Memory B Cell Compartment David J

Development and Maintenance of a B220− Memory B Cell Compartment David J. Driver, Louise J. McHeyzer-Williams, Melinda Cool, Daniel B. Stetson and Michael G. McHeyzer-Williams This information is current as of September 29, 2021. J Immunol 2001; 167:1393-1405; ; doi: 10.4049/jimmunol.167.3.1393 http://www.jimmunol.org/content/167/3/1393 Downloaded from References This article cites 63 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/167/3/1393.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 29, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Development and Maintenance of a B220؊ Memory B Cell Compartment1 David J. Driver, Louise J. McHeyzer-Williams, Melinda Cool, Daniel B. Stetson,2 and Michael G. McHeyzer-Williams3 We have recently demonstrated that a novel somatically mutated B220؊ memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220؉NP؉ memory B cells produce large numbers of B220؊NP؉ B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220؊ memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220؊NP؉ B cells in the spleen to maximal numbers ,wk after initial Ag exposure. Like their B220؉ counterparts, the B220؊ B cells initially appear unmutated at days 5–7; however 3 the majority rapidly accumulate affinity increasing mutations by days 9–14 of the primary immune response. More extensive cell Downloaded from surface phenotype (GL7؊BLA-1؊CD24؊CD43؉) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220؊CD11b؉NP؉ B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220؊ memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow. The Journal of Immunology, 2001, 167: 1393–1405. http://www.jimmunol.org/ cell memory is characterized by the rapid appearance B220Ϫ memory B cell subset emerges following secondary chal- of high-affinity Abs in response to secondary Ag challenge as a major cellular component of the Ag-specific memory B B lenge. This accelerated humoral recall response is due to cell response and contributes significantly to the rapid appearance the expansion of affinity-matured memory response precursors of high-affinity Ab. and their rapid differentiation into plasma cells (1). We have Although the B220Ϫ subset clearly emerges upon secondary recently identified two distinct subsets of isotype-switched challenge, it is not known how this novel subset of B cell memory (IgMϪIgDϪIgGϩ) and somatically mutated memory B cells that develops. Although our previous work provides some indication by guest on September 29, 2021 do not secrete Ab, but can give rise to plasma cells upon adoptive for both subsets of specific memory responders before secondary transfer with Ag (2). One of these subsets expresses high levels of Ag challenge, cell numbers are very low at this point and are not the B lineage marker B220 (the B cell isoform of CD45R; RA3- significantly greater than the adjuvant-only controls (2). In addi- 6B2 binding) and CD19. These B220ϩ memory B cells expand to tion, adoptive transfer experiments in this study demonstrate a hi- peak levels by day 4 following secondary Ag challenge and persist erarchical parent-progeny relationship in which B220ϩ memory B in the spleen for at least 6 wk. Although the second memory subset cells produce nonsecreting B220Ϫ B cell intermediates that then does express CD45R (30-F11 binding), they do not express the B Ϫ cell isoform B220 or CD19. These novel B220Ϫ memory respond- give rise to plasma cells. This pattern suggests that the B220 ers emerge in the spleen with kinetics similar to those of the population seen to expand upon secondary challenge could actu- ϩ B220ϩ subset, but persist long term at much higher frequencies. ally be a product of the B220 memory B cell subset. Thus, ques- Ϫ This unique B220Ϫ memory subset also comprises Ͼ95% of the tions regarding the development of these unique B220 memory B Ag-specific B cells in the bone marrow (BM).4 Thus, a novel cells cannot be answered simply by extrapolation from our previous findings on memory responders and their progeny. Since both memory B cell subsets express somatically mutated Ig, it is likely that their precursors originate in the germinal center Department of Immunology, Duke University Medical Center, Durham, NC 27710 (GC) reaction during the primary response (3, 4). Although there Received for publication September 5, 2000. Accepted for publication May 16, 2001. have been reports of somatic mutation in the absence of GC for- The costs of publication of this article were defrayed in part by the payment of page mation in lymphotoxin ␣- (5) and Lyn-deficient animals (6), so- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. matic mutation in both cases was reported only after tertiary im- 1 This work was supported by grants from the National Institutes of Health (AI47231 munization, and GC have subsequently been observed in the and AI40215) and an Arthritis Foundation Biomedical Grant. mesenteric lymph nodes of the lymphotoxin ␣Ϫ/Ϫ mice (7). The 2 Current address: Departments of Medicine and Microbiology/Immunology, Howard greater weight of evidence supports the GC microenvironment as Hughes Medical Institute, University of California, San Francisco, CA 94143. the site of somatic mutation and affinity-based selection in the 3 Address correspondence and reprint requests to Dr. Michael McHeyzer-Williams, Department of Immunology, Duke University Medical Center, Box 3010, Durham, development of B cell memory (8–14). Therefore, we propose that NC 27710. E-mail address: [email protected] the B220Ϫ memory B cells either 1) develop in the GC during the 4 Abbreviations used in this paper: BM, bone marrow; GC, germinal center; PI, pro- primary response, 2) are the progeny of primary response GC B pidium iodide; NP, (4-hyroxy-3-nitrophenyl)acetyl; KLH, keyhole limpet hemocya- ϩ nin; HSA, heat- stable Ag; PI, propidium iodide; PNA, peanut agglutinin; MZ, mar- cells, or 3) are the progeny of B220 memory B cells and arise ginal zone; TR, Texas Red; APC, allophycocyanin; CR, complement receptor. only as a consequence of Ag recall. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1394 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY To address this issue directly, we used the Th cell-dependent F11), anti-CD19 (FITC-1D3), anti-CD21 (FITC-7G6), and anti-CD22 response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP) in (FITC-Cy34.1). Cells were washed twice in FACS wash and resuspended ␮ C57BL/6 mice (15). The primary response to NP in these mice is in FACS wash containing 2 g/ml propidium iodide (PI) for analysis. Cells were analyzed using a modified dual laser FACStarPlus (BD Im- dominated by B cells that express the VH186.2 H chain and the munocytometry Systems, Mountain View, CA) capable of simultaneous ␭ ϩ VL 1 L chain genes (16–19). NP GC B cells reach maximum seven-parameter acquisition and fluorescence overlap compensation across numbers by day 7 of the primary splenic response and persist at lasers. Files were acquired using CellQuest software (BD Biosciences, these levels for at least 3 wk in the spleen (16). Somatic hyper- Mountain View, CA), analyzed using FlowJo software (Tree Star, San Carlos, CA), and displayed as 5% probability contours with outliers. mutation has been observed as early as day 6 after priming and progressively increases for the duration of the GC reaction (16, Single-cell mutational analysis 20). GC B cells bind high levels of peanut agglutinin (PNA) (21) cDNA synthesis. Single NP-specific B cells were sorted according to phe- and express B220 (22), GL7 (23), CD24 (heat-stable Ag (HSA)) notype using a five-color flow cytometry strategy with an automatic cell (24), and BLA-1 (25), allowing them to be distinguished from dispensing unit attached to the FACStarPlus using CloneCyt software (BD ϩ Ϫ ϩ ␮ plasma cells, which are characterized as being B220 / CD138 , Biosciences). Individual cells were sorted into 5 l of an oligo(dT)-primed ϩ cDNA reaction mixture (4 U/ml murine leukemia virus-reverse transcrip- and CD43 (26, 27). Affinity-matured plasma cells are also ob- tase (Life Technologies, Rockville, MD) with recommended reverse tran- served to persist at very low frequencies in the BM (28, 29), high- scriptase buffer, 0.5 nM spermidine (Sigma Chemical), 100 ␮g/ml BSA lighting the spleen and BM as two main reservoirs for long-lived (Boehringer Mannheim, Indianapolis, IN), 10 ng/␮l oligo(dT) (BD Bio- Ag-specific B cells.

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