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Modulation of the IgE immune response to BSA by fragments of the antigen
CELLULAR
IMMUNOLOGY
59, 392-398 (1981)
Modulation
of the IgE Immune Response by Fragments of the Antigen
to BSA
1. Suppression by Free Fragments and by Fragments Conjugated to Homologous y-Globulin A. MUCKERHEIDE, A. J. PESCE, AND J. G. MICHAEL Departments of Microbiology and Medicine, University of Cincinnati College of Medicine. Cincinnati, Ohio 45267 Received July 16. 1980; accepted October, 1. 1980 Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction la, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSAprimed cells, indicating carrier-specific suppression. The conjugate of Fraction la with mouse y-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given la-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsivenessto BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous y-globulin.
INTRODUCTION We have previously reported (1) that peptic fragments of bovine serum albumin (BSA)’ suppress IgE immune response to the intact antigen through induction of specific suppressor T cells. These fragments, designated Fraction la, did not elicit an immune response to BSA nor to any new determinants which may have arisen during the cleavage process. We now report that these same peptides when conjugated to homologous MGG are more efficient in suppressing IgE immune response to the intact protein than the free fragments. Our results indicate that the Fraction la-MGG conjugates affect B lymphocytes without involvement of a T suppressor mechanism. MATERIALS
AND METHODS
Animals. Female BDFl (C57 B1/6 X DBA/2) mice, 8-12 weeks of age and female HRS/J mice, 6-8 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, Maine. I Abbreviations used: BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; MGG, mouse y-globulin; NRS, normal rabbit serum; PCA, passive cutaneous anaphylaxis; la-MGG, conjugate of Fraction la and mouse gamma globulin. 392 0008-874918I /060392-07$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
IMMUNOSUPPRESSION
BY PEPTIC FRAGMENTS
OF BSA
393
Antigens. Crystalline BSA was purchased from Armour & Company (Kankakee, Ill.) and was subjected to limited proteolysis for the production of Fraction la as previously described (1). Fraction la was coupled to MGG by means of glutaraldehyde. DNP-BSA and DNP-OVA were prepared according to the method of Little and Eisen (2). Immunizations. Mice were immunized by an intraperitoneal injection of 100 pugof the appropriate antigen mixed with 1 mg Al(OH)j gel prepared as described by Levine and Vaz (3). This relatively large dose of antigen was selected after it was established that lower doses of antigen from 0.1 to 50 pg were not effective in producing a primary IgE immune response. Each experimental group consisted from at least 5 animals and each experiment was repeated twice. Measurement of reaginic antibodies. IgE antibody was titrated by passive cutaneous anaphylaxis (PCA) in hairless (HRS/J) mice. Twofold dilutions of antiserum were injected intracutaneously for passive sensitization, and the PCA reaction was elicited 48-72 hr later by an iv injection of 1 mg antigen in 0.5 ml of 0.25% Evans blue dye. The test was performed at least in duplicate and the titer is expressed as the geometric mean of the highest dilution resulting in PCA reactions 5 mm or greater in diameter. Variation for the same sample was twofold or less; therefore, differences greater than this were taken to be significant. Heating the sera at 56°C for 4 hr completely destroyed PCA reactivity, confirming the antibodies as belonging to the IgE class (4). Preparation of mouse y-globulin. Crude MGG was prepared by precipitation of normal mouse serum obtained from BDF, mice with saturated ammonium sulfate solution added to a final concentration of 33% salt. This was followed by extensive washing and dialysis of MGG against phosphate-buffered saline. Cell separation techniques. A population of B-cell-enriched (T-cell-depleted) spleen cells was prepared by treatment with a 1:10 dilution of anti-Thy-l .2 antiserum prepared in AKR/J mice according to a modification of the technique of Reif and Allen (5) followed by incubation with complement. The nylon wool technique of Julius et al. (6) as modified by Schwartz et al. (7) was used to prepare a lymphocyte population depleted of adherent cells and B cells. Spleen cells suspended in RPM1 1640 (Gibco, Grand Island, N.Y.) containing 5% heat-inactivated normal mouse serum were passed through a column of glass wool and glass beads to remove adherent cells, and the eluted nonadherent cells were applied to a nylon wool column (Fenwall Laboratories, Morton Grove, Ill.). The yield of the effluent (T-cell rich) fraction was 15 to 20% of the cells applied to the column. This low yield is attributed to the necessary absence of fetal bovine serum in the medium. Treatment with anti-Thy-l.2 antiserum and complement resulted in the killing of 95% of the cells as judged by trypan blue exclusion. Irradiation of mice. Mice were exposed to 600 R of X-irradiation by means of a Westinghouse Quadrocondex X-ray therapy machine, 24 hr prior to reconstitution. Cell transfers. Spleen cells, spleen cells enriched for T cells as described above, or spleen cells treated with anti-Thy-l.2 antiserum and complement were transferred in various combinations into irradiated recipients, These mice were immunized within 1 hr of reconstitution. Coupling of Fraction 1a to mouse y-globulin. Mouse y-globulin and the peptide fragments were mixed at concentrations of 10 mg/ml each and purified glutaral-
394
MUCKERHEIDE,
PESCE, AND
MICHAEL
dehyde was added dropwise to a final concentration of 0.4%. The mixture was permitted to react at 25°C for 1 hr and was then extensively dialyzed against normal saline solution so that the unconjugated peptides and glutaraldehyde were removed by dialysis. To verify that the peptides and MGG were conjugated Fraction la was radiolabeled with ‘9 and reacted with MGG in parallel with and under identical conditions as the unlabeled fraction. When rabbit anti-MGG antiserum was added to the putative conjugate, radioactivity was recovered in the precipitate indicating coprecipitation of Fraction la and MGG. Less than 1% of the total radioactivity of labeled Fraction la alone precipitated by the antiserum. Self-polymerization of Fraction la with glutaraldehyde resulted in formation of small size dialyzable material. The radiolabeled conjugate consisted of about 20% of Fraction la peptides and 80% MGG. RESULTS Suppression of the Anti-BSA IgE Response by Fraction la Conjugated to MGG To determine whether the coupling of the suppressive fragments to homologous y-globulin modified their immunosuppressive activity the minimum dosage of conjugate necessary to suppress anti-BSA response was established. Graded doses of la-MGG were administered in saline, iv, to groups of mice 24 hr before immunization. Data presented in Fig. 1 show that a dose as small as 10 pg of the conjugate, containing about 2 pg Fraction 1a was immunosuppressive. In contrast, at least 50 pg of free fragments were required to achieve the same effect. Mechanism of the Suppression Induced by Fraction la We have previously reported that nonimmunogenic peptic fragment of BSA, Fraction la, rendered mice unresponsive to BSA by the induction of specific T Grow
Retreatment 24 Haun eefore BSA lmmunlzath
Amount d Fraction b h Conjugate
A a
2OO#g la-MGG 4ONg loofig b-MGG 2opg
C
5ONg b-MGG
D
IO pg la-MGG
F
Ioo~g-MGG
-
G
2oopg-MGG
-
Ii
100&g
14 Day PCA l’ltera 9-mOF coNlRcn.
IO&g 2lrO
Fraction la
I
50 JQ Fraction la
J
IO fig
K
Nan8
Fraction b I
FIG. 1. The dosage of la-MC% I :320.
needed to suppress the anti-BSA
IgE response. Control
response,
IMMUNOSUPPRESSION
BY PEPTIC FRAGMENTS
395
OF BSA
suppressor cells (1). Repeated attempts to prime mice for an augmented response to either BSA or DNP-BSA by administration of Fraction la met with uniform failure. Such data led us to speculate that there may exist on the BSA molecule unique determinants for suppressor (T,) lymphocytes. In order to investigate this possibility cell transfer experiments using irradiated recipients and hapten-carrier conjugates as antigens were devised. Donor mice were primed with 100 pg of either BSA, Fraction la, DNP-OVA or DNP-BSA, all in aluminum hydroxide gel, 6 weeks before cell transfer, or in the case of potential suppressor cell donors, with 100 /lg of Fraction la in saline either once (1 X) 3 days before cell transfer, or on each of the three successivedays prior to the transfer of cells (3X). Recipient mice were subjected to 600 R of Xirradiation 24 hr before reconstitution and were immunized with 100 pg DNPBSA in 1 mg AI gel within 1 hr of reconstitution. Anti-DNP IgE responses were assayed on Day 10. Data presented in Table 1 show that while BSA- or DNP-BSA-primed spleen cells provided help for DNP-primed B cells (Groups B and D), no such effect was observed when Fraction la-primed spleen cells were employed (Group F). Furthermore, spleen cells from mice treated with Fraction la prevented priming by DNP-BSA (Groups H and I), supporting the hypothesis that the fragments in question induce carrier-specific suppressor cells but no functional helper cells. Mechanism of the Suppression Induced by Fraction la Coupled to MGG Attempts to demonstrate induction of specific suppressor T cells by the conjugate by means of cell transfers into normal, nonirradiated recipients were uniformly TABLE 1 The Response of la-Treated Spleen Cells in Irradiated Recipient Mice Immunized with DNP-BSA” Group
Spleen cells transferred (2 X 10’ of each type) Normal BSA-primed
1O-day PCA’ 5 80
DNP-OVA primed
5
DNP-BSA primed
160
BSA-primed DNP-OVA primed
80
la-primed
5
1a-primed DNA-OVA primed
5
la (IX) treated DNP-BSA primed
5
la (3X) treated DNP-BSA primed
5
a 100 pg DNP-BSA in alum ip. * Immune response reassured by PCA reaction in hairless mice. The immunized mice bled 10 days after DNP-BSA challenge.
396
MUCKERHEIDE,
PESCE, AND MICHAEL TABLE 2
The Response of la-MGG-Treated Spleen Cells in Irradiated Recipient Mice Immunized with BSA” Group
Cells transferred 2 X 10’ normal spleen
IO-day PCAb 10
2 X 10’ BSA primed spleen
640
1.7 X 10’ BSA primed B 1.0 X 10’ BSA primed T
640
1.7 X 10’ BSA primed B 1.0 X 10’ untreated T
80
E
1.4 X 10’ untreated B 1.0 X 10’ BSA primed T
320
F
2 X 10’ la-MGG treated (24 h) BSA primed spleen
G
1.7 X 10’ BSA primed B 1.0 X 10’ la-MGG treated T
80
H
1.7 X 10’ la-MGG treated B 1.0 X 10’ BSA primed T
5
I
1.7 X 10’ la-MGG treated BSA primed B 1.0 X 10’ la-MGG treated BSA primed T
5
5
a 100 pg BSA in alum ip. ’ Immune response measured by PCA reactions in hairless mice. The immunized mice bled 10 days after DNP-BSA challenge.
negative (data not shown). Therefore, cell transfer experiments were designed to investigate the cellular basis of la-MGG-induced suppression. Donor mice were primed with BSA 6 weeks prior to harvesting their spleen cells for transfer to irradiated recipients. The animals were irradiated 24 hr before reconstitution with the appropriate donor cells. Fraction la-treated cells were obtained from animals injected 24 hr earlier with 100 pg of the conjugate. Data presented in Table 2 show that Fraction la-MGGtreated B cells did not reconstitute with BSA-primed T cells (Group H), while in the control group (E) the untreated B cells were quite responsive. Untreated T cells and conjugate treated T cells responded equally, although not optimally, when reconstituted with primed B cells (Groups D and G). Thus, it appears that the suppressive effect of the la-MGG conjugate is exerted primarily on the level of the B cell. DISCUSSION Peptic fragments of BSA, designated Fraction la, were shown to be effective in suppressing the anti-BASA IgE response. A greatly increased effectiveness of this material as a tolerogen was demonstrated when it was conjugated with homologous MGG.
IMMUNOSUPPRESSION
BY PEPTIC
FRAGMENTS
OF BSA
397
Results of our experiments clearly point to two different mechanisms of suppression brought about by peptic fragments of BSA dependent upon whether or not these fragments were conjugated to homologous mouse y-globulin. As previously reported ( 1) Fraction 1a alone effectively renders mice unresponsive to subsequent immunization with BSA and this suppression is a result of the stimulation of antigen-specific suppressor T cells by the fraction. This is evidenced by the ability of Fraction la-treated spleen cells to transfer suppression to normal recipient animals, as well as to suppress the hapten-specific responseto DNP-BSA in irradiated mice reconstituted with DNP- and BSA-primed spleen cells. The failure of Fraction la to prime mice for a secondary response to BSA in viva indicates either the absence of helper cell determinants or that the suppressor determinants assumed the dominant role. On the other hand, the mechanism of suppression by Fraction la-MGG conjugate does not seem to involve suppressor T cells but rather the B lymphocytes. In experiments employing DNP coupled to homologous y-globulin, both T and B cells were implicated and evidence that such conjugates induce receptor blockade of antigen-binding (i.e., B) cells has been presented (8). More recently, Katz and Bore1 (9), in experiments involving transfer of cells tolerized by means of DNP-MGG, again presented evidence for the involvement of B cells in their system. As shown in our earlier work B-cell determinants are retained on Fraction la and it is conceivable that they are involved in la-MGG-induced suppression. Such determinants may bind to BSA-specific lymphocytes, while the y-globulin portion of the conjugate may facilitate this binding (10). Although data reported here deal with suppression of BSA response by its peptic fragments exclusively, in another publication the antigenic specificity of this process was clearly demonstrated (11). Finally, it is to be noted that relatively large doses of BSA have been used for immunization. This was done because BSA is a poor inducer of a primary response in mice. We were, in fact, unable to induce measurable primary IgM or IgG responsesto BSA using our immunizing conditions. Consequently it was not possible to determine whether the same mode of suppression is operative in primary responsesof IgM or IgG. The poor responsivenessto BSA has been reported also by other investigators ( 12). ACKNOWLEDGMENTS We thank Mrs. Susan Riebel for excellent technical assistance and Dr. Victor Pollak for his interest and encouragement. This work was supported in part by National Institutes of Health Grants AM 17 196 and AI 15520.
REFERENCES 1. Muckerheide, A., Pesce, A. J., and Michael, J. G., J. Immunol. 119, 1340, 1977. 2. Little, J. R., and Eisen, H. N. In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. Chase (Ed.), Vol. 1, p. 128. Academic Press, New York, 1967. 3. Levine, B. B., and Vaz, N. M., Int. Arch. Allergy Appl. Immunol. 39, 156, 1970. 4. Schwartz, H. A., and Levine, B. B., J. Immunol. 110, 1638, 1973.
398 5. 6. 7. 8. 9. 10. 11. 12.
MUCKERHEIDE,
PESCE, AND MICHAEL
Reif, A. E., and Allen, J. M. V.. J. Exp. Med. 120, 413, 1964. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. Schwartz, R. H., Jackson, L., and Paul, W. E., J. Immunol. 115, 1330, 1975. Aldo-Benson, M., and Borel, Y. J. Immunol. 112, 1793, 1976. Katz, D. H.. and Bore], Y., J. Immunol. 120, 1824, 1978. Sidman, C. L., and Uranue, E. R., J. Exp. Med. 144, 882, 1976. Dosa, S., Pesce,A.J., Ford, D. J., Muckerheide, A., and Michael, J. G., Immunology 38,509, 1979. Chedid, L., Audibert, F., Lefrancier, P., Choay, J., and Lederer, E., Proc. Nat. Acad. Sci. USA 73, 2472, 1976.
IMMUNOLOGY
59, 392-398 (1981)
Modulation
of the IgE Immune Response by Fragments of the Antigen
to BSA
1. Suppression by Free Fragments and by Fragments Conjugated to Homologous y-Globulin A. MUCKERHEIDE, A. J. PESCE, AND J. G. MICHAEL Departments of Microbiology and Medicine, University of Cincinnati College of Medicine. Cincinnati, Ohio 45267 Received July 16. 1980; accepted October, 1. 1980 Nonimmunogenic peptic fragments of bovine serum albumin (BSA), Fraction la, suppressed immune response to BSA in mice. Splenic T lymphocytes from mice treated with these fragments suppressed the anti-DNP response in irradiated mice reconstituted with DNP-BSAprimed cells, indicating carrier-specific suppression. The conjugate of Fraction la with mouse y-globulin (MGG) was found to be an effective suppressive substance but it did not induce suppressor T cells. B cells from mice given la-MGG were unresponsive to BSA when transferred to irradiated recipients along with either normal or BSA-primed T cells. Thus, unresponsivenessto BSA was mediated by either T or B lymphocytes, depending whether the inducing substance was a free fragment of the antigen or fragments conjugated to homologous y-globulin.
INTRODUCTION We have previously reported (1) that peptic fragments of bovine serum albumin (BSA)’ suppress IgE immune response to the intact antigen through induction of specific suppressor T cells. These fragments, designated Fraction la, did not elicit an immune response to BSA nor to any new determinants which may have arisen during the cleavage process. We now report that these same peptides when conjugated to homologous MGG are more efficient in suppressing IgE immune response to the intact protein than the free fragments. Our results indicate that the Fraction la-MGG conjugates affect B lymphocytes without involvement of a T suppressor mechanism. MATERIALS
AND METHODS
Animals. Female BDFl (C57 B1/6 X DBA/2) mice, 8-12 weeks of age and female HRS/J mice, 6-8 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, Maine. I Abbreviations used: BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; MGG, mouse y-globulin; NRS, normal rabbit serum; PCA, passive cutaneous anaphylaxis; la-MGG, conjugate of Fraction la and mouse gamma globulin. 392 0008-874918I /060392-07$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
IMMUNOSUPPRESSION
BY PEPTIC FRAGMENTS
OF BSA
393
Antigens. Crystalline BSA was purchased from Armour & Company (Kankakee, Ill.) and was subjected to limited proteolysis for the production of Fraction la as previously described (1). Fraction la was coupled to MGG by means of glutaraldehyde. DNP-BSA and DNP-OVA were prepared according to the method of Little and Eisen (2). Immunizations. Mice were immunized by an intraperitoneal injection of 100 pugof the appropriate antigen mixed with 1 mg Al(OH)j gel prepared as described by Levine and Vaz (3). This relatively large dose of antigen was selected after it was established that lower doses of antigen from 0.1 to 50 pg were not effective in producing a primary IgE immune response. Each experimental group consisted from at least 5 animals and each experiment was repeated twice. Measurement of reaginic antibodies. IgE antibody was titrated by passive cutaneous anaphylaxis (PCA) in hairless (HRS/J) mice. Twofold dilutions of antiserum were injected intracutaneously for passive sensitization, and the PCA reaction was elicited 48-72 hr later by an iv injection of 1 mg antigen in 0.5 ml of 0.25% Evans blue dye. The test was performed at least in duplicate and the titer is expressed as the geometric mean of the highest dilution resulting in PCA reactions 5 mm or greater in diameter. Variation for the same sample was twofold or less; therefore, differences greater than this were taken to be significant. Heating the sera at 56°C for 4 hr completely destroyed PCA reactivity, confirming the antibodies as belonging to the IgE class (4). Preparation of mouse y-globulin. Crude MGG was prepared by precipitation of normal mouse serum obtained from BDF, mice with saturated ammonium sulfate solution added to a final concentration of 33% salt. This was followed by extensive washing and dialysis of MGG against phosphate-buffered saline. Cell separation techniques. A population of B-cell-enriched (T-cell-depleted) spleen cells was prepared by treatment with a 1:10 dilution of anti-Thy-l .2 antiserum prepared in AKR/J mice according to a modification of the technique of Reif and Allen (5) followed by incubation with complement. The nylon wool technique of Julius et al. (6) as modified by Schwartz et al. (7) was used to prepare a lymphocyte population depleted of adherent cells and B cells. Spleen cells suspended in RPM1 1640 (Gibco, Grand Island, N.Y.) containing 5% heat-inactivated normal mouse serum were passed through a column of glass wool and glass beads to remove adherent cells, and the eluted nonadherent cells were applied to a nylon wool column (Fenwall Laboratories, Morton Grove, Ill.). The yield of the effluent (T-cell rich) fraction was 15 to 20% of the cells applied to the column. This low yield is attributed to the necessary absence of fetal bovine serum in the medium. Treatment with anti-Thy-l.2 antiserum and complement resulted in the killing of 95% of the cells as judged by trypan blue exclusion. Irradiation of mice. Mice were exposed to 600 R of X-irradiation by means of a Westinghouse Quadrocondex X-ray therapy machine, 24 hr prior to reconstitution. Cell transfers. Spleen cells, spleen cells enriched for T cells as described above, or spleen cells treated with anti-Thy-l.2 antiserum and complement were transferred in various combinations into irradiated recipients, These mice were immunized within 1 hr of reconstitution. Coupling of Fraction 1a to mouse y-globulin. Mouse y-globulin and the peptide fragments were mixed at concentrations of 10 mg/ml each and purified glutaral-
394
MUCKERHEIDE,
PESCE, AND
MICHAEL
dehyde was added dropwise to a final concentration of 0.4%. The mixture was permitted to react at 25°C for 1 hr and was then extensively dialyzed against normal saline solution so that the unconjugated peptides and glutaraldehyde were removed by dialysis. To verify that the peptides and MGG were conjugated Fraction la was radiolabeled with ‘9 and reacted with MGG in parallel with and under identical conditions as the unlabeled fraction. When rabbit anti-MGG antiserum was added to the putative conjugate, radioactivity was recovered in the precipitate indicating coprecipitation of Fraction la and MGG. Less than 1% of the total radioactivity of labeled Fraction la alone precipitated by the antiserum. Self-polymerization of Fraction la with glutaraldehyde resulted in formation of small size dialyzable material. The radiolabeled conjugate consisted of about 20% of Fraction la peptides and 80% MGG. RESULTS Suppression of the Anti-BSA IgE Response by Fraction la Conjugated to MGG To determine whether the coupling of the suppressive fragments to homologous y-globulin modified their immunosuppressive activity the minimum dosage of conjugate necessary to suppress anti-BSA response was established. Graded doses of la-MGG were administered in saline, iv, to groups of mice 24 hr before immunization. Data presented in Fig. 1 show that a dose as small as 10 pg of the conjugate, containing about 2 pg Fraction 1a was immunosuppressive. In contrast, at least 50 pg of free fragments were required to achieve the same effect. Mechanism of the Suppression Induced by Fraction la We have previously reported that nonimmunogenic peptic fragment of BSA, Fraction la, rendered mice unresponsive to BSA by the induction of specific T Grow
Retreatment 24 Haun eefore BSA lmmunlzath
Amount d Fraction b h Conjugate
A a
2OO#g la-MGG 4ONg loofig b-MGG 2opg
C
5ONg b-MGG
D
IO pg la-MGG
F
Ioo~g-MGG
-
G
2oopg-MGG
-
Ii
100&g
14 Day PCA l’ltera 9-mOF coNlRcn.
IO&g 2lrO
Fraction la
I
50 JQ Fraction la
J
IO fig
K
Nan8
Fraction b I
FIG. 1. The dosage of la-MC% I :320.
needed to suppress the anti-BSA
IgE response. Control
response,
IMMUNOSUPPRESSION
BY PEPTIC FRAGMENTS
395
OF BSA
suppressor cells (1). Repeated attempts to prime mice for an augmented response to either BSA or DNP-BSA by administration of Fraction la met with uniform failure. Such data led us to speculate that there may exist on the BSA molecule unique determinants for suppressor (T,) lymphocytes. In order to investigate this possibility cell transfer experiments using irradiated recipients and hapten-carrier conjugates as antigens were devised. Donor mice were primed with 100 pg of either BSA, Fraction la, DNP-OVA or DNP-BSA, all in aluminum hydroxide gel, 6 weeks before cell transfer, or in the case of potential suppressor cell donors, with 100 /lg of Fraction la in saline either once (1 X) 3 days before cell transfer, or on each of the three successivedays prior to the transfer of cells (3X). Recipient mice were subjected to 600 R of Xirradiation 24 hr before reconstitution and were immunized with 100 pg DNPBSA in 1 mg AI gel within 1 hr of reconstitution. Anti-DNP IgE responses were assayed on Day 10. Data presented in Table 1 show that while BSA- or DNP-BSA-primed spleen cells provided help for DNP-primed B cells (Groups B and D), no such effect was observed when Fraction la-primed spleen cells were employed (Group F). Furthermore, spleen cells from mice treated with Fraction la prevented priming by DNP-BSA (Groups H and I), supporting the hypothesis that the fragments in question induce carrier-specific suppressor cells but no functional helper cells. Mechanism of the Suppression Induced by Fraction la Coupled to MGG Attempts to demonstrate induction of specific suppressor T cells by the conjugate by means of cell transfers into normal, nonirradiated recipients were uniformly TABLE 1 The Response of la-Treated Spleen Cells in Irradiated Recipient Mice Immunized with DNP-BSA” Group
Spleen cells transferred (2 X 10’ of each type) Normal BSA-primed
1O-day PCA’ 5 80
DNP-OVA primed
5
DNP-BSA primed
160
BSA-primed DNP-OVA primed
80
la-primed
5
1a-primed DNA-OVA primed
5
la (IX) treated DNP-BSA primed
5
la (3X) treated DNP-BSA primed
5
a 100 pg DNP-BSA in alum ip. * Immune response reassured by PCA reaction in hairless mice. The immunized mice bled 10 days after DNP-BSA challenge.
396
MUCKERHEIDE,
PESCE, AND MICHAEL TABLE 2
The Response of la-MGG-Treated Spleen Cells in Irradiated Recipient Mice Immunized with BSA” Group
Cells transferred 2 X 10’ normal spleen
IO-day PCAb 10
2 X 10’ BSA primed spleen
640
1.7 X 10’ BSA primed B 1.0 X 10’ BSA primed T
640
1.7 X 10’ BSA primed B 1.0 X 10’ untreated T
80
E
1.4 X 10’ untreated B 1.0 X 10’ BSA primed T
320
F
2 X 10’ la-MGG treated (24 h) BSA primed spleen
G
1.7 X 10’ BSA primed B 1.0 X 10’ la-MGG treated T
80
H
1.7 X 10’ la-MGG treated B 1.0 X 10’ BSA primed T
5
I
1.7 X 10’ la-MGG treated BSA primed B 1.0 X 10’ la-MGG treated BSA primed T
5
5
a 100 pg BSA in alum ip. ’ Immune response measured by PCA reactions in hairless mice. The immunized mice bled 10 days after DNP-BSA challenge.
negative (data not shown). Therefore, cell transfer experiments were designed to investigate the cellular basis of la-MGG-induced suppression. Donor mice were primed with BSA 6 weeks prior to harvesting their spleen cells for transfer to irradiated recipients. The animals were irradiated 24 hr before reconstitution with the appropriate donor cells. Fraction la-treated cells were obtained from animals injected 24 hr earlier with 100 pg of the conjugate. Data presented in Table 2 show that Fraction la-MGGtreated B cells did not reconstitute with BSA-primed T cells (Group H), while in the control group (E) the untreated B cells were quite responsive. Untreated T cells and conjugate treated T cells responded equally, although not optimally, when reconstituted with primed B cells (Groups D and G). Thus, it appears that the suppressive effect of the la-MGG conjugate is exerted primarily on the level of the B cell. DISCUSSION Peptic fragments of BSA, designated Fraction la, were shown to be effective in suppressing the anti-BASA IgE response. A greatly increased effectiveness of this material as a tolerogen was demonstrated when it was conjugated with homologous MGG.
IMMUNOSUPPRESSION
BY PEPTIC
FRAGMENTS
OF BSA
397
Results of our experiments clearly point to two different mechanisms of suppression brought about by peptic fragments of BSA dependent upon whether or not these fragments were conjugated to homologous mouse y-globulin. As previously reported ( 1) Fraction 1a alone effectively renders mice unresponsive to subsequent immunization with BSA and this suppression is a result of the stimulation of antigen-specific suppressor T cells by the fraction. This is evidenced by the ability of Fraction la-treated spleen cells to transfer suppression to normal recipient animals, as well as to suppress the hapten-specific responseto DNP-BSA in irradiated mice reconstituted with DNP- and BSA-primed spleen cells. The failure of Fraction la to prime mice for a secondary response to BSA in viva indicates either the absence of helper cell determinants or that the suppressor determinants assumed the dominant role. On the other hand, the mechanism of suppression by Fraction la-MGG conjugate does not seem to involve suppressor T cells but rather the B lymphocytes. In experiments employing DNP coupled to homologous y-globulin, both T and B cells were implicated and evidence that such conjugates induce receptor blockade of antigen-binding (i.e., B) cells has been presented (8). More recently, Katz and Bore1 (9), in experiments involving transfer of cells tolerized by means of DNP-MGG, again presented evidence for the involvement of B cells in their system. As shown in our earlier work B-cell determinants are retained on Fraction la and it is conceivable that they are involved in la-MGG-induced suppression. Such determinants may bind to BSA-specific lymphocytes, while the y-globulin portion of the conjugate may facilitate this binding (10). Although data reported here deal with suppression of BSA response by its peptic fragments exclusively, in another publication the antigenic specificity of this process was clearly demonstrated (11). Finally, it is to be noted that relatively large doses of BSA have been used for immunization. This was done because BSA is a poor inducer of a primary response in mice. We were, in fact, unable to induce measurable primary IgM or IgG responsesto BSA using our immunizing conditions. Consequently it was not possible to determine whether the same mode of suppression is operative in primary responsesof IgM or IgG. The poor responsivenessto BSA has been reported also by other investigators ( 12). ACKNOWLEDGMENTS We thank Mrs. Susan Riebel for excellent technical assistance and Dr. Victor Pollak for his interest and encouragement. This work was supported in part by National Institutes of Health Grants AM 17 196 and AI 15520.
REFERENCES 1. Muckerheide, A., Pesce, A. J., and Michael, J. G., J. Immunol. 119, 1340, 1977. 2. Little, J. R., and Eisen, H. N. In “Methods in Immunology and Immunochemistry” (C. A. Williams and M. Chase (Ed.), Vol. 1, p. 128. Academic Press, New York, 1967. 3. Levine, B. B., and Vaz, N. M., Int. Arch. Allergy Appl. Immunol. 39, 156, 1970. 4. Schwartz, H. A., and Levine, B. B., J. Immunol. 110, 1638, 1973.
398 5. 6. 7. 8. 9. 10. 11. 12.
MUCKERHEIDE,
PESCE, AND MICHAEL
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