Tolerance Induction in TxXBT and TxXB Mice

Immunologv, 1975, 28, 943.

Tolerance Induction in TxXBT and TxXB Mice

S. MURAMATSU, T. AMAGAI AND Y. KATSURA

Department of Zoology, Faculty of Science, and Department of Bacteriology and Serology,Chest Disease Research Institute, Kyoto University, Kyoto 606, Japan

(Received 12th September 1974; acceptedfor publication 21st October 1974)

Summary. Adult normal mice and those mice which had been thymectomized,X-irradiated, and reconstituted either with bone marrow cells and thymus cells(TxXBT) or with bone marrow cells alone (TxXB), were given varying amounts(0.1-5 mg) of deaggregated soluble bovine serum albumin (sBSA). They werechallenged 10 days later with an immunogenic form of BSA. TxXB mice weresupplemented with normal thymus cells 3 days before the challenge. With any doseof sBSA, TxXBT and normal mice were made tolerant. Only 5 mg of sBSA, thehighest dose in these experiments, was effective in inducing the tolerance in TxXBmice. The simultaneous administration of 5 mg of sBSA and 0-1 mg of alum-precipitated BSA plus 0-01 mg ofendotoxin resulted in the priming in TxXBT micebut induced tolerance in TxXB mice. These results indicate that: (a) B-celltolerance could be induced independently of the influence of T cells; (b) thetolerogen susceptibility ofB cells may be lower than that ofT cells; (c) such a weaklyimmunogenic agent as the mixture of tolerogen and immunogen could eitheractivate the antibody response in the presence ofT cells or induce B-cell tolerancein the absence of T cells.

INTRODUCTION

According to the clonal selection theory (Burnet, 1959), the induction ofimmunologicaltolerance to self-components is one of the most crucial steps in the establishment ofacquired immunity. One of the most important events in current immunology may bethe discovery of the co-operation between thymus-derived T cells and bone marrow-derived B cells in the antibody response to the majority of antigens, so called T cell-dependent antigens (Claman, Chaperon and Triplett, 1966; Mitchell and Miller, 1968).Thus, some workers have suggested that such a collaboration may be prerequisite also fortolerance induction in B cells to these antigens (Gershon and Kondo, 1970, 1971; Feld-mann, 1973; McCullagh, 1973).

It has been elucidated that both T and B cells can be made tolerant to T cell-dependentantigens, and that T cells are more sensitive to tolerogens than B cells (Chiller, Habichtand Weigle, 1971; Mitchison, 1971; Katsura, Kawaguchi and Muramatsu, 1972; Chillerand Weigle, 1973a; Schrader, 1974). For example, we reported (Katsura, Kawaguchi andMuramatsu, 1972) that T cells in mouse spleen were rendered tolerant to bovine serumalbumin (BSA) by multiple weekly injections of 0-1 mg of deaggregated BSA and thatsplenic B cells were tolerized only by a high dose of the tolerogen at 5 mg or more.

Correspondence: Dr S. Muramatsu, Department of Zoology, Faculty of Science, Kitashirakawa-Oiwakecho,Sakyo-ku, Kyoto, 606, Japan.

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S. Muramatsu, T. Amagai and r. Katsura

Concerning the apparently high susceptibility of T cells in vivo to tolerogens in com-parison with B cells, the following three possibilities may be envisaged: (a) the differencein tolerogen sensitivity between T and B cells is due to their inherent characters so thatthey can be tolerized independently of each other; (b) although T and B cells are notintrinsically different from each other in tolerogen sensitivity, tolerogen-influenced T cellsinterfere with the induction of B-cell tolerance; (c) B-cell tolerance cannot be inducedby means of tolerogen alone without the aid of tolerogen-influenced T cells, in a man-ner similar to the antibody response to the same antigen.The present experiments were undertaken to pick the most plausible of these three

possibilities, using normal, TxXBT and TxXB mice. Effects of varying amounts ofdeaggregated BSA on these mice were examined. The results seem to support supposition(a) mentioned above. The administration of the tolerogenic BSA in combination with theimmunogenic form ofBSA was also performed. It was indicated that positively activated Tcells may sometimes prevent B cells from being otherwise suppressed by a massive dose oftolerogen.

MATERIALS AND METHODS

MiceFemale inbred DDD mice (supplied from the Central Animal Laboratory, School of

Medicine, Kyoto University) were used.

AntigenCrystalline bovine serum albumin (BSA) (Miles Laboratories, Incorporated, Kankankee,

Illinois) was used throughout these experiments. Deaggregated BSA solution was preparedby ultracentrifugation (105,000 g for 2 hours) of 1 per cent BSA in physiological saline.The top one third portion ofthe supernatant (sBSA) was used as tolerogen (Katsura, 1972b;Katsura et al., 1972). The concentration ofBSA in the sBSA fraction was determined photo-metrically at 280 nm. sBSA thus prepared was properly diluted with physiological salineand 0-5 ml ofthe solution (1 ml in the case of the 5 mg injection) was injected intravenouslyvia a tail vein. Alum-precipitated BSA (AP-BSA) was prepared by the addition of2 volumesof 10 per cent potassium alum to 7 volumes of 0 5 per cent BSA, followed by the neutral-ization with 10 per cent sodium carbonate to pH 6-3 (Katsura, 1972a). The precipitatewas washed three times by centrifugation and resuspended in physiological saline. Forimmunization of mice, 0 5 ml of the AP-BSA suspension containing 0-01 mg of a bacterialendotoxin (ET) (lipopolysaccharide B, extracted from Escherichia coli 0111: B4, DifcoLaboratories, Detroit, Michigan) was injected either intravenously or intraperito-neally.

ThymectomyYoung adult mice, 6 weeks old, were thymectomized under Nembutal anaesthesia

(sodium pentobarbital injection, Abbott Laboratories, North Chicago, Illinois) essentiallyfollowing the method of Miller (1960). At the time of the last bleeding, all the micewere autopsied. No thymic remnants were found in any animals used in theseexperiments.

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Preparation of cell suspensionThymus and bone marrow cell donors were syngeneic female mice of 6 weeks of age.

Mice were killed by exsanguination from carotid arteries. Eagle's minimum essentialmedium (Nissui Seiyaku Company, Tokyo, Japan) was used for suspending and washingcells. Heparin (heparin sodium injection, Takeda Yakuhin Company, Osaka, Japan) wasadded to the medium at a concentration of 5 u/ml to avoid cell adhesion or aggregation.Thymus, bone marrow and spleen cell suspensions were prepared following the methodpreviously described (Katsura et al., 1972). Cell suspensions, 05 ml for either spleen orbone marrow cells and 1 ml for the suspension containing thymus cells, were injected intomice intravenously.

X-irradiationMice were placed in perforated polystyrene boxes on a turn-table and received whole

body irradiation with a therapeutic X-ray emitter (Toshiba Electric Company, Tokyo,Japan). The irradiation was performed at 200 kV and 25 mA, at a target distance 44 cm,with a filter of 0.5 mm Cu and 0 5 mm Al, at the dose rate of 100 R/min.

TxXBT and TxXB miceThymectomized mice were rested for 3 weeks and irrated with 750 R. A number of

mice were injected with 107 bone marrow cells (TxXB), and the others were injected with107 bone marrow cells and 8 x 107 thymus cells (TxXBT). These mice were employed inexperiments after an additional 3 weeks.

Testfor antibodyMice were repeatedly bled, about 0-1 ml per bleeding, via the retro-orbital plexus.

Anti-BSA titration was performed by a passive haemagglutination test according to themethod previously described (Katsura, 1972a; Hosono and Muramatsu, 1972). Briefly,glutaraldehyde-fixed mouse erythrocytes were conjugated with BSA by means of bis-diazotized benzidine to prepare the indicator red cells. The test was performed using amicrotitration device. Each V-shaped depression in the titration trays contained 0-025ml of diluted sera and 0-025 ml of the indicator red cell suspension. Mercaptoethanol(2-ME) resistant antibody titres were determined by the method of Scott and Gershon(1970) with slight modification. After the total antibody titres were read, titration trayswere shaken horizontally to resuspend the red cells and 0-025 ml of 0- I M 2-ME was addedto each well. After incubation at 370 for 1 hour, the trays were shaken again and left stand-ing overnight at room temperature. As Scott and Gershon (1970) pointed out, it wasconfirmed in a preliminary test that this method was valid for the same results as when2-ME was added at the beginning of titration. In these experiments, however, 2-ME-sensitive antibodies were scarcely detected. Therefore, only total antibody titres are shownin the following section.

RESULTSCONDITIONS FOR THE IMMUNIZATION OF TxXB MICE

The tolerogenicity ofsBSA should be estimated by the reduction in the level of response

K

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S. Muramatsu, T. Amagai and r. Katsurato the challenge immunization in ways which could elicit a high degree of antibodyresponse in untolerized normal, TxXBT and TxXB mice. Several forms of BSA, such asheat-denatured aggregates, alum-precipitates (AP-BSA), or water-in-oil emulsion, withor without several kinds of adjuvants, such as killed Mycobacterium tuberculosis H37Rv,Bordetella pertussis vaccine, Klebsiella pneumoniae capsular polysaccharide (a gift from DrI. Nakashima, Nagoya University), or Escherichia coli endotoxin (ET), were examined.Although both normal and TxXBT mice revealed very strong, or modestly high responsesto immunogenic stimulation, only very weak or undetectable responses occurred in TxXBmice. The supplementation ofT cells, therefore, seemed necessary in TxXB mice to elicitan antibody response to BSA.The immunizing condition effective in eliciting the antibody response in TxXBT mice

and also in TxXB mice was looked for, the latter being supplemented with 8 x 1O' normalthymus cells (termed Tc in this paper) tentatively at the time of immunization. Amongdifferent results which will be reported elsewhere (Amagai and Muramatsu, manuscriptin preparation), we recognized that the administration ofAP-BSA, together with ET, was

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FIG. 1. Antibody response of TxXBT and TxXB mice immunized with an intravenous injection of0-1 mg ofAP-BSA+ 0.01 mg of ET, followed 10 days later by an intraperitoneal injection of 0-5 mg ofAP-BSA+ O10I mg of ET. TxXBT and TxXB mice were either (a) uninoculated or (b) inoculated withTc on the day of, (c) 3 days before, or (d) 5 days before the primary immunization. Circles and trianglesrepresent TxXBT and TxXB mice, respectively. Each point refers to the geometric mean titre of anumber ofmice (shown in parentheses beside each curve). Each bar represents the standard error of themean.

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most adequate for the present purpose. Thus it was decided that mice would be given theprimary intravenous injection of 0 1 mg of AP-BSA and 0-01 mg of ET followed by thesecondary intraperitoneal injection 10 days later with 05 mg ofAP-BSA and 0 01 mg of ET.The next problem to be settled was the most adequate timing ofTc injection in relation

to the primary immunization. Hence, the examination was carried out in which Tc weregiven on the day of, 3 days before, or 5 days before the immunization of TxXBT andTxXB mice (Fig. 1). Without the aid of Tc, TxXB mice revealed only a very feeble re-sponse, whereas TxXBT mice responded by giving modestly high titre antibodies (Fig. 1 a).

6-Week-old mice

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FIG. 2. Experimental design for assessing tolerance induction by sBSA. Tx = thymectomy; X = 750 RX-ray; Bm = 107 bone marrow cells; Th and Tc = 8 x 107 thymus cells. Symbols at the bottom will beemployed for each subgroup in Figs 3-6.

The addition of Tc evoked the significant level of antibody response in TxXB mice. Theresponse tended to increase as the interval between the Tc injection and the primaryimmunization was lengthened (Fig. lb, c and d). The responses ofTxXB mice in Fig. 1cand 1 d did not differ so much from each other, being considerably higher than that in Fig.1 b, and the response of TxXB mice in Fig. 1 c was nearly at the same level as that ofTxXBT mice in Fig. 1a. Since such a long interval as 5 days seemed undesirable for thepresent purpose, it was decided that Tc would be given 3 days before the primary im-munization in the following experiments. Although Fig. 1 indicated that the responses ofTxXBT mice were slightly elevated by the addition of Tc, this seemed insignificant withreference to the results in the following experiments.

EFFECTS OF VARYING DOSES OF sBSAAn outline of the experiment to estimate the tolerogenicity of a given dose of sBSA is

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represented in Fig. 2. TxXBT, TxXB and normal mice were divided into four subgroupseach, subgroups 1 and 2 receiving sBSA and subgroups 3 and 4 serving as sBSA-untreatedcontrols. Seven days after a single or the final injection of sBSA, subgroups 2 and 4 weresupplemented with Tc. An additional 3 days and a further 10 days later, all themice were challenged according to the standard immunization procedure. Bleedings

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FIG. 3. Antibody response of (a) TxXBT, (b) TxXB and (c) normal mice either untreated (circles),or treated with a single injection of 0-1 mg of sBSA (squares) or three weekly injections of 0.1 mg ofsBSA (triangles). Immunizing procedures are the same as in Fig. 1. Open symbols and closed symbolsrefer, respectively, to mice receiving Tc 3 days before the primary immunization and those uninocu-lated. Each point refers to the geometric mean titre of a number of mice (shown in parentheses besideeach curve). Decreasing number ofmice in a subgroup is due to the death ofsome of them by the end ofthe experiment. Each bar represents the standard error of the mean.

were performed 7, 10, 15, 20 and 25 days after the primary challenge immuni-zation. Antisera obtained in one experiment were stocked at -200 until titrated alltogether.The results are illustrated in Figs 3-6 and their essential points are shown in Fig. 9.

TxXBT, TxXB and normal mice were given a single injection of 0 1 mg ofsBSA (Fig. 3),three weekly injections of 0 1 mg (Fig. 3), a single injection of 1 mg (Fig. 4), injections of0-1 mg and 1 mg at an interval of 3 days (Fig. 5), or a single injection of 5 mg(Fig. 6).The outcome of these experiments were as follows: (1) at any dose, sBSA manifested its

suppressive effect on TxXBT and normal mice; (2) only the dose of 5 mg was effective

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FIG. 4. Effect of a single injection of 1 mg sBSA on (a) TxXBT, (b) TxXB3 and (c) normal mice. sBSA-treated mice and untreated mice are shown by triangles and circles, respectively. Immunizing pro-cedures are the same as in Fig. 1. Open symbols and closed symbols refer, respectively, to mice receivingTc 3 days before the primary immunization, and those uninoculated. Each point refers to the geo-metric mean titre ofa number ofmice (shown in parentheses beside each curve). Decreasing number ofmice in a subgroup is due to the death of some of them by the end of the experiment. Each bar rep-resents the standard error of the mean.

for TxXB mice; (3) the tolerance induced in TxXBT and normal mice was always in-complete, resulting in the hyporesponsiveness to the challenge immunization; (4) thesuppression caused by sBSA in TxXBT mice could be reversed more or less by thesupplementation of Tc; (5) Tc could barely influence the tolerant state in normalmice.

CONFIRMATION OF B-CELL TOLERANCE BY 5 mg OF sBSA IN TxXB MICE

Although a single injection of 5 mg of sBSA appeared to induce B-cell tolerance inTxXB mice, the possibility could not be excluded that the result might be ascribed not toB-cell tolerance but actually to the effect ofsBSA still retained in the mice on the Tc popu-lation. To check this point, the following experiment was carried out. Twelve TxXB micewere divided into three groups of four mice each. Among them, groups 1 and 2 wereinjected with 5 mg ofsBSA and group 3 was uninjected. Seven days later, spleens of all themice were removed and prepared as single cell suspensions. These suspensions were trans-ferred into syngeneic female recipients (13 weeks of age, 750 R X-irradiated on the pre-vious day, one donor spleen into one recipient). The mean number of donor spleen cellswas about 5 x 107. At that time, the cell suspensions from mice in groups 1 and 3 weresupplemented with Tc. The recipients were challenged according to the standard pro-cedure 3 and 13 days after cell transfer.

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FIG. 5. Antibody response of (a) TxXBT, (b) TxXB and (c) normal mice either untreated (circles) ortreated with sBSA (triangles) prior to the challenge immunization. sBSA was administered in twoinjections, first 1 mg, followed 3 days later by 0-1 mg. Immunizing procedures are the same as in Fig. 1.Open symbols and closed symbols refer, respectively, to mice receiving Tc 3 days before theprimary immunization, and those uninoculated. Each point refers to the geometric mean titre of anumber ofmice (shown in parentheses beside each curve). Each bar represents the standard error of themean.

Responses of the recipients are shown in Fig. 7. The hyporesponsiveness was seen in therecipient ofspleen cells from sBSA-injected TxXB mice, irrespective ofthe supplementationwith Tc. This indicated that B cells in donor TxXB mice were actually affected by 5 mgof sBSA, since Tc were inoculated under the sBSA-free condition. The rise in antibodytitres in the recipients of tolerized spleen cells with or without Tc at day 25 may be attri-butable to the recovery in the immune activity of the recipients themselves from radiationinjuries.

EFFECT OF A COMBINED ADMINISTRATION OF TOLEROGEN AND IMMUNOGEN

Instead of sBSA injection in the experimental design in Fig. 2, mice were injectedintravenously with 0-1 mg of AP-BSA plus 0-01 mg of ET followed immediately byintravenous injection of 5 mg of sBSA. Uninjected mice served as controls. Other pro-cedures were the same as in Fig. 2. The results are shown in Fig. 8.Opposing results were obtained with TxXBT mice and TxXB mice. TxXBT mice

injected with AP-BSA+ET+sBSA revealed the enhanced response to the challenge im-munization as compared with the control mice. This indicated that the simultaneousinjection of the tolerogen and the immunogen could positively stimulate these mice.However, such an injection still exerted a suppressive effect on TxXB mice. The response

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FIG. 6. Effect of a single injection of 5 mg of sBSA on (a) TxXBT, (b) TxXB and (c) normal mice.sBSA-treated mice and untreated mice are represented by squares and circles, respectively. Immunizingprocedures are the same as in Fig. 1. Open symbols and closed symbols refer, respectively, to micereceiving Tc 3 days before the primary immunization, and those uninoculated. Each point refers to thegeometric mean titre ofa number of mice (shown in parentheses beside each curve). Each bar representsthe standard error of the mean.

of the pretreated mice to the challenge immunization was significantly lower than that ofthe untreated mice even with the aid ofTc supplementation. In normal mice, the primaryresponse of the control mice to the challenge immunization was remarkably high, so

that the priming effect of the pretreatment was not as conspicuous as in TxXBTmice.

INTEGRATED REPRESENTATION OF THE RESULTS IN THESE EXPERIMENTS

Since the demonstration in Figs 3-6 and Fig. 8 may possibly be too intricate to allowa complete view of the present study, we have compiled the essentials in Fig. 9. The valuesof the ordinate indicate the mean differences between the log2 titres of the experi-mental groups and those of the control groups: in each experiment, mean log2 titresof the control group at days 15, 20, and 25 were subtracted from those of the experimentalgroup, and the mean of these differences was then calculated. Zero+ 1 in these valuescould be regarded as insignificant. Negative values distinctly less than -1 may indicate theinduction of tolerance by pretreatment with antigen prior to challenge, and positivevalues greater than + 1 may indicate the priming effect of pretreatment. In TxXBT andnormal mice, only groups without Tc supplementation were subjected to the calculation.

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FIG. 7. Cell transfer experiment to examine the B-cell tolerance in TxXB mice injected with 5 mg ofsBSA. TxXB mice were either given a single injection of 5 mg sBSA (donors for mice shown by triangles)or uninjected (donors for mice shown by circles). One week later, their spleen cells were transferred into750 R X-irradiated syngeneic recipients together with Tc (0, A) or without Tc (A). Three days later,all the recipients were immunized according to the standard procedure. Each point refers to the geo-metric mean titre of a number of mice (shown in parentheses beside each curve). Each bar representsthe standard error of the mean.

On the other hand, only animals receiving Tc were involved in the calculation in TxXBmice.Although the values calculated as described above may be merely arbitrary, Fig. 9

seems a convenient method for surveying and reviewing the present study. It is shown thatthe tolerance induction with sBSA is achieved more easily in TxXBT and normal micethan in TxXB mice. Conversely, the combined administration of sBSA and theimmunogen is shown to be immunogenic in TxXBT mice but still tolerogenic in TxXBmice.

DISCUSSION

As mentioned before, the difference in the tolerogen sensitivity between T cells and Bcells in the tolerance induction in vivo to the T cell-dependent antigens may be attributableto one of the following three possibilities: (a) the high sensitivity of T cells and the lowsensitivity ofB cells are their inherent characteristics, and these cells behave independentlyof each other in tolerance induction; (b) although antigen sensitivity is not differentbetween T and B cells, the former interferes with the latter in tolerance induction; (c)the induction of B-cell tolerance requires 'helper action' of T cells, as does the positiveantibody response. The present experimental results seem to give evidence in favour of

952


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FIG. 8. Effect of the simultaneous administration of 0 1 mg of AP-BSA+0-01 mg of ET (immuno-genic agent) and 5 mg of sBSA (tolerogen at high dose) on (a) TxXBT, (b) TxXB and (c) normalmice. The treated mice and the untreated mice are indicated by triangles and circles, respectively.Open symbols refer to the animals supplemented with Tc 3 days before the primary challenge immun-ization. The procedures of the primary and the secondary challenge immunizations are the same as inFig. 1. Each point refers to the geometric mean titre of a number of mice (shown in parentheses besideeach curve). Decreasing numbers of mice in some subgroups are due to the death of some of them by theend of the experiment. Each bar represents the standard error of the mean.

possibility (a), because TxXBT and normal mice could be tolerized by the administrationofany doses ofsBSA (even a single injection of0 - mg), but TxXB mice were unaffected bysBSA at a dose of 1 mg or less and could be suppressed only by 5 mg of sBSA. If possibility(b) had been the case, TxXB mice would have been made tolerant by low doses of sBSAas well as TxXBT mice, and if (c) had been correct, TxXB mice would not have beensuppressed, even by a high dose of sBSA.

During the preparation of this manuscript, the excellent paper of Schrader (1974) waspublished. He used homozygous athymic nude mice as experimental animals anddeaggregated fowl y-globulin or its conjugates with dinitrophenol as tolerogens, andclearly demonstrated that B-cell tolerance was induced in vivo in the absence of T cellsAlthough our experiments could not exclude the possibility of a very small number ofT cells in TxXB mice, employing nude mice, treating the bone marrow cells-injectedand recipient animals with anti-thymocyte antibodies and the very low responsive-ness of TxXB mice to even twice the immunization with such a potent immunogen asAP-BSA, together with ET, seems to eliminate any significant activity of T cells in theTxXB mice.

Gershon and Kondo (1970, 1971) insisted on the dependence of B-cell tolerance oncertain specific factor(s) (termed IgY) derived from T cells responding to the antigen,based on an experiment in which they injected 2-5 x 1010 sheep erythrocytes over a 1-month period into TxXBT and TxXB mice. The suppressive effect of the antigen over-

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FIG. 9. An integrated representation of the essentials of the present experiments, compiled from the datain Figs 3-6 and Fig. 8. Open columns, hatched columns, and closed columns refer to normal, TxXBTand TxXB mice respectively. Negative values less than -1 may represent tolerance induction andpositive values greater than + 1 may represent the priming effect of antigen injection given prior to theprimary immunization.

loading on B cells was observed only in TxXBT mice. Feldmann (1973) reported that Bcells were inactivated by the complex of T cell-derived immunoglobulin (termed JgT)with monomeric antigens under an in vitro condition deprived of macrophages. We assume,however, that the results of Gershon and Kondo may be ascribed to the effect of positivelyactivated T cells or their products developing as a suppressive regulator for the increasingimmune response, and that the experimental conditions of Feldmann was an artificial one,in the sense that a macrophage-free condition cannot be found in intact animals. Scott(1973) looked for some positive signs in adult rats during the period of tolerance inductionwith deaggregated sheep y-globulin as tolerogen. He could not find any enhanced synthesisof DNA, RNA or proteins, suppressor cells, suppressive factors or blocking antibodies. Theabsence of suppressor cells in tolerance induction was also reported by Chiller and Weigle(1973a) .The implications are that the induction of tolerance to a B-cell population may be

independent of any intervention of T cells, if such a nonimmunogenic antigen as sBSA isemployed as tolerogen. If a weakly immunogenic agent is administered to animals, how-ever, T cells may sometimes be activated, and the activated T cells may protect B cellsfrom being suppressed. This has clearly been demonstrated in the present experiment.

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Thus, the injection of a large amount of sBSA (5 mg), a sufficient dose for B-cell toler-ance, in combination with a relatively small amount of AP-BSA (0.1 mg), together with0 01 mg of ET, resulted in the production of an antibody response in TxXBT mice, butstill induced tolerance in TxXB mice. The occurrence of an essentially similar phenomenonhas been suggested by Schrader (1974). According to Mitchison (1968), a single orseveral injections of uncentrifuged fluid BSA at certain doses, even exceeding 10 mg,could be immunogenic for adult mice. With reference to our present findings that sBSAwas always tolerogenic at any dose more than 0-1 mg, we can assume that his antigenicfluid probably contained some molecular aggregates. Thus, T cells might be activated andprotect B cells from tolerance induction, which should otherwise have occurred with such amassive dose of BSA.

It must be B cells that were tolerized by sBSA in TxXB mice, since TxXB mice receiving5 mg of sBSA were still hyporesponsive to challenge immunization, even with the aid ofTc, in spite of the modestly high response of control TxXB mice untreated with sBSA andsupplemented with Tc. Furthermore, B-cell tolerance was confirmed by a cell transferexperiment as shown in Fig. 7.

Target cells for tolerance induction in TxXBT mice and normal mice have not beendefinitely identified in these experiments. As shown in Figs 3-6 and Fig. 9, TxXBT andnormal mice were made tolerant with sBSA at any dose under examination. These tolerantstates, especially in normal mice, were difficult to reverse completely to untolerized controllevels by the addition of Tc. This appears to suggest that B-cell tolerance was inducedwith ease, even by low doses ofsBSA ineffective in TxXB mice, in the presence ofT cells, aswas suggested by Gershon and Kondo (1970, 1971). It is difficult, however, to definitelyconclude this, since localization efficiency of the inoculated Tc in lymphoid tissues of therecipients should be taken into consideration.The inability to reverse the tolerant state to human y-globulin by the adoptive transfer

of syngeneic spleen or thymus cells into tolerant mice was reported by Chiller and Weigle(1973a). Similar observations were made by Miller and Mitchell (1970) in attempts toterminate the cyclophosphamide-induced specific unresponsiveness of mice to sheeperythrocytes. These observations, especially the latter, seem to suggest that a large portionof inoculated cells could not localize in the lymphoid tissues ofthe recipients. This may alsobe the case in the present experiment, since we have observed (unpublished results)histologically that thymus-dependent areas (Parrott, Sousa and East, 1966) of thelymphoid tissues are very densely occupied by lymphoid cells in normal mice and fairlydensely in TxXBT mice, in contrast to the relative emptiness of these areas in TxXBmice.

Probably, the most efficient localization ofTc may be achieved in TxXB mice, resultingin the modestly high antibody response caused by the co-operation of these cells with pre-existing B cells in lymphoid tissues. Conversely, Tc could hardly be taken up by lymphoidtissues of normal mice, so that the tolerant state was not reversed at all. A number of Tcseem to enter the tissues in tolerant TxXBT mice, so that the responsiveness in thetolerant mice is more or less improved. The improvement caused by Tc, however, does notnecessarily mean that the target cells for tolerance induction must be exclusively T cells,since the appearance of the tolerant state may be determined by a delicate balance be-tween the tolerization and the activation of T and B cells. For instance, if a very smallnumber of T cells and B cells not only escaped from tolerance induction but were alsopositively activated by a very small fraction of the immunogenic form of antigen in the

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956 S. Muramatsu, T. Amagai and r. Katsura

tolerogen preparation, Tc would more or less improve the immune responsiveness of thesemice. t

With reference to our previous findings (Katsura et al., 1972), although the strain ofmice used was different from that in the present experiment, it seems reasonable to assumethat the major target cells for sBSA at doses of 1 mg or less in TxXBT and normal micemay be T cells, and that B cells in these mice may first be tolerized by a higher dose of thetolerogen, as well as in TxXB mice. Further experiments for ascertaining this are inprogress.The present investigation allows us to allude to the interaction among macrophages, T

cells and B cells (Mosier and Coppleson, 1968; Mitchison, 1969; Gorczynski, Miller andPhillips, 1971; Hamaoka and Kitagawa, 1971; Feldmann, 1972; Katz and Unanue, 1973).From our findings that TxXB mice could barely respond, even to such a stronglyimmunogenic agent as AP-BSA with ET, and that they were tolerized not only by ahigh dose of the tolerogen but also by a simultaneous administration of the immunogenand the tolerogen, it can be envisaged that both macrophages and T cells may beprerequisites for the activation of B cells, as far as the present experimental systems areconcerned.

Thus, when the immunogen and the tolerogen were made to co-exist in vivo, t e formermight be trapped by macrophages to prepare the immunogenic stimulus for T ce4ls and thelatter might bind directly to T and B cells to give the tolerogenic stimulus for these cells.The competition between these two kinds of stimuli seemed to occur on T cells and someportion of T cells could be activated. The activated T cells might release the factor topositively excite B cells. This factor may be non-specific to the antigen, as was suggested byseveral investigators (Dutton, Falkoff, Hirst, Hoffmann, Kappler, Kettman, Lesley andVann, 1971; Osborne and Katz, 1973; Gorczynski, Miller and Phillips, 1973; Chiller andWeigle, 1973b; Schrader, 1973). The4Tmunogenic stimulus derived from macrophagesseemed inert for B-cell activation. Therefore, in the absence of T cells, B cells wereinevitably affected by the tolerogenic stimulus alone. In order to obtain the evidence forthis assumption, in vitro investigations on the tolerance induction with T cell-dependentantigens using isolated pure populations of macrophages, T cells and B cells will berequired.

ACKNOWLEDGMENTS

The technical assistance of Ms Yuko Takaoki is gratefully acknowledged. We thank alsoMs Chieko Taniguchi for typing of the manuscript and Ms Fusae Takimoto for secretarialassistance.

This research was supported by grant number 811109 from the Ministry of Education.

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Tolerance Induction in TxXBT and TxXB Mice

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