TMA-specific first-order T-suppressor hybridoma

CELLULAR

IMMUNOLOGY

97,419-432 (1986)

TMA-Specific

First-Order T-Suppressor

Hybridoma

I. Characterization of the Hybridoma-Derived Single-Chain Inducer Suppressor Factor, TsF,’ GOL WMER

JENDRISAK,*

JOANN TRIAL,?

AND

CLIFFORDJ. BELLONE*

*Department of Microbiology, St. Louis University School of Medicine, St. Louis, Missouri 63104; and fDepartment of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110 Received September 9, 1985; accepted October 15. 1985 Experiments describedin this report will characterizea monoclonal phenyltrimethylammonium (TMA) specific, first-order T-suppressor factor (TsF,) produced by a T-cell hybridoma, 8A.3. The hybridoma expressed the Thy-l, Lyt-1, Lyt-2 antigens as well as cross-reactive idiotypic (CRI) determinants but did not express I-J encoded epitopes. It was also found to bear determinants recognized by a monoclonal antibody raised against single-chain CAT-specific TsF, . The hybridomaderived factor was capable of suppressing primary in vitro trinitrophenol (TNP)-specific responsesinduced with the Brucella abortus antigen, conjugated with TMA and TNP haptens (TMA-BA-TNP). In addition, in vivo administration of 8A.3 culture supematant resulted in the specific suppression of TMA-specific delayed-type hypersensitivity (DTH) responses.Analysis of this factor revealed it to be an induction-phase, antigen-binding, CRI+, and I-J+ single chain polypeptide. Our results represent only the second such described single chain, antigen binding, I-J+ suppressorfactor derived from a monoclonal T-cell hybridoma. o 1986 Academic PBS, IUC. INTRODUCTION

Negative regulation of antigen-specific immune responsesinvolves a series of suppressor T cells (Ts)~ and their factors (TsF) (l-3). The sequence of cellular events which results in the suppression of delayed-type hypersensitivity (DTH) responsesto the phenyltrimethylammonium (TMA) hapten have been identified (4-7). Basically, three distinct Ts cells are involved. The first set of Ts, termed Ts,, are induced by antigen and also bind antigen, bear idiotypic receptors, and function during the induction phase of the immune response (6). The factor extracted from the Ts, cells ’ Supported by U.S. Public Health Services,National Institutes of Health Grant AI-131 15. ’ Abbreviations used: Anti-Id, Anti-idiotypic antisera; ABA, azobenzenearsonate;BA, Brucella abortus; BBS, borate-buffered saline; BSA, bovine serum albumin; BSS, balanced salt solution; CRI, cross-reactive idiotype(s); DMEM, Dulbecco’s modified Eagle medium; DPBS, Dulbecco’s phosphatebuffered saline; DTH, delayed-type hypersensitivity; DTT, dithiothreitol; CAT, L-glutamic acid60-L-aIanine%.-tyrosine’o; FITC, fluorescein; IBC, idiotype-binding capacity; Id, idiotype(s); IEF, isoelectric focusing; PFC, plaque-forming cells; RP-HPLC, reversephase-high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyactylamide gel electrophoresis;SRBC, sheeperythrocyte(s); Th, T helper cells; TMA, ttimethylaminoaniline; TNP, trinitrophenol; Ts, T suppressorcells; Ts, , Tsr , Tss indicate first-order Ts, second-order Ts, and third order Ts; TsFi , TsF,, Indicate Ts factors from Tsi , Tsz; tyr(TMA), L-tyrosine-pazophenyltrimethylammonium. 419 0008-8749186$3.00 Copyright Q 1986 by Academic Press,Inc. All rigbw of reproduction in any form -cd.

420

JENDRISAK, TRIAL, AND BELLONE

(TsF,) has been partially characterized and found to be an apparent single chain, inducer molecule which binds antigen and bears idiotypic as well as I-J encoded determinants (7). Inducer factors, or TsF, , have been described in several antigen systems,including L-glutamic acid6’-L-alanine3’-L-tyrosine (GAT), azobenzenearsonate (ABA), nitrophenyl-acetyl (NP), and sheep red blood cell (SRBC) systems (8-19). All of these factors functionally and serologically resembleone another to some extent; the majority bind to antigen and bear idiotypic as well as I-J encoded determinants. Despite these similarities, the composition of the inducer factor still remains controversial. The ABA, SRBC, and NP specific TsF, consist of two polypeptide chains, one of which binds to antigen and bears idiotype while the second chain bears I-J encoded determinants (12, 15-19). In contrast, the GAT (11) and TMA TsFi (7) are single chain, antigen-binding, Id+ and I-J+ inducer factors. The nature of the single-chain GAT-TsF, has been extensively studied at the biochemical and molecular levels (20, 21). Since TMA-TsFi is also an apparent single chain molecule, biochemical and genetic analysis of this factor will be necessaryto gain more insight into the nature of these interesting single chain antigen-binding suppressor molecules. To accomplish this, Ts, hybridomas which would constitutively produce TMA-specific TsF, are needed. In this manuscript, we describe a TMA-specific Ts hybridoma, derived from the fusion of splenic T cells from Tsi-bearing mice with the HAT-sensitive, AKR thymoma, BW5 147. The data will demonstrate that the hybridoma-derived TsF behaves in an identical fashion to the conventionally derived single-chain TMA-TsFI. Since it is only the second described hybridoma-derived single chain suppressor factor it should prove valuable in elucidating both structure and function for this interesting class of immune modulators. MATERIALS

AND METHODS

Mice. Male A/J (H-2a, Igh-le) mice were obtained from the Jackson Laboratory; Bar Harbor, Maine and were 6 to 8 weeks of age when used in these experiments. Antigen preparation. A 1 M solution of trimethylaminoaniline (TMA) (Bachem Inc.; Torrance, Calif,) and azobenzenearsonate(ABA) (Sigma Chemical Co.; St. Louis, MO.) diazonium salts were prepared as previously described (7). Trinitrophenylated Brucellu abortus (TNP-BA) was prepared as follows: BA (U.S. Department of Agriculture Animal and Plant Health Inspection Services;Ames, Iowa) at approximately 10’ ’ organisms/ml were washed three times in sterile borate-buffered saline (BBS) pH 9.0 and 1 ml of packed BA was resuspendedin 30 ml BBS. The suspension was mixed with 1.05 g of 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Eastman Kodak Company; Rochester, N.Y.) the pH readjusted to 9.0 with 6 N NaOH, and the reaction allowed to proceed for 4 hr at 4°C while gently stirring. The conjugate was washed three times in balanced salt solution (BSS) and diluted to 20% (v/v) in sterile RPM1 1640 (Gibco Laboratories; Grand Island, N.Y.) containing 100 U/ml penicillin, 50 &ml streptomycin (Gibco Laboratories), and 2 &ml gentamicin (Schering Corp.; Kenilworth, N.J.). The TMA-BA-TNP and ABA-BA-TNP were prepared as follows: diazotized TMA or ABA (10 mM final concentration) was added to a 10% suspension of BA in BBS, the pH was adjusted to 9.0, and the mixture was incubated for 4 hr at 4°C. The conjugates were washed three times in BBS and further conjugated with TNP as de-

HYBRIDOMA-DERIVED

TMA-TsF,

421

scribed above. The procedure for obtaining syngeneic spleen cells free of erythrocytes and coupling with the diazonium salt of TMA has been described (22). In brief, syngeneic spleen cells devoid of erythrocytes were coupled with diazotized TMA at a final concentration of 10 mM at pH 8.2. Establishment of TMA-specljic Tsl hybridomas. Naive A/J mice were inoculated intraperitoneally with 100 pg L-tyrosine-pazophenyltrimethylammonium [tyr(TMA)] (Biosearch; San Rafael, Calif.) in FCA containing Hs7Ra, (Difco Laboratories; Detroit, Mich.). Spleen cells were obtained from these mice 2 weeks later and T cells were enriched by using affinity-purified rabbit anti-mouse Ig (Gateway Immunosera; St. Louis. MO.) coated plates as described previously (6). Ts hybridomas were derived by the somatic cell fusion of these T cells with HAT-sensitive AKR thymoma, BW5 147, with polyethyleneglycol (PEG 1500, MA Bioproducts; Walkersville, Md.) according to the technique of Kapp et al. (10). Cells were cultured in HAT-selection media that contained 15%horse serum for 2 weeks,followed by maintenance in Dulbecco’s modified Eagle’smedium (DMEM) containing 100 U/ml penicillin, 50 &ml streptomycin, 2 pug/mlgentamicin, and supplemented with 1 mM sodium pyruvate, 2 m&f L-ghtamine, nonessential amino acids, and 15%horse serum. The hybridomas were initially screenedby immunofluorescence (23) and cells which expressedI-Jk and surface TsF, (as detected by monoclonal anti-TsF, antibodies, 1248A4.10) were cloned by limiting dilution. Twenty-five clones were established and screenedonce more by immunofluorescence. Analysis of cell surjke antigens by immunofluorescence. Cells were harvested and washed with PBS containing 0.1% BSA, 0.2% sodium azide, and 25 &ml dextran sulfate. For fluorescence microscopy, 5 X lo4 cells were placed in individual wells of a 96-well U-bottom plate (Limbro, Flow Laboratories, Inc.; McLean, Va.) and 25100 ~1of antibody was added to each well. After reaction at 4°C for 30 min, the cells were washed three times by filling the wells with the PBS solution, centrifuging the plates at 250g for 5 min, and shaking out the fluid. Cells were resuspendedon a vortex prior to each wash. The secondary antibodies used were FITC-(Fab’)2 fragments of goat anti-mouse IgG, goat anti-rabbit IgG, and goat anti-rat IgG (Cappel Laboratories; Cochranville, Pa.) diluted 1:5 and used at 4 pi/well. Cells were incubated and washed as above, and 2% paraformaldehyde (Eastman Kodak Co.; Rochester,N.Y.) containing 0.005% Evan’s blue stain (Fisher Scientific Co.; Fairlawn, N.J.) was added to each well. Cells were placed on microscopic slides for viewing under a Zeiss microscope equipped with a high-pressure mercury lamp and epifluorescence condenser. Preparation of T cell suppressorfactors (TsF). The supernatants from 8A.3 and 5A. 11 were used as the source of TsFl . All supernatants were collected from cultures at a density of 1 X lo6 cells/ml. The culture supematant of another continuous T-cell hybridoma (8A. 16) lacking the ability to produce TsF, established in the same fusion, was used as the control culture supernatant. The preparation of conventional, secondorder TsF (TsF*) from spleen cells of 6-week tyr(TMA)-primed mice has been described (24). Assayfor suppressor activity of culture supernatant on delayed-type hypersensitivity responses. Briefly, for the induction of TMA-specific DTH responses,3 X lo7 freshly coupled syngeneic SC were injected subcutaneously (SC)into separate sites on the dorsal flank of the animal (24). Five days after immunization, mice were challenged with 30 ~1of 10 mM diazotized TMA in saline in the right footpad. The contralateral footpad remained uninjected and served as the control. The footpad thickness was

422

JENDRISAK, TRIAL, AND BELLONE

measured with a Mitutoyo Micrometer (Schlesingersfor Tools Ltd.; Brooklyn, N.Y.) 24 hr after the challenge. The magnitude of the DTH reaction was expressedas the increment of swelling between the challenged and unchallenged control footpad. Nonimmunized mice challenged similarly served as the negative control. For specificity experiments, mice were injected sc with a total volume of 0.3 ml of 10 mM TNBS dissolved in BSS, pH 7.4, into two separate sites on the dorsal flank (25). Five days later, the mice were challenged by injecting 30 ~1 of 10 mM TNBS (pH 7.4) solution into the right footpad. The footpad thickness was measured 24 hr later. To test the functional activity of the hybridoma-derived suppressor factor, the mice received a single iv injection of 0.5 ml culture supematant on Day 0. Immunoadsorption of hybridoma-derived TsF. The TMA and ABA substituted Sepharose 4B were prepared as previously described by Cheseboro and Metzger (26). Anti-I-Jk, anti-I-Jq, anti-Id, 1248A4.10, normal Rabbit Ig (NRIg), and normal rat Ig (NRat Ig) immunoadsorbent columns were prepared aspreviously described (27): 18% sodium sulfate precipitates of monoclonal anti-I-Jk (KY76), monoclonal anti-I-Jq (KY35.4) (provided by Dr. Vera Hauptfeld, Washington University; St. Louis, MO.) (28), normal rabbit Ig, normal rat Ig, rabbit anti-idiotypic antiserum (29, 30), and the monoclonal antibody, 1248A4.10 (31) (provided by Dr. Craig Sorensen, Washington University; St. Louis, MO.) were coupled to Sepharose4B at 1 mg/ml of protein/ml of packed beads. The hybridoma supernatant was absorbed with the various immunoadsorbents in the following way: briefly, 2 ml of hybridoma-supematant was incubated with 0.5 ml of packed Sepharosebeadspreviously equilibrated with Dulbecco’s phosphate-bufferedsaline (DPBS) containing 0.1% BSA. The mixture was gently rocked at 4°C for 1 hr, spun, and the filtrate collected. The beads were washed twice with DPBS and the bound material was eluted by incubating the beads with 2 ml of 2 M KC1 for 1 hr at 4°C on a rocker. The eluted material was collected by sedimenting the beads. Both the filtrate and eluate materials were dialyzed against distilled Hz0 for 1 hr followed by dialysis against DPBS overnight. The material was filter sterilized and used the same day. Reduction of TsF,. The hybridoma supematant and I/ 10 dilution of conventionally derived TsF2 were reduced with 5 nGt4 dithiothreitol (DTT) (Sigma Chemical; St. Louis, MO.) for 45 min at room temperature under an atmosphere of saturated N2 (7, 32). This mixture was then adsorbed, in the presence of DTT, with anti-I-Jk antibodies coupled to Sepharose beads which were preequilibrated with 5 r&t4 DTT. After a 1 hr incubation, the filtrate was collected, the columns were washed, and the bound material was eluted with 2 M KCl. All the preparations were dialyzed against distilled HZ0 for 1 hr and overnight against DPBS, filter sterilized, and used the same day. Culture system and hemolytic plaque assay. A single cell suspension was prepared from normal A/J spleens and resuspended at 1 X lo6 cells/O.5 ml in RPM1 1640 containing 10 mM Hepes, 100 U/ml penicillin, 50 pg/ml of streptomycin, 2 &ml gentamycin, and supplemented with 1 mit4 sodium pyruvate, 4 mM L-glutamine, nonessential amino acids, 5 X 10e6it4 2-mercaptoethanol, and 5% heat-inactivated fetal calf serum (Lot 12103075; Hazelton Dutchland Inc.; Denver, Colo.). The cells were incubated with 1% (v/v) of antigen for 3 days at 37°C in an atmosphere of 10% COZ, 7% OZ. and 83% Nz in 24-well Falcon tissue-culture plates. After the incubation period, triplicate wells were pooled and assayedfor TNP-specific direct plaque-forming cell (PFC) responses by the slide modification of Jeme’s hemolytic plaque assay.

HYBRIDOMA-DERIVED

TMA-TsF,

423

CRI+PFC were determined by the addition of 10 ~1 of rabbit anti-Id (10 pg IBC/ml) in to the plaquing medium (29, 30). The presented data are from one representative of at least three separateexperiments. All the conditions were run in duplicate (three wells were pooled per condition). Statistical analysis. All data were analyzed for significance by using a two-tailed Student’s t test. Percentagesuppression was calculated by using the formula: % Suppression =

Positive control - experimental group x loo * Positive control - negative control

The number of CRVPFC was calculated by the difference in plaque number (total PFC) in the absence of anti-Id minus the plaque number in the presence of anti-Id. The percentage of CRI+PFC was calculated as: 1 _ Total PFC (with anti-Id) x 100. Total PFC (no anti-Id) RESULTS AND DISCUSSION Production of Ts, Hybridomas TMA-specific TsFl secreting hybridomas were produced by the fusion of T cells from the spleens of Tsl-bearing mice with the HAT-sensitive, AKR thymoma, BW5147. The hybridomas were initially screened by immunofluorescence for the presenceof TMA-specific CRI, I-J encoded determinants, and epitopes detectable by the monoclonal antibody 1248A4.10 (28) which defines single-chain TsFi in the GAT system. The hybridomas which were I-J+, CRI+, and A4.10+ were cloned by limiting dilution and screenedagain for the presenceof these surface antigens. As seenin Table 1, all the hybridomas are T-cell derived since they expressthe Thy- 1, Lyt- 1 and some, the Lyt-2 antigens. However, I-J encoded determinants could not be detected on the clones. Several other investigators have also noted the lack or eventual loss of I-J encoded determinants on the surface of in vitro maintained Ts hybridomas ( 13, 15, 33, 34). For further study, two hybridomas, 8A.3 and 5A.11 which are CRI+ and A4.10+ were selected for the analysis of their secretedproducts. Culture supematant from the CRI- and A4. lo- hybridoma 8A. 16 was used as control. Cross-suppressionof TNP-Specijc Antibody Responseswith Hybridoma-Derived Suppressor Factor After selecting the hybridomas on the basis of surface phenotype, their ability to synthesize and secrete biologically active suppressor factors was examined using an in vitro antibody assay. Previously, we were able to show that TMA-specific TsFl activity can be monitored by its ability to cross-suppresstrinitrophenol (TNP)-specific plaque-forming cell (PFC) responsesin culture stimulated by Bruiella abortus coupled with the TMA and TNP haptens (TMA-BA-TNP) (35, 36). Further analysis of this assay demonstrated that the in vitro stimulation of the naive A/J spleen cells with TMA-BA-TNP and other similar conjugates (TNP-BA, ABA-BA-TNP) resulted in TNP-specific PFC responses of which 20-35% of the PFC were found to bear the cross-reactive idiotype(s) (CRI) normally associated with anti-TMA antibodies. The addition of TMA-specific TsFi only in the TMA-BA-TNP containing cultures resulted

424

JENDRISAK, TRIAL, AND BELLONE TABLE 1 Surface Phenotype of Tsi Hybridomas” % Fluorescent cells

Specificity

Antibody

8A.3

8A.5

8A.9

8A.16

5A.6

5A.11

Thy. 1

T24”

92

98

91

81

84

100

Lyt- 1 Lyt-2

7.313c 6.12’

15 14

15 84

88 92

91 0

81 10

91 4

I-J’ 1-J’ I-J’ 1-J’

WF8A12.8d WF8C12.8d WF8D2.3d KY 76’

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

Single-chain TsFl

1248A4.10’

85

31

25

18

42

71

Anti-Id

R66

84

25

20

17

31

74

u The Ts hybridomas were placed into low-density culture (0.1 X 106cells/ml) in fresh medium. Cells were harvested 24 hr later and stained with the indicated reagents. bAmmonium sulfate precipitate of culture supematant; cells from Salk Institute, developed by Dr. Ian Trowbridge. c Ammonium sulfate precipitate of culture supematant; cells obtained from Dr. Noel Warner. d Ammonium sulfate precipitate of culture supematant; obtained from Dr. Carl Waltenbaugh. ’ Ammonium sulfate precipitate of culture supematant; obtained from Dr. Vera Hauptfeld. ‘Ammonium sulfate precipitate of culture supematant obtained from Dr. Craig M. Sorensen. 8 Rabbit anti-idiotypic antiserum (30, 3 1).

in the cross-suppression of 30-75% of the total anti-TNP and complete suppression of the CRI+PFC responses.The data further demonstrated that an induction-phase, TMA-binding, CRI+ and I-J+ single chain factor was responsible for this suppression (35, 36). The suppressor activity and specificity of the 8A.3 and 5A. 11 derived factors were tested, utilizing the described in vitro antibody assay.Culture supernatants from 8A. 16 was used as control. As seenin Table 2, both 8A.3 and 5A. 11 supernatants completely suppressesthe CRI+PFC component of the response in addition to significantly reducing the overall anti-TNP PFC only in the TMA-BA-TNP-stimulated cultures. The inability of these supematants to suppressresponsesin ABA-BA-TNP-stimulated cultures demonstrates their antigen specificity. In contrast, the control supematant from 8A.16 had no effect. This observed profile of suppression with hybridoma-derived TsFi is very similar to that of the conventionally derived TsFi (35, 36). As we have previously discussed (35), several mechanisms may account for the in vitro suppression of antibody responsesby the TMA-specific TsFi. Since there is a preferential suppression of the CRI+PFC, one possibility is the induction of an Id-specific second-order TsZ cell. TMA-TsF, mediated induction of such Tsz cells in vivo in the presenceof antigen has been previously reported (7). The activated Tsz cell or its product TsF2, may effect suppression by direct interaction with the CRI+ TSS population, which in turn, may preferentially inactivate Id-specific T helper cells resulting in the loss of CRI+TNP PFC responses.The Id-specific TsZ, CRI+ antigenspecific Ts~and Id-specific T helper cells have been described in the TMA system and

HYBRIDOMA-DERIVED

425

TMA-TsF,

TABLE 2 Antigen Specificity of the Hybridoma-Derived TsF,’ Antigen in culture

Hybridoma supemate None

Anti-TNP PFC/ culture + SEb

Suppression’ (%I

Percentage CRI+d

-

33 0 0 29

16+ 2

TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP

Media 8A.3 5A.11 8A.16

324 + 108 + 123 f 279 f

ABA-BA-TNP ABA-BA-TNP ABA-BA-TNP ABA-BA-TNP

Media 8A.3 5A.11 8A.16

385 f 25 425 f 31 354 f 22 405 zk29

20 17 24 11

70 65 12 9 9 0

14 14 17 14

’ Hybridoma supematants from 8A.3, 8A. 16, and 5A.11 at a l/IO final dilution were added to 1 X lo6 naive A/J spleen cell cultures containing TMA-BA-TNP or ABA-BA-TNP antigens at initiation. Cultures were assayed3 days after antigen challenge for direct TNP-specific PFC responses. b Data are expressedas PFC/culture and are from one representative of at least three separateexperiments for this and all subsequent presented data. All the conditions were run in duplicate (three wells were pooled per condition). ’ Percentage suppression was calculated as described under Materials and Methods. In all experiments, 30-40% suppression is considered significant (P < 0.001) since background suppressionbeyond 14%is not observed in control cultures. d See Materials and Methods for the calculation of percentage CR1 + PFC.

all appear to network via Id-anti-Id interactions (6,7,37). Alternatively, or in addition, activated TsJ may inhibit PFC responsesby interaction with B cells via antigen focusing mechanisms and the subsequent release of nonspecific suppressor factors (38). This mechanism would explain the observed mitigation of 20-35% of the CRI-negative component of the anti-TNP PFC response and may in fact be responsible for the suppression of the CRI+ PFC as well. Although the mechanisms operating in vitro have yet to be elucidated, the assayhas proven to be a reliable monitor of TsF, activity. To further characterize the biologically active factor present in these supernatants, 8A.3 derived suppressor factor was used throughout the remaining experiments while supernatant from 8A. 16 was used as a control. To test whether the hybridomaderived TsFl functions in a dose-dependentmanner, various dilutions of 8A.3 culture supernatant were added to TMA-BA-TNP-stimulated cultures. As seenin Table 3, suppressionof the total anti-TNP and the CRI+ component of the PFC responseby 8.3 supernatant was dosedependent. Becausesome nonspecific suppression was seen with high concentrations of 8A. 16 supematant, a l/IO final dilution was routinely used throughout these studies. Hybridoma-Derived TsF Is Efective Only at the Induction Phase We have previously characterized the TMA-TsFi as being a first-order, induction phase suppressor factor because of its ability to suppressonly during the early stages of the immune response (6). To determine at which phase of the immune response the hybridomaderived TsF was functional, the 8A.3 supematant was added to cultures

426

JENDRISAK, TRIAL, AND BELLONE TABLE 3 Dose Responseof Hybridoma-Derived TsFi ’

Antigen in culture

Hybridoma supemate

Dilution of supemate

None

None

-

TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP

None 8A.3 8A.3 8A.3 8A.3 8A.3

l/5 l/l0 l/20 l/50 l/l00

TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP TMA-BA-TNP

8A.16 8A.16 8A.16 8A.16 8A.16

lb l/l0 l/20 l/50 l/l00

Anti-TNP PFC/ culture + SE

Suppression (%)

Percentage CRI+

-

-

276 k 32 74* 14 126 zk 10 144 + 12 266 + 14 346 ?I 26

77 57 50 4 0

18 0 0 0 23 39

214 f 240* 306 + 336 f 306 +

23 14 0 0 0

39 39 29 34 34

12+

1

14 1 6 20 10

’ Hybridoma supematants of 8A.3 and 8A. 16 at various final dilutions were added to 1 X lob naive A/J spleencell cultures containing TMA-BA-TNP antigen at initiation. Cultures were assayed3 days atkr antigen challengefor direct TNP-specific PFC responses.The percentagesuppressionand CRI + PFC were determined as described under Materials and Methods.

on Days 0, 1, or 2 after addition of antigen (Table 4). As was expected, the 8A.3 derived TsF only suppressedwhen added at initiation of cultures, whereasthe addition on Days 1 or 2 had no effect on the antibody responses.Again, 8A.16 culture supernatant had no effect. Therefore, the suppressor material produced by the 8A.3 hybridoma behaves as an induction-phase suppressor factor.

TABLE 4 Hybridoma-Derived TsFi Is an Induction PhaseSuppressorFactor’ Hybridoma supemate

Day of TsF, addition

Anti TNP PFC/ culture f SE

Suppression (%I

Percentage CRI+

Media

-

310 f 54

-

30

8A.3 8A.3 8A.3

0 1 2

152 + 69 275 f 24 336 f 43

51 11 0

0 22 34

8A.16 8A.16 8A.16

0 1 2

273 k 12 290 + 10 341 f 30

12 6 0

41 28 30

’ The hybridoma supematants of 8A.3 and 8A. 16 at l/IO final dilution were added to 1 X lo6 naive A/J spleen cell cultures containing TMA-BA-TNP antigen at Days 0, 1, and 2. Cultures were assayed3 days alter antigen challenge for direct TNP-specific PFC responses.The percentage suppression and CRI+PFC were determined as described under Materials and Methods.

HYBRIDOMA-DERIVED

421

TMA-TsF,

Activity of Antigen-Binding Hybridoma-Derived TsF Using both in vivo and in vitro assays(7, 35, 36), we have previously shown the TMA-TsF, to be a single-chain factor which binds the TMA ligand and bears idiotypic as well as I-J determinants. To serologically characterize and to determine the binding specificities of the hybridoma-produced suppressor factor, supernatants from 8A.3 were passedover various antigen and immunoadsorbent columns. First, we examined the antigen-binding specificities of this factor utilizing TMA and ABA coupled Sepharose 4B columns as indicated in Table 5. The supernatants were passedover the columns and both the effluents and 2 M KC1 eluates were collected and tested for their ability to suppressthe total anti-TNP as well as the CRI+ component of the PFC response. The results indicate 8A.3-derived suppressor factor specifically bound to and eluted from the TMA columns. The binding specificity of this factor was further demonstrated by the inability of ABA-Sepharose columns to retain the suppressor activity. These results demonstrate that an antigen (TMA)-binding suppressor factor is responsible for mediating the observed suppression. Hybridoma-Derived TsF Are I-p’, CRI’, and A4.10’ Since the surface fluorescence data indicated the 8A.3 cells to be CRIt, as well as A4. lo’, and studies with conventional TsF, demonstrated the presenceof these same determinants as well as I-J, appropriate immunoadsorbents were utilized to determine the serological properties of the 8A.3 derived TsF. The following antibodies or Igs coupled to Sepharose4B were used monoclonal anti-I-Jk (KY 76) antibody, monoclonal anti-I-Jq (KY 35.4), rabbit anti-Id, monoclonal anti-single chain TsFi (1248A4. lo), normal rabbit Ig (NRIg), and normal rat Ig (NRat Ig). As seenin Table 6, the biologically active component was specifically bound to and eluted from anti-I-Jk and anti-Id columns. In contrast, the suppressor activity was found only in the effluent fractions of the irrelevant anti-I-Jq antibody and normal rabbit Ig columns. In data not shown, the TsF, could also be bound to and eluted TABLE 5 Hybridoma-Derived TsFr Binds to TMA” Hybridoma supernate

Column

Fraction

Anti-TNP PFC culture + SE

Suppression (W)

Percentage CRI+

Media

None

None

512 k43

-

8A.3

None

Unfractionated

332 k 22

36

0

8A.3 8A.3

TMA-Seph TMA-Seph

Effluent Eluate

524 z!z24 324 + 20

0 38

24 0

8A.3 8A.3

ABA-Seph ABA-Seph

Effluent Eluate

351 f 28 634 f 10

32 0

0 25

25

a The hybridoma supematant of 8A.3 was passedover antigen columns as indicated. The unfractionated and column effluent and eluate (l/l0 final dilution) materials were added to 1 X 106naive A/J spleen cell cultures containing TMA-BA-TNP antigen at initiation. Cultures were assayed3 days atIer antigen challenge for direct TNP-specific PFC responses.The percentagesuppressionand CRJ+PFCwere determined asdescribed under Materials and Methods.

428

JENDRISAK, TRIAL, AND BELLONE TABLE 6 Hybridoma-Derived TsFi Bears I-J and CR1 Dcterminantsn

Hybridoma supemate

Column

Fraction

Anti-TNP PFC/ culture f SE

None

-

-

576 f 24

-

-

340 + 8

Suppression 6)

Percentage CRI+ 24

8A.3

None

41

0

8A.3 8A.3

Anti 1-J’ Anti 1-J’

Effluent Eluate

574 * 30 352 + 8

0 39

25 0

8A.3 8A.3

Anti I-J9 Anti I-Jq

Effluent Eluate

356 + 24 550 f 70

38 5

0 29

8A.3 8A.3

Anti-Id Anti-Id

Effluent Eluate

620 + 16 378 ?I 14

0 35

32 0

8A.3 8A.3

NRIg NRIg

Effluent Eluate

396 + 8 551 + 73

31 4

0 24

‘The hybridoma supematant of 8A.3 was passed over immunoadsorbent columns as indicated. The unfmctionated, effluent, and eluate (at a l/IO final dilution) materials were added to 1 X lo6 naive A/J spleen cell cultums containing TMA-BA-TNP antigen at initiation. Cultures were assayed3 days athx antigen challenge for direct TNP-specific PFC responses.The percentagesuppressionand CRI+PFC were determined as described under Materials and Methods.

from the monoclonal anti-I-Jk, WF8 which was used to phenotype the surface of 8A.3 (see Table 1). Despite the absence of surface I-J on the 8A.3 hybridoma, the data clearly demonstratesthe presenceof I-J as well as CRI determinants on the hybridomaderived suppressor factor. Since the monoclonal antibody 1248A4.10 reacts with both GAT-TsF, and conventional TMA-TsFr (unpublished observations) we next tested whether the 8A.3 TABLE 7 Hybridoma-Derived TsF, Binds 1248A4.10 Antibody” Hybridoma supemate

Column

Fraction

Anti TNP PFC/ culture + SE

Media

-

-

365 + 11

-

30

8A.3

-

-

199 * 21

44

2

Suppression (%I

Percentage CRI+

8A.3 8A.3

1248A4.10 1248A4.10

Effluent Eluate

3161t 4 241 + 15

14 34

34 2

8A.3 8A.3

NRat Ig NRat Ig

Effluent Eluate

229 t- 13 364 f 6

37 0

1 30

a The hybridoma supematant of 8A.3 was passed over immunoadsorbent columns as indicated. The unfractionated, effluent, and eluate materials (at a l/10 final dilution) were added to 1 X ld naive A/J spleen cell cultures containing TMA-BA-TNP antigen at initiation. Cultures were assayed3 days after antigen challenge for direct TNP-specific PFC msponses.The percentagesuppressionand CRI+PFC were determined as described under Materials and Methods.

HYBRIDOMA-DERIVED

429

TMA-TsF,

derived factor also bears the 1248A4.10 determinant. Accordingly, the 1248A4.10 antibody was coupled to Sepharose4B and the supematant from 8A.3 passed over this immunoadsorbent column as well asthe control normal rat-Ig (NRat Ig) adsorbent. As seen in Table 7, the suppressor activity was specifically adsorbed out and eluted only from the 1248A4.10 column. The specificitiy of 1248A4.10 monoclonal antibody for 8A.3-derived TsF was demonstrated by the inability of suppressor activity to bind to the NRat Ig column. The ability of 1248A4.10 monoclonal antibody to recognize another first-order TsF which differs in both antigen specificity and I-J determinants of a different haplotype suggeststhat it recognizesa common or “isotypic” determinant which is shared among single-chain suppressorfactors. These results collectively indicate that the 8A.3-derived factor bear I-Jk, CRI, and A4.10 determinants. This is in concordance with the data

Reduced

BA.3 TsF 1 (+)

44% suppression 100% SUppreSsiOn

of TNP PFC Of CRI+PFC

Anti-I-.tkcolumn

A Effluent

I-)

Eluate

(+I

4% suppression

01 TNP PFC

45% s”ppreSSionTNP

0% suppression

of CRI+PFC

100% suppression

PFC 01 CRI+PFC

\,f,“e”t+El”ate,+/ 30% suppression

of TNP PFC

100% suppression

01 CRFPFC

Reduced TMA-TsF2 (+) 54% suppression 01 TNP PFC 100% suppression

of CRI+PFC

Anti-l-.tkcolwm I

4

Effluent

+

Eluate

(-1

0% suppression

of TNP PFC

OX suppression

of CRI+PFC

’

0% suppression 0% suppression

(-) 01 TNP PFC of CRI+PFC

Effluent + Etuate t+/ 35% suppression 01 TNP PFC 95% suppression

01 CRI+PFC

FIG. 1. Hybridomaderived TsF, is a single chain factor. The hybridomaderived TMA-TsFI and conventional TMA-TsF, were reduced with 5 mM DTT and absorbed on the anti-I-J’ Sepharosecolumns in the presenceof DTT. The effluent and eluate were tested for their activity in the in vitro antibody assay.

430

JENDRISAK, TRIAL, AND BELLONE

obtained with conventional TsF, and correlates with the surface immunofluorescence of 8A.3. 8A.3-TsF Is a Single Chain Molecule We have previously shown that conventionally derived TMA TsF, appears to be a single-polypeptide chain molecule (7). To seeif the hybridoma-derived TsF was also a single-chain factor, supernatant from 8A.3 cells were reduced with 5 mM DTT and passedover anti-I-Jk columns in the presenceof DTT. The two-chain conventionally derived TMA-specific TsF2 was also subjected to the sametreatment (7). The effluents and eluates were extensively dialyzed and tested for suppressor activity in the in vitro antibody assay.As seen in Fig. 1, reduction of the hybridomaderived factor does not affect its ability to suppress the total TNP specific and the CRI+PFC component of the response, whereas reduction and separation of the two-chain TsF2 abolished its suppressor activity. The activity of the TsF2 was restored only when the effluent and eluate fractions were added together into cultures. Therefore, these results strongly suggestthat the hybridoma-derived factor is a single chain molecule. Suppression of DTH Responsesby the Hybridoma-Derived TsF, Since previous work on the characterization of the conventional TMA-specific TsFi was determined using an in vivo DTH assay, the ability of the hybridoma-derived suppressor factor to shut down TMA-specific DTH responseswas examined. To test for specific suppressor activity 0.5 ml of culture supernatant from 8A.3 or the control supernatant from 8A. 16 were injected into mice which were then primed with syngeneic TMA-coupled SC or TNBS to induce DTH responses. As shown in Table 8, TsF derived from 8A. 3 significantly suppressedTMA responses,whereas8A. 16 supematant TABLE 8 TsFi SuppressesTMA-Specific DTH Immunization”

Treatment b

Response(mm + SEM)’

TMA-SC TMA-SC TMA-SC Negative control’

None 8A.3SN 8A. 16SN None

0.90 f 0.36 k 0.925 + 0.28 k

TNBS TNBS TNBS Negative control

None 8A.3SN 8A.16SN None

0.89 0.85 0.75 0.17

0.01 0.02 0.01 0.01

+ 0.02 kO.01 zk0.02 kO.01

Suppressiond (%) 75 0 5 19 -

a A/J mice were immunized sc either with 3 X 10’ TMA-SC or with 0.30 ml of 10 mM TNBS in BSS, pH 7.4. b 0.5 ml of SN from 8A.3 or 8A. 16 was injected iv on Day 0. c Five days after immunization, the mice were challenged in the footpads either with 30 pl of 10 n&f diazotized TMA in saline or with 30 pl of 10 mM TNBS in BSS, pH 7.4. Footpad swelling responsewas measured 24 hr after the challenge. d Percentagesuppressionwas calculated as described under Materials and Methods. Four mice per group were tested. pNegative controls indicate normal A/J mice challenged.

HYBRIDOMA-DERIVED

TMA-TsF,

431

had no effect. The inability of 8A.3-derived TsF to suppress TNBS-induced DTH responsesonce again demonstrates its antigen specificity. Becausethe 8A.3-derived suppressor factor shuts down in vivo DTH and in vitro antibody responsesin a manner similar to conventional TsF, and since these experiments demonstrated this suppressorfactor to be an induction phase, antigen-binding, I-J+, CRI+, A4.10+ single-chain molecule, we conclude that 8A.3 representsan inducer suppressor (Ts,) hybridoma which produces biologically active TMA-specific TsFl . Although there are a number of reports describing antigen-specific factors, few single chain TsF have been characterized to any extent (20,2 1,39). The single chain factors are particularly intriguing since they bind antigen and bear determinants (I-J and CRI) which are encoded by geneslocated on different chromosomes. Detailed study of this single chain factor at the protein and molecular level should allow us to reconcile the serological data with the protein structure and genetic composition. Like the earlier studies with immunoglobulins, analysis of several numbers of first-order TsF within a family will be necessarybefore structure-function relationships can be understood. Additionally, these studies should lend insight into the nature of antigen recognition units at the suppressor T cell level. Isolation and characterization of the hybridomaderived TMA-TsFi is currently underway using a variety of biochemical techniques (i.e., SDS-PAGE, RP-HPLC, IEF). To date, the results indicate that the biochemical properties of TMA-TsF, and the reported GAT-TsF, (20, 21) to be remarkably similar (40). ACKNOWLEDGMENTS We thank Dr. Vera Hauptfeld, Dr. Craig Sorensen,Dr. Ian Trowbridge, Dr. Carl Waltenbaugh, and Dr. Noel Warner for their generous gift of antibodies. We also thank Dr. Judith Kapp for helpful discussions and Dr. Sundararajan Jayaraman for helpful discussionsand the review of this manuscript. Also, to thank Ms. Carole Vogelgesangfor her excellent secretarial assistanceand Tom Handel for technical assistance.

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