Antibody Wars: Extreme Diversity Patricia J. Gearhart J Immunol 2006; 177:4235-4236; ; This information is current as doi: 10.4049/jimmunol.177.7.4235 of October 1, 2021. http://www.jimmunol.org/content/177/7/4235

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Antibody Wars: Extreme Diversity Patricia J. Gearhart1

People have pondered the Ab diversity problem for amazing, the V was encoded by three separate gene seg- years as the universe of potential Ags was expand- ments—variable, diversity, and joining—that were represented ing relentlessly. Research showed that Abs could by multiple members in the germline. Therefore, Ab diversity not only bind to pathogens such as bacteria, toxins, could be explained by combinatorial joining of gene segments and viruses, but also to common , like egg albumin, and within the three families. An added bonus was that random nu- synthetic chemical structures, like dinitrophenyl, that humans cleotides were added during the joining process to further in- had never seen before. If the repertoire of Abs was unlimited, crease variability. Marrying both somatic and germline theories, how was the genetic information for all these proteins encoded gene segment joining should be enough to generate loads of di-

in the DNA? Two polar hypotheses emerged by 1970: the so- verse Abs, and everyone was satisfied. Downloaded from matic theory, which states that a few inherited underwent However, it soon became apparent that B cells continued to high rates of somatic mutation to encode many Abs (champi- mutate their Ab genes, even after rearrangement. Additional oned by Weigert and Cohn; Ref. 1), and the germline theory, substitutions were found that were not encoded in which states that many genes existed to encode each Ab (sup- the germline members and were not located near the sites of ported by Hood; Ref. 2). The somaticists argued that there was joining. These substitutions were completely random and

not enough DNA to encode all the Ab specificities, which most tended to accumulate in hypervariable regions. Rearranged Ab http://www.jimmunol.org/ immunologists agreed was in the range of 106 unique mole- genes from Ag-activated B cells then were sequenced exten- cules. The germliners countered that there was not enough time sively, and the data revealed an incredible number of mutations to generate a full repertoire somatically, and why do it ran- within the V gene and its flanking DNA. Both the high fre- domly? Each side presented compelling data based on quency and restricted targeting of these mutations were unprec- sequences, and the dialog was so fascinating that every immu- edented in biology, and the somatic hypermutation theory was nologist had an opinion. The debate reached its zenith in the resurrected. 1970s, when spin-off theories emerged: the translocation hy- The Brenner and Milstein paper suddenly looked prescient. pothesis, which states that V genes could recombine next to C Molecular biologists validated the concept of DNA cleavage genes (3); a complicated branched DNA model, in which RNA and gap formation in other repair processes, and the recent dis- by guest on October 1, 2021 polymerase is shunted onto different DNA tracks (4); the gene covery of low-fidelity DNA polymerases would allow the syn- interaction theory, where two sets of genes encode the frame- thesis of many mutations in a short region. In fact, the hypoth- work and hypervariable regions of V genes (5); the permutation esis that error-prone DNA repair causes Ab hypermutation is theory, which posits that germline genes undergo predeter- now dogma. In an interesting twist, Milstein originally devel- mined mutations (6); and the minigene hypothesis, where four oped hybridoma technology to study the hypermutation pro- sets of genes encode each of the four framework regions (7). cess in B cells. The creation of hybridomas secreting mAbs of Buried in this plethora of theories was a short paper titled choice was a huge success and earned him the Nobel Prize in “Origin of Antibody Variation” by Brenner and Milstein (8), 1984. Milstein went on to show that random mutations located which described a hypermutation mechanism acting on a few in hypervariable regions are selected over time to produce high- genes. A simple figure accompanied this hypothesis, which affinity Abs (9). This is the fundamental basis of immunization showed a DNA motif located in the middle of a gene that is and the raison d’eˆtre for hypermutation. recognized by a cleaving , analogous to a restriction en- However, the story did not end until the molecular mecha- zyme cutting at a site. The DNA is then degraded 3Ј to 5Ј by an nism was solved. Going back to Fig. 1 in Ref. 8, what was the exonuclease, and an error-prone polymerase fills in the gap with cleaving enzyme that started it? Discoveries by Tasuku Honjo occasional errors. Thus, the left half is variable, and the right and Michael Neuberger a few years ago unequivocally showed half is constant. The authors acknowledged that their error- that a new protein, activation-induced deaminase (AID),2 ini- prone repair model would be difficult to prove, but believed it tiates hypermutation by modifying Ig genes. AID is not a cleav- was consistent with variable and constant regions. ing enzyme but acts on DNA by deaminating cytosine to uracil. Meanwhile, molecular biology was being unleashed in full Uracil is an inappropriate base and is recognized by error-prone force on Ab genes, and it was soon discovered that V and C DNA repair pathways to generate both DNA strand breaks and genes indeed are separately encoded in the DNA. Even more synthesize mutations, exactly as shown in the figure. AID also initiates H chain class switch recombination, which ties to- gether V gene diversity with C gene diversity. In a poignant 1 Address correspondence and reprint requests to Dr. Patricia J. Gearhart, Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail address: [email protected] 2 Abbreviation used in this paper: AID, activation-induced deaminase.

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memory, Neuberger recalls that he showed Milstein, who was References very ill, the draft figures for the first paper suggesting that AID 1. Weigert, M. G., I. M. Cesari, S. J. Yonkovich, and M. Cohn. 1970. Variability in the ␭ deaminates cytosine in DNA. Milstein wanted to continue the light chain sequences of mouse antibody. Nature 228: 1045–1047. 2. Hood, L., and D. W. Talmage. 1970. Mechanism of antibody diversity: germ line discussion the following week that, for him, never came. basis for variability. Science 168: 325–334. It is tempting to speculate about that early time when Francis 3. Gally, J. A., and G. M. Edelman. 1970. Somatic translocation of antibody genes. Nature 227: 341–348. Crick, Sydney Brenner, and Ce´sar Milstein were together in the 4. Smithies, O. 1970. Pathways through networks of branched DNA. Science 169: Laboratory of Molecular Biology in Cambridge. Apparently, 882–883. Crick suggested that Brenner and Milstein collaborate and 5. Capra, J. D., and T. J. Kindt. 1975. Antibody diversity: can more than one gene en- code each variable region? Immunogenetics 1: 417–427. write a letter to Nature about their ideas on how diversity was 6. Sigal, N. H., P. J. Gearhart, J. L. Press, and N. R. Klinman. 1976. Late acquisition of generated. As Aaron Klug commented, the most remarkable a germ line antibody specificity. Nature 259: 51–52. fact is that they wrote the letter together at all since it was the 7. Kabat, E. A., T. T. Wu, and H. Bilofsky. 1978. Variable region genes for the immu- noglobulin framework are assembled from small segments of DNA—a hypothesis. sole example of a joint effort between them. Proc. Natl. Acad. Sci. USA 75: 2429–2433. 8. Brenner, S., and C. Milstein. 1966. Origin of antibody variation. Nature 211: Acknowledgments 242–243. 9. Griffiths, G. M., C. Berek, M. Kaartinen, and C. Milstein. 1984. Somatic mutation I thank Michael Neuberger for sharing his biographical recollections and and the maturation of immune response to 2-phenyl oxazolone. Nature 312: thoughts about Ce´sar Milstein. 271–275. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021