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Establishing Anergy as a Bona Fide In Vivo Mechanism of Tolerance Andrew Getahun, Shannon K. O'Neill and John C. Cambier This information is current as J Immunol 2009; 183:5439-5441; ; of October 2, 2021. doi: 10.4049/jimmunol.0990088 http://www.jimmunol.org/content/183/9/5439

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Pillars of Immunology

Establishing Anergy as a Bona Fide In Vivo Mechanism of B Cell Tolerance Andrew Getahun, Shannon K. O’Neill, and John C. Cambier1

he pioneering work of Owen (1) and Burnet and Fen- However, these experiments did not discriminate whether ner (2) in the late 1940s originated the concept of this was due to or to the induction of a state T . Subsequent experiments con- in which the cell persisted but was simply unresponsive to ducted by Billingham et al. (3) first demonstrated the active Ag. To address this question, Pike and Nossal used a fluo- induction of immune tolerance using a skin graft rejection rescein (Flu2)-human gamma globulin (HGG) tolerization model. These authors consequently coined the phrase “ac- protocol in conjunction with analysis of both the capacity to tively acquired immunological tolerance.” The findings of produce Ab responses and enumeration of the Flu-specific B Billingham et al. were put into structural perspective by cells (13). They found that in mice treated neonatally or in Downloaded from David Talmage’s hypothesis of cell selection (4) and Bur- utero with high doses of Flu-HGG, there was a significant net’s theory (5), both published in 1957, reduction in the number of Flu-binding cells, suggesting which provided a framework for understanding how it might clonal abortion/deletion. However, when they used low Ag be possible to maintain tolerance to self while remaining re- doses they found no reduction in the number of Flu-binding sponsive to foreign Ags. Specifically, if one B cell encoded a cells or their avidity profiles; nevertheless, the cells did not single specificity, deletion of self-reactive cells would provide produce Abs upon stimulation. They coined the term clonal http://www.jimmunol.org/ tolerance while sparing responses to other Ags. An important “anergy” to describe this B cell unresponsiveness. qualification of the clonal selection theory was proposed by Although the conclusions of Pike and Nossal would be Joshua Lederberg (6), who postulated that tra- proven correct, there were some caveats in their experimental verse a developmental stage during which they are uniquely approach. For example, their reported Ab-forming cell pre- sensitive to tolerance induction. Of course we now recognize cursor frequency was much lower than would have been that this principle applies to so-called “,” predicted based on the Ag-binding cell frequency. Therefore wherein autoreactive cells are silenced by deletion (T and B) the differentiated daughters of Ag-binding cells remaining and receptor editing (B) that occur in the and after tolerance induction may never have been capable of by guest on October 2, 2021 . Gradually, additional silencing mechanisms were producing measurable anti-Flu responses, e.g., prior to tol- defined, such as those involving suppressor/regulatory T erance induction. Moreover, like all previous B cell tolerance cells, idiotypic regulatory networks, and anergy. Silencing of studies, the approach involved tolerization with exogenous autoreactive B cells by anergy was first established unequiv- foreign Ag in contrast to the physiological situation in which ocally by the Pillars of Immunology article (7) that is the sub- the self-Ag would be present throughout the ontogeny of au- ject of this commentary. toreactive B cells. Finally, the tolerogen used in the studies, Among the earliest indications that B cells per se could be Flu-HGG, would bind the inhibitory IgG constant receptor rendered tolerant were the experiments of Siskind and col- Fc␥RIIB expressed by the B cells, and this could suppress the leagues in 1963 (8) in which pneumococcal polysaccharides subsequent immune response. Thus, although Pike and were used as tolerogens/Ags. The “thymus independence” of Nossal originated the concept of anergy, Chris Goodnow responses to this Ag made it unlikely that T cells were in- and his mentor Anthony Basten (7) proved that anergy is volved in silencing the response. Subsequent in vitro studies operative in the silencing of autoreactive B cells in vivo. using isolated B cells clearly demonstrated that these cells As reported in the featured publication, Goodnow and could be rendered tolerant (9–12). Consistent with the hy- Basten et al. (7) took advantage of the then newly developed pothesis of Lederberg, these studies also showed that imma- transgenesis technology to express hen egg lysozyme (HEL) ture B cells were more sensitive to tolerance induction than as a neo-autoantigen in mice. The Ag was expressed under a mature B cells, requiring 1000-fold less Ag than mature metallothionein promoter to facilitate expression from em- splenic B cells to achieve tolerance (9). bryonic to adult life. Furthermore, use of this promoter al- In early studies, the mechanism by which B cells were lowed the modulation of Ag expression by exposure to heavy tolerized was often referred to as clonal inactivation/abor- metals. In these mice (ML5 line), the expression of HEL in- tion. It was shown that the frequency of Ag-specific, Ab-pro- duced tolerance in both HEL-specific B and popula- ducing cells fell drastically following tolerization treatments. tions, confirming that it was recognized as self. ML5 mice

2 Abbreviations used in this paper: Flu, fluorescein; HEL, hen egg lysozyme; HGG, human gammaglobulin. 1 Address correspondence and reprint requests to Dr. John C. Cambier, 1400 Jackson Street, K803, Denver, CO 80206. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0990088 5440 PILLARS OF IMMUNOLOGY were then crossed with a B cell Ag receptor transgenic line volves some durable form of reprogramming or is reversible. (MD3) that expressed a BCR with a high affinity for HEL. The transfer of anergic B cells into an environment lacking The IgM and IgD of the MD3 mice were of the Igha HEL gradually (over days) results in loss of the anergic phe- that, when on an Ighb background, allowed tracking of the notype and reacquisition of the ability to produce Ab re- MD3 B cells and production of IgMa by these cells or their sponses to HEL (18). Complementary studies using the daughters. lower affinity Ars/A1 model indicated that the anergic phe- The results of the experiment were striking. The presence notype is lost within minutes of the dissociation of autoan- of HEL did not lead to the deletion of most MD3 B cells; in tigen (19). Although the delay in the loss of anergy upon the fact, they were present in the periphery in nearly normal transfer of MD3 ϫ ML5 B cells could be interpreted as re- numbers but exhibited reduced surface IgM levels. Interest- sulting from a requirement for the decay of a transcription- ingly, IgD expression remained high. Although MD3 mice ally regulated repressor, other data suggests this might be due had high spontaneous IgMa anti-HEL titers, MD3 ϫ ML5 to the relatively high affinity of the MD3 for HEL ϭ ϫ 9 Ϫ1 double transgenic mice produced barely detectable levels of (Ka 2 10 M ) and thus slow dissociation of HEL from IgMa anti-HEL Ab. Moreover, in an adoptive transfer exper- Ag receptors. Taken together, these data are most consistent iment in which either MD3 or MD3 ϫ ML5 spleen cells with a requirement for the constant transduction of a BCR were transferred into sublethally irradiated C57BL/6 mice signal to maintain anergy (19). ϫ together with HRBC-primed T cells, HRBC-HEL immuni- The MD3 ML5 system established anergy as a mechanism of B cell tolerance. However, the model is unphysiologic in its

zation led to drastically lower anti-HEL responses in recipi- Downloaded from ents of MD3 ϫ ML5 spleen cells than in recipients of MD3 reliance on transgenes and an Ag receptor possessing extremely spleen cells. high autoantigen affinity. Thus the studies did not establish ab- These results grounded the theory. A year solutely the contribution of this mechanism to physiologic B later, David Nemazee and his colleagues would demonstrate cell tolerance in the wild-type repertoire. Goodnow did com- pare the phenotype of the B cells in MD3 ϫ ML5 mice with clonal deletion of autoreactive B cells in a BCR transgenic those in wild-type C57BL/6 mice and concluded that a popu- model specific for MHC class I (14). This study would be http://www.jimmunol.org/ lation of IgMlowIgDhigh B cells exists in a normal repertoire, followed by another paper by Hartley, Basten, and Good- suggesting that this population could contain anergic B cells now showing that B cells that became anergic in the presence (16). Later studies by our laboratory and that of Darise Farris of soluble HEL were deleted when the MD3 mouse was using a more extensive anergic cell marker set demonstrated crossed to a mouse expressing HEL in a membrane-bound the existence of anergic “An1” cells in the normal B cell rep- form (15). This result underscored the importance in B cell ertoire (20, 21). More recently, an anergic B cell population fate determination of the form of self-Ag that is encountered has been identified in humans (22). Considering the estima- (i.e., monomeric/oligomeric soluble vs highly multivalent tion that up to 75% of newly formed B cells are autoreactive membrane bound). As stated by Hartley et al., “. . . signaling (23), the size of the An1 population, and the reduced life by guest on October 2, 2021 through the B-cell receptor is not an all-or-none span of anergic cells, it appears that as many as 50% of newly event, but…quantitative differences in the degree of recep- produced B cells may be destined to become anergic (20). tor crosslinking may bring about qualitatively different cel- Thus, anergy is probably a major mechanism of tolerance lular responses.” and, particularly given its reversible nature, its failure is a Subsequent studies using the HEL-anti-HEL and other likely source of autoreactive cells that participate in the de- transgenic models provided a number of additional impor- velopment of . tant insights regarding B cell anergy. By using transgenic models, it was possible to determine the relationship be- Disclosures tween the degree of receptor occupancy (a function of recep- The authors have no financial conflict of interest. tor affinity, autoantigen concentration, and total avidity) and the fate of autoreactive B cells. In the anti-HEL model, Ͻ References occupancy of 5% of receptors led to development of B cells 1. Owen, R. D. 1945. Immunogenetic consequences of vascular anastomoses between that were “ignorant” of Ag and Ͼ5–45% receptor occu- bovine twins. Science 102: 400–401. 2. Burnet, F. M., and F. Fenner. 1949. The Production of , 2nd Ed. Macmillan, pancy to anergy, whereas high occupancy, particularly in Melbourne. conditions of high Ag avidity, led to deletion (16). In an- 3. Billingham, R. E., L. Brent, and P. B. Medawar. 1953. Actively acquaired tolerance of other transgenic anergy model in which B cells were specific foreign cells. Nature 172: 603–606. 4. Talmage, D. W. 1957. and immunology. Annu. Rev. Med. 8: 239–256. for arsonate but displayed low-affinity crossreactivity for 5. Burnet, F. M. 1957. A modification of Jerne’s theory of antibody production using the ssDNA, the so-called Ars/A1 model, occupancy of ϳ20% of concept of clonal selection. Aust. J. Exp. Biol. Med. Sci. 20: 67–69. 6. Lederberg, J. 1959. Genes and antibodies. Science 129: 1649–1653. receptors led to anergy (17). 7. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, Another key question related to potential changes in toler- R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al. 1998. Altered immunoglobulin expression and functional silencing of self- ance susceptibility during development was this: did the Leder- reactive B lymphocytes in transgenic mice. Nature 334: 676–682. berg hypothesis apply to anergy? Regardless of their maturation 8. Siskind, G. W., P. Y. Paterson, and L. Thomas. 1963. Induction of unresponsiveness and in newborn and adult mice with pneumococcal polysaccharide. J. Im- state, the transfer of nontolerant MD3 anti-HEL B cells into munol. 90: 929–934. . an environment with appropriate levels of HEL led to anergy 9. Cambier, J. C., J. R. Kettman, E. S. Vitetta, and J. W. Uhr. 1976. Differntial suscep- tibility of neonatal and adult murine spleen cells to in vitro induction of B-cell toler- (16). ance. J. Exp. Med. 144: 293–297. Another important question that is now being debated, 10. Metcalf, E. S., and N. R. Klinman. 1976. In vitro tolerance induction of neonatal murine B cells. J. Exp. Med. 143: 1327–1340. particularly in view of reported genetic reprogramming in 11. Nossal, G. J., and B. L. Pike. 1975. Evidence for the clonal abortion theory of B- anergic T cells, is whether the induction of B cell anergy in- tolerance. J. Exp. Med. 141: 904–917. The Journal of Immunology 5441

12. Stocker, J. W. 1977. Tolerance induction in maturing B cells. Immunology 32: 18. Goodnow, C. C., R. Brink, and E. Adams. 1991. Breakdown of self-tolerance in an- 283–290. ergic B lymphocytes. Nature 352: 532–536. 13. Nossal, G. J., and B. L. Pike. 1980. Clonal anergy: persistance in tolerant mice of 19. Gauld, S. B., R. J. Benschop, K. T. Merrell, and J. C. Cambier. 2005. Maintenance of antigen-binding B lymphocytes incapable of responding to antigen or mitogen. Proc. B cell anergy requires constant antigen receptor occupancy and signaling. Nat. Immu- Natl. Acad. Sci. USA 77: 1602–1606. nol. 6: 1160–1167. 14. Nemazee, D. A., and K. Burki. 1989. Clonal deletion of B lymphocytes in a transgenic 20. Merrell, K. T., R. J. Benschop, S. B. Gauld, K. Aviszus, d. Decote-Ricardo, mouse bearing anti-MHC class I antibody genes. Nature 337: 562–5066. L. J. Wysocki, and J. C. Cambier. 2006. Identification of anergic B cells within a 15. Hartley, S. B., J. Crosbie, R. Brink, A. B. Kantor, A. Basten, and C. C. Goodnow. wild-type repertoire. Immunity 25: 953–962. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound . Nature 353: 765–769. 21. Teague, B. N., Y. Pan, P. A. Mudd, B. Nakken, Q. Zhang, P. Szodoray, 16. Goodnow, C. C., J. Crosbie, H. Jorgensen, R. A. Brink, and A. Basten. 1989. X. Kim-Howard, P. C. Wilson, and A. D. Farris. 2007. Cutting edge: transitional Induction of self-tolerance in mature peripheral B lymphocytes. Nature 342: T3 B cells do not give rise to mature B cells, have undergone selection, and are 385–391. reduced in murine lupus. J. Immunol. 178: 7511–7515. 17. Benschop, R. J., K. Aviszus, X. Zhang, T. Manser, J. C. Cambier, and 22. Duty, J. A., P. Szodoray, N. Y. Zheng, K. A. Koelsch, Q. Zhang, M. Swiatkowski, L. J. Wysocki. 2001. Activation and anergy in bone marrow B cells of a novel M. Mathias, L. Garman, C. Helms, B. Nakken, et al. 2009. Functional anergy in immunoglobulin transgenic mouse that is both specific and autoreactive. a subpopulation of naive B cells from healthy humans that express autoreactive Immunity 14: 33–43. immunoglobulin receptors. J. Exp. Med. 206: 139–151. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021