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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

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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 © 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.

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. sciences), 200 ␮M each dNTP (Boehringer Mannheim), 1 mM DTT (Pro- ␮ In the current study, we clearly demonstrate that the B220ϪNPϩ mega, Madison, WI), 220 U/ml RNAsin (Promega), 100 g/ml Esche- richia coli tRNA (Boehringer Mannheim), and 1% Triton X-100) in low B cells emerge as a distinct cellular consequence of the primary profile 72-well microtiter plates (Robbins Scientific, Mountain View, CA) Downloaded from ϩ ϩ immune response. Although CD138 NP plasma cells reach peak as described previously (2). Reactions were incubated at 37°C for 90 min numbers by day 5 and B220ϩNPϩ GC B cells reach peak numbers and stored at Ϫ80°C until analysis was performed. by day 7, the B220ϪNPϩ B cells emerge more gradually to peak Nested PCR. The first 35 cycles were as follows: 25-␮l reactions con- ϩ ϩ Ϫ ϩ ␮ levels by day 21. Both B220 NP B cells and B220 NP B cells taining 2 l of cDNA from individual single-cell cDNA reactions were set up using the following conditions: 2 U/ml Taq polymerase with the rec- persist in the spleen at similar numbers for at least 8 wk postprim- ommended 1ϫ reaction buffer (Promega), 0.1 mM of each dNTP (Boehr-

ing. The kinetics of appearance and pattern of somatic mutation in inger Mannheim), and varying concentrations of primers and MgCl2 as http://www.jimmunol.org/ Ϫ ␮ Ј the B220 B cell population are consistent with these cells being follows. L chain was 1 mM MgCl2 and 0.8 M LAM.ext3(sense) (5 - an affinity-selected cellular product of the GC reaction. The B220Ϫ TACTCTCTCTCCTGGCTCTCAGCTC-3Ј) and LAM.ext3(antisense) ϩ (5Ј-GTTGTTGCTCTGTTTGGAAGGCTGG-3Ј). H chain was 2 mM subset appears abruptly and dominates the BM NP B cell com- ␮ Ј MgCl2 and 0.8 M 186.2.ext(sense) (5 -CTCTTCTTGGCAGCAACAGC- partment at day 7 after priming, but does not reach peak cell num- 3Ј) and IgG1ext.(antisense) (5Ј-GCTGCTCAGAGTGTAGAGGTC-3Ј). Ϫ bers until day 21. The major division of B220 B cells previously The second 35 cycles were as follows: 1 ␮l of product from the first- reported in the memory response (IgGϩCD11bϩϩ and IgEϩ) also round PCR was used in a 25-␮l reaction with primers nested medially to develops after initial priming. Cell surface phenotype and local- the first-round primers using the following conditions: 2 U/ml Taq poly- Ϫ merase with the recommended reaction buffer (Promega), 0.1 mM of each ization in situ indicate that the B220 B cells are not found within dNTP (Boehringer Mannheim), and varying concentrations of primers and ␮ by guest on September 29, 2021 the GC and are most likely the recirculating cellular outcomes of MgCl2 as follows. L chain was 1 mM MgCl2 and 0.8 M LAM.sense(int) the GC reaction. Finally, the B220Ϫ subset lacks key signaling (5Ј-CCATTTCCCAGGCTGTTGTG-3Ј) and LAM.anti(int) (5Ј-CTC Ј ␮ molecules (CD19 and CD22), which may indicate unique require- CATACCCTGAGTGACAG-3 ). H chain was 2 mM MgCl2 and 0.8 M Ј Ј ␮ ments for reactivation of these cells upon Ag recall. Thus, the 186.2.int(sense) (5 -GTGTCCACTCCCAGGTCCAAC-3 ) and 0.8 M Ϫ IgG1.int(antisense) (5Ј-GTTCCAGGTCACTGTCACTG-3Ј). Each set of B220 population initially develops in the primary response, un- PCR cycles began with a 95°C incubation for 5 min followed by 40 cycles dergoing mutation and affinity-driven selection in the GC, and per- of 95°C for 15 s, 50°C for 45 s, and 72°C for 90 s and ends with a 5-min sists as a major component of the post-GC memory B cell 72°C incubation. For every 10 PCR, two negative controls (wells contain- compartment. ing cDNA reaction mix into which no cells were sorted) were processed along side experimental samples through both rounds of PCR to control for Materials and Methods contamination during sample processing. DNA sequencing. To screen second-round PCR products for positives, 5 Isolation of NP-specific B cells ␮l of each reaction was run on a 1.5% agarose gel. Samples that yielded a Female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) at 6–10 single band of the correct size were run over a CL-6B Sepharose column wk of age were immunized i.p. with 400 ␮g NP-keyhole limpet hemocy- (Pharmacia, Piscataway, NJ) to separate PCR products from primers and ␮ anin (KLH) in Ribi adjuvant (Corixa, Hamilton, MT). For analysis of the were then directly sequenced as described previously (2). Briefly, 4 lof ␮ memory response, mice were reimmunized with the same dose and route of each PCR product was mixed with 4 l of Dye Terminator Ready Reaction Ag in adjuvant 8 wk postprimary immunization. Spleens were excised and Mix (Perkin-Elmer, Norwalk, CT) and primer. Primers used and volumes Ј BM was aspirated from both femurs at various time points following pri- are as follows: for L chain amplification, 1.6 pmol of LAM.seq (5 -GGCT Ј Ј mary immunization, and single-cell suspensions were prepared as previ- GTTGTGACTCAGGAAT-3 ) and for H chain, 1.6 pmol of 186.2.seq (5 - Ј ously described (2). The Duke University Institutional Animal Care and CCACTCCCAGGTCCAACTG-3 ). Samples were subjected to a linear Use Committee approved all animal use. amplification protocol consisting of 25 cycles of 96°C for 10 s, 50°C for Cells were stained for flow cytometry on ice for 45 min in FACS wash 5 s, and 60°C for 4 min using a 9600 GeneAmp PCR system (PBS ϩ 5% FCS) at 4 ϫ 108/ml with fluorophore-conjugated mAbs or (PerkinElmer). Sequencing reaction products were ethanol precipitated and fluorophore-conjugated Ag. Abs used for flow cytometry are as follows separated on a 6.5% acrylamide gel using an Applied Biosystems 373 (purchased from BD PharMingen unless otherwise noted): anti-CD4 sequencing system and processed using Applied Biosystems Prism Se- (Cy5PE-H129.19), anti-CD8 (Cy5PE-53-6.7), anti-macrophage (Cy5PE- quence 2.1.2 for collection and analysis (PerkinElmer). F4/80; Caltag, South San Francisco, CA), anti-IgD (Texas Red (TR)-11.26; In situ immunofluorescence Ab from F. Finkelman, Immunology Division, University of Cincinnati Medical Center, Cincinnati, OH), allophycocyanin (APC)-conjugated NP Spleens from mice 14 days postprimary immunization were snap frozen in (20 ␮g NP:1 mg APC), anti-CD45R/B220 (FITC/PE-RA3-6B2), anti- OCT embedding compound (Miles Labs, Elkhart, IN) and stored at Ϫ80°C CD138/syndecan (FITC/PE-281.2), anti-IgE (FITC-R35-72), anti-CD11b/ until use. Six-micrometer-thick sections were cut using a cryostat mic- MAC1 (FITC/PE-M1/70), anti-GL7 (FITC-GL7), FITC-PNA (Vector Lab- rotome (Leica, Deerfield, IL) and placed onto gelatin-coated slides, air oratories, Burlingame, CA), anti-BLA-1 (FITC-R53 from L. Herzenberg, dried, acetone fixed for 10 min, and stored at Ϫ80°C until use, as previ- Genetic Department, Stanford University, Stanford, CA), anti-CD24/HAS ously described (30). Sections were rehydrated with PBS (pH 7.4) for 5 (FITC-M1/69), anti-CD43 (PE-S7; Caltag, South San Francisco, CA), anti- min before blocking for 30 min at room temperature in blocking buffer CD79b/Ig␤ (FITC-HM79b), anti-CD45/leukocyte common Ag (FITC-30- (PBS containing 10% FCS and 10% (w/v) skim milk powder). Sections The Journal of Immunology 1395 were then stained with APC-conjugated NP in blocking buffer overnight at and reassembled using Adobe Photoshop software (Adobe Systems, Moun- room temperature. After washing with PBS, sections were blocked for 30 tain View, CA). min at room temperature in blocking buffer containing 50% (v/v) anti-FcR (2.4G2 hybridoma supernatant). Alternatively, sections to be stained with Results biotin reagents were blocked with blocking buffer containing 50% anti-FcR Development of an Ag-specific B cell response in the spleen and 10 ␮g/ml streptavidin (Sigma, St. Louis, MO) for 30 min at room temperature, rinsed, and blocked with blocking buffer containing 2 ␮M Using flow cytometry, we can directly quantitate the emergence of biotin (Pierce, Rockford, IL) for 30 min. Sections were then stained with Ag-specific B cells in the spleens of mice immunized i.p. with combinations of the following Abs, as indicated in Fig. 6, for1hatroom NP-KLH in Ribi adjuvant. Our strategy focuses on PIϪ, CD4Ϫ, temperature: TR-11.26 (anti-IgD), FITC-RA3.6B2 (anti-B220), FITC/bi- Ϫ Ϫ otin-M1/70 (anti-CD11b), FITC-R35-72 (anti-IgE), and biotin-281.2 (anti- and CD8 , F4/80 cells (Fig. 1A, left panel) that bind NP and CD138). Biotin-stained sections were then stained with neutravidin-rho- have down-regulated IgM (data not shown) and IgD upon activa- damine (Molecular Probes, Eugene, OR) for1hatroom temperature. tion in vivo (Fig. 1A, upper left quadrant of NP/IgD plots; an Sections were washed in PBS and mounted in VectorSheild (Vector Lab- NPϩIgDϩ population also appears that is not further characterized oratories). Data were acquired using a Zeiss Axiovert LSM 410 microscope system ( Zeiss, Oberkochen, Germany) with each signal collected serially in this study). By day 3 postprimary immunization, the NP-specific in the first detector using LSM 3.95 software and ϫ10 or ϫ40 objectives. B cell population emerges significantly above baseline levels of The three separate images were optimized for signal-to-noise, colorized, day0(p Ͻ 0.001; one-way ANOVA) and KLH alone in adjuvant Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. The emergence of three subsets of Ag-specific B cells in the spleen during the primary response. Female C57BL/6 mice immunized i.p. with 400 ␮g of NP-KLH in Ribi adjuvant were sacrificed at various times postimmunization and spleens were harvested and labeled for flow cytometry. A, The splenic NP-binding B cell population. PI-positive events (dead cells), T cells, and macrophages (as detected by Cy5-PE labeled anti-CD4, CD8, and F4/80) were excluded from further analysis and light scatter gates were broad so as not to exclude large B cell blasts. Remaining cells were analyzed for their expression of IgD (TR-11.26) and the ability to bind NP (APC-NP); the upper left quadrant of each plot indicates the level of fluorescence considered to be NPϩIgDϪ. B, Cellular dynamics of the total NP-binding IgDϪ B cell population. Total numbers of NP-binding IgDϪ B cells in the spleen were calculated using frequencies obtained via flow cytometry and total splenic cell numbers obtained upon sacrifice of the animal. Each time point is composed of at least three animals with the means Ϯ SEM displayed. C, Three subsets of Ag-specific B cells. The activated, Ag-specific B cell population (PIϪ, CD4Ϫ, CD8Ϫ, F4/80Ϫ, IgDϪ,NPϩ) was analyzed for the expression of CD138 (PE-281.2) and B220 (FITC-RA3-6B2) at the indicated days of the response. D, Cellular dynamics of the three NP-binding B cell subsets. Numbers of NP-specific B cells of the indicated phenotypes were calculated as in B at the indicated days of the response. Each time point represents the mean Ϯ SEM of at least three animals. 1396 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY controls ( p Ͻ 0.05; Fig. 1B). An initial plateau in cell numbers is of the memory response (Fig. 2C)). As expected, few cells from reached by day 7 (0.7% of total spleen cells) that is maintained any subset are mutated during the first week of the primary re- through day 14. A second increase in the number of NPϩIgDϪ sponse (27%, n ϭ 87; Fig. 2, A and D). The B220Ϫ subset was cells occurs from days 14 to 21 ( p Ͻ 0.01; 1.4% of total spleen at seen to emerge initially unmutated, possessing somewhat fewer day 21). The number of Ag-specific B cells markedly declined total mutations (0.05%), mutations per mutated sequence (1.0), over the succeeding week (90% decline in NPϩ cells from day 21 and replacement mutations (0%) than either the CD138ϩ or B220ϩ to day 28; 0.2% of total spleen at day 28) to levels that are main- subsets during the first week of the response (Fig. 2D). tained as a quiescent memory population for at least 8 wk (0.1% of During the second week of the response, an increase in the fre- total spleen; no change from days 28 to 56, p Ͼ 0.05). The number quency of mutated cells (53%, n ϭ 75; Fig. 2, B and E) can be seen of NPϩ cells detected 56 days postprimary immunization in this across all subsets. The B220Ϫ subset is now seen to be mutated to study was greater than previously reported (2). This is due to the a similar level as the CD138ϩ and B220ϩ subsets (2.6 mutations use of an NP-APC reagent with an 8-fold reduced ratio of NP:APC per mutated sequence vs 2.3 and 2.1, respectively). Most notably, that is capable of detecting a greater number of NPϩ B cells with- the percentage of replacement mutations in complementarity-de- out an increase in background staining. Thus, two waves of cel- termining region (CDR) 1 and 2 is similarly high across all subsets lular expansion occur in the spleen during the first 3 wk of the (range, 82–90%), a broad indication of Ag-driven selection (17). primary response, with a quiescent memory population established CD138ϩ memory responders were also sequenced (Fig. 2C), as we by day 28. have shown that B220Ϫ memory cells can give rise to CD138ϩ

Ϫ Ab-secreting cells upon adoptive transfer (2). These memory re-

Induction and maintenance of a B220 memory B cell sponders serve as an indication of the level and pattern of mutation Downloaded from population expected following affinity maturation in vivo. Although the fre- Expression of the cell surface markers B220 and CD138 divides quency of mutation is greater than that seen during the second the NPϩIgDϪ B cell population into three cellular subsets (Fig. 1, week of the primary response, the pattern of mutation and the C and D). NP-specific Ab-secreting cells express intermediate lev- percentage of replacement mutations in these memory responders els of B220 and high levels of CD138 (2, 16, 27). This CD138ϩ is similar to that of all subsets in the primary response (Fig. 2, C Ϫ population emerges rapidly and reaches maximal numbers by the and E). Thus, while the B220 B cells initially emerge unmutated, http://www.jimmunol.org/ fifth day of the response (Fig. 1, C and D). There is a significant they develop a frequency and pattern of mutations that is similar to decrease in CD138ϩ B cells by day 14 ( p Ͻ 0.05) and a return to the CD138ϩ and B220ϩ subsets as well as the CD138ϩ memory baseline levels by day 28. NP-specific GC B cells express high responders, providing evidence for a GC phase in their levels of B220 and do not express CD138 (2, 16, 27). As predicted development. ϩ from kinetic studies of the GC reaction (20, 31), this B220 pop- Ϫ ulation reaches peak cell numbers by day 7 and is maintained The B220 population expresses mutated Ig H chain genes through day 21 (Fig. 1D). After a significant decline from days 21 Although mutation is indicative of B cell memory and GC expe- to 28 ( p Ͻ 0.05), the B220ϩ population persists for at least 8 wk rience, affinity maturation offers the more reliable indicator. A mu- (no change from days 28 to 56; p Ͼ 0.05) as a post-GC B220ϩ tation resulting in a tryptophan to leucine change at position 33 of by guest on September 29, 2021 memory B cell population. CDR1 in the VH186.2 H chain gene confers a 10-fold increase in An NP-specific B220ϪCD138Ϫ B cell compartment clearly affinity for NP (32) and can serve as a marker of high-affinity emerges during the primary response, with kinetics that are distinct NP-specific B cells. As observed in the L chain, few H chain mu- from either the CD138ϩ or B220ϩ subsets (Fig. 1, C and D). By tations are observed among all three subsets during the first week the end of the first week of the response, this B220Ϫ population of the response and the position 33 change is absent (Fig. 3, A and accounts for nearly 50% of all NP-specific cells in the spleen (Fig. D). During the second week of the response, all three subsets are 1D). The second increase in the number of NP-specific B cells observed to accumulate extensive mutation in their H chain loci between days 14 and 21 is attributed to this B220Ϫ population, (Fig. 3, B and E). The B220Ϫ subset has a greater number of accounting for 85% of all NP-specific cells in the spleen by day 21. mutations per mutated sequence than either the CD138ϩ or B220ϩ Similar to the other populations, there was a decline in the B220Ϫ subsets (6.4 vs 2.7 and 5.2, respectively; Fig. 3E). The majority of population from days 21 to 28 to levels that are maintained for at CD138ϩ and B220Ϫ cells posses the affinity-enhancing Trp to Leu least 8 wk (no change from days 28 to 56; p Ͼ 0.05). Although the mutation (75 and 68%, respectively; Fig. 3E), implying Ag-driven B220Ϫ subset dominates the quiescent memory compartment fol- selection. Although the B220Ϫ subset has fewer mutations per lowing secondary immunization (2), the B220Ϫ and B220ϩ pop- mutated sequence than the CD138ϩ memory cells (6.4 vs 12; Fig. ulations comprise similar proportions of the quiescent memory 3E), both subsets are similar in the percentage of replacement mu- compartment in the spleen 8 wk postprimary immunization (ϳ5: tations and position 33 change. Thus, the B220Ϫ subset develops 4:1 proportion of B220Ϫ:B220ϩ:CD138ϩ). Thus, the B220Ϫ Ag- mutated H chain loci with evidence for Ag-driven selection, which specific memory B cell population develops in the primary re- is a molecular hallmark of GC experience and B cell memory. sponse and is maintained as a major component of B cell memory. Highly mutated B220Ϫ sequences do not represent non-

Ϫ VH186.2 J558 family member H chain genes, as a comparison of The B220 population expresses mutated Ig L chain genes our total data set (n ϭ 139) with the germline sequences of all To evaluate whether B220Ϫ memory B cell development involves known J558 family members (n ϭ 67; kindly supplied by G. Kel- a GC phase, we assayed for the presence and prevalence of somatic soe, Immunology Department, Duke University Medical Center, hypermutation in single NP-specific B cells. The vast majority Durham, NC) revealed that the vast majority (99%) are more con- Ͼ ␭ ( 80%) of NP-specific responders in each subset express the L sistent with being mutated VH186.2 genes than other J558 family chain (data not shown). Individual NP-specific B cells from each members (non-VH186.2 sequences were omitted from subsequent subset were sorted for cDNA synthesis, L chain-specific RT-PCR, analyses). The frequency of obtaining a VH186.2-specific PCR and DNA sequence analysis (representative sequences shown in product was consistently 50–65% of the ␭1 L chain frequency on Fig. 2, A–C; sequences are grouped by subset within the first (Fig. a per cell basis, even for the CD138ϩ memory responders (65% ␭

2A) or second week (Fig. 2B) of the primary response or ﬁrst week vs 28% VH186.2), as it is possible that non-VH186.2 genes are The Journal of Immunology 1397

FIGURE 2. Each of the three subsets of NP-spe- ciﬁc B cell populations express somatically mutated ␭1 L chains. Representative V␭1 L chain sequences

and predicted amino acid sequences from days 5 to Downloaded from 7(A)or9to14(B) of the primary response or days 3–7 of the secondary response (C). Sequences are listed in chronological order from the indicated day of the response and are grouped according to phe- ;notype as follows: CD138؉, B220ϩ/ϪCD138ϩ B220ϩ, B220ϩCD138Ϫ; B220Ϫ, B220ϪCD138Ϫ; ؉ ϩ Ϫ ϩ CD138 memory responders, B220 / CD138 http://www.jimmunol.org/ cells from the secondary response. Single (PIϪ, CD4Ϫ, CD8Ϫ, F4/80Ϫ, IgDϪ,NPϩ) cells of each subset were sorted from animals at various days of the response into a cDNA reaction mixture, subjected to two rounds of PCR using nested ␭1L chain-speciﬁc primers, and the PCR products were sequenced. Frequency of obtaining a PCR product for each population is as follows: CD138ϩ, 63%; B220ϩ, 50%; B220Ϫ, 10%; and CD138ϩ memory, by guest on September 29, 2021 65%. Sequences shown are from individual cells and are representative of the entire data set. Nucleotides found to differ from the germline sequences are indicated by letters (dashes represent germline sequence) and the predicted amino acids are shown above. Summary of L chain mutations from days 5 to7(D; n ϭ 87) and days 9 to 14 (E; n ϭ 75) of the primary response. # cells, The total number of L chain sequences obtained from single cells with the proportion of sequences that were mutated and non- mutated shown in parenthesis. % total, The percentage of the total number of base pairs sequenced that were mutated (reliable sequence from positions 26 to 89 for all sequences). # per mut seq, The number of mutations per mutated sequence and % replace, the percentage of mutations in CDR1 and 2 that are replacement mutations. 1398 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Each of the three subsets of NP-speciﬁc B cells express somatically mutated H chains during the primary response. A–C, Representative H chain sequences, displayed as described for Fig. 2, A–C. Single NPϩ cells of each subtype were sorted from animals during the primary response into a cDNA reaction mixture, subjected to two rounds of nested PCR using VH186.2 H chain-speciﬁc primers, and the PCR products were sequenced. Frequency of obtaining a PCR product for each population is as follows: CD138ϩ, 42%; B220ϩ,17%; B220Ϫ, 6%; and CD138ϩ memory, 28%. Sequences shown are The Journal of Immunology 1399 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. The B220Ϫ subset is the dominant NP-specific B cell population in the BM. Animals were immunized as detailed for Fig. 1; BM harvested by aspirating both femurs and cells were labeled for flow cytometry. A, The NP-binding B cell population in the BM. Initial exclusion of PI-positive cells, T cells, and macrophages and gating of NPϩ IgDϪ B cells was performed as in Fig. 1A. B, Cellular dynamics of the NP-binding B cell compartment. Total numbers of NP-binding IgDϪ B cells were calculated using frequencies obtained via flow cytometry and total BM cell numbers obtained after sacrificing animals. Each time point is representative of at least three animals with the means Ϯ SEM displayed. C, Identifying subsets of Ag-specific B cells. NP-specific B cells (PIϪ, CD4Ϫ, CD8Ϫ, F4/80Ϫ, IgDϪ,NPϩ) were analyzed for the expression of CD138 (PE-281.2) and B220 (FITC-RA3-6B2) at the days of the response indicated. D, Cellular dynamics of the three NP-binding B cell subsets. Numbers of cells with the indicated phenotypes were calculated as in B at the indicated days of the response. Each time point represents the mean Ϯ SEM of at least three animals.

expressed and not efficiently amplified at the single-cell level. This The B220Ϫ B cell population dominates the Ag-specific trend is exaggerated among the B220Ϫ population, as it is com- population in the BM posed of ϳ50% IgEϩ B cells, which will not cross-react with the ϩ IgG-directed primers used in this study. The B220Ϫ population We next analyzed the emergence and phenotype of the NP pop- may also express lower levels of Ig mRNA than their plasma cell ulation in the BM, an established site of Ag-specific B cell migra- ϩ Ϫ Ϫ or GC counterparts. Nevertheless, these mutational analyses tion (1, 28, 29, 33–35). By day 7, an NP IgD (also IgM ; data clearly show that the B220Ϫ subset initially emerges unmutated not shown) population emerges abruptly above background levels and develops somatic hypermutations with evidence for increased to near maximal numbers (Fig. 4, A and B; 0.4% total BM). There affinity for Ag, providing strong evidence for affinity-driven selec- is a gradual increase in NP-specific B cells from day 7 until max- tion and GC experience in the development of the B220Ϫ B cell imal numbers are reached at day 21 (0.9% total BM). Cell numbers compartment. then decrease to levels that are maintained for at least 8 wk (20%

from individual cells and are representative of the entire data set. Summary of H chain mutations from days 5 to 7 (D; n ϭ 32) and days 9 to 14 (E; n ϭ 77) of the primary response. # cells, % total, # per mut seq, and % replace as in the legend to Fig. 2 and % W33L, the percentage of mutated sequences containing the affinity-enhancing amino acid change of tryptophan to leucine at position 33 (reliable sequence from positions 19 to 94 for all sequences). 1400 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY of maximal levels). Thus, the B220Ϫ population accounts for the vast majority of NP-specific B cells in the BM throughout both the primary response and the quiescent memory phase (Ͼ90%; Fig. 4, C and D) and develops with kinetics that are distinct from their expansion upon secondary challenge. The B220ϩ/ϪCD138ϩ population accounts for only a small portion of the NP-specific B cells in the BM (Ͻ0.003% of total BM through day 28), which is similar to reported frequencies of Ab-secreting cells in the BM (28, 29). These data are consistent with a migration of B220Ϫ NP-specific B cells to the BM following primary immunization, where they persist as a substantial cellular reservoir for Ag-specific B cell memory.

The B220Ϫ population develops as two cellular subsets Expression of the integrin CD11b and surface IgE divides the B220Ϫ memory population into two cellular subsets, CD11bϩϩIgEϪ and CD11bϩIgEϩ (2), but the balance of these subsets in the quiescent memory compartment and their development during the primary response is not known. The cellular com- Downloaded from position of the B220Ϫ population is most clearly investigated within the BM, where the B220Ϫ population accounts for Ͼ90% of NP-binding B cells during the primary response (Fig. 4C). Al- though the background of the CD11bϩϩIgEϪ subset is 10-fold greater than that of the CD11bϩIgEϩ subset, both B220Ϫ subsets

clearly emerge by day 7 in the BM (Fig. 5A). These two subsets http://www.jimmunol.org/ persist at substantial levels for at least 8 wk. Both subsets of the B220Ϫ population also emerge rapidly in the spleen, with a CD11bϪ IgEϪ population composed of the B220ϩ and the CD138ϩ populations present as well (Fig. 5B). Interestingly, the second increase in NPϩ cells in the spleen from days 14 to 21 is attributed mainly to the CD11bϩϩIgEϪ subset. Thus, the two cellular subsets of B220Ϫ memory B cells develop during the primary response, not as a consequence of secondary encounter with Ag, and make up similar fractions of the quiescent memory by guest on September 29, 2021 compartment.

The B220Ϫ subset has a non-GC phenotype FIGURE 5. The B220- population emerges as two distinct cellular sub- Phenotypic markers can been used to discern GC cells from Ab- sets. A, The B220Ϫ BM population is composed of a CD11bϩϩ and IgEϩ secreting cells and non-GC cells in the spleen by flow cytometry. subset. PI, CD4, CD8, F4/80Ϫ, IgDϪ,NPϩ cells were analyzed for the ϩϩ ϩ Both the CD11b and IgE subsets bind higher levels of PNA expression of CD11b (PE-M1/70) and IgE (FITC-R35-72) at the indicated than resting B cells, which does not allow us to distinguish them days postprimary immunization. Total cell numbers of each population from GC cells (Fig. 6A). The phenotype of the CD11bϩϩ and were calculated using frequency estimates obtained via flow cytometry and ϩ total BM cell numbers. B, The B220Ϫ population in the spleen is composed IgE subsets in the spleen and BM are identical, and samples from ϩϩ ϩ Ϫ Ϫ ϩ the BM are displayed here due to the absence of the B220ϩ and of a CD11b and IgE subset. PI, CD4, CD8, F4/80 , IgD ,NP cells ϩ in the spleen were analyzed for their expression of CD11b and IgE and cell CD138 subsets. GL7 has more recently been used to identify GC numbers of each population were determined as above. B cells (23) and is highly expressed on a major fraction of B220ϩ GC cells and is not expressed on either the CD11bϩϩ or IgEϩ subsets (Fig. 6B). BLA-1 has also been shown recently to identify specific GC as IgDϪ regions that bind NP (red) within IgDϩNPϪ GC B cells (25) and although it is highly expressed on B220ϩ B cell follicles (cyan; Fig. 7, A–C). B220 was observed to be cells, it is present at greatly reduced levels on either CD11bϩϩ or expressed on an overwhelming majority of IgDϪNPϩ GC B cells IgEϩ cells (Fig. 6C). CD24 (HSA) has been used previously to (red, Fig. 7D; yellow Fig. 7E), suggesting that the NPϩB220Ϫ label GC B cells (24) and is also expressed on B220ϩ cells but not population is not found in the GC at day 14. This is consistent with on CD11bϩϩ or IgEϩ cells (Fig. 6D). GC B cells have been shown both our phenotypic analyses and previous studies reporting that to lack CD43 (leukosialin or Ly-48) (25), which is expressed at GC B cells are B220ϩ (25). Serial sections through the same GC high levels on both CD11bϩϩ and IgEϩ subsets (Fig. 6E). Thus, show that the NPϩCD11bϩ subset is not found in the NPϩ GC, although mutational analyses indicate that the B220Ϫ subset has IgDϩ B cell follicle, or CD11bϩ MZ (MZ; Fig. 7F). The splenic transited the GC, phenotypic analysis indicates that B220Ϫ B cells red pulp is the area outside of the CD11bϩ MZ and B220ϩ B cell are not found in the GC, implying that the down-regulation of follicles and can be seen readily at lower power (interspersed B220 and expression of CD11b or IgE are post-GC events. B220ϪIgDϪ regions in Fig. 7A). The red pulp contains very few ϩ ϩ ϩ Ϫ B220 B cells and the majority of NP CD138 cells at day 14 The B220 subset is not found in GC in situ (36, 37) (example shown in Fig. 7G). The majority of In situ analysis allows us to asses GC localization without the NPϩB220ϪCD11bϩ cells at day 14 were also found to be scattered tissue disaggregation required for flow cytometric analysis. Using throughout the red pulp. Fig. 7H shows an example of the three-color laser scanning confocal microscopy, we can detect Ag- NPϩB220ϪCD11bϩ subset found in the red pulp (colocalizes as The Journal of Immunology 1401 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. The B220Ϫ B cell subsets have a non-GC phenotype. Small resting B cells are gated as PIϪ, forward scatter and side scatter small [CD4, CD8, F4/80]Ϫ,NPϪ, IgDϩ, and B220ϩ. CD138ϩ and B220ϩ plots are from spleens 7 days postprimary immunization and have been gated as PIϪ [CD4, CD8, F4/80]Ϫ, IgDϪ NPϩ and either CD138ϩ or B220ϩ. CD11bϩϩ and IgEϩ events are from BM samples 14 days postprimary immunization and have been gated as PIϪ [CD4, CD8, F4/80]Ϫ, IgDϪ, and NPϩ and either CD11bϩϩ IgEϪ or CD11bϩ IgEϩ. Mean ﬂuorescence intensities are shown for each plot. The expression of PNA (A), GL7 (B), BLA-1 (C), CD24 (D), and CD43 (E) is shown for each population. The levels of BLA-1 and CD24 on [CD4, CD8, F4/80]ϩ (primarily T cells) are shown in white as negative controls.

yellow and not associated with the B220ϩ follicle). The high- populations and at low to negative levels on both the CD138ϩ and lighted area is shown in greater detail in Fig. 7I with IgEϩ populations (Fig. 8B). CD19, the signal transduction com- NPϩB220ϪCD11bϩ cells indicated by yellow arrows. These data ponent of complement receptor (CR) 2, is expressed at high levels are consistent with our phenotypic analyses and together clearly on B220ϩ B cells, down-regulated on the CD138ϩ population, and demonstrate that the B220Ϫ subset is not found in the GC at day absent on both the CD11bϩϩ and IgEϩ subsets (Fig. 8C). The 14, a time at which this subset is mutated with evidence for se- expression of CD21, the complement-binding component of CR2, lection. Thus, the B220Ϫ subset appears to be a product of the GC, differs from that of CD19. CD21 was expressed at high levels on which undergoes a phenotypic alteration (down-regulation of the CD11bϩϩ subset and down-regulated on the CD138ϩ and B220 and expression of CD11b or IgE) upon exit from the GC. IgEϩ populations (Fig. 8D). CD22, a negative regulator of BCR- ϩ ϩϩ ϩ mediated signaling, was expressed at high levels on the B220 The phenotype of the CD11b and IgE B cell subsets imply population and down-regulated on the CD138ϩ and B220Ϫ pop- unique responses to Ag ulations (Fig. 8E). Together, these results predict that the B220ϩ Ϫ Ag-driven B cell expansion depends not only on signals transmit- and both B220 memory B cell populations may differ in their ted through the B cell receptor (BCR), but also on the contribution activation requirements and the ability to respond to Ag and high- of regulatory molecules at the cell surface. An isoform of the ty- lights a functional division of B cell memory. rosine phosphatase CD45 is expressed at high levels on B220ϩ B cells, at intermediate levels on the CD11bϩϩ and CD138ϩ popu- Discussion lations, and at low to negative levels on the IgEϩ subset (as de- We have recently shown that a novel B220Ϫ memory population tected by the Ab 30F.11, capable of recognizing all forms of expands upon secondary challenge to dominate the memory re- CD45; Fig. 8A). CD79b/Ig␤, the signal transduction component of sponse (2). How this subset develops is not clear and cannot be the BCR, is expressed at high levels on the B220ϩ and CD11bϩϩ inferred from our previous work. Here, we show that this novel 1402 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. In situ localization reveals that the B220Ϫ subset is not found in the GC. Spleens from mice sacrificed 14 days postprimary injection with NP-KLH were frozen and cryosections were prepared and stained for imaging via laser scanning confocal microscopy. A, NP binding and the splenic microenvironment. The ϫ10 image was stained with TR-IgD (cyan), FITC-B220 (green), and APC-NP (red). GCs can be seen as IgDϪ B220ϩ (green) and NP-specific GCs as IgDϪ B220ϩ NPϩ (colocalize as yellow/orange). B, Schematic of GC highlighted in Fig. 6A. Indicated are the red pulp (RP), MZ, B cell follicle (B foll), and GC. C, IgD expression and Ag binding delineates the GC. Image of highlighted NP-specific GC in Fig. 6A stained with TR-IgD (cyan), APC-NP (red), and FITC-B220 (green; shown in Fig. 6D). B cell follicle is seen as IgDϩ NPϪ (cyan) and GC as IgDϪNPϩ (red). D and E, GC B cells are B220ϩ. In the GC shown in Fig. 6C, IgDϪB220ϩ GC cells are seen as red (D) and NPϩB220ϩ GC cells are seen as yellow (E). F, The CD11bϩNPϩ subset is not found in the GC. Serial section through the GC highlighted in Fig. 6A with APC-NP (red) and CD11b (green) staining displayed. The MZ is highlighted by CD11b expression. G, Ab-secreting cells in the red pulp. Image of red pulp area of the spleen with rhodamine-CD138 (red), The Journal of Immunology 1403 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 8. The B220Ϫ B cell subsets lack B cell signaling molecules. Small resting B cells, CD138ϩ, B220ϩ, CD11bϩϩ, and IgEϩ populations were gated as described in the legend to Fig. 7. The level of each molecule of interest on [CD4, CD8, F4/80]ϩ (primarily T cells) is shown in white as negative controls; the negative level of the isotype-matched anti-CD21 Ab is shown as the negative for staining with the anti-CD45 Ab. The expression of CD45 (A), CD79b (B), CD19 (C), CD21 (D), and CD22 (E) is shown for each population with mean ﬂuorescence intensity shown on each plot.

B220Ϫ subset, and its composite CD11bϩϩ and IgEϩ subsets, de- experience and status as memory cells. Somatic hypermutation velops and persists following initial encounter with Ag in both the in the absence of histological GC has been reported (5, 6); how- spleen and BM. Mutational analysis indicates that the B220Ϫ pop- ever, the greater weight of evidence indicates that mutation pro- ulation has a GC phase in its development, whereas phenotypic ceeds in the GC microenvironment (8, 17, 38) and the loss of GC and in situ analysis indicates that the B220Ϫ subset is not found in in numerous animal models decreases mutational activity (9–14). the GC, implying that the loss of B220 is a post-GC event. This Thus, the somatic hypermutation and evidence for selection ob- recirculating subset localizes to the red pulp of the spleen, not the served among the B220Ϫ subset as early as 9 days postprimary MZ and dominates the Ag-specific B cell compartment in the BM. immunization most likely indicate a GC phase in their develop- Phenotypic analyses distinguish the B220Ϫ population from other ment and not occurring outside of the GC environment. The af- atypical B cell subsets and indicates that these memory B cells finity-enhancing position 33 change is an indication of Ag-driven may display unique responses to Ag recall. This current study selection (32) and is seen among the majority of B220Ϫ B cells Ϫ quantifies the emergence of a novel post-GC B220 B cell pop- (68%), indicating that recruitment into the B220Ϫ compartment is ulation following primary immunization, demonstrating the devel- affinity based. The B220ϩ B cells have a lower penetrance of this opment and maintenance of B cell memory in multiple, distinct affinity-increasing change at days 9–14 in vivo, suggesting that cellular compartments. many of these cells are still undergoing selection within the GC Ϫ Ϫ environment. The phenotype of the B220 subset also indicates Affinity maturation of the B220 population that they are not found in the GC, implying that the loss of B220 Somatic hypermutation of immunoglobulin genes within the is a post-GC event. Thus, the B220Ϫ population has a GC phase in B220Ϫ population provides the clearest evidence of their GC its development during the primary response, and the decision to

APC-NP (green), and FITC-B220 (cyan) displayed. Ab-secreting cells appear CD138ϩB220Ϫ (red), a subset of which are NP specific (colocalize as yellow). H, The B220ϪCD11bϩ subset is found in the red pulp. Image of a red pulp area with rhodamine-CD11b (red), APC-NP (green), and FITC-B220 (cyan) shown. Boxed area is enlarged in I where B220ϪCD11bϩNPϩ cells colocalize as yellow and are indicated by arrows. 1404 DEVELOPMENT OF Ag-SPECIFIC B CELL MEMORY exit the GC and down-regulate B220 may be based on affinity The possibility of the IgEϩ subset being non-B cells such as for Ag. mast cells or eosinophils with NP-specific Ig bound to the surface is unlikely based on a number of observations. Mast cells and Localization of memory B cells eosinophils circulate in a resting or immature state until they are The B220Ϫ population is a major component of the splenic recruited to sites of local tissue reactions or allergic inflammation post-GC B cell memory compartment. In situ, the CD11bϩϩ sub- (primarily mucosal and connective tissues for mast cells and air- set is not found in the GC at day 14, but is seen primarily in the red ways for eosinophils) where they mature to an effector state and pulp. One surprising result from these localization studies is the have a life span of only days (51–54). In contrast, the IgEϩ B cell lack of Ag-specific B cells in the MZ of the spleen at day 14, as subset is found in large numbers in the BM and persists at levels MacLennan and colleagues (37) have shown that the MZ in rats is 10–100-fold over background in the spleen and BM for at least 8 a major site of memory B cell localization. The large MZ popu- wk in the absence of further antigenic challenge. In addition, elec- lation they describe is seen when animals are primed with carrier tron microscopy studies of sorted NPϩ B220Ϫ B cells did not and reimmunized with hapten carrier, thus rendering T cell help reveal any cells with eosinophil or mast cell morphology (data not nonlimiting. However, when they use a single priming protocol, as shown). Phenotypically, the IgEϩ subset does not express the Fc⑀ in our study, the MZ population is dramatically reduced when receptor CD23 (data not shown). Finally, although the IgEϩ subset compared with carrier-primed animals and therefore may have es- lacks a number of typical B cell markers, most notably CD79b, caped our detection. The red pulp localization of the splenic CD19, CD21, and B220, they are phenotypically most similar to B220Ϫ B cells is more consistent with a recirculating memory B the CD138ϩ Ab-secreting B cell population in these same respects. cell compartment primed for Ag surveillance and a rapid recall Downloaded from response. Response to Ag recall The BM is another site of Ag-specific B cell localization (1, 28, Cell surface phenotype can offer insight into the activation require- 29, 33–35). Previous studies have focused primarily on the long- ments of B cell memory subsets, which are thought to differ from lived Ab-secreting population. This population is also observed in their naive counterparts in their activation requirements and pro- the current study, accounting for Ͻ0.003% of the total BM through liferative capacity (55–57). The CD11bϩϩ subset expresses sur- day 28, which is consistent with previous studies using the NP face IgG, expresses high levels of CD79b, and proliferates upon http://www.jimmunol.org/ Ϫ system (28, 29). The B220 population, however, represents the transfer with Ag into RAG1Ϫ/Ϫ mice (2), implying that this mem- vast majority of memory cells in the BM, accounting for nearly 1% ory population can respond to Ag via BCR-mediated interactions. of total BM cells at day 21. Although the rapid emergence and However, the role of coreceptor-Ag interactions appears to differ Ϫ peak of the B220 population is consistent with migration from among CD11bϩϩ and B220ϩ memory cells. Although the the spleen, it is not clear why this population would preferentially CD11bϩϩ subset expresses the complement-binding component of migrate to the BM. One possibility is that this migration is guided CR2, CD21, it lacks the signal transduction component, CD19, ␤ by the up-regulation of CD11b and expression of the 2 integrin responsible for augmenting the BCR-mediated signal. The expres- unique to this population. It is also possible that this specialized sion of complement CR3 (CD11b/CD18), however, may indicate microenvironment contains the requisite growth factors for long- that this subset requires a different set of Ag-complement interac- by guest on September 29, 2021 Ϫ term survival of the B220 B cell compartment. Nevertheless, the tions. The CD11bϩϩ subset does not express CD22, which acts as Ϫ BM appears to provide a significant reservoir of these B220 a negative regulator via recruitment of SHP1 (58–62). However, memory response precursors. this lack of CD22 may not have a significant effect on the activation of the CD11bϩϩ subset in the absence of CD19, as a recent Phenotype of memory B cells study has shown that CD22 exerts its negative effects by acting ϩϩ The CD11b subset is a novel B cell population distinct from primarily upon CD19 (63). Thus, the surface phenotypes of the ϩ previously reported atypical B cell populations. The CD11b sub- individual B cell memory subsets may predict unique and special- set shares some phenotypic characteristics with the B-1 B cell ized activation requirements for each subset. population, but they differ in that splenic B-1 cells are CD5ϩ, ϩ/Ϫ ϩ/Ϫ ϩ ϩ/Ϫ Ϫ B220 , HSA , IgM , IgD , and CD11b , whereas the Acknowledgments CD11bϩϩ subset is CD5Ϫ, B220Ϫ, HSAϪ, IgMϪ, IgDϪ, We thank John Mikszta, Rebecca Pogue Caley, Gabriel Bikah, Michael and CD11bϩϩ (Refs. 39–42 and data not shown). In addition, the ϩϩ D. Eisenbraun, Joanne Fanelli Panus, and Garnett Kelsoe for helpful com- CD11b subset dominates the Ag-specific population in the BM, ments and discussion, Jessica Ebright for assistance with sequencing, and while few B-1 cells are found in adult BM (40). Moreover, it has the Duke Comprehensive Cancer Center Flow Cytometry Shared Resource. been shown that B-1 B cells do not respond to the T-dependent We also thank Fred Finkelman for the gift of the 11.26 Ab and Leonore hapten NP used in this study (43). A population of biphenotypic Herzenberg for the anti-BLA-1 reagent. cells with characteristics of both macrophages and B cells has also been recently described (44, 45). Although this population ex- References ϩ ϩ ϩ ϩ ϩ presses CD11b, they are B220 , IgM , IgD , CD5 , and F4/80 , 1. McHeyzer-Williams, M. G., and R. Ahmed. 1999. B cell memory and the long- whereas the CD11bϩϩ population does not express any of these lived plasma cell. Curr. Opin. Immunol. 11:172. Ϫ Ϫ 2. 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