cell

FIG. 1. Schematic representation of T-cell interactions leading to immune suppression in the TMA system. The single-chain TsF, when given along with the antigen tyr (TMA) + FCA induces an intermediary Id-specific T% population (I). Immunization with TMA-SC results in the production of DTH-mediating T cells as well as the final-effecter Ts3 population. The interaction between the Tsr and the haptenspecific Tss is both H-2 and Igh restricted. Once triggered by Id-specific TsF, , the Tss produces antigennonspecific suppression (9, 12, 14,20). In contrast, when the TsFi alone is introduced into the system, a unique T-cell population is induced (II). The factor-induced cell type requires the presence of a nonspecific accessory T-cell population for its function. The interaction of these complementing cell types is not subjected to any genetic constraint. Moreover, the factor-induced cell acts across H-2 and Igh barriers, provided there is an I-J match between the accessory population and the cells of the recipient mice. Such an interaction results in an antigen-specific suppression.

accessory T-cell population. Such a cell complementation results in antigen-specific and I-J-restricted immune suppression (see Fig. 1). This newly described system appears to be different from known suppressor pathways in several ways. In the presence of antigen, TMA-TsF, induces an Id-specific Tsz cell as described (12); several lines of evidence make it apparent that the herein described TsFi-induced T-cell population is clearly different from the Id-specific TsZ . First, the transfer of Id-specific Ts2 into normal DTH-primed A/J recipients results in immune suppression (9, 12, 14), while the same protocol using the TsF,-induced T-cell population alone fails to do so (Table 1). Further, Id-specific Tsz are restricted in their interaction with target cells by both allotype and H-2 gene products (9). This is clearly not the case with the TsF,-induced population since these cells can act across allotype as well as H-2 barriers in order to effect suppression (Tables 5, 6). Finally, the previously described TsZ binds to Ig-associated Id determinants (9, 12), while the TsF,-induced population does not bind Id or antigen (unpublished data). Thus, these cells differ markedly from the Id-specific Ts2-cell type in both binding and functional properties. In addition the TsF,-induced T cell does not appear to be the final effector Ts3cell type described earlier in the TMA system (14, 20). While the Ts3 population is genetically restricted (9) there is no such restriction imposed on the function of the TsF,-induced cell type (Tables 5,6). Furthermore, the Ts3 bears hapten (TMA)-binding receptors (20) in contrast to the TsF,-induced population which binds only to TMA-TsF, molecules, and not to Ig-associated Id or to the TMA hapten (unpublished data). Thus the TsF,-induced cell type does not represent either the intermediary Id-specific Tsz or the final effector Ts3. The accessory T-cell population required for the function of TsF1-induced cells also does not appear to resemble the Id-specific Ts2 population. While TsF, plus antigen is required for the induction of Id-specific Ts2 ( 12), the accessory population does not require any specific antigenic signal (Table 3). Furthermore, unlike the Id-specific Tsz, the nonspecifically induced T cells do not bind Id (unpublished data).

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CELLS OF SUPPRESSION

361

Several lines of evidence indicate that the nonspecifically induced population also does not represent the Ts3 population. The induction of TMA-specific Tss requires immunization with specific immunogen (20) in contrast to the accessory population which clearly does not require a specific induction signal (Table 3). Moreover, the Ts3 is restricted by both H-2 and allotype-linked gene products for its interaction with Id-specific TsZ (9), while there is no genetic restriction observed between the two complementing cell types. However, the nonspecific T cell is restricted by only I-J subregion products to function in host recipient mice (Table 6). Thus, the I-J restricted, nonspecific T-cell population is clearly different from the Id-specific Ts2 as well as the hapten-specific Ts3. In the Id-nondefined immune systems, the final effector Ts, called the T acceptor (Tact) cell, has been shown to be induced in a nonspecific manner (21). Although the accessory T cell is also induced nonspecifically, it is clearly different from the Tact cell. In the case of Tact-mediated immune suppression, genetic restriction is imparted only by the TsF carried by the Tact and not by the Tact itself (2 1). In our system only the nonspecific T cells have to be genetically matched with the recipients in order to bring about suppression by TsFi-induced T cells. Finally, the complementing cell types described in this report also appear to be different from other suppressor systems which involve synergistic interactions between two different T-cell populations to produce hapten-binding efferent-acting supressor molecules (TsF,) (22-25). In these systems the factors produced by the interacting T-cell subsets in turn have to interact with other cell types to subsequently produce suppression. However, in our system the complementing cell types produce suppression in CY-treated (Ts3-, Tacc-depleted) mice (Table 7) suggesting that these cells do not require interaction with subsequent cell types. Baxevanis et al. (26) have described a suppressor system specific for lactate dehydrogenase B which also involves two interacting T cells as does ours. In their system, the antigen induced Ts cell requires interaction with a nonspecific T cell to become fully functional. As in our system, the interaction between these two T cells is not genetically restricted. However, the interaction of the antigen-induced Ts cell with the target-proliferating T cells is E-restricted. This is in contrast to the genetic restriction observed in our system in that I-J restriction was seen only between the nonspecific T cell and the target cell(s) in the recipients and not between the TsF,-induced cells and the target cell(s) (Fig. 1). Thus, our system appears to be different from other suppressor systems which also involve complementing cell types. Though the present study does not clarify the role of antigen for the induction of Id-specific Tsz by the TMA-TsF, ( 12) it does, however, reveal that the very same TsF, in the complete absence of antigen follows a different pathway to produce suppression. This suggests that the type of Ts subpopulation as well as its fine specificity are heavily influenced by the mode of TsF, presentation to presursor cells. Further work will be necessary to determine the nature and function of such factor-presentation involved in the selection of Ts subpopulations as well as their fine specificity from the available repertoire. In conclusion, we have shown that the administration of TMA-TsF1 in the absence of antigen induced a novel type of T cell which requires the participation of an I-J-restricted nonspecific T cell for function. Because of the unique functional properties exhibited by these complementing T cells, further study is required to elucidate the mechanisms(s) by which these cells produce immune sup pression. Hybridomas or cell lines representing these cell types should provide sufficient material to analyze the nature and products of these complementing cell types.

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AND BELLONE

ACKNOWLEDGMENT The secretarial assistance of Miss Carole Vogelgesang is highly appreciated.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2 1. 22. 23. 24. 25. 26.

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