Katz / Benacerraf Immunological Tolerance

Mechanisms and Potential Therapeutic Applications
1. Auflage 2014
ISBN: 978-1-4832-7343-3
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: Adobe DRM (»Systemvoraussetzungen)

E-Book, Englisch, 660 Seiten

Immunological Tolerance: Mechanisms and Potential Therapeutic Applications documents the proceedings of a conference held at Brook Lodge, Michigan, April 27-May 1, 1974. The conference brought together many of the investigators who have actively contributed to furthering knowledge and understanding of immunological tolerance. It will be immediately clear to the reader that the conference was structured in a way to consider phenomena of tolerance and immune suppression as interrelated entities with a certain degree of emphasis on the possible common cellular mechanisms involved. The volume contains 36 contributions presented during the seven sessions of the conference. The papers presented in Session I examined T-cell tolerance. The presentations in Session II focused on B cell tolerance. The papers in Sessions III and IV focused on the mechanisms of B cell and T cell tolerance, respectively. Session V dealt with the activity of suppressor cells as a mechanism of tolerance. The papers in Session VI investigated the suppressive activity of antibody and antigen-antibody complexes. In Session VII a final General Discussion was held in order to identify what has been established concerning the phenomenology and mechanisms of specific immunological tolerance, what are the major unresolved issues, and what approaches appear most promising to answer these questions.

Autoren/Hrsg.

Katz, David H.

Benacerraf, Baruj

Inhaltsverzeichnis

1;Front Cover;1
2;Immunological Tolerance: Mechanisms and Potential Therapeutic Applications;4
3;Copyright Page;5
4;Table of Contents;6
5;PARTICIPANTS;10
6;PREFACE;14
7;SESSION I:
T CELL TOLERANCE;18
7.1;CHAPTER 1.
FACTORS INFLUENCING THE RATE OF INDUCTION OF TOLERANCE BY BOVINE GAMMA GLOBULIN;20
7.1.1;INTRODUCTION;20
7.1.2;MATERIALS AND METHODS;21
7.1.3;RESULTS;22
7.1.4;DISCUSSION;25
7.1.5;ACKNOWLEDGMENT;28
7.1.6;REFERENCES;28
7.1.7;DISCUSSION FOLLOWING DAVID DRESSER;40
7.2;CHAPTER 2.
INTERACTIONS OF T AND B LYMPHOCYTES IN SELF-TOLERANCE AND AUTOIMMUNITY;42
7.2.1;REFERENCES;62
7.2.2;DISCUSSION FOLLOWING ANTHONY ALLISON;73
7.3;CHAPTER 3. THE CELLULAR BASIS FOR ESTABLISHING TOLERANCE OR IMMUNITY TO BOVINE .-GLOBULIN IN MICE;78
7.3.1;REFERENCES;82
7.4;CHAPTER 4. THE ROLE OF ACCESSORY AND THYMUS-DERIVED CELLS IN RESISTANCE TO TOLERANCE INDUCTION;84
7.4.1;INTRODUCTION;84
7.4.2;RESULTS;85
7.4.3;ACKNOWLEDGMENTS;98
7.4.4;REFERENCES;98
7.5;CHAPTER 5.
SUPPRESSOR ACTIVITY IN MICE TOLERANT OF HEMOCYANIN;104
7.5.1;ABBREVIATIONS;104
7.5.2;INTRODUCTION;104
7.5.3;MATERIALS AND METHODS;105
7.5.4;DISCUSSION;112
7.5.5;ACKNOWLEDGMENT;114
7.5.6;REFERENCES;114
7.6;CHAPTER 6.
SPECIFIC SUPPRESSION OF THE IMMUNE RESPONSE BY T CELLS;124
7.6.1;REFERENCES;136
7.7;CHAPTER 7. TOLERANCE TO CONTACT SENSITIVITY — A ROLE FOR SUPPRESSOR T CELLS?;140
7.7.1;SUMMARY;143
7.7.2;ACKNOWLEDGMENTS;144
7.7.3;REFERENCES;144
7.8;GENERAL DISCUSSION - SESSION I: T CELL TOLERANCE;146
8;SESSION II: B CELL TOLERANCE;156
8.1;CHAPTER 8. ACTIVATION OF AND TOLERANCE INDUCTION IN DNP-SPECIFIC B CELLS: ANALYSIS WITH THREE DISTINCT DNP-CARRIER CONJUGATES;158
8.1.1;INTRODUCTION;158
8.1.2;ACKNOWLEDGMENTS;174
8.1.3;REFERENCES;174
8.1.4;DISCUSSION FOLLOWING WILLIAM PAUL;176
8.2;CHAPTER 9. THREE CATEGORIES OF B CELL TOLERANCE INDUCED BY POLYSACCHARIDES: CHARACTERISTICS, INTERRELATIONSHIPS AND GENERAL IMPLICATIONS;180
8.2.1;GENERAL CONSIDERATIONS;188
8.2.2;REFERENCES;191
8.2.3;DISCUSSION FOLLOWING JAMES HOWARD;197
8.2.4;EFFECT OF TOLERANCE ON ANTIBODY-BINDING AFFINITY;198
8.2.5;ACKNOWLEDGMENTS;202
8.2.6;REFERENCES;202
8.3;CHAPTER 10.
HAPTEN-SPECIFIC TOLERANCE INDUCED BY THE DNP DERIVATIVE OF D-GLUTAMIC ACID AND D-LYSINE (D-GL) COPOLYMER;206
8.3.1;INTRODUCTION;206
8.3.2;TOLERANCE INDUCTION IN INTACT ANIMALS;207
8.3.3;TOLERANCE INDUCTION IN ADOPTIVE TRANSFER SYSTEMS;208
8.3.4;INDUCTION OF DNP-SPECIFIC TOLERANCE IN BOTH IgE AND IgG ANTIBODY CLASSES BY DNP-D-GL;211
8.3.5;ACKNOWLEDGMENTS;212
8.3.6;REFERENCES;212
8.4;CHAPTER 11. TOLERANCE IN ISOLATED B-CELL POPULATIONS IDENTIFIED BY AN ALLOTYPE-MARKER: ROLE OF T-CELLS IN INDUCTION;220
8.4.1;EXPERIMENTS AND RESULTS;221
8.4.2;DISCUSSION;222
8.4.3;ACKNOWLEDGEMENTS;223
8.4.4;REFERENCES;223
8.5;CHAPTER 12.
B CELL TOLERANCE INDUCED BY NON-METABOLIZED OR TOXIC ANTIGENS;228
8.5.1;CONCLUSIONS;235
8.5.2;REFERENCES;237
8.6;CHAPTER 13. EFFECT OF EPITOPE DENSITY ON THE INDUCTION OF TOLERANCE IN VITRO;242
8.6.1;INTRODUCTION;242
8.6.2;METHODS;243
8.6.3;RESULTS;243
8.6.4;DISCUSSION;246
8.6.5;SUMMARY;247
8.6.6;REFERENCES;247
8.7;GENERAL DISCUSSION - SESSION II: B CELL TOLERANCE;250
9;SESSION III:
MECHANISMS OF B CELL TOLERANCE;264
9.1;CHAPTER 14. REVERSIBLE AND IRREVERSIBLE B CELL TOLERANCE: DISTINGUISHING PROPERTIES AND MECHANISMS;266
9.1.1;INTRODUCTION;266
9.1.2;RAPIDLY IRREVERSIBLE B CELL TOLERANCE INDUCED BY DNP-D-GL;267
9.1.3;REVERSIBLE B CELL INACTIVATION;277
9.1.4;CONCLUSIONS;281
9.1.5;ACKNOWLEDGMENTS;283
9.1.6;REFERENCES;284
9.1.7;DISCUSSION FOLLOWING DAVID KATZ;294
9.2;CHAPTER 15.
HIGH-DOSE TOLERANCE IN MICE DEFICIENT IN REACTIVE T CELLS;300
9.2.1;INTRODUCTION;300
9.2.2;DISCUSSION AND SPECULATION;307
9.2.3;REFERENCES;310
9.2.4;DISCUSSION FOLLOWING GRAHAM MITCHELL;314
9.3;CHAPTER 16. FUNCTIONAL RELATIONSHIPS IN ANTIGEN-RECEPTOR INTERACTIONS IN B LYMPHOCYTES;318
9.3.1;REFERENCES;324
9.4;CHAPTER 17. B CELL TOLERANCE IN VITRO THE FATE OF THE TOLERANT CELL AND ITS CONTROL BY HORMONES;328
9.4.1;SUMMARY;332
9.4.2;ACKNOWLEDGMENTS;333
9.4.3;REFERENCES;333
9.5;CHAPTER 18. GRAFT-VERSUS-HOST REACTIONS, TUMOR SPECIFIC IMMUNITY AND SELF TOLERANCE;336
9.5.1;INTRODUCTION;336
9.5.2;MATERIALS AND METHODS;336
9.5.3;RESULTS;338
9.5.4;CONCLUSIONS;340
9.5.5;ACKNOWLEDGMENTS;342
9.5.6;REFERENCES;342
9.6;CHAPTER 19.
RECEPTOR BLOCKADE BY TOLEROGEN: ONE EXPLANATION OF TOLERANCE;350
9.6.1;INTRODUCTION;350
9.6.2;ACKNOWLEDGEMENTS;354
9.6.3;REFERENCES;354
9.7;GENERAL DISCUSSION - SESSION III: IMECHANISMS OF B CELL TOLERANCE;360
10;SESSION IV:
MECHANISMS OF T CELL TOLERANCE;366
10.1;CHAPTER 20. UNIFYING CONCEPTS IN TOLERANCE INDUCTION FOR VARIOUS T AND B CELL SUB-POPULATIONS;368
10.1.1;SUMMARY;378
10.1.2;ACKNOWLEDGMENTS;379
10.1.3;REFERENCES;379
10.1.4;DISCUSSION FOLLOWING G. J. V. NOSSAL;384
10.2;CHAPTER 21. CELLULAR PARAMETERS OF THE TOLERANT STATE INDUCED TO HUMAN . GLOBULIN IN MICE AND OF ITS MODULATION BY BACTERIAL LIPOPOLYSACCHARIDES;390
10.2.1;CELLULAR PARAMETERS OF A TOLERANT STATE IN MICE TO HUMAN . GLOBULIN;392
10.2.2;THE EFFECT OF LIPOPOLYSACCHARIDE ON THE TOLERANT STATE INDUCED TO HGG;399
10.2.3;ACKNOWLEDGMENTS;404
10.2.4;FOOTNOTES TO THE TITLE PAGE;404
10.2.5;REFERENCES;405
10.2.6;DISCUSSION FOLLOWING JACOUES CHILLER;408
10.3;CHAPTER 22. EFFECT OF ANTIGEN STRUCTURE ON THE INDUCTION OF TOLERANCE IN T CELLS;414
10.3.1;INTRODUCTION;414
10.3.2;METHODS;414
10.3.3;RESULTS;414
10.3.4;DISCUSSION;418
10.3.5;SUMMARY;418
10.3.6;ACKNOWLEDGMENTS;419
10.3.7;REFERENCES;419
10.4;CHAPTER 23. INDICATIONS OF ACTIVE SUPPRESSION IN MOUSE CARRIERS OF LYMPHOCYTIC CHORIOMENINGITIS VIRUS;420
10.4.1;THE CYTOTOXIC ASSAY;420
10.4.2;STATUS OF ANTIGEN REACTIVE CELLS IN CARRIER MICE;421
10.4.3;ALLOGENEIC ABROGATION OF T CELL TOLERANCE;421
10.4.4;EVIDENCE FOR ACTIVE SUPPRESSION;423
10.4.5;CONCLUSIONS;426
10.4.6;ACKNOWLEDGMENTS;427
10.4.7;REFERENCES;427
10.5;CHAPTER 24. INTERACTIONS BETWEEN T CELLS WHICH RESULT IN SUPPRESSION OF T CELL FUNCTIONS;430
10.5.1;INTRODUCTION;430
10.5.2;SUPPRESSIVE INTERACTIONS BETWEEN T CELLS;430
10.5.3;SUPPRESSIVE EFFECTS OF SPLEEN-SEEKING T CELLS;431
10.5.4;DIFFERENCES BETWEEN SPLEEN AND LYMPH NODE CELLS;432
10.5.5;EFFECTS OF CYTOTOXIC DRUGS;433
10.5.6;RELATION TO TOLERANCE;434
10.5.7;ACKNOWLEDGMENTS;434
10.5.8;REFERENCES;434
10.6;GENERAL DISCUSSION - SESSION IV: MECHANISMS OF T CELL TOLERANCE;438
11;SESSION V: ACTIVITY OF SUPPRESSOR CELLS AS A MECHANISM OF TOLERANCE;446
11.1;CHAPTER 25.
ACTIVITY OF SUPPRESSOR CELLS AS A MECHANISM OF TOLERANCE;448
11.1.1;REFERENCES;453
11.2;CHAPTER 26. LACK OF ACTIVITY OF CONTRA-SUPPRESSOR T CELLS AS A MECHANISM OF TOLERANCE;458
11.2.1;SUMMARY;475
11.2.2;ACKNOWLEDGMENTS;476
11.2.3;REFERENCES;476
11.2.4;DISCUSSION FOLLOWING RICHARD GERSHON;483
11.3;CHAPTER 27. THE MODE AND SITES OF ACTION OF SUPPRESSOR T CELLS IN THE ANTIGEN-INDUCED DIFFERENTIATION OF B CELLS;488
11.3.1;REFERENCES;502
11.3.2;DISCUSSION FOLLOWING TOMIO TADA;507
11.4;CHAPTER 28. THE ROLE OF SUPPRESSOR T CELLS IN THE DEVELOPMENT OF LOW-DOSE PARALYSIS TO TYPE III PNEUMOCOCCAL POLYSACCARIDE;510
11.4.1;INTRODUCTION;510
11.4.2;MATERIALS AND METHODS;510
11.4.3;RESULTS;511
11.4.4;DISCUSSION;517
11.4.5;REFERENCES;518
11.5;CHAPTER 29. ARE SUPPRESSOR CELLS RESPONSIBLE FOR TOLERANCE TO A SOLUBLE ANTIGEN?;520
11.5.1;REFERENCES;522
11.6;CHAPTER 30. STIMULATION OF SPECIFIC SUPPRESSOR T CELLS BY THE TERPOLYMER L-GLUTAMIC ACID60-L-ALANINE30-L-TYROSINE10 IN GENETIC NONRESPONDER MICE;524
11.6.1;ACKNOWLEDGMENTS;530
11.6.2;REFERENCES;530
11.7;CHAPTER 31.
MECHANISM OF ALLOTYPE SUPPRESSION IN MICE;536
11.7.1;ACKNOWLEDGMENTS;545
11.7.2;REFERENCES;545
11.8;CHAPTER 32.
REMARKS ON ALLOTYPE SUPPRESSION IN RABBITS;548
11.8.1;INTRODUCTION;548
11.8.2;COMPARISONS OF THE SUPPRESSION PHENOMENON IN MICE AND RABBITS;548
11.8.3;REFERENCES;551
11.9;GENERAL DISCUSSION - SESSION V: ACTIVITY OF SUPPRESSOR CELLS AS A MECHANISM OF TOLERANCE;554
12;SESSION VI: SUPPRESSIVE ACTIVITY OF ANTIBODY AND ANTIGEN- ANTIBODY COMPLEXES;558
12.1;CHAPTER 33. IS THERE A UNIFYING CONCEPT FOR DIVERSE MODELS OF TOLERANCE INDUCTION?;560
12.1.1;INTRODUCTION;560
12.1.2;IN VIVO MECHANISMS THAT MEDIATE TOLERANCE;561
12.1.3;THE IMMUNOCOMPETENT CELL THE TARGET OF TOLERANCE-MEDIATING MECHANISMS;565
12.1.4;CONDITIONS AT THE SINGLE CELL LEVEL WHICH BRING ABOUT TOLERANCE INDUCTION;566
12.1.5;A UNIFYING CONCEPT;570
12.1.6;SUMMARY;572
12.1.7;ACKNOWLEDGMENTS;573
12.1.8;REFERENCES;573
12.1.9;DISCUSSION FOLLOWING ERWIN DIENER;579
12.2;CHAPTER 34. BLOCKING FACTORS IN TUMOR IMMUNITY AND ALLOGRAFT TOLERANCE;582
12.2.1;SUMMARY;592
12.2.2;ACKNOWLEDGMENTS;592
12.2.3;REFERENCES;592
12.3;CHAPTER 35. REGRESSOR SERUM BLOCKING AT TARGET CELL LEVEL, NOT AT LYMPHOCYTE LEVEL, INTERACTS WITH TARGET CELLS TO PRODUCE BLOCKING FACTOR;596
12.3.1;ON THE NATURE OF BLOCKING FACTORS;597
12.3.2;DISCUSSION FOLLOWING KARL-ERIK HELLSTRÖM;599
12.3.3;ANTIBODY-MEDIATED TOLERANCE;604
12.3.4;ACKNOWLEDGMENTS;606
12.3.5;REFERENCES;606
12.4;CHAPTER 36. IgE ANTIBODY-SPECIFIC ABROGATION OF AN ESTABLISHED IMMUNE RESPONSE IN MICE BY MODIFIED ANTIGENS;608
12.4.1;REFERENCES;617
12.5;GENERAL DISCUSSION - SESSION VI: SUPPRESSIVE ACTIVITY OF ANTIBODY and ANTIGEN-ANTIBODY COMPLEXES;622
13;SESSION VII:
SUMMARY AND POTENTIAL THERAPEUTIC APPLICATIONS;626
13.1;GENERAL DISCUSSION - SESSION VII;628
13.2;CLOSING COMMENTS SIR MACFARLANE BURNET;660

Leseproben

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 B?G-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-?G 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. B?G 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 B?G coated SRBC and an anti-?G1 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 B?G 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 B?G by painting them with DNFB. Subsequently, the mice were challenged...