HAPTEN-SPECIFIC T CELL RESPONSES TO
4-HYDROXY-3-NITROPHENYL ACETYL
XI.

Pseudogenetic Restrictions ofHybridoma Suppressor Factors*
BY KENJI OKUDA, MUTSUHIKO MINAMI, DAVID H. SHERR, AND
MARTIN E . DORF
From the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

The sequence of cellular interactions resulting in the suppression of cellular andhumoral immune responses to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP)' has
been well characterized (1-3). At least three distinct subpopulations of NP-induced T
lymphocytes have been identified in this suppressor cell circuit (1, 2). The first cell
population, termed Ts' or Ts t, functions during the induction phase of the immune
response and bears an antigen-specific idiotypic receptor (1, 4) . The Ts' populationinduces a second population of suppressor cells, known as Ts' or Ts2 (1, 5) . This Ts'
population functions during the effector phase of the immune response (after antigen
priming) and bears an anti-idiotypic receptor (1, 2). The target of the Ts' cells are an
antigen-primed Ts3 population, which is thought to represent the final effector
suppressor cells (2) . The conclusions derived from the NP system are consistent withmost other observations relating to the cellular interactions involved in immune
suppression (6) . Thus, the NP system may represent a general model that elucidates
the overall pathway of immunoregulation .
To analyze the Ts antigen receptors and the mechanisms ofcellular communication
between the various cell populations, we have prepared a series of monoclonal T cellhybridomas (7) . These hybrids correspond to the Ts' population and produce abiologically active T cell-derived suppressor factor (TsF) that can substitute for theTs' population . This report characterizes the TsF, the genetic restrictions of this factor,
and the second-order suppressor cells induced by the factor . The results extend
previous work in the NP system and yield a more complete model of the mechanismof immune suppression. The data further demonstrate that hybridoma TsF can
suppress both cellular and humoral immune responses.
Materials and Methods

Mice. All mice were either purchased from The Jackson Laboratory, Bar Harbor, Maine,or were bred in the animal facilities at Harvard Medical School, Boston, Mass . Mice were used
* Supported by grant AI-16677 from the National Institutes of Health, grant PCM-80-04573 from the
National Science Foundation, and a grant from the Cancer Research Institute .
'Abbreviations used in this paper: CS, cutaneous sensitivity ; DMSO, dimethyl sulfoxide ; DNFB, 2,4din itrofluorobenzene ; FCS, fetal calf serum ; GAT, polypeptide of L-glutamic acid, L-alanine and L-tyrosine ;HBSS, Hanks' balanced salt solution ; KLH, keyhole limpet hemocyanin ; NP, 4-hydroxy-3-nitrophenyl
acetyl hapten ; NPb, common idiotype on C57BL anti-NP antibodies; NP-0-Su, NP-0-succinimide ester ;PFC, plaque-forming cells ; SRBC, sheep erythrocytes ; Ts', first order suppressor T cells ; Ts', second ordersuppressor T cells ; Ts3, third order suppressor T cells ; TsF, T cell-derived suppressor factor.

468

J. Exp . MED. ©The Rockefeller University Press " 0022-1007/81/08/0468/12 $1 .00
Volume 154

August 1981

468-479

K. OKUDA, M . MINAMI, D . H . SHERR, AND M . E . DORF

469

at 3-12 mo of age, and were maintained on laboratory chow and acidified, chlorinated water
ad libitum .
Antigens. NP-0-succinimide (NP-0-Su) was purchased from Biosearch Co ., San Rafael,
Calif Dimethyl sulfoxide (DMSO) was purchased from Fisher Scientific Co ., Pittsburgh, Pa .
NP-keyhole limpet hemocyanin (KLH) and NP-Ficoll were conjugated by the reaction of NP0-Su with AECM"-Ficoll (Biosearch Co.) or KLH (Sigma Chemical Co ., St . Louis, Mo .) as
described previously (3) . The NP-KLH and NP-Ficoll had an average of 30 and 17 NP groups
per 100,000 mol wt, respectively .
Antigen-specific T Cell Hybridoms and TsF The methods for preparation of antigen-specific
T cell hybridomas have been described previously (7) . Briefly, antigen-specific suppressor T
cells were hybridized with BW5147 tumor cells . The hybrids with I-J and NPb idiotypic cellsurface markers, i .e., CKB-17, CKB-39, and B6-29, were selected for further analysis . The B629 cells were cloned . The cell lines and clones derived from the parent lines were periodicallypassed over antigen plates and the NP-adherent cells were used to maintain the cell line . All of
the hybridomas were cultured in RPMI-1640 containing 8% fetal calf serum (FCS) . The TsF
employed in the present experiments was collected from the cultured supernate of the hybridomas, when their cell numbers were ^-7 X 105 cells/ml in the above medium . Our previous
results (7) indicated that the CKB (Ighb, H-2k) -derived hybrids, designated CKB-17 and CKB39, and the C57BL/6 hybrid, B6-29, possess I -J and NPb idiotypic determinants .
Adsorption and Elution of TsF. The methods of adsorption and elution of TsF using proteinconjugated Sepharose 4B columns were described in detail previously (7) .
Assay for the Ability of TsF to Induce Ts'. To make the Ts' cells for in vivo studies, the TsF (0.3ml) from each hybridoma was injected into naive mice intravenously for 4 d . On day 6, spleensfrom these mice were removed, and a single cell suspension was prepared in Hanks' balanced
salt solution (HBSS) . 4 X 10 7 spleen cells were washed with HBSS three times and injected into
NP- or dinitrofluorobenzene (DNFB)-primed mice . These recipient mice were then challenged
with NP or DNFB antigen and the swelling responses were measured 24 h later . To make theTs' suppressor cells for in vitro studies, 0 .2 ml TsF was injected into normal C57BL/6 miceintravenously for 5 d . 1 d after the last injection, spleen cells from these mice were removed
under sterile conditions, teased, and washed extensively, and 5 X 105 viable cells were added toresponder cultures, which had been challenged 4 d earlier with NP-Ficoll or sheep erythrocytes(SRBC) . 1 d later, responder cells were assayed for NP and SRBC-specific plaque-forming cells(PFC) .
Assay for Suppressive Activity of TsF on NP-mediated Cutaneous Sensitivity (CS) Responses. The
assay of CS responses was described elsewhere (8) . Briefly, each animal was primed subcutaneously with 7 mg of NP-0-Su in DMSO . Soon after priming, 0 .3-0 .4 ml of each hybridoma
supernate, BW5147-cultured supernate, or fractionated samples were injected intravenously .
Unless indicated otherwise, these intravenous injections were given for 4 d beginning on the
day of antigen priming .
6 d after immunization, 24 lyg of NP-0-S'u in 0.025 ml of phosphate-buffered saline, pH 7 .2,
was injected into the left footpad. Footpad swelling was measured 24 h after challenge . Swelling
was determined as the difference, in units of 10 -3 cm, between the left and right footpad
thickness .
In Vitro Responder Cultures. C57BL/6 (B6) mice were immunized intraperitoneally with 150
g,g NP-KLH with 25% pertussis vaccine. 4 wk later, their spleens were removed and teased intoa single cell suspension under sterile conditions . Cells were washed and resuspended in MishellDutton media containing 10% FCS . 7 .5 X 106 viable NP-primed cells were added to each wellof Linbro culture plates . Cultures were challenged with 100 ng NP-Ficoll in 20 ,ul . A primary
SRBC response was obtained by the addition of 2 X 106 washed SRBC to culture wells
containing 7 .5 X 106 normal, unprimed spleen cells . All cultures were incubated in rocking
boxes at 37 ° C in a 10% C0 2 atmosphere and were fed daily with 0.1 ml supplementary
medium . 4 d after challenge, 5 X 105 viable control or suppressor cells were added . 5 d after
challenge, duplicate wells were pooled and assayed for total NP- or SRBC-specific PFC responsein triplicate slides .
PFC Assay. NP- or SRBC-specific PFC were assayed as reported previously (3) .
DNFB Responses . CS was induced by two daily paintings on the shaved abdomen with 25

470

PSEUDOGENETIC RESTRICTIONS OF HYBRIDOMA SUPPRESSOR FACTOR

pl of 0.5% DNFB solution (Sigma Chemical Co) in a 4 :1 dilution of acetone:olive oil (2). 6 dafter the last painting, 20 pl of 0 .2% DNFB in the same vehicle was applied to the left ear, andthe ear swelling was measured as the difference between the left and right ear thicknesses .
When the specificity of TsF or Ts' cells were determined on DNFB responses, the injections
were done as for CS responses to NP antigen.
Percent Suppression . The percent suppression in the present study was calculated by thefollowing formula : percent suppression = 100 x ([swelling of BW tumor supernate-injectedgroup - swelling of TsF injected group)/ (swelling of BW tumor supernate-injected group swelling of unprimed group]) .
Data Analysis . Statistical analysis of the experimental data with respect to controls was
performed by the two-tailed Student's t test.

Specificity of Hybridoma TsF.

Results

To assess the potency of the hybridoma-derived factors,

0.3 ml of various dilutions of TsF were injected daily into mice from the day ofpriming with NP-O-Su to the day before antigen challenge . The supernates from theBW5147 cell line, which was the parental tumor used for the hybridization, were used
as a control . The C57BL/6-derived B6-29 factor was injected into C57BL/6 mice,and the CKB-derived factors were injected into H-2- and Igh-compatible 1310.13R
mice. As shown in Table I, TsF derived from the B6-29, CKB-17, and CKB-39 hybrid
cell lines significantly suppressed the NP response when used undiluted or diluted at
1 :10. At 1 :100 dilutions, these factors demonstrated variable levels ofsuppression andthe suppressive activity was no longer observed when the supernates were further
TABLE I

Specificity of Hybridoma Suppressor Factors
Source of TsF

Strain tested

TsF dilution

BW5147
B6-29

C57BL/6
C57BL/6

BW5147
CKB-17

Bl0.BR
Bl0.BRCKB-39B l0 .BR

Undiluted
Undiluted
1 :10
1 :1001 :1,000
Undiluted
Undiluted
1 :10
1 :1001 :1,000
Undiluted
1 :10
1 :1001 :1,000

Swelling Response ± SE*
NP-O-Su

DNFB

15 .3 ± 1 .6 (8)2 .9 ± 1 .2 (8)$3 .4 ± 1 .8 (7)$8.0 t 2 .0 (7)11
15 .2 ± 0 .5 (7)20 .2 ± 2 .1 (8)9.8 ± 2 .0 (5)$
11 .8 t 1 .7 (5)$20 .5 ± 1 .8(4)17 .5 ± 1 .0 (4)8.5 ± 3 .0 (4)11
7 .0 ± 2 .3 (5)$
14 .0 ± 2 .9 (4)21 .8 ± 2 .7 (4)17 ± 1 (4)16 ± 3 (4)
NT§
NT
NT
21 .5 ± 2 .2 (4)20 .5 ± 3 .2 (4)NTNTNT
19 .5 ± 3 .8 (4)
NT
NT
NT

* Each group of mice was primed with NP-O-Su or DNFB, followed by daily intravenous
injections of TsF until the day before antigen challenge . The results are expressed as themean increment of footpad swelling t SE . The number of mice per group is indicated inparentheses. Background swelling in C57BL/6 mice was 3 .2 ± 1 .7 (9) for NP-O-Su and 4± 1 (4) for DNFB . Background swelling in B10.BR mice was 8 .3 ± 1 .7 (8) for NP-O-Su and3 t 1 .2 (4) for DNFB .
$ P < 0 .01 .
§ Not tested .
11 P < 0 .05 .

K. OKUDA, M . MINAMI, D . H . SHERR, AND M . E . DORF

47 1

TABLE II

Immunochemical Characterization of Hybridoma Suppressor Factors
Immunoadsorbent
columnB6-29 fractions
UnboundEluateCKB-17 fractions
UnboundEluateNone
86*61
Anti-1Jb
-22
81
59
9
Anti-Ii"
89
18
371
Anti-Ig
94-20
87
9Anti-NPb
12
46
ND$
ND
-13
17
NP-bovine serum albumin
46
62* The data are expressed as average percent suppression . The results were obtained
from three separate experiments .
$ Not determined .

diluted to 1 :1,000. The inability of these hybridoma-derived factors to suppress
DNFB-induced CS responses demonstrates their antigen specificity (Table I) .
Characterization of TsF. To characterize the biologically active factors produced bythese cells, the B6-29 and CKB-17 hybrids were selected for further analysis. Culture
supernates from these cells were passed over immunoabsorbent columns and theadsorbed fractions were eluted with glycine-HCI buffer, pH 3 .2. The data in Table IIdemonstrate that the suppressive activity was absorbed by anti-idiotype, anti-I-J, andantigen columns. Furthermore, suppressive activity could be specifically recovered byelution from these columns. Controls for these experiments include the ability ofallele-specific anti-I J alloantisera to specifically absorb these factors. Thus, anti-IJb
absorbed the B6-derived factor, whereas anti-l-Jk absorbed the CKB-derived factor.
The suppressor factor lacked conventional immunoglobulin determinants because
polyvalent guinea pig anti-immunoglobulin antisera failed to absorb the suppressive
activity (Table II) . Furthermore, the activity could not be absorbed on an NPb
idiotype-coupled column (data not shown) . Thus, the TsF bear antigen-specificidiotypic receptors and I-J alloantigenic determinants .
Suppression in the Induction vs . Effector Phase. The Ts' and Ts' suppressor cell
populations were initially designated based on their ability to suppress during theinduction or effector phases of the immune response, respectively (1, 4, 5) . To
determine in which phase of the immune response the hybridoma suppressor factors
were functional, they were injected either at the time of antigen priming (induction
phase) or at the time of antigen challenge (effector phase) . BW5147 supernates wereused as controls. As shown in Table III, TsF obtained from the B6-29, CKB-17, andCKB-39 cell lines significantly suppressed NP responses during the induction phase,but not the effector phase, of the response . We have recently identified another 1+
bearing CKB-derived hybridoma cell line (#59) that suppresses NP-specific immune
responses, but unlike the other hybrids, is not lysed by anti-NPb idiotypic antisera (7) .
In contrast to the B6-29, CKB-17, and CKB-39 TsF, the supernates from CKB-59
cells failed to suppress during the induction phase but demonstrated significant
suppression during the effector phase of the NP CS response (Table III) . Further
characterization of the CKB-59 factor will be reported elsewhere .
Ability of Suppressor Factor to Generate a Ts' Population. The ability of B6-29, CKB-17,
and CKB-39 hybridoma suppressor factors to function only during the induction

472

PSEUDOGENETIC RESTRICTIONS OF HYBRIDOMA SUPPRESSOR FACTORTABLE III

Comparison of the Ability of TsF to Suppress in the Induction vs . Effector
Phases of the CS Response
TsF source

Strain tested

BW5147136-29
BW5147
CKB-17
CKB-39
CKB-59C57BL/6
C57BL/6
B10.BR
B10 .BR
B10 .BRB l0.BR

NP-0-Su-induced foodpad swelling ±SE
Induction phase

Effector phase

30 .0 t 1 .8 (4)13 .8 ± 0 .6 (4)$
15 .2 t 1 .3 (6)4 .0 ± 1 .9 (5)$
9 .3 t 1 .1 (10)§
13 .8 ± 0.5 (6)30.0 ± 1 .8 (4)31 .0 t 3 .5 (4)19.3 ± 1 .3 (6)15 .3 ± 1 .1 (4)21 .7 ± 2 .4 (6)5 .2 ± 3 .0 (6)$* 0 .4 ml TsF was injected either on the day of and the day after antigen
priming (induction phase) or the day before and the day of antigen challenge(effector phase) . Background swelling was <10 units .
$ Significant suppression, P < 0 .01 .
§ Significant suppression, P < 0 .05 .
TABLE IV
Ability of TsF to Generate Antigen-specific Effector Phase Ts In Vivo
TsF
sourceStrain for Ts'
generation

BW5147136-29C57BL/6
C57BL/6
C57BL/6
B10.BR
B10.BR
BIO.BR
B l0.A (2R)
B 10.A (2R)
B l0 .A (2R)

BW5147
CKB-39
BW5147
CKB-17 .

NP-0-Su responses

DNFB responses

NP-0-Supriming

Footpad swelling ±SE

DNFB
priming

Ear swelling t SE

+
+
+
+
+
+
-

14 .3 ± 1 .5 (4)4 .8 t 2 .3 (5)$2 .2 t 2.3 (5)20 .4 ± 2 .2 (8)10 .7 ± 1 .8 (9)$4 .7±1 .3(9)21 .8 t 4.3 (4)9 .6 ± 1 .9 (5)$2 .4 t 1 .6 (5)

+
+
+
+
+
+
-

23 .7 t 1 .8 (4)24 .4 t 1 .7 (5)3 .6 t 0 .7 (8)14 .5 ± 3 .1 (4)17 .8 ± 1 .4 (4)1 .810.9(4)17 .8 t 1 .4 (4)18 .7 ± 1 .7 (3)1 .8 t 0 .9 (4)* 0 .3 ml TsF was injected into each mouse on days 0, 1, 2, and 3 . On day 6, 4 X 107 spleen cells from thesemice were transferred intravenously into syngeneic recipients, which had previously been primed with
NP-0-Su or DNFB .
$ Significant suppression, P < 0 .05 .

phase of the immune response suggested that these factors may be capable of inducing
the production of Ts'. To test this prediction, 0.3 ml of suppressor factor was injected
intravenously into Igh-matched naive recipients on days 0, 1, 2, and 3. On day 6, 4
X 107 washed spleen cells were adoptively transferred intravenously into syngeneic
recipients that had previously been primed with NP-O-Su or DNFB. The recipientswere challenged ^-1 h after cell transfer . As shown in Table IV, the B6-29-,
CKB-17-, and CKB-39-derived suppressor factors generated a second population of
suppressor cells that specifically suppressed NP responses . Thus, the Ts' generated byintravenous administration of hybridoma suppressor factor demonstrated 62-79%
suppression of NP-induced CS responses and no suppression of DNFB responses . It is
unlikely that the minute amounts of suppressor factor that may have been passively
carried over into the transfer recipients caused this suppression, because the adoptive
transfers were performed just before antigen challenge (effector phase) and previous

K. OKUDA, M . MINAMI, D. H . SHERR, AND M . E . DORF

47 3

data (Table III) demonstrated that these suppressor factors would not function in theeffector phase . Furthermore, complete H-2 compatibility between the cell producing
the suppressor factor and the strain used to generate the effector phase suppressor
cells is not required . Thus, the CKB-17-derived factor stimulated Ts' in B10 .A(2R)
mice. The CKB and B10.A(2R) strains share the H-2K through I-E regions of theH-2 complex but differ at the alleles for the I-C, S, and D regions .
Suppression of In Vitro PFC Responses. The previous series of experiments demonstrated the ability of hybridoma suppressor factor to modulate cellular immune
responses . Because we have also demonstrated the specific suppression of NP-specific
PFC responses (3, 9), we sought to determine whether hybridoma suppressor factor
was also active on these in vitro responses . For these experiments, B6-29 or control
hybridoma suppressor factors were administered intravenously into normal C57BL/6
recipients over a 5-d period . On day 6, spleen cells from these donors were washedand 5 X 10 5 cells were added to NP-Ficoll or SRBC-stimulated Mishell-Dutton
cultures . Duplicate wells were harvested 1 d later . As shown in Table V, the B6-29generated suppressor cells specifically suppressed the in vitro NP response and showedno suppression of the in vitro SRBC response . The control hybridoma factors used inthese experiments were derived from the same fusion as the B6-29 cell line but lacked
TABLE V
Ability of B6-29 TsF to Induce Ts' That Suppress In Vitro NP-specific PFC
Responses *PFC responses$

Experiment

TsF source

Strain for Ts'
induction

NP

SRBC

1

B6-29
Control
B6-29
Control

C5713L/6
C57BL/6
C5713L/6
C57BL/6

350 (70)1,200
325 (64)
1,250

1,300 (-42)
950
800(-28)625

2* B6-29 or control hybridoma suppressor factors were given intravenously tonormal C57BL/6 mice for 5 consecutive d . On day 6, 5 X 105 spleen cells
from these mice were added to 7 .5 X 106 responder cells challenged 4 dearlier with NP-Ficoll or SRBC . Duplicate wells were harvested on day 7
and NP- or SRBC-specific PFC were assayed .
$ Percent suppression is indicated in parentheses .
TABLE VI
Genetic Restriction of TsF Factors
TsF
source
B6-29
CKB-17
CKB-39Percent suppression of NP-O-Su CS responses
C57BL/6(H-2b , lghb)

B .C-8(H-2 b , Igha)

C3H(H-2 k, Ighi)

96$
87
108

7
ND§
ND

16
2
6

BIO .B R(H-2b, lgh~
101
94
93

CWB
(H-2 b, Igh)
ND
91
117

* Each group of mice was primed with NP-O-Su, followed by intravenous injection of
0.3 ml of undiluted TsF until the day before antigen challenge .
$ The results are expressed as average percent suppression. The results were collectedfrom several independent experiments .
§ Not determined .

474

PSEUDOGENETIC RESTRICTIONS OF HYBRIDOMA SUPPRESSOR FACTOR

functional activity . BW5147 supernates have also been used as controls in someexperiments .
Genetic Restriction of Hybridoma Suppressor Factors. We were previously unable todetermine the genetic restrictions of the Ts' cell population because of the potential
complicating allogeneic effects between the suppressor cells and the histoincompatible
recipients. Hybridoma suppressor factors derived from Ts' cells should not induce
potential allogeneic effects and consequently these materials were tested in H-2and/or Igh-incompatible strains . As summarized in Table VI, the B6-29 supernates
induced nearly complete suppression of the syngeneic C57BL/6 (H-2 b , Igh b) andH-2-incompatible, Igh-matched B10 .BR (H-2 k, Igh b) NP-induced CS responses . This
indicated that H-2 identity was not a requirement for suppressor factor function. In
contrast, the B6-29 suppressor factor failed to induce suppression in Igh congenic
B.C-8 (H-2b , Igha) mice. This indicates that homology between the suppressor factor
and the recipient strain for genes linked to the heavy-chain allotype complex (Igh) is
required to observe immune suppression . Similarly, the CKB-derived CKB-17 andCKB-39 hybridoma suppressor factors suppressed NP responses in H-2- and Ighmatched BIO .B R mice and in B6 and CWB mice (H-2 b , Ighb), which differ at theH-2 complex, but failed to suppress Igh congenic C3H (H-2k , Igh') mice (Table VI) .
Igh-V Restriction of TsF. To determine which region of the Igh complex controls
the restriction of TsF, the Igh-congenic BALB/c, C .B-20, and BAB/ 14 strains were
tested. These strains differ with respect to their Igh-C and Igh-V alleles . As indicatedin Table VII, the B6-29 and CKB-17 hybridoma suppressor factors inhibited NP-0Su CS responses in the C.B-20 strain (Igh-V b , Igh-Cb) but not in BALB/c mice (IghV a, Igh-Ca) . To determine which region of the Igh complex controls this genetic
restriction, BAB/14 mice were tested. The BAB/14 strain is also Igh congenic andbears a recombinant haplotype in which the Igh-V alleles were derived from the
BALB/c strain and the Igh-C alleles are identical to those of the C.B-20 strain . As
indicated in Table VII, the hybridoma suppressor factors failed to suppress BAB/14
mice, indicating that homology of the Igh-C region is not sufficient to obtainsuppression . These results suggest that the Igh-V genes control the apparent genetic
restrictions of these hybridoma suppressor factors .
Genetic Restriction of Cells Generated by Hybridoma Suppressor Factor. The inability ofIgh mismatched recipients of hybridoma suppressor factor to generate suppression
TABLE VII

Igh-V Restriction of Hybridoma Suppressor Factor*
TsF
sourceNP-O-Supriming

BW5147116-29
CKB-17
BW5147+++-

Strains tested
BALB/c(Igh-V'-Igh-C')

C .B-20(Igh-Vb-Igh-Cb)

BAB/14(Igh-Va -Igh-C')

19 .2 ± 1 .9 (5)$
21 .6 ± 2 .6 (5)19 .7 ± 3.0 (6)3 .6 ± 0 .5 (5)13.6 t 2 .0 (5)4.8 t 1 .4 (6)$5 .8 t 2 .1 (5)$2 .2 t 1 .2 (5)18 .0 ± 2 .4 (4)18 .2 ± 3 .4 (5)17 .3 ± 3 .2 (3)4 .0 t 0 .6 (5)" Mice were given 0 .3 ml of TsF from the day of NP-O-Su priming until the day
before antigen challenge .
The data are expressed as mean footpad swelling response ± SE in units of 10-3 cm .
The number of mice tested is indicated in parentheses . Significant suppression, P< 0 .05 .

K . OKUDA, M . MINAMI, D . H . SHERR, AND M . E . DORF

475

TABLE VIII
Genetic Restrictions of Ts' Cells Induced by Hybridoma Suppressor Factor*

TsF source

Strain for Ts'
generation

BW5147
CKB-17
BW5147
CKB-39
BW5147
CKB-39
BW5147
CKB-17
CKB-39
BW5147116-29
CKB-17

C3H
C3H
C3H
C3H
BIO.BR
B10 .BR
C57BL/6
C57BL/6
C57BL/6(1110 x BIO.BR)Ft(B10 x RMBR)F,(1110 X BIO.BR)F,

Strains used to test Ts" activity
CKB(H-2k, igh")

BIO.B R
(H-2", Igh°)

C3H(H-2k, Igh')C57RL/6(H-2', igh")

CWR(H-2t', Igh")

22 .6 t 2 .8 (5)t
8 .5 t 2 .7 (4)$
23 .6 t 1 .7 (5)12 .5 t 0.6 (4)$
ND
ND
ND
ND
ND
ND
ND
ND

21 .0 t 1 .4(4)9.5 t 1 .0 (4)$18.8 t 1 .5 (6)7 .0 t 2 .4 (5)$19.0- 1 .1 (4)12 .5 t 0 .9 (4)$23 .8 3 1 .8 (5)25 .0 t 1 .8 (5)25.2 t 1 .2 (5)21 .0 t 2 .9 (5)7 .6 t 0 .7 (5)$8.2 t 2 .3 (5)$20.0 t 3 .8 (5)20 .5 t 1 .0 (4)20.0 t 1 .5 (6)22 .5 t 2 .1 (6)22.2 t 2 .6 (5)22.7 t 1 .9 (5)14.8 t 1 .0(6)15 .6 t 0 .9 (6)14 .6 t 1 .2 (6)
ND
ND
ND

18 .3 t 2 .2 (3)16 .8 ± 1 .3(4)20 .4 t 2 .3 (5)19 .4 t 2 .4 (5)26.0 t 4.8 (6)26.6 t 2 .3 (5)29 .5 t 1 .1 (4)8 .7 t 1 .4 (4)$
10 .6 t 1 .4 (5)$18 .0 f 2.1 (5)6 .0 t 2 .6 (5)t12.0 t 2 .8 (4)ND§
ND
20 .3 t 2 .8 (4)23 .0 t 2 .8 (4)
ND
ND
ND
ND
ND
ND
ND
ND

` Normal mice were given 4 daily injections of 0 .3 ml TsF. After 2 d, these Ts` donors were killed and 4 X l0' splenocytes were transferred on

the day of antigen challenge to previously NN-O-Su primed recipients .
The data are expressed as the mean footpad swelling responses t SE in units of 10 -'' in . The number of mice is indicated in parentheses .
Background responses were <10 units of swelling .
§ Not determined .

could be attributed either to : (a) a requirement for Igh homology between the
suppressor factor and the Ts' cell populations ; or (b) a requirement for homology
between suppressor factor or the cells it generates, with another cell population
functioning further along the suppressor pathway, such as the Ts3 or the target cell of
the suppression . Therefore, the following experiment was performed to distinguishthese alternatives . CKB-17 and CKB-39 supernates were given to Igh-incompatible
C3H mice. AS indicated previously, CKB (H-2 k, Igh b) mice are congenic with C3H(H-2k , Igh3) at the Igh complex . From Table VI it was noted that these suppressor
factors would not function in the C3H strain. Furthermore, when the splenocytes from
C3H mice that had received TsF were transferred into previously NP-primed C3H
recipients, they also failed to induce suppression (Table VIII) . However, when spleencells from C3H mice that had received CKB suppressor factor were adoptivelytransferred into CKB or B10 .BR recipients during the effector phase of the CS
response, significant levels of suppression were noted (Table VIII) . This indicates thatthe hybridoma suppressor factors generate Ts' in Igh-incompatible recipients, butthese cells would only function in strains that have the same Igh genes as those used
to induce the suppressor factor, i .e ., the Ts' population is Igh restricted .
To determine whether the hybridoma suppressor factor-induced Ts' population
was also H-2 restricted, the suppressor factor-induced C3H Ts' cells were also
6transferred into Igh-compatible, H-2-incompatible B6 or CWB recipients (H-2 , Igh b) .
As shown in Table VIII, these Ts' cells would not suppress the NP responses in eitherof these strains . Because the CKB and CWB strains are congenic and presumably
only differ at the H-2 complex, the data suggest that the Ts' population is both H-2
and Igh restricted . To confirm these findings, a reciprocal experiment was performed
in which CKB-39 supernates were given to B lO .B R mice, H-2-compatible, Ighincompatible C3H mice, or H-2-incompatible, Igh-compatible C57BL/6 mice . Again,
only the B10 .BR mice were suppressed, which suggests a dual requirement for both
H-2 and Igh compatibility.
When CKB-17 or CKB-39 TsF was given to C57BL/6 mice to generate Ts', the476

PSEUDOGENETIC RESTRICTIONS OF HYBRIDOMA SUPPRESSOR FACTOR

suppressive activity was only noted when the Ts' were transferred into H-2- and Ighcompatible C57BL/6 recipients. Finally, to demonstrate that the Ts' cells were notrejected, B6-29 or CKB-17 TsF was given to (B10 X B10 .BR)FI mice; these Fl animals
were then used as Ts' donors (Table VIII) . The Fl cells could transfer suppression toeither haploidentical parental recipient, but did not transfer suppression into H-2incompatible B10 .D2 mice (data not shown). Finally, we have also demonstrated thatthymocytes from CKB-17 TsF-injected mice can adoptively transfer suppression,indicating that the Ts' cells are of thymus-derived origin .
Discussion
The development of functional suppressor T cell hybrids will permit analysis of the
T cell antigen receptor, antigen-specific TsF, and the mechanisms by which TsF
modulates cellular and/or humoral immunity . Such hybrids also represent a constantsource of monoclonal material for cell biologists and molecular geneticists to probe
the mechanisms of cellular activation and differentiation at the subcellular and DNA
levels .
In the past few years, several TsF-producing suppressor cell hybrids have been
prepared (7, 10-13) . Some of these hybrids were used to produce TsF for immunochemical analysis (12, 14) . The present study focused on the mechanisms of cellularinteractions in the suppressor cell pathway . The NP model was chosen because it is
one of the most thoroughly characterized suppressor cell systems . In the NP system,
three distinct suppressor T cell populations have been defined and the suppression
affects both humoral and cellular immunity (1-3) .
The B6-29, CKB-17, and CKB-39 hybrids characterized in this report represent theTs' population . The evidence supporting this conclusion includes : (a) the cells used
for fusion were purified by the same methods that are used to enrich the Ts' cell
population, and (6) the hybrids have the same cell surface phenotype as the Ts'
population . Furthermore, the factor produced by these hybridoma cells has all theproperties of Ts'-derived factor (TsFi). Thus, the B6-29, CKB-17, and CKB-39
hybridoma factors function during the induction phase of the immune response, bearidiotypic and I -J determinants, and induce Ts', which function during the effector
phase of the response . These properties resemble those of other Ts' populations or Ts'derived factors described in other systems (6) .
However, in contrast to other reports, the hybridoma suppressor factors can generate
Ts' cells in the absence of antigen (15, 16) . In the NP system, we previously
demonstrated that transfers of purified Ts' cells into naive recipients would not
generate Ts' unless antigen was also added (1) . The present observations can bereconciled with the previous results by assuming that antigen is required for the
expansion or differentiation of the Ts' population to generate sufficient factor toinduce functional second order suppressor cells . However, in the poly(L-Glu,L-Ala,LTyr) (GAT) system, TsF' will not generate Ts' in the absence of antigen (15, 16) . It is
not clear why this disparity exists, but it should be noted that in the NP system thesecond order Ts' cells have an anti-idiotypic specificity (1), whereas the specificity of
the Ts' cells in the GAT system has not been defined . Preliminary data suggest thatthe cells generated with GAT-TsF may not possess anti-idiotypic receptors, but appear
to carry idiotypic receptors (Waltenbaugh, personal communication) .
When tested directly for suppressive activity, there is an apparent Igh restriction inK . OKUDA, M . MINAMI, D . H . SHERR, AND M . E . DORF

477

TsF function, without any indication of any H-2 restrictions (Table VI) . This apparent
Igh restriction involves genes in the Igh-V region of the Igh complex (Table VII) .
However, this is only a pseudogenetic restriction, because these factors can still
function, as demonstrated by their ability to generate Ts' cells in Igh-incompatible
recipients . But to observe the activity of these Ts' cells, they must be transferred backto an Igh-compatible strain (Table VIII) . Thus, CKB-17 or CKB-39 factors would
not suppress the NP-0-Su response of Igh-incompatible C3H mice, but cells from
C3H mice that had been treated with these factors could suppress NP-0-Su responses
when transferred back to CKB or B l0 .BR recipients (Table VIII) . Thus, there appear
to be no H-2 or Igh restrictions on the ability of TsF to generate Ts', but thepseudogenetic restriction reflects the requirement for an idiotype-matched Igh-Vcompatible target cell for expression of suppressive activity . Pseudogenetic restrictions
have also been observed in two other suppressor cell systems . Sunday et al . (17)observed that anti-T cell receptor antibodies would only directly suppress Igh-compatible strains . However, suppressor cells were also generated in Igh-incompatible
strains, but their activity could only be observed by adoptively transferring these cells
back to an Igh-compatible strain. Similarly, Sy et al. (18) noted a pseudogenetic
restriction in the ability of idiotype-coupled cells to directly induce suppression in an
Igh-incompatible recipient . These authors also demonstrated that suppressor cells
were generated by adoptively transferring cells into Igh-matched recipients . Thus, inall of these systems, the pseudogenetic restriction appears to reflect a requirement forhomology of second order suppressor cells, which bear anti-idiotypic receptors, withanother cell population (presumably bearing idiotypic receptors) that functions laterin the suppressor pathway . Indeed, we have previously demonstrated a requirement
for a third suppressor cell population (Ts3) in the NP system, which must be Ighcompatible with the Ts' population (2) . This Ts'-Ts3 interaction was not only
restricted by Igh genes but also by genes in the H-2 complex (2) . In this study, CKB39 TsF induced Ts' in C3H mice, which suppress CKB (H-2k) mice, but failed tosuppress H-2 congenic CWB (H-2) mice (Table VIII) . Similarly, Ts' generated in
C3H mice would transfer suppression to B 10 .BR (H-2k) mice but not to Igh-compatible, H-2-incompatible C57BL (H-2~ mice (Table VIII) . Thus, the Ts' populationinduced with hybridoma suppressor factor demonstrated an apparent dual restriction
for both Igh and H-2 genes . We have previously reported a similar dual restriction in
the activity of the antigen-induced effector phase suppressor cell population (5) .
However, we must remain cautious when interpreting these observations, because one
or both of these apparent genetic restrictions may reflect another pseudogenetic
restriction between cells or factors functioning at latter stages in the suppressor
pathway .
Another purpose of the present study was to determine whether a single Ts' cell
could modulate both cellular and humoral responses . Thus, B6-29 hybridoma suppressor factor was used to generate Ts' cells, which suppressed PFC responses when
added during the effector phase of the Mishell-Dutton culture (Table V) . Products
from a single suppressor T cell clone can modulate both cellular and humoral
responses . Furthermore, it should be noted that the NP-0-Su CS responses are
mediated by at least three distinct T cell populations. One of the effector populations
is genetically restricted by genes in the I region of the H-2 complex, and the other two
populations are restricted by genes in the H-2K and H-2D regions (8, 19) . The latter

478

PSEUDOGENETIC RESTRICTIONS OF HYBRIDOMA SUPPRESSOR FACTOR

populations are considered "killer cell-like" because they bear the Lyt-2 phenotype
and react with NP-coupled H-2K or H-2D compatible cells but not NP-conjugatedproteins (20) . In contrast, the NP-specific H-21-restricted CS clones respond to NP
protein conjugates and lack the Lyt-2 marker (20) . Because in many experiments the
hybridoma suppressor factor completely inhibited the CS response (e .g., Tables I and
VI), these factors must suppress multiple subpopulations of T cells, including thosemediating H-2I and those mediating H-2K/H-2D restricted NP-specific responses .
We must still determine whether the hybrid cells are producing a single product thatsuppresses all of these immune responses (perhaps by activating a single Ts' thatindirectly inhibits a common helper cell required for all of these responses), or if
several distinct but related products are produced by these hybridoma cells . Further
analysis of these suppressor cell hybrids and their factors will clarify these issues andwill help to further characterize the mechanisms of immune regulation .
Summary
Suppressor factor derived from three different murine T cell hybridomas werecharacterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous
sensitivity responses . The factors bind antigen and bear I-J and idiotypic determinants,
but lack conventional immunoglobulin constant-region determinants . The factors
function during the induction phase of the immune response, by inducing a second
population of suppressor cells (Ts') . Suppressor factor can inhibit both cellular andplaque-forming cell responses in appropriate strains of mice. These hybridoma
suppressor factors directly suppress strains of mice that are Igh-V homologous withthe strain producing the factor. Thus, there is an apparent Igh-V restriction in theactivity of these factors . However, this is a pseudogenetic restriction because thesefactors generate second order suppressor cells (Ts') in Igh-incompatible mice, but inorder to express the suppressive activity, the cells must be adoptively transferred into
recipients that are Igh compatible with the strain producing the suppressor factor .
Finally, it was shown that the factor-induced Ts' population is under an apparent
dual genetic restriction . Thus, Igh and H-2 homology is required in order for the Ts'
population to express its suppressive activity .
We thank Dr. Shyr-Te Ju for preparing the immunoadsorbent columns and Ms . Teresa
Greenberg and Ms. Nancy Axelrod for their unfailing secretarial support .
Receivedfor publication 27 April 1981 .

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