A second chance for bad B cells

G.J.V.

IMMUNOLOGICAL TOLERANCE

NOSSAL

A second chance for bad B cells B lymphocytes bearing receptors for self antigens do not develop. New results suggest that they are allowed a second attempt at receptor gene rearrangement, rather than being killed immediately. The toughest problem for the immune system is that it does not ‘know’ what it will be required to recognize. It can respond to millions of antigens, including synthetic molecules that have never previously existed. Antibody molecules can successfully discriminate between not only microbial species but also strains and sub-strains, and can also recognize many different molecules within each microbe. Meanwhile, the immune system ‘ignores’ all the molecules that are ‘self. Instead of being encoded by an enormous number of genes, the necessary numbers of antibodies are generated by a combinatorial system of diversification. During the development of B lymphocytes in the bone marrow, a special recombinase machinery shuffles the minigenes that encode antibody chains. Each antibody is made up of heavy (H) and light (L) chains, each of which COIItributes to the recognition of the antigen, and in each case, the ‘one gene ~ one polypeptide chain’ rule has been broken. Heavy chains are encoded by four minigenes, V D, J and C, and light chains by three, r/; J and C The C minigene is constant for a particular immunoglobulin (Ig) type and so does not contribute to antibody diversity. r/; D and Jgenes lie in arrays, separated from each other by a considerable distance, The process of gene shuffling occurs in a precise order: first a particular D gene translocates to a particular JH, then a V, gene is selected to move closer to DJH Once the full VHDJH assembly is ready, the process starts on light chain genes. There are two type of L chains, x and h. The cell begins light chain assembly by translocating a KA to aJ, and then it either stops or translocates a 1,: to

a JL Having assembled two functional Ig-chain genes, the B cell displays the end product, an antibody molecule, as an antigen receptor on its cell surface. As this process occurs separately in each cell, an enormous diversity of B cells results. The rules of the game allow only one specific receptor per cell, so the B-cell population is really a huge repertoire of specific recognition units. The induction of an antibody response involves the selective activation, multiplication and differentiation of those few B cells that happen to have receptors that fit the antigen in question; these B cells soon mass-produce monoclonal antibodies that are identical to the B-cell’s unique receptor. As the minigene-shuffling and chain-pairing events occur in pre-lymphocytes at random (more or less), B cells must sometimes be generated that have receptors directed against autologous molecules, ‘self antigens’. The mechanisms that constitute ‘immunological tolerance’ collectively prevent the immune system from destroying the body [l] Clearly, one element of tolerance is to avoid making anti-self antibodies, and indeed, if self-tolerance breaks down, one variety of autoimmune disease that results is directly due to the production of autoantibodies - examples of this type of disease include haemolytic anaemia, myasthenia gravis and systemic lupus erythematosus (SIX). 460

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So what prevents healthy individuals from making autoantibodies? As immune induction frequently requires help from T cells, ensuring the removal of autoreactive T cells is one way to prevent autoantibody production. In addition, XI toantigens that ‘catch’ a potentially reactive B cell while ii is still immature can silence that B cell directly, either by C’I/ suring its death or by delivering a non-lethal, negative signal that inactivates the cell, creating a state of clonal anergy [ 2 1, In the last few years, research on tolerance in the B-cell population has been helped enormously by methods that introduce foreign genes into mice using transgenic technol o&y. One can create an ‘immunological nightmare’ mouse. in which nearly all B cells are made to express transgenei encoding antibodies against a genuine self-molecule, OIagainst a ‘neo-self molecule that is itself transgenically imposed on the animal. Such mice do not explode with autoimmunity - they become tolerant, either by send ing B cells into an anergic state (31 or by deleting them altogether 141. The greater the intensity of B-cell surface-& cross-linking, induced by binding to specific antigen, the more likely the latter fate. Because most of the B cells in a transgenic mouse have identical receptors, their fate can be tracked in detail, for example, by using a labelled monoclonal antibody directed against the common receptor. It has recently become al>parent that the unwanted, anti-self B cells often do not die immediately on encountering self antigen. Rather, their maturation is arrested and some of them, realizing the error of their ways, attempt to reform by constructing a modified or edited antigen receptor [ 5-81. Important cellular events in this process have been documented by Hartley et al. [ 51. In their transgenic mouse model, B cells carry transgenes for the chains of an antibody with high affinity for hen egg lysozyme (HEL). The mice are also transgenic for a membrane-bound form of HEL (mHEL) so that, as immature B cells develop from pre-B cells in the bone marrow, they immediately contact an array of their antigenic target, which is present as an integral membrane protein on adjacent cells. Without such an insult, normal immature B cells undergo maturation within the marrow, in a process that takes about two days, during which the cells acquire a variety of cell surface molecules that are necessary for their function as mature B cells ~ for example, molecules important in ensuring proper migratory patterns, and receptors for a variety of regulatory signals. Eventually, normal B cells leave the marrow and enter the organs of antibody pro duction, which include the spleen and lymph nodes, In the doubly transgenic mice, no B cells with transgenic H and L chains can be found in these lymphoid organs. However, within the bone marrow, immature self-reactive an&HEL B cells can be found in considerable numbers. A maturational arrest, presumably induced in response to encountering the neo-self antigen, somehow stops them from acquiring the 1993,

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receptors and adhesion molecules that are important for activation and migration. Extensive cell death is not apparent within the bone marrow of the doubly transgenic mice, and bromodeoxyuridine labelling studies show that the whole population of immature B cells turns over within two to three days, as in normal mice, The absence of mature B cells in lymphoid organs shows that the cells must die, either within the marrow or very shortly after emigration. So the elimination of the ‘bad’ B cells must occur in two steps: first, development is arrested and then, after a slight delay, the cells die. It is possible to rescue the anti-self B cells in the doubly transgenic mice [ 51. Immature B cells can be placed in tissue culture, with or without the target antigen. In the absence of antigen, the cells develop into mature B cells. So, the in viva contact with antigen does not deliver an irreversible signal leading to death. A further fascinating experiment that shed light on the fate of anti-self B cells involves the introduction of an ‘antideath’ gene into mice [9]. Animals transgenic for the bcl-2 gene, expressed under the control of an H-chain enhancer, have B cells with a greatly extended lifespan. These mice can be mated with the anti-HEL mice, generating double-transgenics that express only anti-HEL antibodies. The bone marrow from these doubly transgenic progeny can then be transplanted into mHEL transgenic

mice, where the bone marrow cells will encounter the ubiquitous membrane-bound neo-self antigen. These reconstituted mice showed an enormous accumulation of immature anti-self B cells, both in the marrow and in the blood and spleen, but no mature anti-self B cells were found anywhere. So, bcl-2 had not altered the maturation arrest, but it had markedly delayed the elimination of the arrested cells. When these arrested cells were cultured in an antigen-free environment, they matured and could be induced to produce anti-HEL antibodies [5]. What might the arrested cells be doing while awaiting a delayed death? To find out, we must mm to another model system, recently described by Tiegs et al. [ 61. In their systern, B cells are transgenic for specific Ig chains directed against a class 1 major histocompatibility (MHC) *antigen, H-2K. If the transgenic mouse also bears this MHC antigen, immature anti-self B cells appear in the bone marrow but not in the periphery. These cells up-regulate the expression of two genes, RAG1 and KAG2, which are critically involved in the minigene shuffling process, and then alter the specificity of their surface antigen receptors. Tiegs et al. [6] present strong evidence that these RAGexpressing cells are in fact trying to replace the x light chain encoded by the transgene with a newly formed h chain ~ the h chain arises as a result of a PX to & translocation in the endogenous h chain gene array. As a

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Fig. 1. Receptor editing allows some selfreactive B cells to escape maturational arrest. B cells expressing strongly selfreactive surface antibodies (dark pink) are deleted after a maturational delay, while weaker self-reactive B cells (light pink) enter a potentially reversible state of anergy. The maturationally arrested cells attempt to re-initiate Ig gene translocation; if successful in this ‘receptor editing’, leading to the surface expression of non-self-reactive Ig, the maturational arrest is reversed and the cell progresses to normal B-cell status.

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cells bearing transgenic H chains and endogenously encoded hL chains, no longer displaying antiH.2K specificity, escape from the maturational arrest, leave the bone marrow, and behave like normal mature B cells. The ‘bad’ B cells have tried again, and in some cases have succeeded in reforming to become useful members of the B-cell population (Fig. 1). A similar conclusion was reached by Gay et al. [7], using a system that shows some intriguing differences. In this case, the antigen is DNA, antibodies against which occur frequently in SLE.Anti-DNA antibodies fall into two classes, those reacting only with single-stranded (ss) DNA and those that react with both ssDNA and double-stranded (ds) DNA; the antibodies that recognize both ss- and dsDNA are of higher affinity. Mice were made transgenic for the H- and L-chain genes of an anti-dsDNA antibody, derived from a mouse suffering from SLE. Interestingly, such mice do not make any B cells with anti-dsDNA specificity - not even immature B cells in the bone marrow. Young mice, as a consequence, have very few B cells of any sort. Adult mice, however, have essentially normal numbers of B cells, which express the transgenic H chain with an endogenous L chain. These are clearly ‘try again’ variants, cells that have expelled the transgene and inserted a ‘good’ endogenous L-chain gene. The authors [7] suggest that the editing of the receptor might occur at the level of H/L pairing, the L chain of the autoreactive antibody being displaced by an endogenous L chain that generates a non-autoreactive receptor. Both the transgenic and endogenous, edited (newly rearranged) I,chain genes are transcribed, but in the cases in which the endogenous 1,chain fits better with the H chain, and is not of an anti-DNA type, the B cell progresses and becomes normal. Only some L chains have the required characteristics, and so only a limited set of V, genes are expressed in these ‘trying to be good’ B cells. It is not yet clear why B cells in this model begin the receptor editing process at the very earliest stages of surface receptor expression - no immature anti-dsDNA B cells are ever seen in the marrow - in contrast to the other models in which immature ‘bad’ B cells can easily be found. This is rather unexpected, ;1s the autoantigen in question, DNA, is intracellular and presumably only transiently present in a form accessible to the surface of B cells. One might therefore have expected some additional delay in deletion, rather than accelerated receptor editing or cell death. A further contribution from the Weigert group [S] attacks the problem in a slightly different way, with only the H chain of the anti-dsDNA antibody introduced as a transgene; the B cells are left to express endogenous Lchain genes. An extensive repertoire analysis of the B cells of these mice was performed. Again, no B cells with anti-dsDNA specificity were allowed to emerge, although 7 out of 44 monoclonal antibodies had anti-ssDNA (low affinity) activity. All expressed the transgenic H chain, but the L-chain repertoire used was very restricted. Only 9 of more than 20 possible Vx chains were used and 6 of the genes accounted for 33 out of 44 observed sequences; a single Vgene was encountered in 16 of 44 cases. Calculations suggest that the total VL-gene repertoire used in these mice consisted of only 37 genes, although the mouse has about 200 in its genome. So, the many H- and

consequence,

some

-_ L-chain combinations that could have given rise to an,:. &DNA activity must have been removed from the repel toire. The study design does not permit one to say ho,\ many of the emergent B cells are ‘try again’ variants, but iii significance lies in the fact that removal from the repertoirL. is demonstrated in a context in which one of the chains is endogenous and not transgenically imposed. The mechanisms underlying negative signalling in B cells, and subsequent receptor editing, are undoubtedly corn. plex. It is tempting to speculate that the inhibitory patlj~ way of signaliing following surface-Ig cross-linking differs somehow from the activating pathway, for example by ~5~ ing a particular receptor-associated tyrosine kinase. There are at least four members of the Src family that are activattxcl on cross-linking the B-cell’s surface IgM: Blk, Fyn, Lyn :~tld Lck. In all B-cell lines that express Blk, IgM cross-lir&:ii!:; causes growth inhibition, whereas in cell lines that do .‘(rt express Blk, no inhibition is observed [lo]. Moreover, :.b. moval of Blk by antisense deoxyribonucleotides prevci;;,s IgM-dependent growth inhibition. All cell lines that al-e resistant to negative signalling express Fyn, but removing Fyn with antisense does not confer inhibition in response to IgM cross-linking. The mechanisms determining dela\:Ld versus immediate cell death, and regulating the time-w dow during which receptor editing can occur, are e\c :, more obscure. At least the various transgenic models no\<, available, with their relatively homogeneous B-cell pop”lations, should allow vigorous biochemical exploration of these issues. References Nosw

and cellular aspects of immunologic toler1991, 202~729-737. NOSSAL GJV:Cellular mechanismsof immunological tolerance. Annu h’ev lmmunoll983, 1:3+62. ance.

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functional silencing of self-reactive B lymphocytes in transgenic mice. iVatuw 1988. 334:676X&2. NE?VIAZE DA, B~JKKI K: Clonal deletion of B lymphocytes in a transgenie mouse bearing anti-MHC class I antibody genes IVdure 1989, 337:562&566.

HARTLE’i SB, COOKE MP, FULCI”!H

DA, IIARRIS AW, CORY S, I~ILSTES 4

Goon~~ow CC: Elimination of self-reactive B Ivmohocvtes oroceeds in two stages: arrested development anh cell diath.‘&ll 1993, 72~325-335. ~‘IEGS SL, RUSSELL DM, NFMAZEE D: Receptor editing in self-reactive bone marrow B cells. J /3@ Med 1993, 177:100~1020. GAY D, SAUNDERS T. CAMPER S, WEIGERT M: Receptor ending: an anpreach by autoreactive B cells to escape t;erance. .I&JJ Mid 1993, 177:99’+1008. RAOIC MZ, ER~KSON J, LITY+TN S, WEICEKT M: B lymphocytes may escape tolerance by revising their antigen receptors.JE.upMed 1993, 177:1165-1173. STKA?;SERA, W~~ITI’INGJUM S, V,UJX DL, BATH ML, ADAMS JM, Cow S, Ims AW: EnforcedBCL2 expressionin B-lymphoid cells pro-

longs antibody responses and elicits autoimmune Natl Acad Sci USA 1991, 88~661-8665. YAO X-R, X01-r DW: Expression of protein tyrosine Ig complex of anti-p-resistant B-cell lymphomas: role kinase in signaILing growth arrest and apoptosis. 1993, 132:1-24.

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G.J.V. Nossal, The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Victoria 3050, Australia.