Peripheral B Cell Tolerance and Function in Transgenic Mice Expressing an IgD Superantigen This information is current as Bao Hoa Duong, Takayuki Ota, Djemel Aït-Azzouzene, of October 2, 2021. Miyo Aoki-Ota, José Luis Vela, Christoph Huber, Kevin Walsh, Amanda L. Gavin and David Nemazee J Immunol 2010; 184:4143-4158; Prepublished online 15 March 2010; doi: 10.4049/jimmunol.0903564 http://www.jimmunol.org/content/184/8/4143 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/03/15/jimmunol.090356 Material 4.DC1 http://www.jimmunol.org/ References This article cites 120 articles, 67 of which you can access for free at: http://www.jimmunol.org/content/184/8/4143.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! 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The Journal of Immunology Peripheral B Cell Tolerance and Function in Transgenic Mice Expressing an IgD Superantigen Bao Hoa Duong,*,† Takayuki Ota,* Djemel Aı¨t-Azzouzene,*,1 Miyo Aoki-Ota,* Jose´ Luis Vela,*,†,2 Christoph Huber,* Kevin Walsh,* Amanda L. Gavin,* and David Nemazee* Transitional B cells turn over rapidly in vivo and are sensitive to apoptosis upon BCR ligation in vitro. However, little direct evidence addresses their tolerance sensitivity in vivo. A key marker used to distinguish these cells is IgD, which, through alternative RNA splicing of H chain transcripts, begins to be coexpressed with IgM at this stage. IgD is also expressed at high levels on naive follicular (B-2) and at lower levels on marginal zone and B-1 B cells. In this study, mice were generated to ubiquitously express a membrane- bound IgD-superantigen. These mice supported virtually no B-2 development, a greatly reduced marginal zone B cell population, but a relatively normal B-1 compartment. B cell development in the spleen abruptly halted at the transitional B cell population 1 to 2 Downloaded from stage, a block that could not be rescued by either Bcl-2 or BAFF overexpression. The developmentally arrested B cells appeared less mature and turned over more rapidly than nontransgenic T2 cells, exhibiting neither conventional features of anergy nor appreciable receptor editing. Paradoxically, type-2 T-independent responses were more robust in the transgenic mice, although T-dependent responses were reduced and had skewed IgL and IgH isotype usages. Nevertheless, an augmented memory response to secondary challenge was evident. The transgenic mice also had increased serum IgM, but diminished IgG, levels mirrored + by the increased numbers of IgM plasma cells. This model should facilitate studies of peripheral B cell tolerance, with the http://www.jimmunol.org/ advantages of allowing analysis of polyclonal populations, and of B cells naturally lacking IgD. The Journal of Immunology, 2010, 184: 4143–4158. ince shortly after the discovery of IgD, its main role in the of IgD expression to be regulated by RNA splicing is evolutionarily immune system has been believed to be as a BCR (1–6) (for ancient and probably predates the process of class switching (14). S review see Ref. 7). IgD is not appreciably expressed by Surface expression of IgD varies with B cell subset. It is highest early developing B lymphocytes of the bone marrow (BM) or end in long-lived recirculating B cells that are abundant in follicles of stage Ab-secreting plasma cells. Instead, through alternative RNA the spleen (SP) and lymph nodes (LNs), on which mIgD levels can by guest on October 2, 2021 splicing of primary IgH locus transcripts (8, 9) IgD is coexpressed exceed those of mIgM by 10-fold (24). IgD is expressed at lower with IgM as a membrane Ig (mIg) by most mature, quiescent B levels on B-1 cells, which are abundant in the peritoneal cavity cells in mammals (1, 2, 10) and most other vertebrate species (11– (PC), lamina propria, and pleural tissues (25), and on distinct B 14) [although not in rabbits and birds (15–17)]. Alternative RNA cells of the splenic marginal zone (MZ) (26). IgD is also expressed splicing ensures that on any given cell, the Ag specificity of IgM on many recently generated semimature transitional B cells found and IgD molecules is identical (18–21) and allows coexpression of in the SP and BM, most of which are short lived and express CD93 IgD and IgM at different relative abundances (22, 23). This ability (27–31). B cells initially transitioning from the BM to the SP are CD212CD232IgDlo/2 and are referred to as transitional B cell population 1 (T1) cells. Upon maturation to the T2 stage, all three *Department of Immunology and Microbial Science and †Doctoral Program in surface molecules are upregulated along with CD62L. T3 and Chemical and Biological Sciences, Kellogg School of Science and Technology, naive follicular (B-2) cells continue to retain high surface IgD The Scripps Research Institute, La Jolla, CA 92037 (sIgD) expression while downregulating their sIgM (31). IgD 1 Current address: Neostasis/Biogen Idec, San Diego, CA. expression is therefore an important marker for B cell de- 2 Current address: La Jolla Institute for Allergy and Immunology, La Jolla, CA. velopmental stage and maturation. However, IgM and IgD can Received for publication November 3, 2009. Accepted for publication February 9, also be functionally redundant. For example, in IgM2/2 mice and 2010. in IgD transgenic (Tg) mice, IgD can substitute for IgM in pro- This work was supported by National Institutes of Health Grant RO1AI059714 moting early B cell development (32, 33). and by Training Grant T32AI007606. K.W. was supported by Grant AI74564 to M. Oldstone. Immature B cells and B cells from fetal and neonatal mice express Address correspondence and reprint requests to Prof. David Nemazee, The Scripps exclusively IgM (1, 2, 34) and tend to be more susceptible to im- Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail mune tolerance than are more mature cells that coexpress IgD (35, address: [email protected] 36), correlations that prompted the hypothesis that IgD expression The online version of this article contains supplemental material. could protect B cells from immune tolerance (37, 38). However, Abbreviations used in this paper: ASC, Ab-secreting cell; BM, bone marrow; cIgM, mature IgD+ B cells encountering foreign or self-ligands can be cytoplasmic IgM; DTg, double transgenic; HEL, hen egg lysozyme; LN, lymph node; MHC II, MHC class II; MZ, marginal zone; mIg, membrane Ig; PB, Pacific Blue; PC, rendered tolerant by clonal elimination or functional inactivation peritoneal cavity; p-Tyr, phosphotyrosine; pUb, part of the ubiquitin promoter; sIg, (33, 39–42). Moreover, functionally hyporesponsive anergic B cells surface Ig; SP, spleen; T, transitional B cell population; TD, T cell-dependent; Tg, are characterized by high levels of mIgD combined with low levels transgenic; TI-2, T cell-independent type 2; TNP-Hy, trinitrophenol-hemocyanin. of mIgM and generally turn over rapidly when competing non- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 tolerant B cells are present (43–45). On the other hand, IgD www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903564 4144 TOLERANCE IN IgD SUPERANTIGEN MICE expression is correlated with developmental maturation and the loss cag gac ctg gc-39); and AMS9 VH R(59-atc acc tcc cgg gtt tct ggg ggc tgt of the ability of B cells to undergo self-Ag–induced receptor ed- tgt ttc agc tga gga gac ggt gac cgc ggt ccc tgt gcc-39). iting (46, 47). However, transitional cells might retain the capacity IgDa-macroself mice to edit given the appropriate microenvironment (48, 49). Animal studies have been reviewed and approved by the The Scripps Like mIgM, mIgD can transmit signals through the associated Research Institute institutional review committee. All IgDa-macroself Tg signal transducers Ig-a/b (CD79a/b) (50–52), triggering a tyrosine mice described in this work had been bred and maintained on C57BL/6 kinase cascade and the activation of additional signaling path- background congenic for IgHa/a alleles (B6.Cg-Igha Thy1a Gpi1a/J, stock number 001317, JAX) for at least eight generations. In some experiments, ways. Early biochemical changes triggered by IgM and IgD in a/a mature B cells are biochemically similar, although signaling they were also bred to overexpress BAFF-R but retain IgH alleles. BAFF Tg mice have been described (66). To genotype for the presence of transgene through IgD may be more prolonged (53). However, upon acti- in each breeding, 35 cycles of PCR were performed using AMS9.1 VL CDR3 vation by Ag, IgD expression on B cells is lost (54–56). With rare S(59-cca aca atc tta taa tct att ca-39) and AMS9.1 VH CDR3 AS (59-cag aca exceptions, IgD is not expressed by plasma cells, although the tcg aag tac cag taa t-39) primers and the following conditions: melting (94˚C), secretory domain of IgD is intact and conserved in most species annealing (58˚C), and elongation (72˚C) for 40 s each.