Immune response gene control of antibody specificity

CELLULAR

69, 128-137

IMMUNOLOGY

Immune

Response

Gene

ANTONIO CAMPOS-NETO,* Sidney

Farber

Cancer Received

(1982)

Control

HERBERT

Institute, January

Harvard

of Antibody

LEVINE, Medical

29, 1982;

AND STUART

School,

accepted

Specificity’

Boston,

February

F. SCHLOSSMAN

Massachusetts

02115

11, 1982

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F, (2 X 13) guinea pigs, immunized with the cYDnp-Lys,, produce both T cells and antibody which are equally discriminatory for c~Dnp-Lyse. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with oDnp-Lyss do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F, (2 X 13) environment, PLL Ir genenegative B cells are provided with F, (2 X 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.

INTRODUCTION A considerable amount of information exists indicating that both T and B cells bear antigenic recognition structures as defined by anti-idiotypic antibody and by isolation of V gene products (l-5). Similarly, studies with Dnp-oligolysines in guinea pigs also indicate a comparable repertoire of specificities in both T and B cells (6-15). In the guinea pigs, the immune response to synthetic poly-L-lysine polymers (PLL)3 is controlled by major histocompatibility (MHC) immune response (Ir) genes ( 16, 17). Responder guinea pigs immunized with Dnp-oligolysines develop T-cell-mediated immune responses and antibodies which are both highly specific for the immunizing antigen and can readily discriminate closely related Dnp-oligolysines (10). In contrast, nonresponder animals lack a specific T-cell response to DNP-oligolysines and produce antibody which is only Dnp-specific and as such cannot differentiate Dnp-oligolysines from dinitrophenol. It has been suggested that in the guinea pig, the cellular locus of the histocom’ Supported by NIH Grant 5R Al 12069-08, NC1 Grant CA 19589-06, and by Capes, Brazil. * Present address: Departamento de Imunologia, Instituto de Microbiologia da UFRJ, Caixa Postal 68040, Rio de Janeiro, R.J., Brazil. ’ Abbreviations used: Dnp, 2,4-dinitrophenyl; Lys, L-lysine; Ala, L-alanine; “B” guinea pigs, guinea pigs deprived of T cells; PLL, synthetic poly-L-lysine polymers; Ir gene, immune response gene; CFA, complete Freund’s adjuvant; PBS, phosphate-buffered saline; MHC, major histocompatibility; PFC, plaque-forming cells. 128 0008-8749/82/070128-10$02.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Ir GENE

CONTROL

OF ANTIBODY

SPECIFICITY

129

patibility-linked Ir gene is expressed primarily at the level of the T cells and macrophages (18, 19). This suggestion is based on the experimental evidence that in vitro proliferation of primed T cells to macrophages pulsed with antigens under Ir gene control required MHC identity between the lymphocytes and the macrophages used for primary sensitization. However, in experiments in which the alloreactive clones of T cells were eliminated (20) or selected antigen-reactive T-cell clones were used (2 1), no such restriction could be observed; i.e., the antigen presentation is unimpaired in nonresponder macrophages. Thus, it is possible that the failure of nonresponder guinea pigs to make responder-type antibody is a consequence of either suppression or absence in these animals of specific B- or T-cell clones or a combination of both. Recently (15) we have shown that PLL Ir genepositive guinea pigs (strain 2) deprived of T cells by adult thymectomy, lethal irradiation, and reconstitution with syngeneic bone marrow cells behaved like PLL Ir gene-negative animals after immunization with aDnp-Lysg. These animals failed to produce T-cell responses and the anti-Dnp antibody synthesized, although primarily IgG2 and restricted in heterogeneity, could not discriminate the immunizing antigen from dinitrophenol. Reconstitution of these “B” guinea pigs with normal syngeneic T cells restored their ability to develop T-cell-mediated reactions and their capacity to make highly specific antibody. These observations suggested the possibility that nonresponder animals had the identical B-cell repertoire of the responders and that their incapability to produce discriminatory antibody was therefore due to a defect in their specific T-cell clones. In the present studies we attempted to investigate this possibility. The results indicated that B cells from strain 13 guinea pigs (nonresponder), if provided with help of PLL Ir gene-positive T cells from F, (2 X 13) guinea pigs, have the ability to produce antibodies of high specificity, similar if not identical to the ones produced by B cells of strain 2 (responder) guinea pigs. These studies support the notion that the cellular locus for PLL Ir gene expression in the guinea pig is limited to T cells and not possessed by antigen-presenting cells or B lymphocytes.

MATERIALS Animals

AND

METHODS

and Immunizations

Inbred strain 2, strain 13, and F, (2 X 13) guinea pigs (both sexes), weighing 400 to 500 g and raised in our animal facilities, were used in all studies. Each animal was injected with 150 pg of antigen in phosphate-buffered saline (PBS) emulsified with an equal volume of complete Freund’s adjuvant (CFA) containing 1 mg/ml of Mycobacterium tuberculosis H,,Ra (Difco Laboratories, Detroit, Mich.). Each guinea pig received a total of 1 ml of the CFA-antigen emulsion into the four footpads and neck. Peptides Synthetic Dnp-oligolysine peptide antigens were prepared as described previously (11, 14). For this study the following peptides were used: aDnp-Lys3, &np-Lys,, aDnp-Lys,, LuDnp-Lysg, aDnp-Lys,S, tDnp-Lys,, tDnp-LysB, tDnp-Lysg, and Lys4Ala,-Lys (Dnp).

130

CAMPOS-NETO,

In Vitro Antigen-Znduced

LEVINE,

Incorporation

AND

SCHLOSSMAN

of [‘HI Thymidine

Immunized guinea pigs were killed by bulbar dislocation between 2 and 3 weeks after immunization. Inguinal and axillary lymph nodes were aseptically removed and teased in RPM1 1640 (Gibco, Grand Island, N.Y.), supplemented with 1% Lglutamine (200 mM), 1% penicillin-streptomycin (5000 units penicillin and 5000 pg streptomycin/ml), and 10% normal guinea pig serum (Rockland, Gilbertsville, Pa.). The cells were counted and the viability was assessed by trypan blue exclusion. Dead cells were removed as described previously (22). Viability greater than 95% was invariably obtained. For antigen-induced proliferation 2 X lo5 cells in a volume of 0.2 ml were cultured in round-bottomed Microtest II plates (Falcon Plastics, Oxnard, Calif.). Cultures were performed in triplicate, in the presence of various concentrations of the homologous (immunizing) and heterologous antigens. After 48 hr of incubation at 37°C in a humidified atmosphere of 95% air and 5% CO*, the cultures were pulsed with 20 ~1 of RPM1 solution containing 0.2 @i of [3H]thymidine (sp act 1.9 Ci/mmol, SchwarzMann, Orangeburg, N.Y.). After an additional 24-hr incubation, the cultures were harvested on a MASH II apparatus (Microbiological Associates, Bethesda, Md.). The incorporation of [3H]thymidine was measured by scintillation spectroscopy. Results are expressed as stimulation index which is the ratio of [3H]thymidine incorporation (cpm) by 2 X lo5 cells in the presence of antigen to incorporation (cpm) by unstimulated cultures. Antibody

Response

The production of anti-Dnp antibody was assayed by direct and indirect passive hemagglutination by using dinitrophenylated sheep red blood cells (SRBC) and rabbit anti-guinea pig immunoglobulin. The Dnp was coupled to the erythrocytes by conjugation of N-( 2,4-dinitrophenyl)-P-alanylglycyl azide (Regis Chemical, Norton Grove, Ill.) to fresh SRBC as described (23). In some experiments a hemolytic plaque assay was performed as described previously (24). Antibody

Specificity

The specificity of the antibodies was measured by fluorescence quenching. Free binding energy of interaction between purified anti-Dnp antibody and Dnp-oligolysines was calculated as described previously (25, 26). The purification of the antiDnp antibody was performed with a Dnp-HSA-bromoacetyl-cellulose immunoabsorbant column as previously reported (27). The concentration of purified antibody was evaluated by spectrophotometry. The value of the average intrinsic association constant, K,, was obtained and converted to -AF,, the standard free energy, by the formula -AE’, = RT In KO. Values for -AF’, had confidence limits at the 0.05 level between +- 150 cal/mol and duplicate titrations for the same antibody and the same antigen done on different days did not differ by more than 100 cal/mol. Hence, -aFO values differing by more than 200 cal/mol were considered significant. B Guinea Pigs

B guinea pigs were prepared by thymectomy of normal adult strain 2 and F, (2 X 13) guinea pigs and lethal irradiation (1000 rad) on Day 15. Immediately

Ir GENE

CONTROL

OF ANTIBODY

131

SPECIFICITY

following irradiation the animals were reconstituted with 100 X lo6 syngeneic or semisyngeneic bone marrow cells. Thirty days later the guinea pigs were immunized as described above. Purification

of T Cells

T cells were obtained by passing spleen and lymph node cells through a Sephadex G-200 rabbit anti-guinea pig column as described previously (24). For the majority of the experiments the resulting T cells were further treated with specific anti-Ia antiserum and guinea pig complement for 45 min in order to eliminate any residual B cells. The anti-Ia antisera were produced in inbred strains 2 and 13 as described (28). RESULTS Specificity

of the in Vitro Response

to Dnp-Oligopeptides

Strain 13 (nonresponder) and F, (2 X 13) (responder) guinea pigs were immunized with aDnp-Lys9, and tested 2-3 weeks following immunization. Both lymph nodes and sera were obtained from these animals. The specificity of the purified antihapten antibody was studied by fluorescence quenching. The specificity of cellular immune response was analyzed by antigen-induced incorporation of [ ‘Hlthymidine. As expected, lymph node cells from strain 13 guinea pigs did not incorporate thymidine when stimulated in culture with aDnp-Lys9 or related peptides. Nevertheless these cells responded very well to PPD and concanavalin A (data not shown). The anti-Dnp antibody purified from these strain 13 guinea pigs could not discriminate aDnp-Lys, from related peptides as is shown in Fig. 1. The binding energy for Dnp-Lys, was comparable to that obtained for six other related

m

d, D~P-LY~~

a

Q, Dnp-Lysis

0

d, Drip-

0

6. Dnp-Lysg

0

DnvOH

Lys5

d, Drip - Lyse -

Q, DIP-

EXPERIMENT FIG. 1. Binding energies (-AF,) from strain 13 guinea pigs.

Lys,-

LYS~

I

EXPERIMENT

of Dnp-oligolysines

2 and dinitrophenol

Alo3

-Lys(Dnp)

EXPERIMENT to anti-olDnp-Lys,

3 antibody

132

CAMPOS-NETO,

I Q,Dnp-Lys5 III Q,Dnp - Lyse I Q,Dnp-Lys9

LEVINE,

SCHLOSSMAN

I Q,Dnp-Lyslo Ei E, Dnp- Lyss I Lys4-Ala3Lys(Dnp)

- A. Cells

Experbment

AND

ii3 Dnp-OH

8. Antibody

I

Experiment

2

Experiment

I

Experiment

2

FIG. 2. Proliferative response of lymph node cells (A) and binding energies (-AF,) of purified antibody (B) from F, (2 X 13) guinea pigs immunized with aDnp-Lys,. The results of incorporation index express the maximal responses obtained to several doses of the peptides used.

Dnp-oligopeptides and for the Drip-OH itself. Thus in strain 13 animals, there is an absence of both specific T- and specific B-cell responses for cyDnp-Lys,. In contrast, lymph node cells obtained from F, (2 X 13) guinea pigs equally to cells from strain 2 animals were strongly stimulated in culture to proliferate in response to the homologous immunizing antigen and closely related peptides (Fig. 2A). It is apparent that aDnp-Lys, produced best stimulation. The incorporation results shown are the maximal responses obtained to several doses of the peptides. As indicated, cYDnp-Lys5, Lys4-Ala,-Lys (Dnp) and Dnp-OH were nonstimulatory, whereas cross-reaction was seen with cuDnp-Lys8, oDnp-LyslO, and CDnp-Lys9. Similarly to the cells, the antibody produced by these F, (2 X 13) guinea pigs could discriminate the immunizing aDnp-Lys, from other related peptides. As shown in Fig. 2B the maximal binding energy is obtained with cuDnp-Ly+. Peptides containing fewer or more lysine residues resulted in a decrease in binding energy compared to cYDnp-Lys9 as well as for EDnp-Lys9, Lys-4-Ala,-Lys (Dnp), and DnpOH. Thus, both in vitro T-cell responses as well as antibody produced in responder animals showed exquisite specificity for the homologous immunizing agent. Ability

of la-Negative

T Cells to Promote

the Emergence of Specific B-Cell clones

Since we have recently shown (15) that T cells reconstituted the specific immune response of strain 2 B guinea pigs, we now investigated the ability of the Ia-negative

Ir GENE

CONTROL

OF ANTIBODY

SPECIFICITY

133

T cells to perform this function. For this purpose, eight adult strain 2 guinea pigs were thymectomized, lethally irradiated, and reconstituted with lo8 bone marrow cells from a single strain 2 donor. One group received intravenously different numbers of untreated Sephadex G-200 anti-F(ab’),-purified strain 2 T ceils. The second group received these same cells, but treated with anti-strain 2 Ia serum plus complement. Two days later all guinea pigs were immunized with cYDnp-Lys9 in CFA. Three weeks later the animals were killed and their immune responses were studied. Guinea pigs which were not reconstituted with T cells did not manifest cell-mediated immune response (delayed-type hypersensitivity to aDnp-Lys9 and to PPD or in vitro proliferation to these antigens (data not shown). The antibody they produced, as expected, could not discriminate cYDnp-Lys, from Dnp-OH (Fig. 3-groups which did not receive T cells). In contrast the animals which received T cells developed cell-mediated responses (in vivo delayed hypersensitivity and in vitro lymphocyte proliferation to antigens and mitogens-data not shown) and the antibody they produced was highly specific for cu-Dnp-Lysg. It is clear from the results shown in Fig. 3 that the reconstitution of the antibody specificity is achieved either with untreated T cells or with Ia-negative T cells. In addition, this specificity starts to be manifested with as few as 5 X IO6 Ia-negative T cells. Ability of B Cells from Strain 13 (Nonresponder) Animals to Cooperate Ir Gene-Positive T Cells from F, (2 X 13) Guinea Pigs

with PLL

We next investigated the ability of B cells from nonresponder animals to cooperate with PLL Ir gene-positive T cells from F, (2 X 13) guinea pigs. Normal F, (2 X 13) guinea pigs were thymectomized, lethally irradiated on Day 15, and reconstituted with 1 X lo8 strain 2 or strain 13 bone marrow cells (Fig. 4). One month later the B animals were given 1 X lo8 normal F, T cells intravenously. The T cells were obtained as already mentioned. In addition, the T cells, transferred to F, B guinea pigs reconstituted with strain 2 bone marrow cells, were treated

NUMBER FIG.

from (B).

OF

T CELLS

3. Binding energies (-MO) of Dnp-oligolysines B guinea pigs reconstituted with normal untreated

(X106)

TRANSFERRED

and dinitrophenol to anti-aDnp-Lys, antibody T cells (A) and with normal Ia-negative T cells

134

CAMPOS-NETO,

LEVINE,

AND

SCHLOSSMAN

FI ( 2 x 13 )

4 THYMECTOMY

DAY

DAY

15

LETHAL

IRRADIATION

(lOOOr)

I

AND

/

DAY 42

13 BONE

,?L.-

DAY 60

MARROW

STRAIN CELLS

I

I

t

RECONSTITUTION WITH NORMAL F1(2xl3) T CELLS TREATED WITH ANTI Ia SERUM (STRAIN 13 ANTI STRAIN 21 AND COMPLEMENT

IMMUNIZATION a, Drip-LYS,

FOR

CELLULAR

FIG. 4. Experimental protocol for the cooperation with PLL Ir gene-positive T cells.

MARROW

RECONSTITUTION WITH NORMAL Fa(Zxl3) T CELLS TREATED WITH ANTI Ia SERUM (STRAIN 2 ANTI STRAIN 13) AND COMPLEMENl

1 ASSAY

2 BONE

ti

DAY 44

WITH

\

i, STRAIN CELLS

RECONSTITUTION

AND

J

WITH

ANTIBODY

of B cells from

SPECIFICITY

PLL

Ir gene-negative

guinea

pigs

with strain 2 anti-strain 13 serum (anti-Ia serum) and complement. T cells transferred to F, B guinea pigs reconstituted with strain 13 bone marrow cells were treated with strain 13-anti-strain 2 serum and complement. These animals as well as those reconstituted with B cells alone were immunized with cYDnp-Lys9 in complete Freund’s adjuvant and the antibody produced was analyzed 15 days later. All animals produced anti-Dnp antibody as assayed by plaque-forming cells (PFC) using Dnp-sheep red blood cells (Dnp-SRBC) as targets. Moreover, all animals produced quantitatively similar amounts of antibodies (about 150 pg/ml), predominantly of IgG, class (defined by specific antisera) and highly restricted in heterogeneity when analyzed by isoelectric focusing (data not shown). In each case the antibody produced by F, B guinea pigs was synthesized by cells of the donor bone marrow cell lineage. Thus, the anti-Dnp PFC from F, B guinea pigs, reconstituted with strain 2 bone marrow cells, were totally abrogated after treatment with strain 13-anti-strain 2 serum and complement. Treatment of these cells with strain 2-anti-strain 13 serum and complement had no effect. The reverse situation occurred with the F1 B guinea pigs reconstituted with strain 13 bone marrow cells. Strain 2-anti-strain 13 serum and complement abolished their anti-Dnp PFC while strain 13-anti-strain 2 serum and complement had no effect (Table 1). As shown in Fig. 5, purified anti-Dnp antibody from F, B guinea pigs reconstituted with either strain 2 or 13 bone marrow cells alone showed no specificity for

Ir GENE

CONTROL

OF ANTIBODY TABLE

Origin Source of bone marrow (B) cells Strain

Strain

’ Normal ’ Means

2

13

of the Anti-Dnp

Antibody-Forming

135

SPECIFlCITY

1 Cells of F, (2 X 13) B Guinea Dnp

Number of F, T cells transferred

Cell treatment

Direct SO6 8 80

Pigs

PFC/lO’

cells Indirect

0

NGPS + C” 13-anti-2 + C 2-anti-13 + C

158 8 169

IO8

NGPS + C 13-anti-2 + C Z-anti-13 + C

110 9 178

178 12 280

0

NGPS + C 13-anti-2 + C 2-anti-13 + C

131 138 23

220 225 35

lo8

NGPS + C 13-anti-2 + C 2-anti-13 + C

85 124 7

289 283 22

guinea pig serum plus complement. of results of three experiments.

the Dnp-peptides. Very interesting was the demonstration that F, T cells could provide equal help for the B cells from Ir gene-positive or -negative animals for the production of highly specific antibodies. DISCUSSION In the present studies we have investigated the cellular location of the PLL Ir gene defect in inbred strain guinea pigs. As is already known, strain 2 guinea pigs immunized with Dnp-oligolysines develop both T cells and antibody which are highly specific and discriminatory for the immunizing peptide. Strain 13 guinea pigs, in contrast, do not develop specific T-cell responses to these antigens and the antibodies they produce are only Dnp specific. These observations suggest that selection of specific B-cell clones is dependent on the presence of specific T cells (15). Strain 2 guinea pigs deprived of T cells by adult thymectomy, lethal irradiation, and reconstitution with syngeneic bone marrow cells produced antibody to aDnp-Lysg, highly restricted in heterogeneity but, similar to the nonresponder type of antibody, did not distinguish the immunizing peptide from closely related ones. However, the B guinea pigs once reconstituted with normal T cells recovered their ability to synthesize very specific antibody. These studies led us to investigate the repertoire of specificity of Ir gene-negative B cells if they could cooperate with Ir gene-positive T cells. This situation was achieved in Fi (2 X 13) chimeric guinea pigs. These animals were deprived of T cells by adult thymectomy, lethal irradiation and reconstitution with bone marrow cells from either strain 2 or 13 donors. In addition, these animals were reconstituted with F, (2 X 13) T cells treated with alloantisera and complement in order to eliminate any residual B cells in the Tcell preparation. Using this protocol, PLL Ir gene-positive T cells can coexist with

136

CAMPOS-NETO,

LEVINE,

0 a,Dnp-Lyss W a, Dnp-Lyso

AND

SCHLOSSMAN

kSi E, Dnp- Lys, fZl Dnp-OH

1.: j :, :. :. y :. ; ; :: ~: :.

No. Of Normal

FI (2X 13) T Cells

Tronsferred

FIG. 5. Binding energies (-AFa) of Dnp-oligolysines and dinitrophenol to anti-cYDnp-Lys9 antibodies from F, (2 X 13) B guinea pigs reconstituted with (A) strain 2 bone marrow cells and (B) strain 13 bone marrow cells. B guinea pigs were prepared by adult thymectomy, lethal irradiation (1000 rad), and bone marrow reconstitution. Before the immunization, half of the animals from each group were inoculated intravenously with 1 X 10’ F, T cells. The antibody specificity was analyzed 15 days later by fluorescence quenching. Results are expressed as the means of three to four experiments.

PLL Ir-negative cells. After immunization with cYDnp-Lysg these animals produced antibody highly restricted in heterogeneity (data not shown) and with the same specificity of the antibody produced by PLL Ir gene-positive animals (Fig. 5). The possibility that the antibody produced by these animals is secreted by contaminating PLL Ir gene-positive B cells in the chimeras could be ruled out for three reasons: first the recipients were lethally irradiated (lClO0 rad); second the F2 (2 X 13) T-cell preparation transferred to these animals was treated with strain 13-anti-strain 2 antiserum (anti-Ia antibody), thus eliminating B cells; and third their anti-Dnp plaque-forming cells were totally abrogated after treatment with strain 2-anti-strain 13 serum and complement. Treatment of these cells with strain 13-anti-strain 2 serum and complement had no effect. These observations appear to differ from those of Katz et al. (29), who found that carrier-primed T and hapten-primed B cells from Dnp-GTL responder and nonresponder mice cooperate in the F, environment only when both sets of .cells come from the responder donor. However, others (30-33) have shown that T cellB cell interactions can occur in a genetically less restricted environment provided that alloaggressive reactions are diminished or abolished. In any event, our results strongly suggest that in the guinea pigs, the PLL Ir

Ir GENE

CONTROL

OF

ANTIBODY

SPECIFICITY

137

gene defect is localized to the T cells and that the repertoire of antibody specificity is similar if not identical in both strain 2 and strain 13 animals. In addition, these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is confined to T cells and not to B lymphocytes. REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

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