Immunological Tolerance: Mechanisms and Potential Therapeutic Applications

Burnet

Session I

T Cell Tolerance

Outline

Chapter 1: FACTORS INFLUENCING THE RATE OF INDUCTION OF TOLERANCE BY BOVINE GAMMA GLOBULIN

Chapter 2: DISCUSSION FOLLOWING DAVID DRESSER

Chapter 3: INTERACTIONS OF T AND B LYMPHOCYTES IN SELF-TOLERANCE AND AUTOIMMUNITY

Chapter 4: DISCUSSION FOLLOWING ANTHONY ALLISON

Chapter 5: THE CELLULAR BASIS FOR ESTABLISHING TOLERANCE OR IMMUNITY TO BOVINE γ-GLOBULIN IN MICE

Chapter 6: THE ROLE OF ACCESSORY AND THYMUS-DERIVED CELLS IN RESISTANCE TO TOLERANCE INDUCTION

Chapter 7: SUPPRESSOR ACTIVITY IN MICE TOLERANT OF HEMOCYANIN

Chapter 8: SPECIFIC SUPPRESSION OF THE IMMUNE RESPONSE BY T CELLS

Chapter 9: TOLERANCE TO CONTACT SENSITIVITY--A ROLE FOR SUPPRESSOR T CELLS?

Chapter 10: GENERAL DISCUSSION - SESSION I: T CELL TOLERANCE

FACTORS INFLUENCING THE RATE OF INDUCTION OF TOLERANCE BY BOVINE GAMMA GLOBULIN

D.W. Dresser,     National Institute for Medical Research, Mill Hill, London NW7 1AA

Publisher Summary

This chapter discusses the factors influencing the rate of induction of tolerance by bovine gamma globulin. The experiments described in the chapter confirm the supposition that observed differences in the rate of induction of immunological tolerance have two main sources: (1) differences of technique and experimental protocol; and (2) differences in the target cells. In the BγG-CBA system used in the experiment, there are gross differences between results derived from elimination, ABC (Farr), and plaque assays. The elimination assay may measure overall responsiveness including all classes of humoral response together with cell-adherent antibodies, whereas the ABC assay may only measure serum-γG antibodies. In contrast, the plaque assay measures the number of cells secreting a soluble antibody irrespective of whether or not that antibody is physiologically stable or functional in vivo. The plaque method only reflects the response in the organ assayed, although the dissection of that response into its component classes is easier and more accurate to carry out than it is for secreted serum antibody.

INTRODUCTION

Antibody production and immunological tolerance are good systems for studying cellular differentiation despite their overt complexity. The advantages of the systems lie in the specific handle afforded by antigen and in the temporal reference point of exposure to antigen or adjuvant. Nossal has pointed out that immunological tolerance can be discussed in terms of a decision between two alternative pathways for an individual lymphocyte (1) (see Fig. 1). Such concepts of stimulation of a differentiating cell, down one or two or more alternative pathways, are commonplace in embryology and epigenetics. Known inducers range from diffusable substances acting at long range to substances dependent on cell-cell contacts (2).

Fig. 1 A simplified diagram to illustrate possible alternative pathways of B cell differentiation in relationship to antigenic and adjuvant (non-specific) stimuli. Neither is the precise timing of the differentiation switch known nor is the number of specific and non-specific hits required by B-cell lines of different classes. The subject is developed further in Fig. 13.

Differences in the rate of entry into a state of immunological unresponsiveness after the administration of tolerogen (tolerance-inducing form of antigen) exist (3–6). Sometimes these differences can be accounted for by different methods of measuring responsiveness (precipitation, antigen elimination, plaques), sometimes to different protocols (intact animals, cell transfers, in vitro) and sometimes to the antigen (immunogenic/non-immunogenic, protein/carbohydrate). However, in a series of classical experiments, Chiller and his colleagues in cell transfer experiments have shown that real differences exist in dose sensitivity and rate of entry into a state of unresponsiveness, between tolerance induction in T and in B lymphocytes (4, 5, 7) and in further experiments differences between spleen and bone marrow B cells have been demonstrated (6).

Since all mechanisms demonstrated in disrupted systems must eventually be related to physiological reality in intact animals, it was decided that a study of factors affecting the rate of induction in intact mice would be made. Experiments have been started in an attempt to measure the induction of tolerance in different lines of B cells; some preliminary results are included here. If observed differences can be ascribed to differences at the cellular level, then it may be possible to gain insight into the variety of tolerance induction mechanisms which may operate in normal development.

MATERIALS AND METHODS

Male mice of the CBA/H strain which were free of Tyzzer’s disease were used in these experiments (8). Thymectomy of 3–5 week old mice and reconstitution with syngeneic fetal liver was carried out as described previously (9). A whole body dose of 800–850 röntgens was delivered from a ⁶⁰Co source at a rate of about 40 röntgens per minute. In some experiments a population of helper T cells sensitive to DNP was raised by painting the shaved belly of the mice with DNFB (10).

Bovine gamma globulin (Ethanol fraction II, Armour, BGG) was refractionated on DEAE cellulose (DE 32; 0.02M phosphate buffer, pH 7.4). This material (B G) was used for tolerance induction after centrifugation at 30,000 Xg for 30–40 minutes or for immunization after either: a) alum precipitation (BA), or b) heat aggregation by a midification of a published method (11) (65° for 2 hours followed by 0° for 2–24 hours and finally, one or two washed in 0.9% NaCl). Purified antibody from bovine or porcine anti-mouse-lymphocyte serum (ALSAb) was prepared from ALS by first preparing a DEAE globulin fraction and then eluting antibody from glutaraldehyde-fixed thymocytes (anti-T) or spleen cells from thymectomized-reconstituted mice (anti-B) (12, 13).

T cells were primed (educated) against B G (EdT) (14) by injecting 70 × 10⁶ CBA thymus cells plus 250 g HaB intravenously into lethally irradiated syngeneic recipients. A week later the spleens of these mice were harvested, and the cells used as a source of EdT (yield 3–4 × 10⁶ cells/spleen).

The LHG assay was carried out using the slide method (15). Class specific developing sera was prepared, absorbed and tested as described earlier (9) but received an additional absorption with B G-Sepharose. For use as target cells sheep erythrocytes (SRBC) were coated with B G by the methodology of Hosono and Maramatsu (11) with the modifications that the CrCl3 is dissolved in 0.9% NaCl (immediately before use), 2.5 mg B G per ml of solution are used and all solutions are kept at 0° until homogenous mixing of all components is obtained. After 1 hour at 37° with very gentle stirring, the coated SRBC are washed three times in Hank’s solution containing 0.5% Difco gelatin (HG). HG was used for suspending cells (PFC and target cells) used in these experiments.

The coprecipitation assay for anti-5563-idiotype antibodies was described by Iverson (10). The antigen-binding capacity (ABC or Farr) assay was a modification of the methodology used by Mitchison and his colleagues for serum albumins (16). For bovine and porcine gamma globulins it is necessary to use ethanol-fractionated material refractionated on DEAE-cellulose. The ammonium sulfate is diluted to 32% saturation (at 4°) with 0.15 M borate buffer at pH 8.4. This ABC assay could be considered as a direct precipitation in high salt concentration: it is about ten fold more sensitive than coprecipitation with a very strong antiserum to mouse gamma globulins, and more than ten fold as sensitive as direct precipitation in 0.15 M borate buffer. BγG was labelled with ¹²⁵I by the standard method (17). Isoelectric focusing (IEF) was carried out by the method of Phillips and Dresser (19, 20) using BγG coated SRBC and an anti-γG1 developing serum in the indicator layer.

RESULTS

Rates of tolerance induction

Different rates of tolerance induction have been reported by workers using the same or similar antigens in the same species of experimental animal. These differences may reflect different experimental protocols, for instance, in experiments in intact animals total responsiveness often declines slowly (3), whereas experiments involving the transfer of cells to irradiated recipients, usually result in a much more rapid rate of tolerance induction (4, 5). In intact animals it is difficult to determine whether it is the T or the B arm of the response which is the primary target of the tolerogen. In the cell transfer protocols which allow in vitro manipulations and various admixing of cells, analysis of tolerance at the cellular level is practicable. It was this latter protocol which allowed Chiller and colleagues to demonstrate differences between T and B and between spleen B and bone marrow B cells.

In Fig. 2 it can be seen that in intact mice the rate of tolerance induction to BγG is slow when responsiveness is measured by the elimination technique and faster when responsiveness is measured by the ABC assay. In experiments in intact animals such as these, it is impossible to know whether it is T cells or B cells or both which are the effective limiting factor causing a decline in responsiveness. If T cell help is a necessity for a response to manifest itself, it should be possible to raise a population of helper cells to a determinant not normally on the tolerogenic molecule and then couple this determinant to the immunogenic challenging form of the antigen. In this protocol it can be argued that an observed unresponsiveness must be due to tolerance induction in B cells (21). As a consequence of this, T cell help to DNP was raised in mice prior to the induction of tolerance to BγG by painting them with DNFB. Subsequently, the mice were challenged with alum-precipitated DNP6BγG and the response to BγG measured. The observed rate of tolerance induction (in B cells) is the same as that seen in mice challenged with BγG (BA) in the normal way.

Fig. 2 A comparison of the rates of tolerance induction as measured by the elimination assay and by the ABC assay. In two groups an alternative source of T-cell help was raised by painting the mice with DNFB; these mice were challenged with 100 µg alum-precipitated DNP6-BγG (or DNP3.2-5563) together with 2 × 10⁹ pertussis organisms. Uncoupled BγG was used in unpainted mice.

The rate of tolerance induction by 5563 idiotype, also measured in DNFB painted mice, was substantially the same as for BγG. The observed difference between elimination and ABC protocols could possibly be due to the two assays measuring different kinds of antibody, each kind (class) manifesting its own rate of tolerance induction. At the level of the B cells, the differences can be in whole or in part due to primary B cell class differences or to a secondary effect reflecting differences in T cell dependence.

Although age has a profound effect on the rate of loss of a state of tolerance (22, 23, 24, 3), it can be seen in Fig. 3 that age has at best a marginal effect on the rate of induction of tolerance. It is possible that very old mice enter a state of tolerance more slowly than younger mice. Mice thymectomized 2 1/2 months previously as young adults but not reconstituted enter a state of tolerance at a similar rate to that seen in intact mice of the same age (Fig. 4). These mice are deficient in T1 cells (25) but retain enough T cells (T2) for controls not injected with tolerogen to mount as good a response to a challenge with immunogenic BγG, as do normal (intact) mice. It is clear from this result that an earlier suggestion that a functional thymus might be necessary for tolerance induction (3), is wrong. Thymectomized-reconstituted control mice fail to respond to immunogenic BγG (see next section).

Fig. 3 The rate of tolerance induction to 1 mg BγG in mice of different ages (indicated in months).

Fig. 4 The lack of effect of adult thymectomy (Tx) without reconstitution on the rate of tolerance induction by 500 µg BγG. The tolerogen was injected 2 1/2 months after thymectomy.

T-dependence

If an antigen is generally only capable of stimulating a class of antibody which is T dependent, then it is important to know if there are any situations where such T dependence can be by-passed. With antigens such as sheep RBC and ØX 174, this can be done by using very high doses of antigen in conjunction with a powerful adjuvant (9, 26). In the response to sheep RBC, the γM response seems to be partially T independent. The dependence of the γG2a and γG1 responses are almost complete in the absence of adjuvant. Coprecipitation tests of anti-BγG-immunized mice show that the response to BγG is more than 90%  γG1. ALSAb (both bovine and porcine) is a highly immunogenic form of soluble gamma globulin. In Figs. 5 and 6 it can be seen that despite the use of very high doses of ALSAb, thymectomized/reconstituted (T−) mice failed to respond to a degree detectable by the ABC assay.

Fig. 5 The inability of soluble BγG or ALSAb (plus 4 × 10⁹ pertussis - all i.p.) to immunize T− (thymectomized reconstituted) mice, T+ indicates sham-reconstituted controls. In a similar experiment using alum-precipitated porcine γG and alum-precipitated porcine ALSAb, both plus pertussis, there was no detectable antibody in T− mice even with a dose of ALSAb of 1 mg.

Fig. 6 Porcine ALS was divided into two aliquots; one was absorbed with CBA thymocytes and the other with spleen cells from T− mice. ALSAb was prepared reciprocally from similar cells after glutaraldehyde fixation. The ALSAb was injected i.p. as a solution (without pertussis, which makes only a slight difference). It can be seen that the anti-T material is more immunogenic than the anti-B. This result is compatible with the view that T-cell help is only marginally concerned with antigen concentration at the surface of the B cell.

Class differences in tolerance induction

Experiments (unpublished) have partly confirmed the suggestion of Hosono and Muramatsu (11) that after immunization with heat-aggregated gamma globulin (HaB), an early peak of developable (indirect) γM plaques can be detected. Somewhat later, at about 12 days after priming, a more conventional γG response was seen by these workers who used a polyspecific-developing serum in their experiments. In Mill Hill CBA mice it has been shown, using highly specific developing sera (anti-µ; anti-γ1; anti-γ2a; anti-γ2b; anti-Fab), that a peak of indirect γM plaques occurs at day 3, followed by an early γG1 peak at day 5 with another γG1 peak at day 10 (plus some γG2). Figure 7 shows that the plaques have different morphologies. Splenic indirect γM plaques are induced best by an intraperitoneal injection of 100 µg alum-precipitated BγG (BA) and the γG plaques by Img HaB injected intravenously. These challenge procedures have been used in the tolerance experiments illustrated in Figs. 8, 9 and 10, where it can be seen that the tempo of tolerance induction (and perhaps in some cases concomitant immunization) differ between indirect γM, early γG1 and late γG1 (and γG2). After a tolerance induction time of 14 days (t.14), the simple direct relationship between the degree of indirect γM tolerance and the dose of tolerogen contrasts with the more complex relationship seen especially clearly in the induction of tolerance in the early γG1 response.

Fig. 7 Anti-BγG plaque morphology, with left to right, indirect γM; early γG1 and late γG1 plaques.

Fig. 8 Induction of tolerance to different doses of BγG after different tolerance induction times, which are indicated (days) on each curve. The geometric mean number of plaques per spleen in 5 groups of 4 animals is indicated on the 100% line; the shaded area represents the standard deviation of the control value expressed as a percentage of the mean. Indirect γM after challenge with 100 µg alum-precipitated BγG i.p.

Fig. 9 As Fig. 8 but early γG1 after 1 mg HaB intravenously.

Fig. 10 As Fig. 9 but late γG1.

Using a cell transfer protocol, spleen cells have been exposed to particle-free BγG in vivo, at two dose levels, for different lengths of time before being transferred with antigen (250 µg HaB) and BγG-educated T cells, intravenously into lethally irradiated syngeneic mice. In this situation it seems likely that any observed tolerance effect will be the result of B cell tolerance. Figure 11 shows that with high doses of tolerogen (3 mg), the γG1 response in the spleen (only a trace of γG2 classes seen in controls) declines rapidly between days 3 and 6, whereas with a lower dose of tolerogen (60 µg), there is evidence of a slight immunization effect. This experiment confirms for BγG a cadence of tolerance induction in B cells previously demonstrated for human γG in other strains of mice.

Fig. 11 The γG1 plaque response in lethally irradiated recipient mice, 11 days after the transfer of spleen cells (70 × 10⁶) from normal or tolerogen-treated donors. The different dose levels of tolerogen and the different times of exposure to tolerogen are indicated on the graph. Each mouse received in addition to spleen cells 250 µg HaB and 3.7 × 10⁶ BγG-educated T cells, i.v.

It would be interesting to know if the early γG1, and the late γG1 responses are made by the same line of cells or are made by two physiologically distinct lines of B cell. The isoelectric spectrum of a population of antibody molecules may be a finger print which could in certain circumstances be a marker for the product of a particular clone of B cells (27). If a similar pattern occurs frequently in different individual animals, such a finger print cannot of course be a clonal marker, although it may be an effective method of detecting a particular sub-class of gamma globulin. Although CBA mice immunized with 32 µg to 3.2 mg of HaB show extremely heterogenous γG1 and γG2a responses, a dose of 3.2 µg stimulates γG1 responses with a reasonably restricted heterogeneity.

Using mice immunized with this low dose of HaB, Dr. J.M. Phillips and I have run day 6 and day 12 sera from individuals side by side of an IEF plate. Figure 12 shows that the same γG1 spectrum seems to predominate in the two bleeds; differences between the spectra of different individuals can be seen as well. If the response to the low dose of antigen used in this experiment is eventually shown to stimulate early and late γG1 in a similar way to higher doses of HaB, then we would conclude that this result is compatible with both early and late γG1 being made by the same line of cells. If this were so, it might imply that the late γG1 is dependent on more than one antigen hit and perhaps could be considered as a secondary type of response.

Fig. 12 The IEF spectra of γG1 anti-BγG antibodies from 4 individual mice primed with 3.2 µg of HaB, i.v. Each pair of spectra represent respectively on the left a day 6 bleed and on the right a day 12 bleed from each individual.

DISCUSSION

The experiments described briefly in this paper confirm the supposition that observed differences in the rate of induction of immunological tolerance (paralysis) have two main sources. These are: 1) differences of technique and experimental protocol; and 2) differences in the target cells.

In the BγG-CBA system used here, there are gross differences between results derived from elimination, ABC (Farr) and plaque assays. The elimination assay may measure overall responsiveness including all classes of humoral response together with cell-adherent antibodies, whereas the ABC assay may only measure serum-γG antibodies. In contrast, the plaque assay measures the number of cells secreting a soluble antibody irrespective of whether or not that antibody is physiologically stable or functional in vivo. Furthermore the plaque method only reflects the response in the organ assayed, although the dissection of that response into its component classes is easier and more accurate to carry out than it is for secreted serum antibody.

A further technical contribution to different rates of tolerance induction lie in the experimental protocols employed. In intact animals feedback loops and cell interactions may exist which could be altered or absent in vitro or in irradiated recipients of populations of washed cells whose normal microanatomical relationships have been disrupted.

The cellular targets of a tolerogen differ. Chiller and Weigle and their colleagues have shown very clearly that tolerance to human γG in T cells and B cells require different doses of tolerogen and that the rates of tolerance induction are different (4, 5 and 7). They have also shown in further cell transfer experiments that a population of B cells in spleen enter a state of unresponsiveness more quickly than a population of B cells in bone marrow (6). This last difference may reflect the state of maturity of B cells in the two organs. Consideration of the maturity of the target cell may be of importance in any consideration of how a tolerogen turns off or kills a line of potentially responsive cells, and also in considerations of the ontogeny of the immune response in relation to aberrations such as autoimmunity. A cell transfer experiment reported in this paper shows that spleen B cells of CBA mice become unresponsive to BγG in a manner similar to that demonstrated for human γG.

The results of experiments in which particle-free (deaggregated) BγG was shown to induce a specific state of immunological tolerance, while animals injected with the same antigen together with a powerful adjuvant induced a state of active immunity, compelled the author to propose a two stimulus model of immunocyte maturation (28, 29). Figure 13a outlines the essentials of such an hypothesis. The adjuvant (non-specific) stimulus is proposed as a necessary addition to the specific antigenic stimulus for triggering the differentiation of antibody-forming cells. The adjuvant stimulus can originate from a substance such as endotoxin or pertussis organisms injected independently but in temporal terms concomitantly with the antigen; or it can originate from the intrinsic adjuvanticity of the antigenic molecule itself. Subsequently, a model was proposed which suggested that the adjuvant stimulus was mitogenic (18, 30, 31); compelling evidence that this mechanism operates the tolerance-immunity switch does not yet exist, although there is recent evidence which is compatible with the hypothesis (32). The general schema outlined above has been elaborated and integrated into a more general immunological theory by Bretscher and Cohn (33, 34).

Fig. 13 This figure illustrates in a simplified form alternative mechanisms of immune induction and tolerance. In the two stimulus (signal) hypothesis (a), the triggering of immune differentiation depends on a specific (antigenic) stimulus accompanied by a non-specific (adjuvant-mitogenic?) stimulus. A cell capable of detecting and receiving an antigenic stimulus (tolerogenic) but which does not receive an adjuvant stimulus is turned off or killed.

Howard and Mitchison in their recent thought provoking review (35) have dismissed the two stimulus (two signal) hypothesis and have suggested an alternative which they believe offers a more satisfactory explanation of the experiments of Diener and Feldmann (36, 37) and of Howard and colleagues (38, 39). Howard and Mitchison’s hypothesis is outlined in simple form in Fig. 13b. They propose that a tolerance stimulus kills off cells which have bound antigen. The difference between simple stimulation of an inbuilt go trigger and killing the cell by some sort of antigenic stangulation is the presence of a cross-linking antibody in the Feldmann/Diener experiments and a sufficient concentration of highly polymerized-polysaccharide antigen in the Howard experiments. A somewhat similar alloallergic killing model of immunological tolerance at the cellular level was proposed many years ago by Chase (40).

It is not impossible that tolerance will turn out to be a generic term covering several mechanisms or alternative pathways, each of which can result in antigen specific immunological unresponsiveness. These alternatives may include: 1) cell modulation in the absence of an adjuvant (mitogenic) stimulus; 2) direct cell killing or opsonation by antigen or by antigen cross linked by certain kinds of antibody; 3) antigen-specific suppressor T cells similar to the mechanism proposed for allotype suppression; and 4) antibody feedback (42, 43). Different mechanisms or combination of mechanisms may operate simultaneously in different kinds of lymphocyte.

The immune mechanism like other systems of cellular differentiation is a Gordian knot which may be resolved at one level by experiments with an Alexandrian protocol, but which will have to be patiently unravelled piece by piece if the matrix of feedback, direct cellular interaction and cellular change are to be understood. A degree of acceptance of the dogma of clonal selection is implicit in both alternatives outlined in Fig. 13; for example, this may be convenient but dangerous. Howard and Mitchison consider that the question of whether tolerance induction means cell killing or cell turn off is an irrelevance from the point of view of the acquisition of a state of antigen specific unresponsiveness. In terms of practical immunology this may be so but to those workers interested in tolerance induction as a complex model of cellular differentiation, albeit one with a convenient handle for its manipulation, the question is indeed relevant. The alternative to killing off unwanted pre-differentiated cells is that cells are switched into another epigenetic pathway before differentiation is irreversible. The alternative pathway may perhaps be one for which the experimenter has at present no convenient means for detection.

ACKNOWLEDGMENT

I thank Miss A.M. Popham for excellent technical assistance.

In contrast, (b), Howard and Mitchison have suggested that adjuvant is largely irrelevant as a decision-making stimulus and that the antigen-receptor is an intrinsic go signal. In their model a state of tolerance is induced by the elimination of clones of responding cells through the cytotoxicity or opsonizing effect of antigen or antigen-antibody complexes.

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DISCUSSION FOLLOWING DAVID DRESSER

G. MÖLLER: In your ALS experiments with anti-T and anti-B sera where only anti-T was immunogenic, did you study whether anti-B induced tolerance?

DRESSER: No.

G. MÖLLER: This is most important because anti-T and anti-B most likely have the same antigenic determinants. Therefore, what appears important is the cell with which the substance binds to rather than differences in structure between immunogens and tolerogens.

DRESSER: I agree that it will be very important to follow this up by doing the appropriate experiment.

TAYLOR: In your experiments do you think tolerance occurred at the T or B cell level or both?

DRESSER: In the experiments using elimination and antigen-binding capacity to measure the response, where fairly large (1/2 − 1 mg) doses of tolerogen were used, the evidence is, I think, that both T and B cells are made tolerant. However, the experiments carried out more recently using the plaque assay and doses of tolerogen as low as 1 µg have not yet been extended far enough to answer your Question.

LESKOWITZ: If I interpreted your data correctly, you demonstrated significant tolerance induction with doses of BGG as low as 1 µg. Yet, tolerance with these doses is not demonstrable by other techniques such as immune clearance. How do you account for these discrepancies?

DRESSER: Mainly because the observed tolerance was in the γ G response. At a tolerogen dose of 1 µg there is absolutely no effect on the indirect γM response. The The elimination assay presumably measures all kinds of antibody irrespective of class.

FELDMANN: I was interested in the difference between the high immunogenicity of ALS eluted from T cells compared with that eluted from B cells, as these experiments remind me of those of Reber and Riethmüller who have reported similar results using anti-Ig antibody. They found that anti-Ig eluted from T cells is highly immunogenic, while that from B cells is not. They interpreted their results as indicating that a helper factor was eluted from T cells, but not from B cells. Have you looked to see if anything elutes with the ALS from T cells; as this could be a helper factor as suggested by Reber and Riethmüller.

DRESSER: No, I have not. However, I must remind you that the immunoabsorbent cells were fixed with 0.25% glutaraldehyde, so if such a factor does exist, it is resistant to this treatment. We certainly ought to investigate this point.

DIENER: Did you test the tolerant cells for the presence of suppressor T cells by treating them with anti-θ serum and complement before transfer?

DRESSER: No. In the transfer experiment shown, T cells were increased by the addition of educated T cells since the question being asked concerned tolerance induction in the B cell line. Anti-θ experiments will certainly have to be done.

MITCHELL: Can you elaborate on the T cell-dependence/independence of indirect IgM PFC and whether you find such cells in the primary response to SRBC rather than BGG?

DRESSER: The concentration of anti-µ serum used in these experiments to develop indirect (anti-BγG)γM plaques completely (>97%) inhibits both background and actively acquired direct plaques to sheep RBC. The experiment to determine the T-dependence of the indirect γM response has not been done yet.

INTERACTIONS OF T AND B LYMPHOCYTES IN SELF-TOLERANCE AND AUTOIMMUNITY

A.C. Allison,     Clinical Research Centre Harrow, Middlesex, England

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This chapter discusses the interactions of T and B lymphocytes in self-tolerance and autoimmunity. With the development of sensitive methods for quantization, notably radioimmunoassay antigens thought to be secluded have been demonstrated in circulating blood. Thus, thyroglobulin is found in serum from normal human newborns and adults in concentrations of about 10–100 ng. per ml. T cells do not help B lymphocytes to recognize antigen and multiply in response to antigen exposure, thereby generating a population of memory cells, but the T cell helper effect facilitates the differentiation of cells of the B lineage into antibody-secreting cells. In the absence of T cell helper effects, antigenic stimulation would build up a relatively large population of memory cells that are not diverted into the population of antibody secreting cells with a finite life span. In general, the balance of proliferation and differentiation in lymphocytes is of key importance both in the immune response and in the induction of tolerance.

Ehrlich (22) with his usual perceptiveness drew attention to the remarkable fact that, although vertebrates can readily be immunized with cells or body fluids from other animals, they do not as a rule make antibodies against their own tissue constituents. He termed the phenomenon horrow autotoxus but was unable to advance any satisfactory explanation for it. While developing the clonal selection theory of immunity, Burnet (9) postulated that autoantigens (self antigens) are either secluded from the immune system or that potential antibody-forming cells exposed to autoantigens early in the course of ontogenetic development are eliminated or inactivated. Autoimmunity was thought to follow the proliferation of forbidden clones of lymphocytes with specificity for autoantigens.

Burnet’s postulates attracted widespread interest but have run into serious difficulties. With the development of sensitive methods for quantitation, notably radioimmunoassay antigens thought to be secluded have been demonstrated in circulating blood. Thus, thyroglobulin is found in serum from normal human newborns and adults in concentrations of about 10–100 ng. per ml (59). Thyroglobulin will be taken as a model autoantigen in this paper because of the ease with which autoantibodies against this protein can be elicited, for example, by immunization with autologous thyroglobulin in the presence of Freund’s complete adjuvant or by immunization with heterologous thyroglobulins in the absence of adjuvant. Formation of autoantibodies against thyroglobulin in experimental animals is often accompanied by thyroiditis. Indeed, in subprimates the main antigen involved in the development of autoimmune thyroiditis is thyroglobulin, although in primates including man, microsomal antigens appear also to be involved.

Such autoimmune reactions are not confined to thyroglobulin; for example, immunization with isologous testicular or brain extracts leads to autoimmune orchitis or encephalomyelitis. It is difficult to understand how such procedures could rapidly induce the proliferation of forbidden clones of lymphocytes able to react with the appropriate autoantigens.

Tolerance in T and B lymphocytes

Several findings of the past few years have allowed reconsideration of the problem. The first and most important is the distinction between thymus-dependent (T) and other (B) lymphocytes; the latter cells and their progeny responsible for antibody synthesis and release while the former participate in cell-mediated immunity and exert helper effects in the formation of antibodies against most antigens (34, 47). The second relevant finding is that, in the absence of adjuvants, administration of native heterologous serum proteins in certain dosage schedules induces specific unresponsiveness so that treated animals are unable to form antibodies when the same proteins are inoculated in a highly immunogenic form together with adjuvants (21). Such specific unresponsiveness can be induced by administration of a high dose or repeated administration of low doses of proteins such as heterologous serum albumin or immunoglobulin. It is shown by Taylor (55) for mice inoculated with bovine serum albumin and by Chiller et al (15) for mice inoculated with human immunoglobulin that low-dose unresponsiveness affects mainly T cells whereas high-dose unresponsiveness affects both T and B cells. This was demonstrated by testing immune responses in irradiated syngeneic mice reconstituted with thymus and lymph node or bone marrow cells from normal and unresponsive donors. Thus, lymph node or bone marrow cells from low-dose unresponsive donors transferred to irradiated recipients together with normal thymus cells allowed the development of good immune responses in the recipients. After a high dose of antigen bone marrow cells also become unresponsive; moreover, unresponsiveness is rapidly induced and is persistent in T lymphocytes and is more slowly induced and transient in B