Commentary Clonal Anergy of B Cells: A Flexible, Reversible, and Quantitative Concept By G.J.V. Nossal From The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia t the conclusion of the 1986 Immunology Congress in termed clonal anergy (7). Support for clonal anergy among A Toronto, the late Georges K6hler presented a sum- T lymphocytes also gradually emerged (8, 9). mary lecture which was, unfortunately, poorly attended. It Enter transgenic mouse technology. As far as B cell tol- dealt with the impact of the new genetics on immunology, erance was concerned, the first results were disappointing. Downloaded from http://rupress.org/jem/article-pdf/183/5/1953/1108328/1953.pdf by guest on 29 September 2021 and, among other wise things, he said that transgenic tech- Antigen-transgenic mice were either non-tolerant or vari- nology was set to revolutionize the way we studied immu- ably tolerant (10, 11). However, antigen-transgenic animals nologic tolerance (1). The greatest barrier to uncovering were not the main game, as they left the investigator with the details of what happened to anti-self cells in the repertoire the task of studying the minority of (unidentifiable) reactive was the heterogeneity oflymphocytes. Cells potentially re- lymphocytes. Things really took off when B (12, 13) or T active with a given self antigen were so rare that most at- (14) cell receptor transgenic mice were used. From the tempts to study their fate retied on inferential rather than viewpoint of the present story (15), the critical model was direct methods. Successful attempts to create mice trans- an ingeniously devised double-transgenic strategy (12; re- genic for both heavy (H) and tight (L) immunoglobutin (Ig) viewed in 16, 17). Mice were rendered transgenic for mono- chains, or for the oe and [3 chains of the recently discovered clonal B cell IgM and IgD receptors with high affinity for T cell receptor (TCR) in theory, at least, had the potential to hen egg lysozyme (HEL) and such mice were mated with create monoclonal mice, i.e., animals in which all or at least mice transgenic for HEL itself. Further features of the model the greater part of the B or T cell population consisted only were a capacity to distinguish transgenic from endogenous of cells bearing the transgenically imposed specificity. Anti- antibody via an allotype marker; the use of various founder idiotypic reagents could identify such cells, or, even more lines constitutively producing various amounts of soluble easily, simple enumeration of B or T cell numbers under HEL (s-HEL); a capacity to raise low HEL levels to high various experimental circumstances could yield information ones through zinc feeding in mice where the metallothio- about the fate of anti-self cells. nein promoter formed part of the HEL transgenic con- How tight K6hler was; but it took some time to prove struct; and comparison of the effects of s-HEL with a it. Burnet first articulated the concept of clonal deletion as membrane-anchored form of the same antigen (m-HEL). the key mechanism of tolerance, postulating that if an en- Some key findings of this model were as follows, s-HEL at counter between a self antigen and a cell reactive with it very low concentrations caused T cell tolerance but with occurred in early life, while the immune system was imma- no discernible effects on the B cell compartment. Higher ture, clonal deletion rather than clonal selection would be concentrations also resulted in T cell tolerance but in addi- the end result (2). Lederberg refined this notion, placing tion caused a non-deletional, clonal anergy type of B cell the transition from paralyzability to inducibility at the level tolerance, readily observed as the great majority of B cells of each differentiating immunocyte, regardless of the age of carried the transgenic Ig. This B cell tolerance was accom- the animal (3). When it first became possible to enumerate panied by a down-modulation of surface IgM and a marked antigen-binding B cells by autoradiographic techniques, early shortening of the B cell's life-span (18). The transgenic B results indeed favored deletion as a tolerance mechanism cells clustered chiefly in follicular areas of the spleen, espe- (4). For T cells, it was still necessary to rely on functional cially the mantle zone of the follicles. Only when the techniques to support repertoire purging as a tolerance mecha- strongly cross-linking signal of transgenic m-HEL was in nism as not even enumeration of antigen-specific precur- place did a deletional type of tolerance, representing clonal sors of effector T cells by in vitro cloning techniques could elimination shortly after Ig expression (while the immature distinguish clonal deletion from some form of effective non- B cell was still in the bone marrow) become manifest. Still, lethal silencing (5). However, the latter concept had articu- the delayed deletion of the anergic cells at 3-4 d does raise late supporters on theoretical grounds (6). Careful work on the question of whether anergy in B cells is entirely discrete antigen-specific B cells, involving both their enumeration from "clonal abortion" (19). and purification, showed that high doses of antigen could The plot thickens as we move to studies that seek to cause clonal deletion, but tolerance could be achieved with track the migration of transgenic B cells after transfer into much lower doses, this non-deletional phenomenon being various kinds of host mice (20). Anti-HEL B cells trans- 1953 j. Exp. Med. The Rockefeller University Press 0022-1007/96/05/1953/04 $2.00 Volume 183 May 1996 1953-1956 ferred into antigen-free hosts homed to lymphoid follicles, stable intracellular dye 5-carboxyfluoresceine diacetate-suc- coming to occupy much the same location as within the cinimidyl ester (CFSE). To give a calibration device com- donor animal. A similar trafficking pattern was noted when paring the fate ofanti-HEL transgenic B cells and normal B the transgenic B cells were transferred to doubly s-HEL-anti- cells, the former were treated with a concentration of CFSE HEL transgenic mice. However, when the anti-HEL (anti- four-fold higher than the latter. Thus, flow cytometry can neo-self) B cells find themselves in a minority among a di- easily distinguish the numbers of the two transferred popu- verse B cell repertoire, and still in the presence of the HEL lations in various organs under different experimental cir- antigen, something different happens. Transferred B cells still cumstances. To study the effects of antigen concentration, appear in the white pulp of the spleen, and soon move to anti-HEL B cells were transferred into different founder the outer part of the T cell trafficking area, the so-called lines, with zinc-induced antigen upregulation, or use of peri-arteriolar lymphocyte sheath or PALS, where they come m-HEL transgenic mice, providing further variables. Trans- to occupy an area just outside the follicle, at the follicle/ fer into non-transgenic mice given HEL by injection served PALS border. They do not penetrate into the follicle, but as a check on s-HEL-transgenic results. To investigate the appear to die within a day or two. influence of effective cognate T cell help on trafficking pat- To understand the interpretation that Cyster et al. (20) terns, survival, and fate of transferred normal or anergic place on these findings, we must first present some further cells, B and T cell cotransfer experiments were performed. curious facts. Newly formed B cells leave the bone marrow CFSE-labeled-anti-HEL B cells were prepulsed with a pep- Downloaded from http://rupress.org/jem/article-pdf/183/5/1953/1108328/1953.pdf by guest on 29 September 2021 in very large numbers every day and home predominantly tide MCC87_103 from moth cytochrome C. At the same to the spleen (21). At this stage of their lives, they are pre- time, CD4 + T cells from a TCg.-transgenic mouse, with dominantly IgM high IgD l~ B cells, and these cells are rather specificity for MCC87_103, preactivated in vivo one day pre- short-lived, having largely disappeared by a week or so. If viously, were also transferred. Appropriate F1 hybrid mice B cells from lymph nodes, from thoracic duct lymph, or with a suitable class II MHC restriction element were used. from other parts of the recirculating lymphocyte pool are The following are the main results of the study. The examined, these possess a different phenotype. They are shortened life-span of anti-HEL B cells on transfer into ei- Ig-M l~ lgD high, recirculating, follicular mantle-seeking and ther the standard s-HEL transgenic or m-HEL transgenic they live for several weeks. These cells are not memory B hosts was confirmed. As neither cell division (which would cells, in that they display no or very few Ig V gene muta- have halved the CFSE labeling) nor migration to lymph tions, are not isotype switched, and do not carry memory if nodes were noted, the cells probably died in situ in the adoptively transferred from immunized mice. Yet they are spleen. Some enlargement of the anti-HEL cells by com- different from virgin, just emerging B cells, in that they ex- parison with cotransferred normal B cells suggested a de- hibit a different and more restricted Ig V gene repertoire gree of activation before death. As noted by Cyster et al. (22). It is as though this population has either been posi- (20), the anti-HEL B cells migrated to the PALS-follicle tively selected by contact with antigen, but not in a way border.