Induction of anti-hapten antibody response by hapten-isologous carrier conjugate

CELLULAR IMMUNOLOGY 18, 396410

Induction of Anti-Hapten

(1975)

Antibody Response by Hapten-lsologous Carrier Conjugate

ii. Specificity of Hapten-Reactive Helper T Cells 1 UKI

YAMASHITA, Institute

TAKESHI

TAKAMI,

AND MASAYASU KITAGAWA

for Cancer Research, Osaka University Medical Dojinrahamadori, Fukushimaku, Osaka, Japan Rece#ived December

School,

31,1974

Anti-hapten antibody production was elicited by the immunization of haptenisologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNPMGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These haptenreactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PABMGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGGprimed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PABMGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity. 1 This work Government.

was supported

by a Grant-in-Aid

396 Copyright 1975 by Academic Press, Inc. All rights o? reproduction in any form reserved.

for

Scientific

Research

from

the Japanese

SPECIFICITYOFHAPTEN-REACTIVE

I-IELPERTCELM

397

INTRODUCTION It has been well established that cooperative interaction between B lymphocytes (derived from bone marrow) and helper T lymphocytes (derived from thymus) is necessary to produce antibody against several antigens in mice (for review, see 1). B lymphocytes are direct precursors of antibody producing cells. T lymphocytes are not precursors of antibody producing cells, but help antibody production of B lymphocytes by cooperative interaction ( 1, 2). B lymphocytes have receptors for antigen on their surface which are immunoglobulin-like and have specificities similar to that of secreted antibody (325). The specificity restrictions of B cell receptors have been made clear through studies using haptenic determinants as antigens (6). T lymphocytes have been reported to also have receptors for antigen (4, 7, S), but the exact nature of the antigen receptors of T cells is not fully understood. The study of the specificity restrictions of T cell receptors has been confronted with difficult problems, because the nature of the antigenic determinant to which T lymphocytes respond is unclear. In a study of the specificity restrictions of helper T celis, it seems very vaIuabIe to use a known structural determinant, a haptenic group. From this standpoint, there have been some attempts to induce hapten-reactive T cells (9-14). HOWever, the specificity restrictions of helper T cells have not been fully studied. In the previous report of this series, we established that hapten-reactive helper T cells could be developed along with hapten-specific B cells by the immunization of hapten-isologous carrier conjugate, and that the former cells could demonstrate their helper activity on anti-hapten antibody production against another haptenic group on the same carrier (15). This system of hapten-reactive helper T cells will give us a very useful tool to analyze the specificity restrictions of helper T cells. In the present study we attempted to analyze the specificity restrictions of helper T cells and to compare it with that of B cells using a double hapten conjugated carrier as antigen. We will demonstrate that hapten-reactive helper T cells can discriminate structural differences among related haptenic groups as well as B cells do, but the specificity restrictions of helper T cells are different from that of B cells. MATERIALS

AND METHODS

Crystalline bacterial cu-amylase (BorA) derived from Bacillus subtilis was purchased from Nagase Sangyo Co. Ltd., bovine serum albumin (BSA) from Sigma Chemical Co. Ltd., human gamma globulin (HGG) (Cohn fraction II) from Nutritional Biochemicals Corporation, and keyhole limpet hemocyanin (KLH) from Mann Research Laboratories. Mouse gamma globulin (MGG) was prepared from pooled serum of normal dd0 mice by salting out with 40% saturated ( NH4)zS04 and by chromatography on diethylaminoethyl cellulose column with 0.015 M, pH 8.0 potassium phosphate buffer. Mouse serum albumin (MSA) was prepared from pooled serum of normal dd0 mice by salting out with 67% saturated (NH*)zSOh, by gelfiltration on Sephadex G-150 column and by chromatography on carboxymethy1 cellulose column with 0.06 M, pH 5.0 acetate buffer. These six antigens are not immunologically cross-reacting when assessed by radio-

398

YAMASHITA,

TAKAMI

AND

KITAGAWA

immunodiffusion antisera.

technique in an agar layer using 1311-labeled antigens and mouse

Haptert-Protein

Conjugates

Each protein was dinitrophenylated to give DNP-protein conjugate according to the method of Eisen et al. ( 16) using 2, 4-dinitrobenzene sulfonate, and coupled with diazotized nz-aminobenzoic acid (MAB), p-aminobenzoic acid (PAB), and p-aminobenzene sulfonic acid (PABS) to give azoprotein according to the method of Tabachnick et al. (17). The following hapten-carrier conjugates were prepared : DNPa-BaA, DNPsBSA, DNPg-KLH, MABs-MGG, PABQMGG, PABSs-MGG, and PABs-MSA. Subscripts refer to the average number of moles of haptenic group per mole of carrier protein, and this was calculated from the absorption readings at 290 and 360 nm of DNP-proteins (18)) and 460 and 500 nm of azoproteins (19) in alkaline solution. In these calculations, the molecular weights of BaA, BSA, HGG, KLH, MGG, and MSA were 50,000, 69,000, 160,000, 100,000, 160,000, and 69,000, respectively. The following double hapten conjugated carriers were also prepared by the same procedure: DNP-HGG-PAB, DNP-KLH-PAB, DNP-MGG-MAB, DNP-MGG-PAB, DNP-MGG-PABS, and DNP-MSA-PAB. Procedures of Adoptive

Cell Transfer

The dd0 albino mice, supplied by the Central Breeding Laboratory of Experimental Animals of Osaka University, were primarily immunized by intraperitoneal injection of 100 rg of hapten-protein conjugate in Freund’s complete adjuvant (FCA) 3-8 wk previously. The primed cells were prepared from the mesenteric, cervical, and axillary lymphonodes and from the spleens of these primed donor mice, suspended in Eagle’s minimum essential medium (MEM), mixed with second antigen in saline and transferred intravenously into 600R X-irradiated recipient mice in the cell number of S-10 x lo7 per recipient. The recipient mice were killed to remove the spleens for plaque forming cell assay 7 days after the cell transfer. Measurement of Anti-Hapten

Antibody

Response

Anti-hapten antibody response was determined by estimating anti-hapten plaque forming cells (PFC) in the spleens of the recipient mice according to a modification of the method of Cunningham et al. (20), using hapten-conjugated sheep red blood cells (SRBC) as target cell. Single cell suspensions in MEM were prepared from the spleens of the recipient mice, treated with 0.83% NHdCl for 10 min, washed three times with MEM, and the total volume finally adjusted to 2 ml per individual spleen with MEM. A 100 ~1 portion of these spleen cell suspensions was mixed with 25 ~1 of hapten-conjugated SRBC suspension (1: 6 dilution of packed cells), 25 ~1 of 2 : 3 diluted guinea pig serum (absorbed with SRBC) as complement source, and 25 ~1 of 1: 20 diluted rabbit anti-Fc of mouse IgG antisera (heat-inactivated and absorbed with SRBC) for the facilitation of indirect PFC, in wells of a plastic microtiter plate. These mixtures were inserted into Cunningham PFC chambers, and the chambers were

SPECIFICITY

OF HAPTEN-REACTIVE

HELPER

399

T CELLS

sealed by warmed paraffin wax and incubated at 37°C for 2 hr. The PFC in the chambers were counted through a magnifying glass, and total numbers of PFC per spleen were calculated. In this assay system, rabbit anti-Fc of mouse IgG antiserum did not inhibit direct PFC. Thus, the total number of PFC expressed per spleen includes both direct and indirect PFC. Hapten-conjugated SRBC was prepared according to a modification of the method described by Kishimoto et al. (21). DNP-BSA-coupled SRBC was prepared as follows: 10 vol of 5% SRBC suspension in saline were mixed with 1 vol of DNP-BSA (10 mg/ml) and 10 vol of 0.5 mM CrCla in saline at room temperature for 60 min, then washed three times with saline, and finally adjusted to 1 : 6 dilution with MEM. MAB-SRBC and PAB-SRBC were prepared by diazocoupling by the following method: 8.0 mg of m-aminobenzoic acid or p-aminobenzoic acid in 2 ml of water was mixed with 0.9 ml of 0.2 N HCI and 1.8 ml of 0.5 M NaN02 and stirred at room temperature for 10 min. 0.6 ml of 0.1 IV urea was then added, and the mixture further stirred at room temperature for 10 min. After adjusting the mixture to 4°C in an ice cold bath, 5.4 ml of 0.005 M, pH 7.3 sodium phosphate-buffered saline contained 1% glucose, 0.9 ml of 0.5 M, pH 7.6 sodium phosphate buffer and 0.6 ml of 0.2 M Na2C03 were added. Finally, 1 ml of a 50% SRBC suspension was added into this mixture and stirred in an ice cold bath for 10 min, washed three times with saline, and then adjusted to a 1 : 6 dilution with MEM. Preparation

of Rabbit

Anti-Mouse

Tlzymocyte

Antiserum

(ATS)

Anti-mouse

thymocyte antiserum was prepared according to the method of Levey One billion thymus cells obtained from C3HJHe mice were injected intravenously into an albino rabbit twice at 2-wk intervals. One week after the second immunization, the rabbit was bled from an ear vein. The serum was inactivated by incubation at 56°C for 50 min. The cytotoxic activity of ATS, obtained as above, was tested in vitro by the trypan blue dye exclusion test in the presence of guinea pig complement. Ninety to 95% of thymus cells, about 40% of spleen cells, and 60% of lymph node cells were killed at a 1 : 64 dilution, but less than 10% of bone marrow lymphocytes were killed even at a 1 : 2 dilution. Accordingly, this ATS preparation can be considered specific for T cells. et al. (22).

RESULTS Anti-Hapten

Antibody

Response

of Hapten-Isologous

Carrier-Primed

Cells

p-Azobenzoate group (PAB) was coupled to isologous carrier, mouse gamma globulin (MGG), and 100 PLgof PAB-MGG in Freund’s complete adjuvant (FCA) was injected intraperitoneally into mice. In accordance with the use of the DNPhaptenic group ( 15)) anti-PAB antibody production was elicited 3 wk after this immunization. The PAB-MGG-primed cells harvested from these donor mice responded to PAB-MGG (Group 1) but not to MGG alone (Group 2) to produce anti-PAB antibody when they were transferred with 50 pug of second antigen intravenously into 600R X-irradiated recipient mice, as shown in Table 1.

400

YAMASHITA,TAKAMIANDKITAGAWA

TABLE ANTI-HAPTEN

Group no.

1 2 3 4

1

ANTIBODY RESPONSE OF HAPTEN-ISOLOGOUS CARRIER-PRIMED CELLS~

Primed cells

7 7 7 7

X 10' PAB-MGG-primed

cells X 10’ PAB-MGG-primed cells X 107 MGG-primed cells X 10’ Nonprimed cells

2nd Ag

Anti-PAB antibody response (PFC/spleen)

PAB-MGG MGG PAB-MGG PAB-MGG

24480 3190 2720 1610

o 7 X 10’ Spleen and lymph node cells taken from mice which were immunized with 100 pg of PAB-MGG or MGG in FCA intraperitoneally 3 wk previously were mixed with 50 pg of each second antigen and transferred intravenously into 600 R X-irradiated recipient mice. Indirect anti-PAB PFC in spleen of the recipient mice was estimated 7 days after the cell transfer. Four donor mice were used and the mean PFC in each group of three to five recipient mice are listed.

Helper Activity of PAB-MGG-Primed Cells for Other Hapten-Specific Using a Double Hapten Conjugated Carrier

B Cells

The anti-hapten antibody response by hapten-isologous carrier conjugate observed above seemed to be due to the occurrence of helper T cells reactive for the haptenic group, and to their helper activity for B cells specific for the same haptenic group, because isologous carrier is thought to be nonimmunogenic (15). In order to confirm the development of PAB-reactive helper cells by the immunization of PAB-MGG, whether or not PAB-MGG-primed cells could demonstrate their helper activity for other hapten-primed B cells through a double hapten conjugated carrier was examined. Spleen and lymph node cells (7 x 10’) taken from mice which were immunized with 100 pg of DNP-KLH in FCA 5 wk previously (for DNP-specific B cells) were mixed with spleen and lymph node cells (6 X 107) taken from mice which were immunized with 100 pg of PAB-MGG in FCA 5 wk previously and challenged with several doses of DNP-MGG-PAB as the second antigen, then transferred intravenously into recipient mice. The helper activity of PAB-MGG-primed cells was assessed by estimating the anti-DNP antibody production of the DNP-primed B cells in the recipient spleens 7 days after the secondary stimulation. As shown in Table 2, DNP-KLH-primed cells were not stimulated to produce anti-DNP antibody with hapten-heterologous carrier conjugate, DNP-MGG-PAB alone (Group 4), but were effectively stimulated in the presence of PAB-MGG-primed cells (Groups 2 and 3). Helper activity of PAB-MGG-primed cells for DNP-primed B cells increased as the dose of second antigen increased from 1 to 100 pg. This helper activity of PAB-MGG-primed cells depended on the PAB haptenic group because PAB-MGG-primed cells could not be replaced by MGG-primed cells, as shown previously ( 15). Accordingly, the development of PAB-reactive cells can be considered to be induced along with that of PAB specific B cells by the immunization of hapten-isologous carrier conjugate (PAB-MGG). Hapten-reactive helper activity was also induced by immunization with another As hapten-isologous carrier conjugate, PAB-mouse serum albumin (MSA). shown in Table 3, DNP-BaA-primed cells (7 x 107) which were immunized 5 wk previously were not stimulated to produce anti-DNP antibody with 100 pg of

SPECIFICITY

OF HAPTEN-REACTIVE

TABLE

HELPER

2

COOPERATIVE INTERACTION BETWEEN DNP-PRIMED Group no.

B cells

1 2

7 x 10’ DNP-KLHprimed cells

3

Helper

401

T CELLS

B CELLS

AND PAB-MGG-PRIMED

Anti-hapten antibody response (PFC/spleen)

2nd Ag

cells

CELLS~

Anti-DNP

Anti-PAB

3360

1200

6 X 107 PAB-MGGprimed cells

DNP-MGG-PAB

1 /.~g

6 X IO7 PAB-MGGprimed cells

DNP-MGG-PAB

10 pg

39360

20640

6 x 10’ PAB-MGGprimed cells

DNP-MGG-PAB

100 pg

66240

36000

DNP-MGG-PAB

100 pg

1920

1440

4

C-1

a 7 X 1Or spleen and lymph node cells taken from mice which were immunized with 100 rg of DNP-KLH in FCA intraperitoneally 5 wk previously were mixed with 6 X lo7 spleen and lymph node cells taken from mice which were immunized with 100 /*g of PAB-MGG in FCA intraperitoneally 5 wk previously and challenged with each second antigen, then transferred intravenously into 600 R X-irradiated recipient mice. Indirect anti-DNP and anti-PAB PFC in spleen of the recipient mice was estimated 7 days after the cell transfer.

DNP-MSA-PAB alone (Group l), but were effectively stimulated in the presence of PAB-MSA-primed cells (6 X lOi) which were immunized 4 wk previously (Group Z), This helper activity of PAB-MSA-primed cells could not be replaced by MSA-primed cells (6 x 107) (Group 3). Efect of Anti-Thywzocyte Primed Cells

Antisera

(ATS)

on the Helper Activity

of PAB-MGG-

To study whether hapten-reactive helper cells are thymus-derived lymphocytes or not, ATS was used to kill T cells in tivo. The in viva treatment of our ATS preparation damaged exclusively T lymphocytes, and not B lymphocytes, as shown TABLE HELPER ACTIVITY Group no.

B cells

1 2 3

Helper

3

OF PAB-MSA-PRIMED cells

2nd Ag

c--j 7 x 10’ DNP-BolAprimed cells

6 X 1Or PAB-MSA-primed 6 X IO’ MSA-primed

CELLS~

cells

cells

Anti-DNP antibody response (PFC/spleen)

DNP-MSA-PAB

310

DNP-MSA-PAB

6170

DNP-MSA-PAB

310

a 7 X 10’ DNP-BLuA-primed cells which were immunized 5 wk previously were challenged with 100 pg of DNP-MSA-PAB in the presence of 6 X 10’ PAB-MSA or MSA-primed cells which were immunized 4 wk previously, then transferred intravenously into recipient mice.

402

YAMASHITA,

TAKAMI

AND

KITAGAWA

in our previous report (15). Mice which were immunized with 100 pg of PABMGG in FCA 4 wk previously were treated three times with intraperitoneal injection of 0.5 ml of ATS or normal rabbit serum (NRS) 6, 4, and 2 days before harvesting the primed cells. PAB-MGG-primed cells (6 x 107) taken from those donor mice were transferred with DNP-BaA-primed cells (7 X 107) which were immunized 5 wk previously and 100 pg of DNP-MGG-PAB intravenously into 600R X-irradiated recipient mice. As shown in Table 4, DNP-BaA-primed cells were clearly stimulated to produce anti-DNP antibody by DNP-MGG-PAB in the presence of PAB-MGG-primed cells from the NRS-treated mice (Group 2)) but this helper activity of PABMGG-primed cells was completely abolished by treatment with ATS (Group 3). Furthermore, the helper activity of PAB-MGG-primed cells was also completely abolished by treatment with AKR anti-8 C3H serum and guinea pig complement, as reported in another article (23). Accordingly, these hapten-reactive helper cells were thymus-derived lymphocytes. Specificity of the PAB-MGG-Primed

Cells

It has been reported by other investigators (13, 14) that hapten-reactive T cells respond not only to the haptenic group but also to the hapten-conjugated carrier site. In order to confirm this point for the helper activity of PAB-MGG-primed cells, the following experiment was carried out using a double hapten conjugated carrier. DNP-primed B cells were mixed with PAB-MGG-primed cells and challenged with several kinds of antigen, then transferred into recipient mice. As shown in Expt I of Table 5, DNP-BcuA-primed cells (7 X 107) were clearly stimulated with 100 pg of DNP-MGG-PAB to produce anti-DNP antibody in the presence of PAB-MGG-primed cells (6 x 10’) (Groups 1 and 2). When 100 pg of DNPTABLE

4

EFFECT OF ATS-TREATMENT ON HELPER ACTIVITY OF PAB-MGG-PRIMED Group no.

B cells

Helper

cells

Anti-DNP antibody response (PFC/spleen)

DNP-MGG-PAB

3360

6 X 107 PAB-MGG-primed cells from NRS-treated mice

DNP-MGG-PAB

30960

6 X 10’ PAB-MGG-primed cells from ATS-treated mice

DNP-MGG-PAB

960

C-1 7 x 10’ DNP-BaAprimed cells

2nd Ag

CELLS”

4 7 X 10’ spleen and lymph node cells taken from mice which were immunized with 100 pg of DNP-I%YA in FCA intraperitoneally 5 wk previously were challenged with 100 pg of DNP-MGGPAB in the presence or absence of 6 X 10’ spleen and lymph node cells taken from mice which were immunized with 100 pg of PAB-MGG in FCA intraperitoneally 4 wk previously and treated with intraperitoneal injection of 0.5 ml of NRS or ATS 6, 4, and 2 days before harvesting the primed cells, then transferred intravenously into recipient mice.

SPECIFICITY

OF HAPTEN-REACTIVE

TABLE SPECIFICITY

Experiment

Group no.

HELPER

5

OF THE PAB-MGG-PRIMED

B cells

Helper

I

6 X 10’ PAB-MGGprimed cells C-1 6 X lo7 PAB-MGGprimed cells

II

107

DNP-KLHprimed cells

Anti-DNP antibody response (PFC/spleen)

DNP-MGG-PAB

3440

DNP-MGG-PAB

207360

DNP-KLH-PAB DNP-KLH-PAB

13600 42400

DNP-MGG

1920

6 X lo7 PAB-MGGprimed cells

DNP-MGG

2880

C-1 5 x

2nd Ag

(-)

7 8

HELPER T CELLS”

cells

(6) 7 x 107 DNP-&Aprimed cells

403

T CELLS

5 X 107 PAB-MGGprimed cells

DNP-MGG-PAB

3120

DNP-MGG-PAB

31280

9

(-)

DNP-HGG-PAB

720

10

5 x 107 PAB-MGGprimed cells

DNP-HGG-PAB

13840

(1In Expt I, 7 X 107 DNP-BcuA-primed cells which were immunized 7 wk previously were challenged with 100 pg of each second antigen in the presence or absence of 6 X 107 PAB-MGGprimed cells which were immunized 5 wk previously. In Expt II, 5 X 107 DNP-KLH-primed cells which were immunized 6 wk previously were challenged with 100 rg of each second antigen in the presence or absence of 5 X 107 PAB-MGG-primed cells which were immunized 4 wk previously.

KLH-PAB was used as the second antigen, the helper activity of PAB-MGGprimed cells was weak although significant (Groups 3 and 4). PAB-MGG-primed cells could not demonstrate their helper activity by DNP-MGG (Groups 5 and 6). As shown in Expt II of Table 5, however, clear helper activity of PAB-MGGprimed cells (5 X 107) for DNP-KLH-primed cells (5 x 107) could be demonstrated using 100 pg of DNP-HGG-PAB to be the same as with DNP-MGG-PAB (Groups 7, 8, 9, and 10). HGG but not KLH resembles MGG in composition, so PAB-MGG and PABHGG may be considered to form a similar microenvironment with regard to the PAB-conjugated carrier site. Accordingly, PAB-MGG-primed helper T cells seem to recognize not only the haptenic group but also structural differences of the hapten conjugated carrier site. This datum confirms the specificity restrictions of T cells reported by other investigators (13, 14). Can PAB-MGG-Primed Groups ?

Helper

T Cells Discriminate

Among Related Haptenic

As demonstrated in the above experiments, the specificity of PAB-MGG-primed cells was thought to be directed not only toward the haptenic group but also to the hapten-conjugated carrier site. Immediately, a question arises as to whether these

404

YAMASHITA,

TAKAMI

AND

KITAGAWA

helper T cells can discriminate among other related haptenic groups. This question was studied by the following experiments. Spleen and lymph node cells (7 x 107) f rom mice which were immunized with 100 pg of DNP-KLH in FCA 5 wk previously were mixed with spleen and lymph node cells (6 X 10’) from mice which were immunized with 100 pg of PAB-MGG or p-azobenzene sulfonate (PABS) -MGG in FCA 8 wk previously and challenged with 100 ,ug of DNP-MGG-PAB or DNP-MGG-PABS, then transferred into recipient mice. As shown in Table 6, DNP-KLH-primed cells were markedly stimulated with DNP-MGG-PAB to produce anti-DNP antibody in the presence of PAB-MGG-primed cells (Group 2)) but weakly in the presence of PABS-MGGprimed cells (Group 3), although a fairly good cross-reaction was observed between PAB- and PABS-reactive helper T cells. When DNP-MGG-PABS was used as the second antigen, similar specificity was observed (Groups 4, 5, and 6). Accordingly, these helper T cells were thought to be able to discriminate the structural difference among related haptenic groups. However, the cross-reaction of this helper activity may be due not only to the cross-reaction of each haptenic group but also to the hapten-conjugated carrier site common to both haptens. of the Specificity Restrictions

Comparison

of Hapten-Specific

B and Helper T Cells

B cells have been known to have immunoglobulin-like receptors on their surface, and demonstrate high specificities of the antigenic determinants, Helper T cells can also discriminate among related haptenic groups as demonstrated in the above experiments. Then, a question arises as to whether B and helper T cells have the same specificity restrictions to the same haptenic groups.

TABLE

6

SPECIFICITY OF HAPTEN-REACTIVE B cells

Expt group

Helper cells

C-1

1 2 3 7 x

107

DNP-KLHprimed cells

HELPER T CELLS” 2nd Ag

Anti-DNP antibody response (PFC/spleen)

DNP-MGG-PAB

960

6 X 10’ PAB-MGGprimed cells

DNP-MGG-PAB

188640

6 X 10’ PABS-MGGprimed cells

DNP-MGGPAB

47520

DNP-MGG-PABS

19680

5

6 X 1Or PAB-MGGprimed cells

DNP-MGG-PABS

203520

6

6 X 1Or PABS-MGGprimed cells

DNP-MGG-PABS

460800

4

(-)

a 7 X 10’ DNP-KLH-primed cells which were immunized 5 wk previously were challenged with 100 rg of DNP-MGG-PAB or DNP-MGG-PABS in the presence of 6 X 10’ PAB-MGGor PABS-MGG-primed cells which were immunized 8 wk previously and then transferred intravenously into recipient mice.

SPECIFICITY

OF HAPTEN-REACTIVE

TABLE

HELPER

7

SPECIFICITY OF HAPTEN-REACTIVE

Expt

B cells

Helper cells

HELPER T CELLS”

2nd Ag

group

1

3

4 5 6

7 x 10’ DNP-KLHprimed cells

Anti-DNP antibody response (PFC/spleen)

DNP-MGG-MAB

5520

DNP-MGG-MAB

72480

6 X 107 PAB-MGGprimed cells

DNP-MGG-AMAB

15680

c--j 6 X 107 MAB-MGGprimed cells

DNP-MGG-PAB

2470

DNP-MGG-PAB

48480

6 X lo7 PAB-MGGprimed cells

DNP-MGG-PAB

102720

C-1 6 X lo7 MAB-MGGprimed cells

2

405

T CELLS

a 7 X 107 DNP-KLH-primed cells which were immunized 5 wk previously were challenged with 100 pg of DNP-MGG-MAB or DNP-MGG-PAB in the presence of 6 X 10’ MAB-MGG- or PAB-MGG-primed cells which were immunized 4 wk previously, then transferred intravenously into recipient mice.

This question was studied by measuring their responsiveness to the same haptenic group. Spleen and lymph node cells (7 x 10’) from mice which were immunized with 100 pg of DNP-KLH in FCA 5 wk previously were mixed with spleen and lymph node cells (6 x 107) from mice which were immunized with 100 rg of m-azobenzoate (MAB)-MGG or PAB-MGG in FCA 4 wk previously and challenged with 100 pg of DNP-MGG-MAB or DNP-MGG-PAB, then transferred into recipient mice. The responsiveness of T cells was assessed by estimating helper activity for anti-DNP antibody production and that of B cells by estimating antiMAB and anti-PAB antibody responses. As shown in Table 7, DNP-KLH-primed cells were clearly stimulated with DNP-MGG-MAB to produce anti-DNP antibody in the presence of MAB-MGGprimed cells (Group 2) but less so in the presence of PAB-MGG-primed cells (Group 3). When DNP-MGG-PAB was used as the second antigen, similar specificity was observed (Groups 4, 5, and 6). Accordingly, azobenzoate-reactive helper T cells are thought to be able to discriminate between meta and para derivatives of the azobenzoate group. To study the specificity restrictions of hapten-specific B cells in the same experiment, anti-MAB and anti-PAB PFC were assayed. As shown in Table 8, MAB-MGG-primed cells were clearly stimulated to produce anti-MAB antibody by MAB-MGG, but hardly by PAB-MGG (Groups 7 and 2). PAB-MGG-primed cells were also stimulated only by PAB-MGG to produce anti-PAB antibody, but hardly by MAB-MGG (Groups 3 and 4). Furthermore, anti-hapten antibody produced in response to the homologous hapten-carrier conjugates reacted only to the homologous hapten-conjugated SRBC to form plaques (Groups 1 and 4).

406

YAMASHITA,

TAKAMI

TABLE

AND

KITAGAWA

8

SPECIFICITY OF HAPTEN~PECIFIC Expt group

1 2 3 4 5 6

Hapten-primed

6 6 6 6 6 6

X X X X X X

2nd Ag

cells

10’ MAB-MGG-primed 107 MAB-MGG-primed 107 PAB-MGG-primed lo7 PAB-MGG-primed lo7 Nonprimed cells lo7 Nonprimed cells

B CELLS”

cells cells cells cells

MAB-MGG PAB-MGG MAB-MGG PAB-MGG MAB-MGG PAB-MGG

Anti-hapten antibody response (PFC/spleen) Anti-MAB

Anti-PAB

14240 1120 2160 1120 2480 ND

3120 6320 1120 27200 ND 1120

n 6 X lo7 each hapten-primed cells which were immunized 4 wk previously or nonprimed cells were challenged with 100 pg of DNP-MGG-MAB or DNP-MGG-PAB and transferred intravenously into recipient mice. Anti-MAB and anti-PAB PFC in spleen of the recipient mice was estimated 7 days after the cell transfer. ND = not done.

Hapten-reactive helper activity demonstrated a fair cross-reaction between the related haptenic groups, but hapten-specific B cells demonstrated only a minimal cross-reaction. Thus, hapten-specificity is much more restricted for hapten-specific B cells than for hapten-reactive helper T cells. DISCUSSION In recent years evidences have accumulated that T cells as well as B cells possess specific receptors for antigen (4, 7, 8). It is well established that the receptors of B cells are immunoglobulin-like, with specificities similar to that of humoral antibody (3, 4, 5). However, the nature and the specificity of T cell receptors is not fully understood and is rather controversial. Some investigators have reported that T cell receptors are immunoglobulin-like and similar to B cell receptors in their specificities (24227). On the other hand, several investigators have suggested that the specificity of T cell receptors is different from that of B cells (28231) and that T cells respond to a different portion of the antigenic site than do B cells (3233). In the study of the specificity restrictions of T cell receptors and the nature of the determinant to which T cells respond, it seems very useful to use a determinant with known structure, a haptenic group. Recently it has been reported that haptenreactive T cells could be developed by 2, 4-dinitrofluorobenzene-painting (9, 10) and also by the immunization of hapten-isologous carrier conjugates in mice (11214). However, it was still unclear whether the hapten-reactive T cells had the same specificity as hapten-specific B cells and humoral antibody or not. In our previous report (15) we established that hapten-reactive helper T cells could be developed along with hapten-specific B cells by the immunization of haptenisologous carrier conjugate in mice, and the former cells could cooperate with other hapten-primed B cells through a double hapten conjugated carrier. We can analyze

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more clearly the specificity restrictions of helper T cells and compare the specificity differences between helper T cells and B cells to the same determinant by using this system. In the present study we attempted to analyze the specificity restrictions of T cells and compare them with those of B cells using a double hapten conjugated carrier as antigen. Anti-PAB antibody response was induced by the immunization of the haptenisologous carrier conjugate PAB-MGG as well as with DNP-MGG (Table 1). Spleen and lymph node cells taken from mice which were immunized with PABMGG could help anti-DNP antibody production of DNP-primed B cells through DNP-MGG-PAB (Table 2). This helper activity was hapten-reactive because the helper activity of PAB-MGG-primed cells could not be replaced by MGG-primed cells [reported in the previous article (15) ] or MSA-primed cells, This haptenreactive helper activity was also developed by the immunization of PAB-MSA (Table 3). Hapten-reactive helper activity was T cell in origin because the helper activity of PAB-MGG-primed cells was completely abolished by treatment with ATS or with anti-8 serum and complement (Table 4). There are some reports that hapten-reactive T cells respond not only to the haptenic group but also to the hapten-conjugated carrier site (13, 14). Rubin et ~2. (13) reported that DNPMSA-reactive T cells had specificity for new antigenic determinants introduced into the protein carrier by the hapten coupling. Moorhead et al. (14) reported that the responsiveness of 4-hydroxy-3-iodo-5-nitrophenyl acetic acid (NIP)-MGGprimed cells to NIP-MGG was not completely inhibited by the addition of NIPepsilon-aminocaproic acid. As shown in Table 5, the hapten-conjugated carrier site fairly contributed to the hapten-reactive helper T cells to demonstrate their helper activity, for DNP-primed B cells were clearly stimulated with DNP-MGGPAB and DNP-HGG-PAB to produce anti-DNP antibody, but only weakly with DNP-KLH-PAB, in the presence of PAB-MGG-primed cells. MGG and KLH are very different in their physicochemical properties. However, MGG and HGG are similar in composition, so PAB groups are thought to conjugate to a similar portion of the carrier protein to form similar microenvironments for the PAB conjugated carrier site. Accordingly, PAB-MGG-primed cells are thought to recognize not only the haptenic group but also structural differences of the hapten-conjugated carrier site. However, these helper cells could discriminate the structural differences among related haptenic groups, and between para and meta derivatives of azoproteins (Tables 6 and 7). These data suggest that the haptenic group has a major role in the development of hapten-reactive helper T cells, although these helper cells responded to the hapten-conjugated carrier complex. The specificity restrictions of T and B cells to the same haptenic group were compared by measuring the responsiveness to the second antigen. T cell activity was assessed by helper activity and B cell activity by anti-hapten antibody production. Table 7 showed that the helper activity demonstrated a fairly strong cross-reaction between meta- and para-derivatives of azoproteins. This cross-reaction might result from the possibility that these helper T cells responded not only to the haptenic group but also to the hapten-conjugated carrier site. However, B cells showed strict hapten-specificity and noncross-reactivity (Table 8). These results suggest that the responsiveness of B cells and helper T cells to the same haptenic group is different. Why did these differences in responsiveness develop ?

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Firstly, it might derive from differences in the sensitivity of the responsiveness of B and helper T cells to the second antigen. That is, the 100 pg of second antigen used in Expts 7 and 8 may be insufficient, so that only helper T cells are able to respond to such doses of second antigen to help B cells, while B cells are not able to respond such doses to produce anti-hapten antibody. As shown in Table 2, however, B cells and helper T cells specific for the PAB determinant equally responded to various doses of second antigen. Accordingly, these differences cannot be thought to derive from sensitivity differences in the responsiveness of B and helper T cells. Secondly, variations in responsiveness may be considered to derive from a difference in the development of B and helper T cells. There is a possibility that the agents which we used to prepare hapten-carrier conjugates contained other agents, and these contaminants stimulated preferentially helper T cells but not B cells. Thus, only hapten-reactive helper T cells may be able to develop and to demonstrate their helper activity with other haptenic groups, while B cells do not. However, differences in the development of B and helper T cells were not observed with the doses of primary antigen which we used, varying from one to 500 pg (34). Furthermore, B and helper T cells equally responded when various numbers of primed cells were used (these data are not here included). Therefore, differences in the development of B and helper T cells using hapten-isologous carrier conjugate as antigen appears not to be a tenable explanation. Thirdly, there is a possibility that the specificity restrictions of T cells are different from that of B cells. We consider that the differences in the responsiveness of B and helper T cells are most likely derived from specificity differences between B and helper T cell receptors. In recent years there have been several reports that the specificity restrictions of T cell receptors are different from that of B cells (28~33). When heterologous protein is used as antigen, however, it is very difficult to discuss the specificity restrictions of B and T cells on the same level because protein antigens have heterogeneous determinants and B and T cells may thus recognize different portions of the same antigen. It seems very useful to use B and T cells specific for the same haptenic group when discussing the specificity restrictions of their receptors. We have established that B and helper T cells reactive for the same haptenic group could develop by the immunization of hapten-isologous carrier conjugate. When we compared the responsiveness of B and helper T cells using this system, results which indicated that the specificity restrictions of B and T cells were different were obtained: B cells were effectively stimulated with only the same haptenconjugated carrier (Table 8), but helper T cells could respond to another haptenconjugated carrier to demonstrate their helper activity (Tables 6 and 7). These variations in the responsiveness of B and helper T cells are thought to derive from the differences in the specificity restrictions of B and T cell receptors. B cell receptors are apparently directed only to the haptenic group, while T cell receptors are directed not only to the haptenic group but also to the hapten-conjugated carrier site. Why are B cell receptors directed only to the haptenic group, while T cell receptors are not? Firstly, the affinity of T cell receptors were thought to be lower than that of B cells. As B cell receptors are possibly higher in affinity, B cells would be stimulated

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with only the haptenic group, but it may be required for the contributions of the hapten-conjugated carrier site, such that T cells with low affinity receptors respond to the haptenic group and are thus stimulated. Secondly, T cell receptors may require a physicochemically different nature of the antigenic site than do B cell receptors. Our data confirmed that the specificity restrictions of T cell receptors were different from those of B cells. However, it is still unknown what determine these differences. Recently, Hammerling et nl. (35) reported about the different specificities of B and T cells. They observed that antigen binding B cells specific for (T, G)-A- -L, were inhibited by (T, G)-A- -L, (Phe, G) -A- -L and (I-I, G)-A- -L but that antigen binding T cells specific for (T, G)-A- -L were inhibited only by (T, G)-A- -L. These data are clearly different from ours. Namely, we suggest that B cells are more hapten specific than helper T cells, but in their data antigen-binding T cells are thought to be more antigen specific than antigen-binding B cells. However, these ostensibly conflicting results may not be discussed at the same level, as the experimental procedures were different from each other. Firstly, there is a difference of antigen used. (T, G)-A- -L is a rather large and heterogeneous antigen. Accordingly, antigen binding T cells may recognize different portions of the determinant than do antigen binding B cells. Secondly, there may be sensitivity differences between the two experimental procedures. They compared it with the technique of antigen binding cells and we compared it with responsiveness. Antigen binding cells are rather large populations, but only a few of them can demonstrate their helper activity. Thirdly, we may be observing divergent phenomena. Elliott et al. (36) reported that antigen binding B cells were precursors of antibody producing cells but antigen binding T cells were not helper cells, so that there may be different mechanisms for antigen binding and helper activity. As T cells are very heterogeneous population, when the specificity restrictions of T cell receptors were studied, the cell lines of T cell subsets must be studied more clearly. Recently, the relationship of T cell receptors and immune response (Ir) gene products linked to the major histocompatibility antigens has been discussed (37, 38). There is a report that antigen-binding B cells were inhibited by anti-IgG serum but antigen-binding T cells were not so inhibited, but were inhibited by anti-H-2 serum (39). Accordingly, antigen binding receptors of T cells may be different from those of B cells (immunoglobulin) . As we showed in this report, the specificity restrictions of T cell receptors were different from those of B cells. T cells may thus recognize antigen by a different mechanism than do B cells. We also showed that T cells can discriminate the structural difference among related haptenic groups as well as B cells do. If the T cell receptors are not immunoglobulin, it will be very interesting to discern exactly how T cells can discriminate those structural differences of antigen. In any case, the specificity restrictions and the nature of the T cell receptors must be further explored in relation to the B cell receptors. Such studies are now being conducted in our laboratory, using hapten-isologous carrier conjugate as antigen.

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ACKNOWLEDGMENTS The authors are deeply grateful to Dr. T. Hamaoka for his helpful discussions. We thank Mr. K. Sonomura and Miss M. Hanada for skilled technical assistance and Miss R. Yamada for excellent secretarial assistance in the preparation of this manuscript.

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