Proc. Natl Acad. Sci. USA
Vol. 78, No. 7, pp. 4557-4561, July 1981

Immunology

Functional association of idiotypic and I-J determinants on theantigen receptor of suppressor T cells(T-cell receptor/hybridoma T cells/suppressor factor)

KENJI OKUDA, MUTSUHIKO MINAMI, SHYR-TE JU, AND MARTIN E. DORF
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115

Communicated by Baruj Bennacerraf, April 15, 1981

ABSTRACTThe serological characteristics of the antigen receptor on 4-hydroxy-3-nitrophenylacetyl (NP) specific suppressor
T cell hybridomas were analyzed. Three T-cell hybrids could be
lysed with anti-idiotype and complement. The reactivity patternobserved from a panel of anti-idiotypic reagents indicated that NPb
determinants were detected on all three hybrid lines. NP conjugates with bovine serum albumin or caproic acid specifically inhibited the complement-mediated lysis of these cells by both anti-

both I-J and idiotypic determinants are intimately associatedwith the antigen receptor on suppressor T lymphocytes.

MATERIALS AND METHODS
Mice. C57BL/6J(B6), C3H/HeJ, and B10.BR mice werepurchased from The Jackson Laboratory (Bar Harbor, ME). Allother inbred strains were bred in the animal facilities of Harvard
Medical School. Mice were used at 2-12 months of age.
Antigens. The preparation of hapten-conjugated proteins has
been described (15). The molar conjugation ratio of haptenicgroups used in this work was: dinitrophenyl (DNP)/bovine
serum albumin, 12:1; NP/albumin, 12:1; and 4-hydroxy-5-iodo3-nitrophenyl (NIP)/albumin, 7:1. The caproic acid (Cap) derivatives of DNP and NP were purchased from Biosearch (SanRaphael, CA). DNP-Cap and NP-Cap were dissolved in phosphate-buffered saline at 10 mM and the pH was adjusted to 7.3
with 1 M NaOH. NP-(CH2)6-NP (0.1 mM) was dissolved in 0.25
M phosphate buffer (pH 7.8) containing 20% dimethyl sulfoxide.
Alloantisera. B10.A(3R) anti-B10.A(5R) (I-Jk), B10.A(5R)
anti-B1O.A(3R) (anti-I-J ), (B10 x LP.RIII)F1 anti-B10.A(4R)(anti-Kk and I-Ak), and (B1O.A(4R) x B10.GD)F1 anti-B10 (antiKb + I-Ab) were produced by immunization with spleen andlymph node cells as described (4, 16). Anti-brain-associated Tcell antigen (anti-BAT) antibody was made by the techniquedescribed by Golub (17). Monoclonal anti-Thy 1.1 and Thy 1.2
antibodies were purchased from New England Nuclear.
Anti-Idiotypic Antisera. C57BL/6, A/J, SJL, and C3H mice
were immunized intraperitoneally with 100 ,g of NP-BGG and
pertussis vaccine (2). Primary anti-NP antibodies were specifically purified by addition ofpooled sera to Sepharose 4B beads(Pharmacia) conjugated with NP-albumin and elution with 0.2
M glycine HCl buffer (pH 2.35) into tubes containing 2 M
Tris HCl at pH 8.0. Guinea pigs were immunized with 100 'gof anti-NP antibodies emulsified with complete Freund's adjuvant, followed by repeated intramuscular and subcutaneous
injections with the same immunogen. Sera were rendered idiotype specific by adsorption with Sepharose 4B beads coupledwith a gamma globulin fraction of MOPC 104E ascites and normal serum from the appropriate mouse strains. The binding of"2I-labeled B6 anti-NP antibodies to its anti-idiotypic antiserum could be strongly inhibited by 1 u1 of B6 anti-NP antiserum but not by 30 A1 of B6 normal mouse serum or MOPC
104E ascites. The idiotypic specificities detected were closely
associated with NP binding sites because 0.06 ,umol of NP-Cap
or NIP-Cap but not 6 pgmol of p-aminoarsanilate completely
inhibited the binding. Furthermore, strain distribution studies
indicated that the anti-idiotypic antiserum defines NPb-idiotypic specificities similar to those previously reported (18).

NPb idiotype and anti-I-J antisera. These hapten conjugates failed
to block lysis by anti-Thy 1 or anti-H-2K antisera on the same target cell populations. The data indicate that both I-J and Igh variable region gene products are intimately involved in the recognition of antigen by suppressor T cells. Finally, the suppressor cellhybrids produce soluble factors that mediate antigen-specificsuppression. The characteristics of the cells and their factors indicate that the hybrids correspond to the Ts' or first-order suppressor cells in the suppressor cell pathway.
Immune responses to specific antigens are controlled by theactivity of suppressor T lymphocytes. The sequence of cellularinteractions resulting in suppression ofthe cellular and humoral
immune responses to antigen has been well characterized in the
4-hydroxy-3-nitrophenylacetyl (NP) system. At least three distinct cell populations have been implicated in this suppressor
cell pathway (1-5), thereby making the NP system one of themost thoroughly characterized of the suppressor cell models.
Furthermore, the conclusions derived from the NP system areconsistent with most other observations concerning the cellularinteractions involved in immune suppression (6-10). However,in spite of considerable data relating to the cell surface phenotype of the T cells involved in immune suppression, little isactually known about the interrelationships of such membranemarkers and the nature of the antigen receptors on these cells.
To investigate the nature of the T cell antigen receptor andto characterize the molecules responsible for the communication between cells, in the suppressor pathway, large numbersof functional suppressor T cells must be obtained. To achieve
this goal we have utilized the techniques of somatic cell hybridization (11). These techniques have been used previously
to prepare monoclonal cell lines that maintain the biologicalactivity of the original suppressor cell population, including theability to secrete biologically active suppressor factors (12-14).
We now describe a series of suppressor cell hybrids that produce soluble products capable of specifically suppressing NP
immune responses. The relationship of these hybrids to theoverall suppressor cell pathway is analyzed. These hybridoma
cell lines also provide a source of monoclonal cells that are used
to probe the T cell antigen receptor. The data demonstrate that
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Abbreviations: NP, 4-hydroxy-3-nitrophenylacetyl; Cap, caproic acid;
DNP, dinitrophenyl; NIP, 4-hydroxy-5-iodo-3-nitrophenyl; NBrP, 4-hydroxy-5-bromo-3-nitrophenyl.
4557

4558

Immunology: Okuda et al.

Anti-idiotypic antisera to B6 anti-4-hydroxyl-5-bromo-3-nitrophenyl (NBrP) and B6 anti-NIP, characterized in a similar fashion, also detected NPb idiotypic specificities.
Anti-NP-i idiotypic antiserum was made in guinea pigs
against NP-1 hybridoma anti-NP antibody (Au, A) secreted bya hybridoma cell line generated by fusion of SJL spleen cells
immunized to NP-Ficoll with the SP2/0 cell line. The antiidiotypic antiserum was adsorbed as described above. The adsorbed antiserum reacted strongly with NP-1 hybridoma antiNP antibody but not with MOPC 104E or MOPC 315 myeloma
protein or SJL normal mouse serum. The specific idiotype binding, termed "NP-1 idiotype," was inhibited completely in thepresence of 0.06 Amol of NP-Cap or NIP-Cap but not 6 Iumolof p-aminoarsanilate. NP-1 idiotypic specificities were sharedby B6, SJL, and A/J anti-NP antisera and were not detectablein seven other mouse strains not bearing Ighb or Ighe alleles.
Additional serological analyses indicated that anti-idiotypic
antisera against B6 anti-NP, B6 anti-NBrP, B6 anti-NIP, andSJL anti-NP crossreactively bound NP-1 hybridoma anti-NP
antibody. However, the binding strength of these anti-idiotypic
antisera were 'Ao to 1A that of anti-idiotypic antiserum to NP1 hybridoma antibody.
Anti-idiotypic antisera against CGAT and hybridoma F17. 148.3
of B6 origin have been described'(i9).
Preparation of Antigen Specific Suppressor T Cells. The
methods for the preparation and enrichment of NP-specific suppressor T cells have been described (1, 3).
Hybridization. Suppressor T-enriched C57BL/6J or CKB
splenic lymphocytes were hybridized with BW5147 T lymphoma cells. The hybridizing method was as described by Taniguchi et al. (12).
To provide an additional specificity control, in some experiments another T cell hybrid, prepared by fusion of BW5147 andT cells from DBA/1 GAT nonresponder mice pretreated-with
GAT to induce suppressorT cells, was studied. This hybrid line,
F65,121 (termed "DI-121" in this report) was the gift ofRonald
N. Germain (Harvard Medical School).
Screening for I-J+ and Id' Hybrids. Soon after the hybrid
cells began to grow, both I-J+ and Id' hybrids were selectedby cytotoxic tests (20). Briefly, 2 ,1 of cell suspension (1 x 106
cells per ml) was treated with 2 ,ul of diluted antisera in L-15
medium (Microbiological Bioproducts, Walkersville, MD) containing 0.5% fetal calf serum. The reaction was carried out atroom temperature in Terasaki microplates (no. 3034, Falcon).
After 20 min, the cells were washed with one drop of serum-free
L-15 medium. Then 2 ,ul of a 1:5 dilution ofselected rabbit complement in serum-free L-15 medium was allowed to react withthese washed cells for 40 min at 37°C. Then these cells were
stained with 0.2% nigrosine and the percentage of dead cells
was determined.
To inhibit the cytotoxicity test, 1 1,u of DNP-albumin, albumin, NP-albumin, or NIP-albumin solution (2 mg/ml) in
phosphate-buffered saline or 0.01 M DNP-Cap, 0.01 M NPCap, or 0.1 mM NP-(CH2)6-NP were mixed with 1 ul of cellsuspension (2 x 106 cells per ml) prior to the addition ofantiserum.

Adsorption and Elution of Suppressor Factor. NP-albumin,
specifically purified antibodies, and gamma globulin of various
antisera were conjugated to CNBr-activated Sepharose 4B
beads according to the manufacturer's protocol. Two to 4 mgof protein was coupled to 1 ml of beads and the product was
packed into a 3-ml syringe. Adsorptions were carried out atroom temperature by repeatedly passing 5 ml of'culture supernatants through the columns. Unbound material was saved.
After each column was washed with 10 ml of phosphate-bufferedsaline, bound materials were eluted with 4.2 ml of 0.2 M gly-

Proc. Natl. Acad. Sci. USA 78 (1981)

cine'HCl (pH3.2) into tubes containing 0.5 ml offetal calf serum
and 0.3 ml of 2 M Tris'HCl (pH 8.0). The pH ofthe eluted fractions was adjusted to 7.4-7.6 when necessary. Fractions werestored at -200C until used.

Assay for Suppressive Activity of Culture Supernatant or
Fractionated Samples on Cutaneous Sensitivity Responses.
The assay has been described in detail (21). Footpad swelling
was determined as the difference, in units of 10-3 cm, between

left and right footpad thicknesses.
Data Analysis. Statistical analysis of the experimental datawith respect to controls utilized the two-tailed Student's t test.
Data are expressed as the mean (±SEM) incremental footpad
swelling.
RESULTS
Serological Analysis of T-Cell Hybridoma Cell Lines. Twoseries offusions using either B6 (H-2b, Ighb) or CKB (H_2k, Ighb)
antigen-enriched suppressor cells and the BW5147 AKR thymoma were analyzed. The cell fusions were performed with
polyethylene glycol, and the hybrids were selected by growthin hypoxanthine/aminopterin/thymidine medium. Of 1050
wells plated, 30% developed hybrids. All hybrids were screened
with anti-I-J alloantisera and subsequently with guinea pig antiNPb idiotype antisera; 16% of the hybrids were specificallylysed with allele-specific anti-I-J alloantisera and 29% of the IJ-bearing hybrids were specifically lysed with anti-NPb idiotype
antisera. For the present study one B6- and two CKB-derived
hybrids that were susceptible to lysis with both anti-I-J and antiNP" antisera were selected. In addition one CKB hybrid that
had I-J determinants but lacked idiotypic determinants was selected as a control.
The specificity of lysis with these reagents is shown in Table
1. The anti-I-Jb antiserum only lysed hybrids of B6 (H-2b) origin;the anti-I-Jk reagent only lysed cells of CKB origin. Several
batches of anti-I-J antisera were used and all gave concordantresults.
The results from adsorption experiments indicated that thelytic activity of the anti-I-Jk antisera could be specifically absorbed with spleen cells from H-2k, but not H-2b-bearing mice.
The specificity ofthe anti-idiotypic serum was demonstrated by
its ability to lyse the B6-29, CKB-39, and CKB-17 hybrids but
not T-cell hybrids produced by fusion with GAT-induced suppressor T lymphocytes. As a reciprocal control, guinea pig antiCGAT idiotype failed to react with the NP hybrids but lysed
the DBA/i-derived GAT suppressor cell hybrid. To verify the
T-cell nature of these hybrids, each cell line was tested with
Table 1. Serological analysis of T-cell hybridomas% specific cytotoxicity*
Antisera
B6-29 CKB-17 CKB-39 CKB-70 D1-121 BW5147
0
0
0
0
5
35
Anti-IjJb

Anti-14J
Anti-NPb

0
30
0
100
89

70
26
5
94
84

81
60
12
0
90

90
0
0
70
88

0
0
45
95
95

5
0
0
11
86

Anti-CGAT
Anti-Thy 1;2
Anti-Thy 1.1
The results are from a single experiment, but each cell line has beentested on at least three separate occasions with comparable results.
Each reagent was diluted 1:10 for use in cytotoxicity assays.
* The data were calculated as 100 x (% experimental lysis - % control
lysis) (% total lysis - % control lysis). Control lysis was 5-20%.
In a large series of experiments with control sera, we observed thatspecific lysis <15% is within control limits and specific lysis >20%
is considered significant.
-Immunology: Okuda et al.

Proc. NatL. Acad. Sci. USA 78 (1981)

for analysis. Culture supernatants from these cells were passed
over immunoadsorbent columns and the adsorbed fractions
were eluted with glycine HCI buffer (pH 3.2). The suppressive
activity was adsorbed by anti-idiotype, anti-I-jb, and NP-albumin columns (Table 2). Furthermore, suppressive activity
could be recovered specifically by elution from these columns.
The suppressor factor lacked conventional immunoglobulin determinants because polyvalent guinea pig anti-immunoglobulin
antisera failed to absorb the activity. Furthermore, a control
anti-I-Jk immunoabsorbent column failed to remove the activityof the H-2"-derived suppressor factor.
Idiotype Analysis of Hybridoma T-Cell Lines. In order tocharacterize which idiotypic determinants were present on the
hybridoma cell lines, the hybrids were screened against a seriesof guinea pig anti-idiotypic reagents prepared against purified
antibodies to NP or to NP derivatives such as NIP or NBrP
(Table 3). The anti-idiotypic reagents prepared against B6 antiNP, anti-NIP, or anti-NBrP were specifically lytic for the B629, CKB-39, and CKB-17 hybrids. In contrast, none ofthe otheranti-idiotypic reagents prepared against purified NP antibodies
derived from other strains of mice could lyse these cell lines.
Furthermore, anti-idiotypic antibody prepared against the NPb
idiotype bearing SJL B cell hybridoma were specifically lytic
for the three functional suppressor T cell hybrids (see below).
As a control, guinea pig anti-idiotypic antibodies specific for the
GAT-specific B-cell hybridoma F17. 148.3 were found to be
nonlytic.
Antigen-Specific Inhibition of Anti-Idiotype- and Anti-IJ-Mediated Lysis. In order to verify the specificity of the complement-mediated lysis with anti-idiotype antisera, we attempted to inhibit this reaction with antigen. Either NPalbumin or NIP-albumin was used to inhibit cytolysis, and
DNP-albumin and albumin were used as controls. Each of these
inhibitors was used at a final concentration of 0.5 mg/ml, andthe antisera were titrated to give 30-80% lysis. Addition ofeither NP-albumin or NIP-albumin specifically inhibited lysis
by anti-idiotype (Table 4). Furthermore, addition of NP- or
NIP-albumin also blocked lysis by anti-I-J alloantisera on the
B6-29, CKB-39, and CKB-17 targets. Neither of these hapten
conjugates blocked anti-I-J cytolysis of the idiotype-negative IJ-bearing CKB-70 cells. In addition, NP-albumin and NIP-albumin could not inhibit complement-mediated cytolysis byanti-H-2K, anti-Thy 1.2, or anti-BAT antiserum.
To determine if the NP-albumin or NIP-albumin inhibitionof anti-idiotype and anti-I-J was due to steric hindrance, we
attempted to block cytolysis with the low molecular weight
mono- and bivalent Cap derivatives NP-Cap and NP-(CH2)6NP. NP-Cap at 10 mM and NP-(CH2)6-NP at 0.1 mM could

Table 2. Immunochemical characterization of B6-29 hybridoma
suppressor factor
ImmunoSource
Column fractionof
NP-O-Su adsorbent
UnboundEluatefactor priming column+
26.8 + 4.6 (5)BW5147+
11.8 ± 2.3 (5)*B6-29+
22.8 ± 2.4 (5) 12.0 ± 1.6 (6)*AntiNPb
B6-29+
Anti-NIPb 25.2 ± 5.0 (5) 12.8 ± 2.1 (4)*B6-29+
10.5 ± 2.0 (4)* 25.0 ± 9.6 (4)B6-29
Anti-Ig+
Anti-IJb
27.4 ± 3.9 (5) 13.8 ± 0.7 (5)*B6-29+
10.8 ± 1.8 (5)* 30.0 ± 6.4 (5)B6-29
Anti-I~Jk+
B6-29
NP-albumin 24.3 ± 3.5 (4) 14.5 ± 1.5 (4)*8.8 ± 1.8 (5)BW5147C57BL/6 mice were primed with NP-O-Su followed by daily intravenous injections ofthe column fractions until the day prior to antigen
challenge. The results are expressed as the mean (±SEM) incrementof footpad swelling. The number of mice per group is indicated in parentheses. The data are from one experiment; a second experimentyielded comparable results.
* Significant suppression, P < 0.05.

monoclonal anti-Thy 1 reagents. With the exception of the
CKB-39 cell line, all were lysed with the anti-Thy 1.2 reagent
which fails to lyse the parental BW5147 line, indicating thatmost ofthese hybrids contained the Thy 1.2 allele derived fromthe suppressor T lymphocyte donor.
The percentage of cells lysed with the anti-I-J and -NP" reagents varied considerably in different experiments, from 20%
to 90%. Similar results were noted with cloned cell lines. Preliminary experiments suggest that this variation is correlatedwith the stage of the cell cycle (22). The percentage ofcells susceptible to lysis with anti-I-J and anti-idiotypic antisera appeared to decrease after prolonged maintenance in cell culture.
Functional Analysis of T-Cell Hybrids. Supernatants fromthe four hybridoma cells were assayed for their ability to inhibit
NP-specific cutaneous sensitivity responses. The B6-29, CKB39, CKB-17, and CKB-70 hybrid supernatants inhibited the
NP-O-Su response 90%, 69%, 59%, and 12%, respectively, if
given daily from the time of antigen priming until the day prior
to antigen challenge. The inhibition by the first three hybrids
was antigen specific in that DNFB contact sensitivity responses
were not inhibited by the same supernatants (the inhibition was
9%, 11%, and 11%, respectively). Controls for these experiments included testing supernatants from the BW5147 tumor
line, which failed to inhibit cutaneous sensitivity responses.
To characterize the biologically active factors present in thesesupernatants, we selected supernatants from the B6-29 hybrid

Table 3. Idiotype analysis of T-cell hybridoma cell lines
code

StrainAnti-NPb
Anti-NIPb
Anti-NBrPb

B6
B6
B6
SJL

Anti-NP-1

Anti-SJL
Anti-A/J

Anti-C3H
Anti-CGAT
Anti-148.3% specific cytotoxicity

Idiotype

Anti-idiotype

SJL
A
C3H
D1.LP
B6

Immunogen
NP
NIP
NBrP
Monoclonalanti-NP
NP
NP
NP
GAT
Monoclonalanti-GAT
See legend to Table 1.

4559

B6-29

CKB-17

CKB-39

CKB-70

30
39
28
38

36
42
26
26

63
53
63
63

0
0
0
0

0
0
11
6
0

0
5
10
5
10

0
0
0
12
0

BW5147
0
0
0
5
0
0
0
0
0

4560

Immunology: Okuda et al.

Proc. NatL Acad. Sci. USA 78 (1981)

Table 4. Antigen-specific inhibition of anti-idiotype and anti-I-J
complement-mediated lysis% specific cytotoxicity
DNPNPNIPCell
Albumin albumin albumin albumin
line
Antiserum
029
529
B6-29 Anti-NPb
5290
Anti-I-Jb
35
70
65
79
70
Anti-Kb
94
94
100
100
Anti-Thy 1.2

CKB-17 Anti-NP"

Anti-NIPb
Anti-I-Jk
Anti-Kk
Anti-Thy 1.2
Anti-BATCKB-39 Anti-NP"

Anti-NIP"
Anti-I-Jk

Anti-Kk
Anti-Thy 1.2

Anti-BAT

38
39
63
63
75
63

36
36
68
79
79
79

0
0
12
78
68
79

5
5
5
75
63
63

47
58
89
79
0
79

57
57
93
79
0
71

0
0
5
89
0
79

6
0
5
95
0
75

Table 5. Hapten inhibition of anti-I-J and anti-NPb lysis% specific cytotoxicity
NPCellNo (CH2)rNP NP-Cap DNP-Cap
inhibitor (0.1 mM) (10 mM) (10 mM)
line
Antiserum37
330
6B6-29 Anti-NP"
33
6
3
48
Anti-NIP"
12
33
13
Anti-I-Jb
37
74
77
82
76
Anti-Kb
58
63
63
Anti-Thy 1.1
76

CKB-17 Anti-NPW

25
28
52
63
57

0
2
5
55
72

0
13
25
81
75

25
25
63
63
69

CKB-39 Anti-NP"

41

Anti-NIP"

36
55
60
63

0
4
9
61
63

10
15
19
69
70

35
31
56
69
72

47.
50
50

39
67
33

33

44

-

-

-

-

0
3
47
58
58

0
3
42
63
63

0
6
65
53
53

0
0
65
53
65

Anti-NIP"
Anti-14k
Anti-Kk
Anti-Thy 1.1

Anti-I-Jk
Anti-Kk
Anti-Thy 1.1

CKB-70 Anti-I~Jk
CKB-70 Anti-NP"
Anti-NIPb
Anti-IJk

0
0
0
0
47
53
37
59
56
Anti-Kk
63
63
59
75
Anti-Thy 1.2
Antisera were titrated in order to obtain 30-80% specific lysis. All
inhibitors were used at a final concentration of 0.5 mg/ml. See legend
to Table 1.
0

0

0
0
47
48
59

block the lysis mediated by either antiTNPb or anti-NIPb antiidiotypic antiserum (Table 5). Furthermore, these compounds
specifically blocked anti-I-J lysis in the B6-29, CKB-17, andCKB-39 cell lines but failed to inhibit anti-I-J lysis of the nonfunctional CKB-70 hybrid or the CKB-59 cell line. Preliminary
analyses suggest that the CKB-59 hybrid represents an effectorphase second-order suppressor T cell and presumably bears ananti-idiotypic receptor. The specificity of the inhibition wasdemonstrated by the inability of NP-Cap or NP-(CH2)6-NP toblock anti-H-2K or anti-Thy 1 lysis and the inability ofDNP-Cap
to block anti-idiotype- or anti-I-J-mediated lysis.
Finally, the data suggest that NP-(CH2)6-NP at 0.1 mM is abetter inhibitor of anti-idiotype and anti-I-J lysis than is a 100fold excess ofmonovalent NP-Cap. Although this trend has been
observed in nearly all experiments, more data are required toevaluate adequately the role of valency in suppressor T-cellantigen interactions.
DISCUSSION
The suppressor cell pathway resulting in the modulation of cellular and humoral NP responses has been well characterized.
The first population of suppressor cells (Ts1 or Ts') bear I-J and
idiotypic determinants and function during the induction phaseof the immune response (1, 3). The Ts, population induces asecond complementary population of suppressor cells (Ts2 orTse) which bear I-J determinants and anti-idiotypic receptors(3). The latter population functions during the effector phaseof the immune response in previously primed animals (2, 3).
However, this second population does not contain the final effector suppressor T cells. The available data suggest that the Ts2cells activate a third population of I-J- and idiotype-bearing suppressor T cells (Ts3) in the immune recipient (4, 9). The B6-29,

Anti-Kk
Anti-Thy 1.1
CKB-59 Anti-NPb

Anti-NIP"
Anti-I-Jk

Anti-Kk
Anti-Thy 1.1
See legend to Tables 1 and 4.

CKB-17, and CKB-39 T-cell hybrids described here appear tocorrespond with the Ts, population. In support of this, we notethat (i) the cells were induced and selected by the same procedures that have been shown previously to generate NP-specific Ts, (1), (ii) these hybrid cells have the same surface phenotype as the Ts, population (4), and (iii) preliminary data
indicate that the supernatants from these hybrid cells can generate a Ts2 population (data not shown).
The relationship of the B cell idiotypic determinants withthose on hybridoma suppressor T cell lines was compared bya panel of anti-idiotypic antisera. The anti-NPb, antiusing
NIP, and anti-NBrPb antisera have similar specificity patterns-i.e., each ofthese guinea pig antisera bind purified antiNP antibodies from Ighb-bearing C57BL/6 mice and the predominant idiotypic determinants detected by these reagents
have a strain distribution similar to that described for the allotype-linked NPb idiotype (18). Another reagent was also capable of specifically lysing the Ts hybrids. Anti-idiotype against
a SJL monoclonal anti-NP hybridoma antibody which possessedsome NPb idiotypic determinants and demonstrated weak binding of NPb ligand could also lyse the three functional T cell hybrids. Because this anti-idiotypic reagent was prepared against
purified monoclonal hybridoma antibodies, it excludes the possibility that this reagent contained contaminating antibodies
against T cell-derived products that may have copurified withthe anti-NP antibodies obtained from conventional immune
serum.

Anti-idiotype reagents prepared against purified anti-NP or
anti-NIP antibodies from C3H or A/J mice (which lack the Ighb
allotype) failed to lyse the NP-specific T cell hybrids. Finally,anti-idiotype prepared against purified SJL anti-NP antibodies,Immunology: Okuda et al.
which carry the Ighb allotype but lack the NPb idiotype due toa defect in the production ofA light chain (18), also failed to lysethese hybrids. Anti-idiotype prepared against these SJL antibodies can bind the NP-1 hybridoma protein which possesses
some of the NPb idiotypic determinants, but this reagent failed
to lyse the T cell hybridoma lines. These findings suggest that
only selected idiotypic determinants on NP-1 hybridoma antiNP antibody are expressed on individual hybrid T cell lines.
The availability of a homogeneous population of antigen-specific suppressor T cells permitted serological analysis of the T
cell's antigen receptor. The ability of NP-albumin or NIP-albumin to inhibit lysis by anti-idiotype specifically verifies the
specificity of this lysis. The finding that NP-albumin or NIPalbumin could also block lysis by anti-I-J alloantisera on hybridsthat possessed both the NPb and I-J antigenic determinants indicated that the I-J determinant is closely associated with theantigen receptor of the T cell. Furthermore, monovalent NPCap and bivalent NP-(CH2)6-NP also were able to block antiidiotype- and anti-I-J-mediated lysis. Because these compounds
are smaller than 500 daltons, the results indicate that (i) there
is a functional association of H-2 and Igh gene products on the
Ts cell surface, (ii) the predominant idiotypic determinants detected on Ts are associated with the combing site, not framework variable region, determinants, and (iii) I-J determinants
are intimately associated with the combining site. It is important
to note that these hapten conjugates failed to inhibit the lysisof anti-I-J antisera on the I-J-bearing but NPb-idiotype-negative
CKB-70 hybridoma. This hybridoma lacks NP-specific suppressor function and presumably lacks an anti-NP receptor.
Furthermore, these NP conjugates could not block anti-H-2Kor anti-Thy 1-mediated lysis.
Finally, among the small number of hybrids tested, there isan apparent correlation between the expression of cell surface
I-J and NPb determinants and the ability of factors from thesehybrids to mediate NP-specific suppression. Only one of thehybridoma factors was characterized. The antigen-binding B629 factor had both I-J and NPb determinants which correlateswith the phenotype of the antigen receptor on the cell fromwhich it was derived. The properties of this factor indicate thatit corresponds to a series of suppressor factors described in othersystems (10).

Proc. Natl. Acad. Sci. USA 78 (1981)

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This work was supported by Grants AI-16677 from the National Institutes of Health and PCM 80-04573 from the National Science
Foundation.
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