Induction of Primary and Inhibition of Secondary Antibody Response to Hapten by Hapten Conjugates of Type 111 Pneumococcal Polysaccharide S. P. LERMAN,' T. 1. KOMANO,~ J. J. R~OND, M. HEIDELBERGER, AND G. J. THORBECKE Deportment of Pathology, Nrw York IJrri.rwsity School of Medicitie, New York, New k'ork 10016 Receit*ed Jiirte 20, 1974 Tri- or dinitrophenylated pneumococcal polysaccharide type 111 (TNP- or DNP- SIII) ) induced a primary 19s anti-TNP response without generating immunological niemory to the hapten in LAFI mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. a6ortirs organisms (DNP-BA) induced both 19s and 7s primary responses and memory to the hapten. Spleen cells from mice immunized with TNP- KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SI11 and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH- primed spleen cells for as short as 5 min at 0°C with 10 pg of TNP-SI11 per milliliter virtually abolished their ability to give 19s and 7s memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help. I NTRODUCTI 0 N Immunization with hapten coupled to a polysaccharide carrier which, by itself, induces thymus-independent immune responses, inay give rise to anti-hapten anti- body (1, 2). The mechanism of cellular interaction involved, if any, is unclear, but it has been suggested that direct mitogenic activity of these carriers for B cells is of prime importance in inducing T-cell independent responses (3, 4). The anti-hapten response may have similar properties as the response to its carrier in that it is limited to antibody production of the 19s class (1, 2) and deficient in the formation of immunological memory (5). On the other hand, hapten conjugates of nonimmunogenic molecules such as a copolymer of D-glutamic acid and D-lysine ( D-GL) (6-8), syngeneic y-globulin (9, lo), and syngeneic red cells 1 Supported by Grant AI-3076 from the United States Public Health Service and in part by 2 Supported by USPHS Training Grant No. GM000127. 3 Recipient of National Institutes of Health Training Grant No. 5GM01668-11 from the Grant QFB13592 AI from the National Science Foundation. National Institute of General Medical Science. 32 1 Copyright 0 1975 by Academic Press, Inc. A11 rights of reprndurtion in any form reserved 322 LERMAN ET AL. do not appear to induce ininiuiie responses, but on the contrary may induce toler- ance to the hapten (11). It has been suggested that "noiii~iiiiiunogei~ic" hapten-carrier conjugates iniglit have important applications in the abrogation of ongoing but tinwanted humoral responses (12). It would be of interest if hapten conjugates could be found which specifically abolish some but not all facets of the immune response to the hapten. For example, B-cell tolerogens such as the dinitrophenylated (DNP) D-GL inhibit antibody production while leaving intact T-cell functions with specificity for the hapten such as delayed and contact hypersensitivity (13, 14). Another promising approach to this problem was initiated by Mitchell et ul. (15), who demonstrated interference with the 7s antibody memory response to DNP-hemocyanin through injection of the DNP-lysine conjugates of pneumococcal polysaccharide type I11 The present studies were undertaken to determine (i) whether the trinitro- phenyl-lysine conjugates of SI11 (TNP-SIII) could induce an immune responye to TNP with generation of immunological memory, and (ii) whether or not the inhibition of 7.5 memory expression observed by Mitchell et al. (15) also enconi- passed 19s memory and could be obtained by in vitro incubation of memory cells. (DNP-SIII) . MATERIALS AND METHODS Animals and Immunizations Male young adult LAFl mice (Jackson Labs, Bar Harbor, ME) were used throughout. Donor mice were immunized by intravenous injection of 0.3 mg TNPla-KLH usually with 10 pg E. coZi endotoxin (Difco Laboratories, Inc., Detroit, MI). In a few experiments donor mice received 0.1 ml of a 0.3% (v/v) suspension (equivalent to 2 x loR) of killed Brucella abortus (BA) organisms. Whenever the interval prior to sacrifice of donor mice exceeded 24 months, they received a second dose intraperitoneally. Donors were used no earlier than 1 month after their last priming injection. Immunizations with TNP and DNP conjugates of SI11 or of BA were always by intravenous injectionlof antigen in dosages as described in the tables, On the day prior to transfer, recipient mice received 630-750 R 13iCs 7-irradiation from a gammator M (Radiation Machinery Corp., Parsippany, N J ) . Antigens DNP,o-SIII and TNPl,o-SIII were prepared by conjugation to SI11 of E-DNP- L-lysine HCI (Sigma Chemical Co., St. Louis, MO) or c-TNP-L-lysine HC1 (Nutritional Biochemicals Corp., Cleveland, OH), respectively, by the cyanogen halide method of Axen and Ernback (16) as described by Mitchell et al. (15). Trinitrophenylated sheep erythrocytes (TNP-SE) were prepared according to the method of Rittenberg and Pratt (17). TNP-BA and DNP-BA were prepared by the method of Little and Eisen (18). Briefly, trinitrobenzene-sulfonic acid (TNBS) was recrystallized from the commercial preparation (Eastman Kodak Co., Rochester, NY) by cooling from hot 1.0 N HCl and washing with cold HCl. To 20 rng of recrystallized TNBS or of dinitrobenzene-sulfonic acid (Eastman Kodak Co., Rochester, NY) dissolved in 2.0 ml of a 2% K2C03 solution were added 2.0 nil of packed RA (U.S. Department of Agriculture) with stirring. Stir- BLOCK OF ANTI-ITAPTEN MEMORY RY 11 .\PTEN-SI11 323 u) v, 104 +I al al v, - a .. V h 103 DNP SIU DNP SIU 0 2 34 5 6 Doys After Injection FIG. 1. Primary anti-TNP responses of LAF, mice expressed as PFC/spleen at various days after injection of DNP-BA (A-A) and TNP-B.4 (A-A) or 1 pg (0-0) and 100 +g (.--e) DNP-SIII. Each point represents the mean I+ SE for 2-9 mice. Shaded area denotes the mean PFC level for normal spleen f SE measured with TNP-sheep erythro- cytes. The numbers of PFC/spleen against sheep erythrocytes were subtracted from all values. ring was continued for an additional 2 hr at rooin temperature and overnight at 0°C. The mixture was dialyzed successively against distilled water, 0.1 M KC~HSO~, and finally 0.15 &I NaC1. Dinitrophenyl groups were conjugated to human gamma globulin (HGG) and keyhole limpet hemocyanin ( KLH) (Mann Laboratories, Orangeburg, NY) (18). The extent of coupling of DNP to HGG, KLH, and SI11 was estimated from the molar extinction coefficient, 17,400, at 360 nm with 100,000 as the unit of molecular weight for the protein and polysaccharide carriers (15). The degree of TNP conjugation was estimated using the molar extinction coefficient, 15,400, at 346 nni. SI11 was prepared as described previously (19), and the amount of poly- saccharide present in each of our hapten-polysaccharide conjugates was deter- mined by the phenol-sulfuric acid reaction of Dubois et al. (20). Cell Transfers The spleens of donor mice were teased into a cell suspension in Medium 199 (Microbiological Associates Inc., Bethesda, MD) and washed twice. Cells were either injected intravenously into irradiated recipients with antigen or were incu- bated with antigen in vitro prior to transfer as described under Results. Assays Numbers of antibody-forming cells per spleen to TNP were enumerated by localized hemolysis in gel of TNP-SE (17). As a control, spleen cell suspensions were also plated against uncoated SE and the values obtained subtracted from those for PFC against TNP-SE. Rabbit anti-mouse Ig was used for development of indirect PFC (21). The numbers of 7s PFC were calculated by subtracting the direct PFC from the indirect PFC. Sera from recipient mice were titrated for anti-TNP and anti-KLH by passive hemagglutination. Indicator cells were tanned cells (22) to which had been adsorbed 0.05 nig TNP13-ovalbumin or KLH per niilliliter of 2.5% tanned SE. 3 24 LERMAN ET AL. RE S 1 1 LT S Primary Response to Hapten Presented on the BA and SIII Carriers Initial studies of responses to a single injection of DNI'-l:A, TNP-GA, DN1'- SIII, and TNP-SI11 showed that all induced a rise in the number of PFC per spleen with anti-TNP specificity, but with large differences in effectiveness between the SI11 and the BA carrier. Figure 1 shows the responses to DNP-BA and TNP-BA. Peak PFC/spleen values were reached on day 3, and the number of antibody-forming cells remained high for several days. TNP-BA always gave a much higher response than three different preparations of DNP-BA, and at the dose used was also more effective than either TNP-KLH or DNP-KLH given as a single intravenous dose of 100 pg. The mean PFC/spleen on day 5 after TNP-KLH was 4175 as compared to 583 for DNP-KLH. * The high immu- nogenicity of BA conjugates might be due to their probable content of endotoxin. Indeed, immunization with DNP-KLH, given together with 50 pg of E. coli endotoxin, resulted in a resporise at least as high as that to DNP-BA. Although the majority of the PFC detected through clay 6 after injection of KLH or BA conjugates were 19S-producing, a significant percentage of animals also showed 7.5 antibody-producing cells on day 6 of the primary response. The response to DNP- or TNP-SIII, on the contrary, did not show a significant 7s component at any time. Peak PFC/spleen values after an optimal (1 pg) dose of DNP-SI11 were reached 011 day 3 and declined thereafter (Fig. 1). Ten micrograms of DNP-SI11 induced a similar response, but that to 100 pg was much lower (Fig. 1). Control LAFl mice injected with 0.1 ml of or of 1W M TNP-lysine containing 1 pg unconjugated SI11 did not show any splenic PFC above background 4 days later. The response to TNP-SI11 was slightly higher than to DNP-SI11 but otherwise was similar. Mean PFC/spleen values on days 4-5 were 3550 after 1 pg, 3218 after 10 pg, and 724 after 100 pg of TNP-SIII. Experiments in which immunization with DNP-SI11 was attempted 1 week after a previous tolerogenic or immunogenic dose of SI11 were difficult to interpret, since both dose levels of SI11 used appeared to lower somewhat the subsequent response to DNP-SIII. Secondary Response in Vivo to Haptens Presented on BA and SIII Carriers Mice reinjected with 1 pg DNP-SI11 3 weeks after a primary injection of 1, 10, or 100 pg DNP-SI11 did not show a secondary response. Direct PFC/spleen values measured 6 days later were no different from those in unchallenged mice and were similar to those seen 5 days after a primary injection (Expt 1, Table 1). 7s PFC were absent. DNP-BA induced a slightly higher response comparable to the one seen on day 6 after a primary injection of this antigen. There was no 4 In this experiment all PFC determinations were made against TNP-SE rather than DNP-SE. That higher PFC values after immunization with TNP as compared to DNP con- jugates were not due to the method of detection used was shown in unpublished observations with Dr. E. Goidl and Dr. G. W. Siskind, Department of Medicine, Cornell University Medical Center, New York, in which PFC detected with DNP-SE as indicator cells were also more numerous after immunization with TNP conjugates. Thus, TNP conjugates appear to have higher immunogenicity for LAF, mice than do DNP conjugates. BLOCK OF ANTI-IIAPTEN MEMORY BY 1I:ZI'TF.N-SIlI 325 Priming Challenging hTean log PFC/spleen j= SE antigen antigen (geometric mean) ______ __ - Expt la Expt 2" Direct Indirect Direct Indirect ~- -- DNP-SI11 (1 pg) DNP-SI11 (10 pg) DNP-SI11 (100 pg) DNP-BA D N P-SI I 1 * DNP-BA TNP-BA None DNP-SI11 D N P- B4 TNP-BA None DNP-SI I I DNP-BA None DNP-SI11 DNP-BA None 3.25 f 0.09 3.39 f 0.15 (1 760) (2425) (3290) (3405) 3.52 f 0.10 3.53 f 0.17 3.18~ 3.1lC (1510) (1300) 3.36 f 0.07 3.19 st 0.13 (2270) (1555) (4535) (4670) 3.66 f 0.10 3.67 f 0.12 3.18" 3.2OC (1 520) ( 1 570 j 3.19 f 0.12 3.23 i 0.08 (1555) (1710) (2785) (2850) 3.1& 3.16e (1510) (1460) 3.45 f 0.07 3.46 3~ 0.06 3.58 f 0.06 3.64 f 0.06 (3830) (4410` 3.69 f 0.08 4.06 f 0.09 (4930) (11,450) 3.14~ 3.39 (1370) (2230) 3.35 f 0.13 3.36 f 0.16 (2255) (2270) (28,150) (44,500) 4.45 f 0.07 4.65 f 0.13 3.30 f 0.12 3.29 & 0.07 (2010) ( 1940) (24,200) (36.480) 4.38 f 0.25 4.56 f 0.22 Interval between injections was 21 days (Expt 1) or 14 days (Expt 2). Numbers of mice in each group varied from 4 (Expt 1) to 2 or 3 (Expt 2). Determination of PFC/spleen was done on day 6 (Expt 1) or day 5 (Expt 2) after challenge. * Challenge dose was 1 pg DNP-SI11 in all experiments. Recipients' spleens in these groups were pooled prior to assay for PF-C. tolerance to DNP in mice previously injected with 100 pg DNP-SI11 since they were still able to respond to DNP-BA, although possibly somewhat less than normal mice. In Expt 2, mice reinjected with DNP-BA and TNP-BA 2 weeks after priming with DNP-SI11 gave similar results. Responses comparable to those 326 LERMAN ET AI.. Donors primed with DNP-BA DNP-SI11 (1 pg) DNP-SI11 (100 fig) Recipients challenged (geometric mean) with Mean log l'FC/spleen f SE .____- Expt 9 Expt @ Direct Indirect Direct Indirect ____ DNP-BA DNP-SI I I * DNP-KLH * None DNP-BA DNP-SI I I None DNP-BA DNP-SI1 I None 2.65 f 0.05 3.68 f 0.09 (442) (4810) (57) (65) 1.76 f 0.45 1.81 i 0.18 2.36 f 0.16 2.26 f 0.08 (229) (182) 3.28 f 0.13 3.21 f 0.06 (1 905) (1620) (1550) (1130) 3.26c 3.08" (1840) (1190) 3.19 f 0.19 3.05 f 0.09 3.22 i 0.08 2.87 f 0.18 (1660) (742) (709) (617) 3.08" NDd (1200) 2.85 f 0.21 2.79 f 0.14 3.11 f 0.04 (1 290) 2.75 =t 0.14 (563) 3.07 f 0.13 (1160) 2.93c (85 1) 3.70 f 0.08 (5200) 2.68 f 0.18 (479) 3.11 f 0.14 (1305) 2.9& (870) a Interval between priming and transfer was 16 weeks for the DNP-BA-primed spleen cells and 4 weeks for DNP-SIII-primed cells in Expt 5, and 3 weeks for UNP-BA-primed spleen cells in Expt 6. Irradiated recipients received 3-3.5 X 10' cells (Expt 5) or 2 X lo7 cells (Expt 6) together with the challenging antigen. Receipients' spleens were assayed for PFC 7 days later (4-5 mice per group). * All challenges with DNP-SI11 employed 1 pg DNP-SI11 except for Expt 5 (donors primed with DNP-BA), where the dose was 50 pg DNP-SIII. Dose of DNP-KLH was 100 pg. Recipients' spleens in these groups were pooled prior to assay for PFC. Not determined. after a primary injection of these antigens were found on day 5 after challenge, and a 7s component was detectable as also seen after the primary injection. In contrast, mice primed with DNP-BA gave a secondary 7s anamnestic response to DNP which was not demonstrable after challenge with DNP-SIII. There was a slightly higher 19s response to DNP-SI11 in these mice than in those primed with DNP-SIII. Adoptive Secondary Responses to DNP-BA and DNP-SIII Since it was conceivable that the presence of antibody to the SI11 carrier molecule in the sera of primed mice was inhibiting the secondary response to BLOCK OF ANTI-HAPTEN MEMORY BY I3APTEN-SI11 327 Recipients Mean log fFC/spleen f SE challenged with (geometric mean) ~- - Expt 31' Expt +a Direct Direct Indirect DNPZrKLHb 3.21 f 0.08 (1623) DNP-RA DNPs- HGG 3.12 f 0.04 (1320) (832) 2.92 f 0.05 DNPds-HCG 1.26 i 0.43 (18) 3.38 f 0.09 (2380) 3.26 f 0.09 (1830) 3.28 f 0.04 (1895) 3.01 f 0.21 (1025) 4.40 f 0.04 (25,200) 3.81 f 0.22 (6460) 3.70 f 0.04 (5020) 3.20 f 0.08 (1 585) Spleen cells were taken 2 weeks (Expt 3) or 3 weeks (Expt 4) after injection of 1 mg DNPw KLH with or without 10 pg E. coli endotoxin. Irradiated recipients received 10' (Expt 3) or 2 X lo7 (Expt 4) spleen cells together with the challenging antigens. Recipients' spleens were assayed for PFC 6 days (Expt 3) or 7 days (Expt 4) later (4-5 mice per group). * Challenge dose of antigen, 0.2 mg DNP-KLH or 0.1 mg DNP-HGG. these conjugates, adoptive secondary responses after transfer of 2-3 x lo7 spleen cells from immunized mice were studied. Table 2 shows the results of these experiments. Cells taken 4 weeks after injection of DNP-SI11 did not respond to either DNP-BA or DNP-SIII, giving similar PFC values in recipient spleens with or without antigenic challenge at the time of transfer. DNP-BA-primed spleen cells showed definite 7s and weak 19s anamnestic responses to DNP-BA but none to DNP-SIII. These data implied that DNP-SI11 was unable to generate memory cells to DNP and, thus, behaved like SI11 alone, which is known to induce 19s responses without generating detectable memory (22). However, the possibility existed that DNP-SI11 had generated memory cells but was unable to challenge them and that DNP-BA could not challenge this memory to DNP-SI11 because of the dif- ference in carrier. Although BA itself is a largely thymus-independent antigen with respect to challenge of memory cells (23), the DNP-BA conjugate might not be equally thymus independent. We therefore tested the capacity of DNP-BA to challenge memory cells generated by DNP-KLH. Spleen cells from DNP-KLH- primed mice were taken 2 weeks (Expt 3, Table 3) or 3 weeks (Expt 4, Table 3) after immunization and challenged upon transfer (2 X 10' cells/mouse) with DNP- KLH, DNP-BA, or DNP-HGG conjugates, DNP-BA was much more efficient than DNP-HGG in challenging these memory cells, although it induced a lower 7s response than the homologous DNP-KLH conjugate. It should, therefore, have been capable of challenging memory cells to DNP-SI11 if such cells had been present. 328 LERMAN ET AL. Ahility of I~NP-.SllI and 7"P-SIII to Block .Idopti7~ 19.S lr~td 7.S ill[.r/rory I< csponscs The previous experiments showed that DNP-311 neither generated nor chal- lenged DNP memory cells. We next examined the ability of these conjugates to hlock the responsiveness of memory cells in 7fizm. Table 4 shows that, when hapten- RA- or hapten-KLH-prinie~l memory cells were transferred with the homologous antigen and varying doses of the hapten-SI11 conjugates, Imth 19s ant1 7s memory cells did not respond to the homologous conjugate. Even as little as 0.5-1 pg of hapten-SI11 resulted in significant inhibition of both responses. In two of the experiments in which this comparison could be made the 19s responses appeared slightly less sensitive to inhibition than the 7.5 responses (Expts 11 and 12, Table 4). When passive hemagglutination titers to TNP of the recipients' sera were compared with values for PFC/spleen, agreement between these two measures of immune responsiveness of the transferred cells was noted. In experiments in which the PFC/spleen values indicated an effective block of the response, the anti-TNP titers of the sera were also greatly reduced, while a less effective block of the PFC/spleen response resulted in a smaller reduction in titer. For example, in three typical experiments, the control recipient groups had mean log2 serum titers ranging from 10.0 to 10.8, the groups which did not receive antigen had mean logz titers of 2.0-2.7, and the mice receiving TNP-KLH with 5-50 pg TNP-SI11 had mean logz titers of 2.3-3.3. Mice receiving a lower dose of the blocking antigen (0.5-1 pg) had titers of 7.4-8.7. Further studies showed that a simple preincubation with hapten-SI11 for as short as 5 min at O'C, followed by washing of the cells and transfer into irradiated recipients together with the homologous conjugate, was sufficient to block partially the response of TNP memory cells (Table 5). Excellent correlation was again found between results obtained for PFC per spleen and for anti-TNP titers in recipients' Sera (Table 6). Incubation at 0°C was no less effective than at 37"C, and prolonging the incubation to 60 min did not increase the degree of inhibition (Table S), nor did increasing the concentration of DSP-SI11 to 200 pg/ml (45% 17s 41 % ) . Although the results were somewhat variable, TKP-SI11 was more effective than DNP-SIII, particularly at the 10 pg/ml level, possibly because of the much higher degree of conjugation of SI11 with TNP than with DNP. IVhile only one experiment was included in Table 5, good inhibition was obtained at this level of TNP-SI11 in several additional experiments. It was noted that both 19s and 7s memory cells were sensitive to the blocking effect of preincubation. Pre- incubation with 40 pg DNP-SIII/ml, followed by washing, was slightly less effective in blocking the subsequent response to TNP-KLH than the simultaneous injection of 0.5 pg of DNP-SI11 (Expt 12, Tables 4 and 5). Additional experiments (not in the tables) showed that incubation of TNP- KLH primed cells with 100 pg/ml of either DNPs-HGG or DNP,,-HGG, followed by washing had no detectable blocking effect on the subsequent adoptive response to TNP-KLH. Controls for Specificity and Suppressor Eflects Specificity of the phenomenon was established by an experiment in which cells primed to burro erythrocytes were exposed for 1 hr at 0°C to 40 pg TNP-SIII/nd. TABLE 4 PERCENT OF RESPONSE TO HOXOLOGOUS ANTKEN REMAINING AFTEK IKJECTION OF BLOCKING ANTIGEN Homologous Blocking % (PFC/spleen) antigen antigen6 challengea (PIT) Expt 8 Expt 11 Expt 12 Expt 7. Expt 9c Expt 10~ 7s 19s 7s 7s 7s 19s 7s 19s 7s ~ + None 100 100 100 100 100 100 100 100 100 (637) (1 660) (1790) (1339) (1501) (16,932) (14,904) (2891) (3 5,329) + 5-50 <1 4 3 3 <1 + 0.5 1 3 20 37 <1 53 16 41 4 1-50 <1 10 <1 <1 <1 None TNP-SI I I (40) 60 75 23 35 3 3 5 23 8 > r (16,932) (14,904) (2891) (35,329) (3289) (26,389) (3529) (28,321) (9671) (13,922) (1356) (1902) ' TNP-SIII (10) 5 12 18 DNP-SI11 (10-20) 60 34 83 52 81 Medium onlyb 60 16 <1 19 2 18 <1 11 <1 3 <1 4 (1 DNP-SI11 (40) 60 28 9 40 61 a Cells were preincubated at 2 X lo7 cells/ml with or without DNP- or TNP-SI11 at O°C as indicated. They were washed twice prior to injection of 1-2 X lo7 * Control cells, not challenged with TNP-KLH after transfer. cells per recipient with 0.1 mg TNP-KLH. PFC/spleen in recipients were determined 7 days after transfer. BLOCK OF ANTI-HAPTEN MEMORY BY HAPTEN-SI11 331 TABLE 6 INHIBITORY EFFECT OF PREINCUBATION WITH TNP-SI11 ON ADOPTIVE ANTI-TNP RUT NOT ON ANTI-KLH RESPONSES OF TNP-KLH-PRIMED SPLEEN CELLS Cells Cells Expt 14 Expt 17 witha with Geometric Mean log, Geonietric 34ean log2 incubated injected __ mean heinagglutination mean heniagglutination PFC/spleen titer PFC/spleen titer anti- anti- TNP KLH anti- anti- TNP KLH Medium only TNP-KLH 31,850 10.8 13.3 25,365 9.6 15.6 Medium only Medium only 606 2.7 7.0 677 4.0 17.7 TNP-SI11 TNP-KLH 1,514 2.8 14.3 4,562 4.8 7.0 a Cells were preincubated at 2 X lo7 cells/ml with or without 40 pg/ml TNP-SI11 for 60 niin at 0°C. They were washed twice prior to injection of 1 X lo7 (Expt 14) or 2 X lo7 (Expt 17) cells per recipient with 0.1 mg TNP-KLH. PFC/spleen vs TNP and serum antibody titers were deter- mined 7 days after transfer. The adoptive memory response of these cells, approximately 6OOO 7s PFC/spleen, was totally unaffected. Furthermore, while serum titers to TNP of recipients were reduced in parallel with numbers of PFC per spleen, the preincubation of donor cells with the blocking antigen had no detectable effect on the response to KLH, the carrier protein, as determined by serum antibody titers (Table 6). Also, transfer of a combination of unincubated TNP-KLH memory cells with an equal number of such cells after incubation with TNP-SI11 and washing gave similar responses to those of unincubated cells showing both the absence of sup- pressor activity and of excess TNP-SI11 with the incubated cells. DT SCUS SI ON The results indicate that DNP-SIII, like DNP-levan (2), induces a primary response to DNP in mice which is dose dependent in the sense that high doses (100 pg) give a lower response than low doses (0.5 pg). The response to the hapten on the SI11 molecule as a "carrier" appears to take on the properties of that to the carrier in two significant respects: (i) 19s but no 7s antibody is detectable in the serum, as is also the case after injection of optimal doses of SI11 alone; and (ii) immunological memory, that is, a higher response to a second injection than to the first, is absent as with SI11 (23). This lack of immunological memory was seen both upon challenge of the intact "primed" mice and in adoptive immune responses. It was not due to an inhibition by serum antibody, since higher doses of the conjugates could not overcome this defect and, on the contrary, made the situation worse. The lack of memory was not only due to the shown inability of DNP-SI11 to challenge 7s memory cells, but also to a lack of 7s memory formation after priming, since memory was equally lacking upon challenge of DNP-SIII- primed cells with either DNP-BA or with DNP-SITI. Although the thymus-independent responses to polysaccharide antigens are frequently ascribed to the ability of these antigens to induce mitotic activity in B lymphocytes (3), the lack of memory formation characterizes the major 332 LERMAN ET AL. deficiency of such responses and is probably due to insufficient proliferation of the I3 cells. Allogeneic effect (25, 26), antilymphocyte serum (27, 28), and coupling to a thymus-dependent carrier (29, 30) may all result in significant increases of the response to SI11 and other T-independent antigens in the mouse, in some cases accompanied by the appearance of 7s serum antibody (26, 28) and memory (31). It remains to be determined whether enhanced proliferation of the B lymphocytes, possibly in germinal centers, constitutes the major difference between the full- blown humoral immune response with resulting 19Sf7S antibody and memory, and the response to "thymus-independent" antigens lacking 7s antibody and memory. In nude, thymusless mice the response to SI11 is normal or supranormal (27) as compared to littermate controls, even though all germinal center formation in nude mice is lacking. It should be noted, of course, that even in primary responses of nude and other mice to SI11 a minor component of 7s antibody- forming cells does occur (31).5 In the absence of T help the proliferation induced by polysaccharide antigens in B lymphocytes may drive all the antigen-sensitive precursors into terminal differentiation without establishment of a memory B-cell line, as suggested by recent studies of Howard and co-workers (32). The inability of TNP-SI11 and DNP-SI11 to challenge memory cells to TNP- KLH was not simply due to the absence of interaction between the B memory cells and TNP-SIII. On the contrary, the hapten-SI11 conjugates interacted so well with the TNP-memory cells that they blocked the response to the homologous antigen. This almost instantaneous abolition of responsiveness in TNP memory cells is in apparent contrast with the ability of these conjugates to induce primary responses to TNP. In view of the observed dose effect in the primary response, where 100 pg induced a lower response than did 1 pg while 1 pg con~pletely inhibited the secondary response to homologous antigen, a quantitative difference between primary and secondary responses suggests itself. It seems likely that such a difference between primary and memory responses is caused by the affinity of the cells for the antigen (TNP group) which is influenced by (i) the numbers of antibody molecules on the surface of the cell (IgM 7s monomers or 7s IgG molecules), and (ii) the avidity of these antibody molecules. It is well known that serum antibody attains a higher average avidity with time after immunization (33), and it is less sure but likely that this property is shared by IgM and IgG (34, 35). Higher affinity of the cells for antigen would result in the concentration of much more antigen on their surface, resulting in a situation akin to the one seen with high conjugates of DNP-polymeric flagellin (36), or with antibody-antigen com- plexes (37, 38) which have been shown to facilitate induction of tolerance in vitro, a tendency attributed to the need for a certain degree of lattice formation on the surface of the cells (39). It has also been suggested by Coutinho et al. (24) that high concentrations of B-cell mitogens on the cell surface may in themselves be inhibitory rather than stimulatory for B-cell mitotic activity. The present results are consistent with (i) a higher avidity of both the IgM and IgG antibody on memory cells and (ii) a complete lack of proliferation and dif- ferentiation once the cells have interacted with too high a concentration of hapten- SIII. Other explanations are also possible. It is conceivable that a relative lack of 6 Similar findings have recently been obtained in chickens (M. D. Grebenau and G. J. Thor- becke, unpublished observations), in which low doses of DNP-SI11 or of SI11 alone induced some mercaptoethanol-resistant serum antibody, but no memory. BLOCK OF ANTI-HAPTEN MEMORY BY HAPTEN-SI11 333 competition for antigen between B cells and macrophages in the primary response results in the stimulation of some B cells by antigen via a macrophage surface, whereas in the secondary response the higher affinity of the cells overrides such an intermediary role for macrophages and the direct interaction between the antigen and B cells causes tolerance. This implies the need for macrophages in thymus-independent responses, which so far has not been established in vitro (40). Tolerance in both unprimed and primed B cells has been induced in vivo and in vitro by a variety of hapten conjugates to nonimmunogenic carriers (6, 11). Under special circumstances soluble hapten conjugates may interfere with the induction of a response in vitro by macrophage-bound hapten conjugates to the same immunogenic carrier (41). The avidity of the receptor on the B cell has been implicated in the sense that higher avidity always leads to a higher sensitivity to tolerance induction or to "blocking" (33, 42). In some such in vitro systems it was shown that several hours of incubation at 37°C were needed (43, 44). In the present study 5-15 mill at 0°C sufficed, but further studies are required to determine whether or not a simple block of the receptors induced in vitro is iollowed by cellular changes at 37°C after transfer into the animal before unrespon- siveness is establishecl. Studies on the cellular mechanism and reversibility of this induction of tolerance in memory cells will be the subject of another publication One striking similarity between induction of tolerance to hapten-polysaccharide conjugates and hapten conjugates to syngeneic substances is that they either do not involve T cells at all or are relatively inefficient at inducing T help via a new anti- genic determinant (46). Nude mice which lack T cells can be rendered tolerant at the B-cell level with relative ease, even to thymus-dependent antigens (37). Fidler and Golub (48) also showed recently that free hapten (TNBS), which probably conjugates in the animals to produce TNP conjugates of syngeneic proteins, may induce tolerance in the absence of added T cells in R cells from fetal liver. The absence of suppressor activity in incubated, blocked cell populations is clearly shown in the present studies. It appears, therefore, that the absence of T help, rather than the presence of T suppressor cells, is crucial in the induction of tolerance in this system and that the sensitivity of B cells to such induction is dependent on the affinity of the cells for the antigen. It can be postulated that a high concentration of antigen directly bound to the B-cell surface induces tolerance or blocking, which is overcome if the cells are made to proliferate greatly by T help. Such a proliferation alone would be expected to dilute the antigen on the surface of B cells and cause a lowering of its concentration to immunogenic levels. This interpretation of tolerance induction at the B-cell le17el is supported by the recent suggestion of Benson and Bore1 (49) that tolerant cells are able to circulate with antigen bound to their surface. This might explain the continued block of receptors and interference with T help by carriers such as SI11 which are non- immunogenic for T cells. In view of recent observations that B cells form caps and strip their surface free of Ig within 1 hr after incubation with antigen (SO), an explanation is needed why blocked cells should keep the antigen on their surface for a time. Perhaps the multipoint surface interaction with antigen can initiate a state similar to the one described by Edelman and co-workers (51) after interaction of cells with tetra- meric concanavalin A involving resistance to cap and patch formation by anti-Ig. (45). 334 LERMAN ET AL. Such changes on the cell surface might be reflected intracellularly by yariations in mediator levels such as cyclic AMP, an increase which may cause inhibition of the immune response (52) and may be important as a signal for induction of tolerance (53). ACKNOWLEDGMENTS We are deeply indebted to Dr. A. Nisonoff, Department of Biochemistry, University of Illinois Medical Center, for generous gifts of TNP-protein conjugates. We also wish to thank Dr. C. E. Watson of the US. 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