Dependence of Antibody Somatic Diversification on Gut-Associated Lymphoid Tissue in Rabbits Michael Vajdy, Periannan Sethupathi and Katherine L. Knight This information is current as J Immunol 1998; 160:2725-2729; ; of October 2, 2021. http://www.jimmunol.org/content/160/6/2725 References This article cites 21 articles, 6 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/160/6/2725.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 2, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Dependence of Antibody Somatic Diversification on Gut-Associated Lymphoid Tissue in Rabbits1,2 Michael Vajdy,3 Periannan Sethupathi, and Katherine L. Knight4 By ;4 to 8 wk of age, the IgH VDJ genes of essentially all rabbit B lymphocytes have undergone somatic diversification. Some of this diversification occurs in the appendix, which is a gut-associated lymphoid tissue (GALT). To determine whether GALT is essential for somatic diversification, we surgically removed the appendix, sacculus rotundus, and Peyer’s patches from neonatal rabbits (designated GALT-less) and examined the extent to which VDJ genes were somatically diversified. We found that the IgM VDJ genes of peripheral B cells from 2- to 5-mo-old GALT-less rabbits had undergone considerably less somatic diversification than those of control rabbits. Further, the percentage of peripheral B cells in the GALT-less rabbits was generally less than that of controls. Our data suggest that, in rabbits, the primary Ab repertoire develops in GALT, and B cell expansion also occurs there. Hence, GALT may function as a mammalian bursal homologue. The Journal of Immunology, 1998, 160: 2725–2729. n mammalian species such as humans, mice, and rabbits, B which the primary Ab repertoire is forming in rabbits (9). To- Downloaded from lymphopoiesis occurs in bone marrow, and the B cells that gether, these studies led us to investigate further whether GALT is I undergo combinatorial joining of Ig variable (V), diversity necessary in rabbits for the somatic diversification of Ig genes that (D), and joining (J) genes leave the bone marrow to seed the pe- results in the establishment of the primary Ab repertoire. riphery (1–3). In humans and mice, combinatorial joining of mul- To determine whether GALT is necessary for generating the tiple V, D, and J gene segments results in a vast array of V(D)J primary Ab repertoire, we performed experiments similar to those genes that form the primary Ab repertoire. This Ab repertoire is of Cooper et al. (11) and surgically removed the organized GALT http://www.jimmunol.org/ expanded further by somatic mutation after Ag stimulation (4). In (i.e., appendix, sacculus rotundus and Peyer’s patches) shortly af- chicken IgH and IgL and in sheep IgL, the primary Ab repertoire ter birth. Then we tested whether, in the absence of organized is formed by combinatorial joining of a limited number of V, D, GALT, VDJ genes would undergo normal levels of somatic diver- and J gene segments followed by somatic diversification of the sification within the first few weeks of life. V(D)J genes in an exogenous Ag-independent manner (5–7). Sim- ilarly, in rabbits, the primary Ab repertoire results from combina- Materials and Methods torial joining of primarily one VH gene, VH1 followed by somatic Removal of GALT from rabbits diversification by ;1 to 3 mo of age (8, 9). The GALT of 10 rabbits was surgically altered in accordance with insti- by guest on October 2, 2021 5 Gut-associated lymphoid tissue (GALT) has a key role in so- tutional guidelines for animal welfare using the following procedure: In matic diversification of V(D)J genes in both chickens and sheep. In nine 1-day-old rabbits, the appendix and the ileocecal junction, in which chicken, before hatching, the VDJ and DJ genes somatically di- the sacculus rotundus develops, were surgically excised, and the ileum was versify by gene conversion in the bursa (5, 10); in sheep, VJ genes religated to the cecum by an end to side anastomosis. The rabbits were rested until 3 to 5 wk of age, at which time the Peyer’s patches of the small somatically diversify in the ileal Peyer’s patch (6, 7). There is intestine were surgically removed using purse-string sutures. After surgery, evidence to suggest that in rabbits, GALT may play a similar role. these rabbits were maintained under conventional conditions in our rabbit Cooper et al. (11) surgically removed the appendix, sacculus ro- colony. We designated these rabbits GALT-less, even though they proba- tundus, and Peyer’s patches from neonatal rabbits and found that bly harbor macroscopically imperceptible lymphoid aggregates in the small and large intestines. With the exception of two rabbits that died of un- after immunization with several Ags, these rabbits responded to known causes at the ages of 3 and 9 mo, the GALT-less rabbits appeared some of the immunogens with decreased Ab production. Further, healthy, with no apparent signs of infection. The growth rate of GALT-less Weinstein et al. (12) showed that VDJ genes undergo somatic di- rabbits was similar to that of control littermates (Fig. 1). versification in the appendix of 6-wk-old rabbits, an age during Control littermates underwent sham surgery at the same times as the GALT-less rabbits; however, on the day of birth, no tissue was removed, whereas at 3 to 5 wk of age, small pieces of small intestine were removed by purse-string suture, leaving the GALT intact. The rabbits were labeled Loyola University Chicago, Stritch School of Medicine, Department of Microbiology so that each littermate had an identical letter (L or M) as well as identical and Immunology, Maywood, IL 60153 numbers preceding the letter, e.g., rabbits 339M1 and 339M2 were litter- Received for publication July 18, 1997. Accepted for publication November 24, 1997. mates. In one rabbit, 150M1, we removed the appendix at birth as a sep- arate test to determine whether somatic diversification occurs only in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance GALT. Surgery performed at 3 mo of age confirmed that the appendix had with 18 U.S.C. Section 1734 solely to indicate this fact. been completely excised. 1 This study was supported by National Institutes of Health Grants AI36907 and Nucleotide sequence analysis AI16611. PBLs were prepared from buffy coats of whole blood as previously de- 2 All sequences reported herein have been deposited in the GenBank database and assigned accession numbers AF029916-AF029969. scribed (9). Erythrocytes were lysed by hypotonic shock treatment, and RNA was prepared using TRIzol according to the manufacturer’s instruc- 3 Current address: Children’s Hospital, Harvard Medical School, Boston, MA 02115. tions (Life Technologies, Grand Island, NY). The cDNA was prepared 4 Address correspondence and reprint requests to Dr. Katherine L. Knight, Loyola from 3 mg of RNA and amplified by PCR (9). The PCR products were University Chicago, Stritch School of Medicine, Department of Microbiology and cloned into M13 mp19, and their nucleotide sequences were determined Immunology, 2160 S. First Ave., Maywood, IL 60153. (13, 14). The sequences were compared with those of germline VH1, VHy 5 Abbreviations used in this paper: GALT, gut-associated lymphoid tissue; MLN, and VHz as well as with the presumed germline sequence of VHx (15); the mesenteric lymph node; L chain, light chain; KLH, keyhole limpet hemocyanin. gene that was used in the VDJ gene rearrangement was identified by its Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 2726 DEPENDENCE OF Ab DIVERSIFICATION ON GALT FIGURE 1. Growth curves of GALT-less and normal rabbits. Downloaded from similarity to the framework regions of these germline genes. Because VH1 To investigate whether the appendix, sacculus rotundus, and is the only gene for which the nucleotide sequence is known for each of the Peyer’s patches taken together are required for somatic diversifi- three IgH allelic types, a1, a2, and a3, only the V 1-utilizing genes are H cation of VDJ genes early in life, we surgically removed all visible reported in this study. GALT; the appendix and sacculus rotundus were removed at birth, Flow cytometric analysis and the Peyer’s patches were removed at ;4 wk of age. When PBLs, isolated from buffy coats as described above, and single cell sus- these GALT-less rabbits were 10 to 12 wk of age, we then ana- http://www.jimmunol.org/ pensions from spleen and mesenteric lymph nodes (MLNs) were stained lyzed the level of somatic diversification of the VDJ genes. We for light chain (L chain) expression with biotinylated goat anti-rabbit L amplified the IgM heavy chain VDJ genes of PBLs by RT-PCR. chain and avidin phycoerythrin or stained for IgM expression with mouse By nucleotide sequence analysis, we found that the V regions of anti-rabbit m-chain mAb and FITC-goat anti-mouse Ig.