Regulation of immune response by autogenous antibody against ...

Proc. Nat. Acad. Sci. USA

Vol. 71, No. 12, pp. 5083-5087, December 1974

Regulation of Immune Response by Autogenous Antibody against Receptor (antibodies against phosphorylcholine/specific suppression)

L. KLUSKENS AND H. KOHLER La Rabida, University of Chicago Institute, Chicago, Illinois 60649

Communicated by Hewson Swift, September 20, 1974 ABSTRACT BALB/c mice repeatedly immunized with Pneumococcus R36A vaccine produce antibodies to phosphorylcholine having the TEPC-15 myeloma idiotype (murine IgA myeloma protein that binds phosphorylcholine). The plaque-forming cell response to phosphorylcholine shows a decrease with repeated immunizations. In contrast, spleen cells from multiply immunized mice responded better in vitro than spleen cells from nonimmunized mice. The serum of animals immunized four or five times agglutinates TEPC-15-coated sheep erythrocytes. Inhibition of hemagglutination shows that the agglutinating activity is directed against the TEPC-15 idiotype. Sera from these mice, when added to cultures of normal spleen cells, specifically suppress the response to phosphorylcholine. The suppressive activity in the serum can be removed by solid absorption with TEPC-15. Evidently, repeated immunization with antigen induces two kinds of antibody responses: one directed against antigen and the other directed against the antibody to the antigen. It is proposed that this "auto" antibody against receptor is involved in the regulation of the immune response.

At least three factors have a specific effect on the production of antibody: antigen, antibody, and antibody against receptor. Serum from immunized animals contains enhancing and suppressing factors that can alter the primary response specifically (1, 2). In several instances, the specific suppressive activity cannot be attributed to either antigen or antibody (3). Antibody directed against the specific receptors (antireceptor antibody) for an antigen has been raised and shown to suppress the antibody response specifically (4-6). Furthermore, it has been shown that animals can make specific antibodies against autologous immunoglobulin (7-9). Therefore, it seems possible that during the immune response to antigen, antibody and its complementary anti-receptor antibody are produced. In the present study we report the formation of an autogenous anti-receptor antibody in BALB/c mice repeatedly immunized with antigen. This "auto" anti-receptor antibody is equivalent in its immunochemical specificity and biological activity to homologous anti-receptor antibody produced in A/He mice (4, 10). The system we used was developed by Cosenza and Kohler. BALB/c mice respond to the hapten phosphorylcholine with a "monoclonal" IgM antibody (11). This antibody is of the same idiotype as the phosphorylcholine-binding protein (T15) produced by the BALB/c myeloma TEPC-15 (4). Antibody to T-15 raised in A/He mice specifically suppresses the response of BALB/c or A/He mice to phosphorylcholine or of their spleen cells in vitro. Thus, antibody against T-15 functions as an anti-receptor antibody, and will be referred to as such (10, 12). Abbreviations: PnC, C-polysaccharide; PFC, plaque-forming cell. 50)83

MATERIALS AND METHODS

Mice. BALB/c females (Cumberland View Farms) 6-8 weeks old were used at the beginning of experiments.

Immunizations. Each immunization was done with 0.2 ml of 0.5% suspension (109 organisms) of heat-killed and formalinized Pneumococci (R36A). Serum antibody and spleen plaque-forming cell responses were measured 4 days after each immunization. Sera were heat-inactivated at 560 for 30 min before they were titered.

Purification of Myeloma Proteins. Phosphorylcholine-binding murine myeloma proteins, TEPC-15, MOPC-167, and McPC 603, were purified by the method of Chesebro and Metzger (13). Briefly, ascites fluid from tumor-bearing mice was mildly reduced with 5 mM dithioerythritol and alkylated with 10 mM iodoacetamide at pH 8.4, 1.0 M Tris buffer. Proteins were purified by affinity chromatography on Sepharose (Pharmacia) diazotized with phosphorylcholine. Proteins were eluted from the column with 10 mM phosphorylcholine in 10 mM Tris, 0.15 M NaCl (pH 7.9). Eluted proteins were concentrated by vacuum dialysis against 0.15 M NaCl. Myeloma proteins that did not bind phosphorylcholine were precipitated by 50% saturated ammonium sulfate from ascites fluid. After precipitation, proteins were redissolved in saline and chromatographed on Sephadex G-200 in 0.15 M NaCI. The first peak was used for polymeric IgA (MOPC460) or IgM (MOPC-104E). Immunoadsorbents. Proteins were conjugated to cyanogen bromide-activated Sepharose 4B (Pharmacia) by the method of Porath et al. (14). Five milligrams of protein were conjugated to 1 ml of packed Sepharose. Partially purified Cpolysaccharide (15) was conjugated to Sepharose by the method of Sherr and Tarakis (16). Serum samples were absorbed on an equal volume of conjugated Sepharose at 370 for 2 hr and then placed in the cold, overnight. Serum was sterilized by passage through a 0.45/Am filter (Millipore, Inc.). Passive Hemagglutination. Heterologous erythrocytes were coated with mouse myeloma proteins or pneumococcal extract (15) by the method of Gold and Fudenberg (17). Briefly, 100 ,Al of 50% suspension of sheep erythrocytes in 0.15 M NaCl were added to 200 ul of a 1-mg/ml of solution of the material used for coating. One hundred microliters of a 1mg/ml of solution of chromium chloride were added, and the mixture was incubated at room temperature for 4 min. The coated cells were then washed three times in physiological saline and suspended in 5% (v/v) fetal calf serum.

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Proc. Nat. Acad. Sci. USA 71

TABLE 1. Responses of multiply immunized BALB/c to phosphorylcholine No. of immuniza- Week of assay tions 1st 3rd 5th 12th 28th

1 2 3 4 5

Antiphosphoryl- AntiT-15 choline phosphorylcholine titer titer (PFC/spleen) Anti-

24,900 18,940 21,200 12,560 8,710

4-

i + i +

1820* 2363 4610 2104 2331

4t 128 4100 2050 2050

0 0 0 16 8

BALB/c mice were immunized intraperitoneally with R36A vaccine. The plaque-forming cell (PFC) responses to phosphorylcholine were measured 4 days after each immunization. Hemagglutination titers were done on sera collected 4 days after each

immunization. * Geometric mean PFC response 4- SEM. There were at least six animals per group, with triplicate determinations for each animal. t The reciprocal of the last dilution of pooled serum that caused

agglutination. Hemagglutination assays were performed in microtiter plates (Cooke Engineering) by serial dilution of antisera in phosphate-buffered saline-0.5% dextrose for titers of antibody against phosphorylcholine or 5% fetal calf serum-saline for titers of antibody against TEPC-15. Titer plates were incubated at 370 for 1 hr and at 40 overnight. The agglutination titer was determined by the pattern of sedimentation. The titer is reported as the reciprocal of the highest dilution of serum that caused dispersed or nonpelleted sedimentation. Hemagglutination inhibition assays were performed on autologous or homologous antisera by the following procedure. Sera were titered against TEPC-15-coated sheep erythrocytes. A 4-fold greater concentration than the last dilution causing agglutination was used in the diluent. Inhibitors were serially diluted in 2-fold steps. TEPC-15-coated erythrocytes were added and incubations were carried out as stated. The last dilution inhibiting agglutination is reported. TABLE 2. Response of mice to phosphorylcholine after the fifth immunization Anti-phosDay of assay 4 6 8

10 12 14 16

Anti-phosphorylcholine phorylcholine Anti-T-15 titer titer (PFC/spleen) 8710

4-

2331*

15,060 4 1560 11,950 + 2284 8670 * 2410 2280 1908

+- 608 + 336 + 332 4 222

2050t 4100 4100 512 1020 128 4100

8

8 8 8 8 16 32

BALB/c mice having received previously four injections of 109 heat-killed pneumococci (R36A) were immunized again 16 weeks after the last immunization. The mice were assayed for plaque-forming cells (PFC) and titers for 16 days after the fifth immunization. The PFC response of mice immunized four times and rested for 16 weeks was 1778 + 623 PFC/spleen. * Mlean PFC response per spleen + SEM. Six mice per group done with four determinations per animal.

t Hemagglutination titer of pooled serum of six mice.

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Immune Responses In Vitro. Cultures were prepared by the methods of Mishell and Dutton (18) with the modification of Cosenza and Kohler (4). A minimal essential medium was supplemented with 10% fetal calf serum. Spleen cells (1.5 X 107) from BALB/c mice were mechanically separated and placed in culture. Sera from immunized BALB/c mice were added to cultures to give a final concentration of 1:200. Two hours after addition of the sera, the cultures were immunized with 5 X 107 pneumococci and either 0.0625% horse erythrocytes or 1 ng of TNP-Ficoll (a gift from D. Mosier, NIH, Bethesda, Md.). Plaque-forming cell responses were assayed 4 days after immunization by a modification of the JerneNordin plaque assay (19). Production of Homologous Anti-Receptor Antibody. Female A/He mice (6-8 weeks old) were immunized subcutaneously with 50 ,ug of purified TEPC-15 protein in complete Freund's adjuvant and methylated bovine serum albumin. After three more immunizations, each with 50 ,ug of T-15 in incomplete Freund's adjuvant, the serum was collected. This serum specifically *suppressed the response to phosphorylcholine in vivo and in vitro and henceforth will be referred to as homologous anti-receptor antibody. RESULTS Response to Multiple Immunizations with R36A. Different groups of BALB/c mice, each consisting of at least six animals, received one to five immunizations with R36A vaccine. The immunizations were given 3, 5, 12, and 28 weeks after the first immunization. Four days after each immunization the plaque-forming cell response to phosphorylcholine was measured and sera were collected for titers. After the fifth immunization, groups of six animals were assayed for plaques and titers at days 4, 6, 8, 13, 12, 14, and 16 after the fifth immunization. Also, cultures prepared from groups of mice having received none, four, or five immunizations were immunized in vitro. The results are presented in Tables 1, 2, and 3. First, the plaque-forming cell and antibody responses to phosphorylcholine stabilize or decline with time and repeated immunizations (Table 1). Second, after the fourth and fifth immunizations, serum-agglutinated sheep erythrocytes coated with TEPC-15 and the level of this activity may have risen as the plaque-forming cell response declined (Tables 1 and 2). Third, the response of spleen cells of multiply immunized mice increased to phosphorylcholine substantially after repeated immunizations (Table 3). The agglutination of T-15-coated erythrocytes suggested the appearance of an antibody which, if specific, would be directed against the receptor for phosphorylcholine. If this were indeed the case, then the appearance of such autogenous anti-receptor antibody might account for the stabilization or decline in response in vivo, since the number of cells competent to respond to phosphorylcholine increased, as judged by the response in vitro. Washing of cells in their preparation for culture may remove autogenous anti-receptor antibody, thus permitting increased responses due to amplification of the clone from previous immunizations. These possibilities were examined in the following ways. Characterization of Sera that Agglutinate TEPC-15 Sheep Erythrocytes. Serum taken from animals after the fourth immunization agglutinated TEPC-15-coated sheep erythrocytes.


Autogenous Anti-Receptor Antibody

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The serum was analyzed with various inhibitors and was compared with homologous anti-receptor antibody obtained from A/He mice. First, we determined that agglutination was indeed caused by antibody. If a polyvalent-free phosphorylcholine antigen such as C-polysaccharide were released from the R36A vaccine and caused agglutination, then free phosphorylcholine should inhibit the agglutination. As seen in Table 4, 10 mM phosphorylcholine did not inhibit agglutination by either serum. Furthermore, if agglutination were due to antigen, then antibody against antigen should neutralize this effect. Antibody against phosphorylcholine raised in chickens, rabbits, or a human myeloma protein with antiphosphorylcholine activity (20) did not inhibit agglutination in either system. From these results we conclude that agglutination of TEPC-15-coated sheep erythrocytes is not caused by a polyvalent antigen but by an antibody. To decide whether this anti-antibody activity was directed against the constant or variable portions of the anti-phosphorylcholine antibodies, the agglutination of sheep erythrocytes coated with nonphosphorylcholine-binding myeloma proteins MOPC-460 (IgA kappa) or MOPC-104E (IgM, lambda) was tested. The serum from multiply immunized mice did not agglutinate sheep erythrocytes coated with these proteins. Apparently, then, the anti-antibody activity is directed against the variable region of the antibodies against phosphoryleholine. Since the anti-phosphorylcholine response in BALB/c mice is predominantly of the TEPC-15 idiotype, TEPC-15 protein should be an effective inhibitor. As seen in Table 4, this is the case; two other phosphorylcholine-binding myeloma proteins of different idiotype (21), MOPC 167 and McPC 603, inhibit agglutination of both sera but at much higher concentrations. Thus, insofar as examined in these ways the antibody against T-15 sheep erythrocytes obtained from multiply immunized mice, i.e., autogenous anti-receptor antibody, was indistinguishable from homologous antireceptor antibody. The similarity in the biological activities between autogenous and homologous anti-receptor antibody was examined in another way in the following experiments. Inhibition of Responses by Serum from Immunized Mice. Antibody against phosphorylcholine given to mice or added to cultures specifically suppresses responses to phosphorylcholine; suppressive activity can be eliminated by absorption with C-polysaccharide-Sepharose (16) but not with T15-Sepharose. Homologous anti-receptor antibody specifically suppresses the response of mice or cultures to phosphorylcholine, and the suppressive activity can be eliminated by absorption with T-15 Sepharose but not by C-polysaccharideSepharose (L. Kluskens, unpublished observations). Therefore, sera from singly or multiply immunized mice were characterized for suppressive activity using these immunoadsorbents. Pooled sera were tested from the mice bled either 4 days after the first immunization or 4 days after the fifth immunization. Both sera suppressed (Fig. 1A). The suppressive activity of the serum obtained after one immunization was decreased by absorption with C-polysaccharide-Sepharose but not by absorption with T-15-Sepharose (Fig. 1B). In contrast, the suppressive activity of serum obtained after five immunizations was decreased by absorption with T-15-Sepharose but not by C-polysaccharide-Sepharose (Fig. 1C). Thus, suppression caused by the early serum can be attributed to antibody against phosphorylcholine, whereas suppression caused

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TABLE 3. Response of spleen cells in vitro obtained from BALB/c mice previously immunized with phosphorylcholine Week of immunization in vitro after the first No. of previous immunization immunizations in vivo None 4 5

28 36

PFC*/1.5 X 107 spleen cells

Phosphorylcholine 591 ± 85 3760 i 507 2700 4- 181

Horse

erythrocytes 1110 4 124 1050 4± 189

1400

4-

119

Spleen cells from BALB/c mice were immunized in vitro with 5 X 107 heat-killed pneumococci R36A. The animals were either not immunized or had previously received four or five immunizations with R36A. The fourth immunization of mice occurred at the 12th week after the first, and the fifth at the 28th week. The cultures were assayed 4 days after the immunization in vitro. * Geometric mean plaque-forming cells (PFC) i SEM.

by the second or late serum can be attributed predominantly to antibody against T-15. In other experiments, absorption of late immune serum with Sepharose coupled to MOPC-167 or McPC 603 did not remove the suppressive activity of either early or late sera. DISCUSSION

In the present study we could detect two kinds of antibody responses after repeated immunization with phosphorylcholine. One kind of antibody was conventionally directed against the immunizing antigen phosphorylcholine; the other kind bound to the idiotype of the antibody against phosphorylcholine, i.e., was autogenous anti-idiotypic antibody. Previously we have used anti-idiotypic antibody to specifically suppress the response to phosphorylcholine in vivo and in vitro (4, 10). Further studies on the mechanism of suppression (22) showed that anti-idiotypic antibody binds to the receptor for phosphorylcholine on responsive lymphocytes and blocks its function. Thus, anti-idiotypic antibody is functionally anti-receptor antibody in the anti-phosphoryleholine response. In the following, we shall discuss the evidence that the autogenous anti-idiotypic antibody induced by antigen is TABLE 4. Inhibition of hemagglutination of TEPC-15 sheep erythrocytes Inhibitor TEPC-15 MOPC-167

McPC-603 MOPC-460

MOPC-104E Phosphorylcholine

Autogenous antireceptor antibody

Homologous antireceptor antibody

0.005 jg 0.310 ug

0.050,ug 0.625,ug

0.310,g

0.625 lug 12.5 jug > 50.0 ,g >10 mM

25.0 yg 25.0 ug > 10 mAI

Hemagglutination of TEPC-15 sheep erythrocytes was by a 1:8 dilution of the pooled sera from multiply immunized BALB/c with phosphorylcholine or a 1:50 dilution of anti-TEPC-15 (homologous anti-receptor antibody) raised in A/He mice. The inhibitors were added in serial 2-fold dilutions. The number of Ug shown is the least amount to completely inhibit hemagglutination.

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DAY 4 SERUM

DAY 200 SERUM


DAY 4 DAY 200 SERUM SERUM

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DAY 4 DAY 200 SERUM SERUM

FIG. 1. Sera from BALB/c mice immunized one or five times with R36A were absorbed on C-polysaccharide-Sepharose or TEPC-15Sepharose. The response of 1.5 X 107 spleen cells to phosphorylcholine was 1555 i 196 plaque-forming cells, to horse erythrocytes 1401 i 335 plaque-forming cells, and to TNP-Ficoll was 1154 + 109 plaque-forming cells. A. The percent of the control response to phosphorylcholine, horse erythrocytes, and TNP in the presence of sera obtained 4 days (day 4) after the first immunization or 4 days after the fifth immunization (day 200). B. The response in the presence of antisera against phosphorylcholine absorbed on C-poiysaccharideSepharose (C) of antisera against phosphorylcholine absorbed on TEPC-15-Sepharose is shown.

"auto"-anti-receptor antibody and might have a biologically important function in the regulation of immunity. The autogenous antibody against antibody directed against phosphorylcholine is detectable in the serum of immunized BALB/c mice after the fourth immunization with phosphorylcholine antigen. This antibody is measured by passive agglutination of sheep erythrocytes coated with TEPC-15 myeloma protein, which is of the same idiotype as the induced antibody to phosphorylcholine in BALB/c mice (4, 11, 23). Inhibition of hemagglutination with various inhibitors indicated that the agglutination was idiotypic-specific. We excluded the possibility of free antigen causing agglutination of antibody (T15)-coated sheep erythrocytes by showing the hapten (phosphorylcholine) does not inhibit the agglutination. Furthermore, if the agglutination were caused by free antigen, the addition of antibody against phosphorylcholine should neutralize this effect. The antibodies against phosphorylcholine used were raised in different species and did not share idiotypes with the BALB/c anti-phosphorylcholine. From these studies we can conclude that the agglutination of T15-sheep erythrocytes by serum from BALB/c repeatedly immunized with phosphorylcholine is not caused by an excess of free antigen. The finding of antibody and anti-antibody activity in the same serum samples from multiply immunized animals suggests that both are in an equilibrium of complexed and free antibody. It could be expected that complex formation would neutralize either anti-phosphorylcholine or anti-receptor antibody titer activity and that no titers of either antibody were detectable. To test the ability to measure both antibodies in complex form, TEPC-15 and anti-TEPC-15 were mixed in different ratios. We found that over a wide range of ratios, net activities of both anti-receptor antibody and anti-phosphorylcholine are measurable by passive hemagglutination. However, at certain ratios of both antibodies, anti-phos-

phorylcholine and anti-anti-phosphorylcholine, some mutual inhibition of titers was observed. Thus, while we can conclusively show the presence of specific anti-antibody activity, we cannot accurately quantitate the amount present in the serum. Precipitates of antibody complexes may be trapped in the kidney; however, this becomes effective only when a minimal concentration of complex is reached in the circulation (24). No histological evidence was found for the presence of complex disease in the kidneys of these animals. A comparison of anti-receptor antibody produced in A/He mice by immunization with TEPC-15 and of "auto" antireceptor antibody induced in BALB/c by phosphorylcholine shows that they are indistinguishable in their immunochemical and biological activities. It has been reported by several researchers that animals can produce anti-idiotypic antibodies against their own immunoglobulins (7-9). Thus, the appearance of "auto"-anti-receptor antibody in BALB/c concurs with these reports. Repeated immunizations as reported here lead to a stabilization or slight decrease of the anti-phosphorylcholine plaqueforming cell responses. In another study, we have observed greater than 90% suppression of the responses to phosphorylcholine in BALB/c mice after repeated immunizations given at weekly intervals (25). Spleen cells from such suppressed animals challenged in vitro gave plaque-forming cell responses greater than normal. Thus, the size of the clone of cells responsive to phosphorylcholine is not reduced in multiply immunized animals, but seems to be reversibly blocked by a factor present in the serum. Precisely the same reversible blockade of responsive cells has been observed in BALB/c mice that had received anti-receptor antibody by passive immunization (22). Serum collected after the fourth immunization can suppress the in vitro response to phosphorylcholine specifically, and this suppressive activity was removed by


(1974)

absorption on TEPC-i5-Sepharose but not by abs6iption-with phosphorylcholine binding proteins of different idiotypes. Simultaneously with the appearance of anti-receptor antibody we find the response to phosphorylcholine decreasing. This decrease may be due to anti-receptor antibody binding to receptors for phosphorylcholine on immunocompetent cells and blocking further interaction with antigen. Since this blockade is reversible, the mode of auto-anti-receptor antibody induced suppression may be analogous to the mechanism of suppression by reagent anti-receptor antibody given to adult mice (10). Taken together, these findings are consistent with the suggestion that autogenous anti-receptor antibody may suppress the response in multiply immunized mice. The biological significance of auto-anti-receptor antibody induced suppression might be to prevent overstimulation of responsive clones and provide protection from clonal senescence in adult animals (11, 26). We have reported that passively administered anti-receptor antibody induces tolerance in neonatal mice (27) by depleting clones of cells responsive to phosphorylcholine (22). Conceivably, neonatal animals produce autogenous anti-receptor antibody directed against receptors for self-antigens and in this way deplete clones reactive with those antigens. We gratefully acknowledge the expert technical assistance of Miss Susan Smyk and Miss Ingrid Nebl. We thank Dr. Donald A. Rowley for his help in preparation of this manuscript. This work was supported by USPHS Grant Al-11080. L.K. is supported by USPHS Training Grant 2-TO5-GM0-1939. H.K. is recipient of 'Research Career Development Award A1-70559. 1. Wigzell, H. (1966) "Antibody synthesis at the cellular level: Antibody induced suppression of 7S antibody synthesis," J. Exp. Med. 124, 953-969. 2. McKearn, T. J. (1974) "Antireceptor antiserum causes specific inhibition of reactivity to rat histocompatibility antigens," Science 183, 94-96. 3. Rowley, D. A., Fitch, F. W., Stuart, F. P., Kohler, H. & Cosenza, H. (1973) "Specific suppression of immune responses," Science 181, 1133-1141. 4. Cosenza, H. & Kohler, H. (1972) "Specific inhibition of plaque formation to' phosphorylcholine by antibody against antibody," Science 176, 1027-1029. 5. Hart, D. A., Wang, A., Pawlak, L. L. & Nisonoff, A. (1972) "Suppression of idiotypic specificities in adult mice by administration of anti-idiotypic antibody," J. Exp. Med. 135, 1293-1300. 6. Eichman, K. (1974) "Idiotype suppression. Influence of the dose and of the effector functions of anti-idiotypic antibody on the production of an idiotype," Eur. J. Immunol. 4, 296301. 7. Sirisinha, S. & Eisen, H. N. (1971) "Autoimmune-like antibodies to the ligand binding sites of myeloma proteins," Proc. Nat. Acad. Sci. USA 68, 3130-3135.

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8. Rodkey, L. S. (1974) "Studies of idiotypic antibodies: Production and characterization of auto anti-idiotypic antisera," J. Exp. Med. 139, 712-720. 9. Yakulis, V., Bhoopalam, N. & Heller, P. (1972) "The production of idiotypic antibodies to BALB/c plasmacytoma globulins, in BALB/c mice," J. Immunol. 108, 1119-1122. 10. Cosenza, H. & Kohler, H. (1972) "Specific suppression of the antibody response by antibodies to receptors," Proc. Nat. Acad. Sci. USA 69, 2701-2705. 11. Lee, W., Cosenza, H. & Kohler, H. (1974) "Clonal restriction of the immune response to phosphorylcholine," Nature 247, 55-57. 12. Cosenza, H. & Kohler, H. (1972) "Antireceptor antibodies: Specific suppression of the immune response," Third Int. Convoc. Immunol., Buffalo (Karger, Basel), pp. 330-339. 13. Chesebro, B. & Metzger, H. (1972) "Affinity labeling of a phosphorylcholine binding mouse myeloma protein," Biochemistry 11, 766-771. 14. Porath, J., Axen, R. & Ernback, S. (1967) "Chemical coupling of proteins to agarose," Nature 215, 1491-1492. 15. Liu, T. & Gotschlich, E. C. (1963) "The chemical composition of pneumococcal C-polysaccharide," J. Biol. Chem. 238, 1928-1934. 16. Sherr, A. & Tarakis, H. (1971) "Hapten binding studies on mouse myeloma proteins with antibody activity," J. Immunol. 106, 1227-1233. 17. Gold, E. R. & Fudenberg, H. H. (1967) "Chromic chloride: -A coupling reagent for passive hemagglutination reactions," J. Immunol. 99, 859-866. 18. Mishell, R. & Dutton, R. W. (1966) "Immunization of normal mouse spleen cell suspensions in vitro," Science 153, 1004-1005. 19. Plotz, P. H., Talal, N. & Asofsky, R. (1960) "Assignment of direct and facilitated hemolytic plaques in mice to specific immunoglobulin classes," J.'Immuno., 100, 744-751. 20. Riesen, W., Noseda, G., Rudicoff, S. & Potter, M. (1973) "An IgM Waldenstrdm with anti-phosphorylcholine specificity," Protides of the Biological Fluids-200th Colloquium, 201-206. 21. Potter, M. & Lieberman, R. (1970) "Common individual antigenic determinants in five of eight BALB/c IgA myeloma proteins that bind phosphorylcholine," J. Exp. Med. 132, 737-751. 22. Kohler, H., Strayer, D. & Kaplan, D. (1974) "Clonal depletion in neonatal tolerance," Science, in press. 23. Claflin, J. L., Lieberman, R. & Davie, J. M. (1974) "Clonal nature of the immnune response to phosphorylcholine," J. Exp. Med. 139, 58-73. 24. Jerne, N. K. (1972) "What precedes clonal selection?" in Ontogeny of Acquired Immunity, CIBA Foundation Symposium 1971 (Associated Scientific Publishers, Amsterdam). 25. Lee, W. F. & Kohler, H. (1974) "Decline and spontaneous recovery of the monoclonal response to phosphorylcholine during repeated immunization," J. Immunol., in press. 26. Williamson, A. R. & Askonas, B. A. (1972) "Senescence of an antibody forming cell clone," Nature 238, 337-339. 27. Strayer, D., Lee, W., Rowley, D. A., Kohler, H. & Cosenza, H. (1974). "Neonatal tolerance induced by anti-receptor antibody," Science, in press.