Maintenance of Long-Lived Plasma Cells and Serological Memory Despite Mature and Memory Depletion during CD20 Immunotherapy in Mice This information is current as of October 5, 2021. David J. DiLillo, Yasuhito Hamaguchi, Yoshihiro Ueda, Kaiyong Yang, Junji Uchida, Karen M. Haas, Garnett Kelsoe and Thomas F. Tedder J Immunol 2008; 180:361-371; ;

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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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Maintenance of Long-Lived Plasma Cells and Serological Memory Despite Mature and Depletion during CD20 Immunotherapy in Mice1

David J. DiLillo,2 Yasuhito Hamaguchi,2 Yoshihiro Ueda, Kaiyong Yang, Junji Uchida, Karen M. Haas, Garnett Kelsoe, and Thomas F. Tedder3

CD20 mAb-mediated B cell depletion is an effective treatment for B cell malignancies and some autoimmune diseases. However, the full effects of B cell depletion on natural, primary, and secondary Ab responses and the maintenance of Ag-specific serum Ig levels are largely unknown. The relationship between memory B cells, long-lived plasma cells, and long-lived humoral immunity also remains controversial. To address the roles of B cell subsets in the longevity of humoral responses, mature B cells were depleted in mice using CD20 mAb. Peritoneal B cell depletion reduced natural and Ag-induced IgM responses. Otherwise, CD20؉ B cell depletion prevented humoral immune responses and class switching and depleted existing and adoptively transferred B cell Downloaded from memory. Nonetheless, B cell depletion did not affect serum Ig levels, Ag-specific Ab titers, or bone marrow Ab-secreting numbers. Coblockade of LFA-1 and VLA-4 adhesion molecules temporarily depleted long-lived plasma cells from the bone marrow. CD20؉ B cell depletion plus LFA-1/VLA-4 mAb treatment significantly prolonged Ag-specific plasma cell depletion from the bone marrow, with a significant decrease in Ag-specific serum IgG. Collectively, these results support previous claims that bone marrow plasma cells are intrinsically long-lived. Furthermore, these studies now demonstrate that mature and memory B cells are not required for maintaining bone marrow plasma cell numbers, but are required for http://www.jimmunol.org/ repopulation of plasma cell-deficient bone marrow. Thereby, depleting mature and memory B cells does not have a dramatic negative effect on preexisting Ab levels. The Journal of Immunology, 2008, 180: 361–371.

he B1, marginal zone, and germinal center B cell subsets 13). The cellular precursors contributing to memory Ab responses contribute to the circulating natural Ab pool, -inde- are classically defined as surface IgMϩIgDϪB220ϩ or IgMϪIgDϪ T pendent (TI)4 IgM Ab responses, and adaptive immunity B220ϩ Ag-binding B cells that do not express syndecan-1 (1–4) by terminal differentiation into plasma cells, the effector (CD138) (12, 14–16). Memory B cells persist in substantial num- cells of humoral memory and immunity (5, 6). Ag activation of bers for at least 8 wk after Ag challenge, rapidly expand during by guest on October 5, 2021 mature B cells leads initially to the development of germinal cen- secondary responses, and terminally differentiate into plasma cells ters, the transient generation of plasmablasts that secrete Ab while (17). Thereby, postgerminal center memory B cells give rise to Ab still dividing, and short-lived plasma cells that secrete Ag-specific isotype-switched plasmablasts and then long-lived plasma cells germline-encoded Abs (7). Extrafollicular plasma cells with half- once outside of germinal centers. lives of 3–5 days appear first in the T cell zones (8) and then Persistent Ag-specific Ab titers are thought to derive from long- migrate to the red pulp of the spleen (9). In germinal centers, lived plasma cells (5, 17). Ab-secreting plasma cells generated in Ag-specific B cells proliferate, diversify their receptors, and give the spleen migrate to the bone marrow where they may persist for rise to pools of long-lived memory B cells (10). Memory B cells the life of the animal without the need for self-replenishment or generated during the second week of primary Ab responses ex- turnover (6, 7, 18–21). In fact, bone marrow cell transfers confer press mutated Ag receptors with enhanced affinities for Ag (11– long-lived Ab production, presumably due to the transfer of long- lived plasma cells (19). Nonetheless, Radbruch et al. (5) state in a recent review, “The biology of plasma cells. . . remains enigmatic Department of Immunology, Duke University Medical Center, Durham, NC 27710 and is of considerable debate. Of particular interest is whether Received for publication August 1, 2007. Accepted for publication October 26, 2007. plasma cells are long-lived and form a pool of memory plasma The costs of publication of this article were defrayed in part by the payment of page cells that is separate from the pool of memory B cells.” In one charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. model for the maintenance of serum Ab levels, primary and sec- ondary immune responses generate a pool of long-lived plasma 1 This study was supported by grants from the National Institutes of Health (CA105001, CA81776, CA96547, AI56363, AI24335, and AI67584), the Arthritis cells that occupy essential survival niches within the bone marrow Foundation, and the Bill and Melinda Gates Foundation. K.M.H. is supported by a where they survive indefinitely, resulting in long-lived serum Ab Special Fellow Award from the and Society. levels (6, 8, 20, 22). In a second model, persisting Ag drives the 2 D.J.D. and Y.H. contributed equally to this study and share first authorship. memory B cell pool to chronically generate plasmablasts and 3 Address correspondence and reprint requests to Dr. Thomas F. Tedder, Box 3010, Department of Immunology, Room 353, Jones Building, Research Drive, Duke Univer- short-lived plasma cells that result in long-lived Ab production sity Medical Center, Durham, NC 27710. E-mail address: [email protected] (23). In a third model, memory cells themselves (24, 25) or pre- 4 Abbreviations used in this paper: TI, T cell independent; ASC, Ab-secreting cell; plasma cell precursors (26) continuously give rise to plasma cells KLH, keyhole limpet hemocyanin; NP-CGG, 4-hydroxy-3-nitrophenyl acetyl conju- in an Ag-independent manner as a means of maintaining serum Ab gated to chicken ␥-globulin; TD, T cell dependent; TNP, 2,4,6-trinitrophenyl. levels for extended time periods. In this third model, Ag-indepen- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 dent cytokines or TLR signals continuously induce a subset of www.jimmunol.org 362 B CELL DEPLETION ABROGATES ADAPTIVE HUMORAL IMMUNITY IN VIVO memory B cells to differentiate into plasma cells with defined or FACSCalibur flow cytometers (BD Biosciences). Background staining half-lives. These three models are not mutually exclusive, but all was determined using unreactive control mAbs (Invitrogen Life Technol- Ն hinge on understanding whether long-lived plasma cells are dis- ogies) with gates positioned to exclude 98% of the cells. Bone marrow and spleen B cell subsets were gated as previously described (32). Frozen tinct from the memory B cell pool or other precursor populations. ϩ sections of spleen were fixed in acetone and stained with FITC-conjugated Mature CD20 B cell depletion is proving to be effective in GL-7, PE-conjugated TCR␤ chain, and biotin-conjugated B220 mAbs fol- treating B cell malignancies and some autoimmune diseases (27, lowed by Alexa Fluor 350-labeled streptavidin at predetermined optimal 28). CD20 is a B cell-specific molecule that is first expressed on dilutions in saline containing 1% BSA. Ab reactivity was visualized by fluorescence microscopy. the cell surface during the pre-B to immature B cell transition, but is presumed to be lost upon plasma cell differentiation (29, 30). ELISAs However, it has been difficult to determine the precise effects of B cell depletion on human B cell subsets and humoral immunity DNP- and TNP-specific Ab levels in individual serum samples were determined in duplicate using Ab isotype-specific ELISA as previously since most CD20 mAb therapy patients are also given immuno- described (38). Sera from TNP-LPS-immunized mice were diluted ϩ suppressive drugs (27). To assess the effects of CD20 B cell 1/400, whereas sera from DNP-Ficoll- and DNP-KLH-immunized mice depletion on germinal center B cells, memory B cells, plasma- were diluted 1/1000 for analysis using ELISA plates coated with DNP- blasts, Ab-forming plasma cells, existing Ab levels, and primary BSA (Calbiochem-Novabiochem) or TNP-BSA (Biosearch Technologies). Relative Ag-specific IgM and IgG titers were determined for each group of and memory Ab responses in mice, B cells were depleted using mice using pooled serum samples with results plotted as OD vs dilution CD20 mAb (30–36). Although mature and memory B cells were (log scale). NP-specific serum Ab was detected by ELISA with IgG or CD20ϩ and were effectively depleted by CD20 mAb treatment in IgG1 anti-NP Ab concentrations estimated by comparisons to standard vivo, CD20ϩ B cell depletion did not deplete long-lived plasma curves generated using the high-affinity NP-specific H33L␥1 mAb (39). cell numbers or negatively affect long-lived serum Ab levels. Thus, The relative affinities of serum NP-specific Ab responses were assessed by Downloaded from comparing the relative amounts of Ab bound to NP5-BSA vs NP25-BSA as the bone marrow plasma cell pool does not require ongoing con- described elsewhere (10). Briefly, half of a 96-well ELISA plate was tributions from the memory B cell pool for its maintenance. coated with NP5-BSA or NP25-BSA, and a set of sera was applied to the plate. OD values of NP5-specific and NP25-specific Abs of each sample Materials and Methods were used to calculate the relative ratio of NP-specific low- to high-affinity Mice, Abs, and reagents Abs. Relative NP-specific IgM and IgG titers were determined for each group of mice using pooled serum samples with results plotted as OD vs http://www.jimmunol.org/ Wild-type C57BL/6 and Rag1Ϫ/Ϫ (B6.129S7-Rag1tm1Mom/J) mice were ob- dilution (log scale). tained from The Jackson Laboratory. Hemizygous transgenic mice express- ing human CD19 (TG-1 line) on a C57BL/6 genetic background were as Adoptive transfer experiments described previously (37, 38). In some experiments, mice received drinking ␮ water containing BrdU (1 mg/ml; Sigma-Aldrich). Mice were housed in a Spleen B cells from mice immunized i.p. with 100 gofNP18-CGG in alum 42 days before harvest were enriched by T cell depletion using pathogen-free barrier facility and were used at 6–8 wk of age. These Ͼ studies were approved by the Duke University Animal Care and Use Thy1.2 mAb-coated magnetic beads (Invitrogen Life Technologies; 94% B220ϩ cells and Ͻ0.7% Thy1.2ϩ cells). Spleen T cells from mice immu- Committee. ␮ Mouse CD20-specific mouse mAbs were as described elsewhere (30). nized i.p. with 100 g of CGG in alum 30 days before harvest were en- riched by B cell depletion using B220 mAb-coated magnetic beads (In- Other mAbs included B220 mAb RA3-6B2 (provided by Dr. R. Coffman, Ͻ ϩ DNAX Corporation, Palo Alto, CA) and CD1d (1B1), CD21 (7G6), CD24 vitrogen Life Technologies and Dynal; 0.8% B220 cells). Equal by guest on October 5, 2021 ϫ 7 Ϫ/Ϫ (M1/69), CD138 (281-2), and GL-7 mAbs from BD Pharmingen. TCR␤ numbers of B and T cells (3 10 ) were injected i.v. into Rag1 mice chain mAb (H57-597) was purchased from eBioscience. Alexa Fluor 350- immediately before i.v. injection of MB20-11 or isotype-matched control mAbs. One day after mAb treatment, all mice were given soluble NP18-CGG conjugated streptavidin was obtained from Invitrogen Life Technologies. A ␮ BrdU Flow (BD Pharmingen) was used to detect intracellular BrdU (100 g) i.v., with sera collected at the times indicated. For ELISPOT assays, labeling. VLA-4 (clone PS/2) and LFA-1 (clone M17/4) blocking mAbs bone marrow and spleen cells were harvested from individual mice 10 days were purified from hybridoma (American Type Culture Collection) tissue after boost immunizations. culture supernatant fluid. Isotype-specific and anti-mouse Ig or IgM sec- ondary Abs were purchased from Southern Biotechnology Associates. ELISPOT assays Immunotherapy The frequency of NP-specific Ab-secreting cells (ASCs) from single-cell splenocyte and bone marrow suspensions were estimated by ELISPOT

Sterile and endotoxin-free CD20 (MB20-11) and isotype-matched control assays using NP6-, NP25-, or NP33-BSA conjugates as previously described mAbs were injected i.v. through lateral tail veins. Mice were given 250 ␮g (10). Bone marrow and spleen cells were added to Immobilon-P Multi- of mAb in 200 ␮l of PBS, unless otherwise indicated. For thioglycolate- screen 96-well plates (Millipore) that were precoated with NP-BSA (5 induced peritonitis, 1 ml of thioglycolate solution (3% w/v; Sigma-Aldrich) ␮g/ml) at either 104,105,or106 cells/well in culture medium (100 ␮l; was injected i.p. 1 day before mAb treatment. In some cases, function- RPMI 1640 containing 10% FCS, 10 mM glutamine, 100 U/ml penicillin/ blocking LFA-1 and VLA-4 mAbs (200 ␮g each) were injected i.p. as streptomycin, and 55 ␮M 2-ME). After incubating the plates for 3 h at indicated. 37°C in a humidified CO2 incubator, the plates were washed three times and incubated with alkaline phosphatase-conjugated polyclonal goat anti- Immunizations mouse IgG or IgG1 Abs (Southern Biotechnology Associates) for1hat Two-month old mice were immunized i.p. with trinitrophenyl (TNP)-LPS room temperature. After washing, the plates were developed using BCIP/ (50 ␮g; Sigma-Aldrich) or DNP-Ficoll (25 ␮g; Biosearch Technologies, NBT substrate (Sigma-Aldrich). Similarly, the total number of splenic and San Rafael, CA) in 200 ␮l PBS. Mice were also immunized i.p. with DNP bone marrow IgG and IgM ASCs was determined using plates coated with conjugated to keyhole limpet hemocyanin (KLH) (100 ␮g; Calbiochem- polyclonal anti-mouse Ig and developed with alkaline phosphatase-conju- Novabiochem) in 200 ␮l of CFA (50% v/v) and were boosted i.p. 28 days gated goat anti-mouse IgM or IgG Abs (Southern Biotechnology Associ- ates). For sorting of cell populations before use in ELISPOT assays, the later with DNP-KLH in 200 ␮l of IFA (50% v/v). In other experiments, ϩ Ϫ mice were immunized i.p. with 100 ␮g of 4-hydroxy-3-nitrophenyl acetyl MACS system (Miltenyi Biotec) was used to purify CD20 and CD20 cells. Cells labeled with biotinylated MB20-11 mAb and anti-biotin mi- conjugated to chicken ␥-globulin (NP-CGG) precipitated in alum as de- ϩ scribed previously (10). Mice were bled before and after immunizations as crobeads were used to positively select CD20 cells. Flow-through cells indicated. were harvested and run a second time over the magnetic column to remove contaminating CD20ϩ cells. The purity of the CD20Ϫ population was Immunofluorescence analysis and immunohistology Ͼ98%, while CD20ϩ cell purity was Ͼ95%. Single-cell leukocyte suspensions were stained on using predetermined CFSE labeling and cell culture optimal concentrations of each primary and secondary Ab for 20–60 min and fixed as described elsewhere (37). Cells with the forward and Purified B cells were differentiated in culture by stimulation with LPS or side scatter properties of were analyzed on FACScan CD40 mAb plus IL-4 as previously described (40). Briefly, T cell-depleted The Journal of Immunology 363

FIGURE 1. Humoral immunity following CD20ϩ B cell depletion. A, Serum Ig levels in CD20 mAb-treated mice. Two-month-old littermate pairs of mice (n Ն 3) were treated with MB20-11 or control (CTL) mAb on day 0. B, Effect of CD20ϩ B cell depletion on inflam- mation-induced immune responses. Two-month-old lit- termate pairs of mice were given thioglycolate solution followed by MB20-11 (n ϭ 10) or control (n ϭ 10) Downloaded from mAb treatment 1 day later on day 0. C, Effect of con- tinuous B cell depletion on serum IgM and IgG levels in CD19-transgenic mice. Mice were treated biweekly with 5 ␮g of CD20 (n ϭ 5) or control (n ϭ 5) mAb every 2 wk for 2 mo. A–C, Values represent mean relative Ig levels (ϮSEM) in individual serum sam-

ples obtained on the days indicated as determined by http://www.jimmunol.org/ isotype-specific ELISA. Differences between CD20 p Ͻ ,ء ,and control mAb-treated mice were significant .p Ͻ 0.01 ,ءء ;0.05 by guest on October 5, 2021

ϩ splenocytes (Ͼ93% B220ϩ) were labeled with 1 ␮M CFSE (CFSE Vy- to 58 days following mAb treatment (Fig. 1A). Thus, CD20 B brant; Invitrogen Life Technologies and Molecular Probes) according to cell depletion did not significantly reduce basal serum Ig levels. the manufacturers’ instructions. B cells were cultured in 24-well flat- Since peritoneal B cells are not efficiently depleted by CD20 bottom tissue culture plates (3 ϫ 106 cells in 1.5 ml of culture medium/ o ␮ mAb treatment (31, 32), their contribution to basal serum Ig levels well) at 37 C with 5% CO2 in the presence of LPS (10 g/ml) or CD40 mAb (1 ␮g/ml. HM40-3, BD Biosciences) in combination with IL-4 was assessed. Thioglycolate given i.p. 1 day before CD20 mAb (100 U/ml, Sigma-Aldrich) for 72 h. CD20 expression levels were as- treatment induces significant peritoneal B cell depletion within 7 sessed by immunofluorescence staining of harvested cells with flow cytometry analysis. days (32). B cell depletion prevented the increase in serum Ig levels that follows thioglycolate-induced inflammation. In addi- Statistical analysis tion, thioglycolate plus CD20 mAb treatment reduced serum IgM All data are shown as means Ϯ SEM. The Student t test was used to ϳ40% from initial levels ( p Ͻ 0.05), while serum IgG1, IgG2b, determine the significance of differences between sample means. IgG3, or IgA levels did not fall below the normal range (Fig. 1B). To assess whether prolonged CD20 mAb treatment might also af- Results fect serum Ig levels, 3-mo-old CD19-transgenic mice were treated ϩ Serum Ig levels following CD20 B cell depletion biweekly with a CD20 mAb dose (5 ␮g) that effectively depletes The influence of B cell depletion on serum Ig levels was assessed circulating and peripheral B cells (34). Serum Ig levels increase at in littermates after CD20 (MB20-11) or control mAb treatments a faster rate than normal in autoimmune-prone CD19-transgenic ϩ (250 ␮g). At this mAb dose, circulating, spleen and lymph node B mice, as described (38). Continuous CD20 B cell depletion did cell numbers were reduced by Ͼ95% by day 2 and began to re- not significantly affect basal IgM or IgG levels, but did prevent the cover on day 57 (data not shown and Refs. 31, 32, and 34). Despite development of hypergammaglobulinemia (Fig. 1C). Thus, effective B cell depletion, serum IgM, IgG, and IgA levels were CD20ϩ B cell depletion significantly attenuated increases in serum similar for both CD20 and control mAb-treated littermates for up Ig, but had minimal effects on basal serum Ig levels. 364 B CELL DEPLETION ABROGATES ADAPTIVE HUMORAL IMMUNITY IN VIVO

FIGURE 2. TD and TI Ab responses in CD20ϩ B cell-depleted mice. A, Effect of B cell depletion on DNP-KLH Ab responses. B, Effect of thioglycolate treatment and B cell depletion on DNP-KLH Ab re- sponses. A and B, Mice were retreated with mAb on day 21 and challenged with DNP-KLH on day 28. Lower Downloaded from panels, The relative titers of DNP-specific IgM and IgG Ab in pooled sera from immunized mice with back- ground measured using RagϪ/Ϫ mouse serum. C and D, Effect of B cell depletion and thioglycolate treatment on TNP-LPS Ab responses. E, Effect of B cell depletion on DNP-Ficoll Ab responses. A–E, Littermate pairs of mice were treated with MB20-11 or control mAb on day Ϫ7 http://www.jimmunol.org/ with serum harvested before immunization (arrow) on days 0. Hapten-specific Ab levels in individual serum samples harvested on the indicated days were measured by ELISA. Values represent mean (ϮSEM) relative OD units obtained using individual sera from five mice of each group. Differences between CD20 or control mAb- .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء ,treated mice were significant by guest on October 5, 2021

Ab responses following CD20ϩ B cell depletion (Fig. 2A). When mice were treated with CD20 mAb plus thiogly- Ab responses to the T cell-dependent (TD) Ag DNP-KLH were as- colate, primary IgM responses to DNP were significantly de- sessed in mice treated with CD20 mAb 7 days before immunization. creased, with 6-fold lower IgM Ab titers on day 7 and 77-fold CD20 and control mAb-treated littermates generated similar primary lower IgG titers on day 28 (Fig. 2B). Secondary IgM and IgG titers IgM Ab responses 7 days after DNP-KLH immunization (Fig. 2A). were 30- and 36-fold lower on day 35, respectively. Thus, initial However, IgM Ab responses in CD20 mAb-treated mice decayed TD IgM Ab responses progressed normally after B cell depletion rapidly between 2 and 4 wk following immunization relative to due predominantly to peritoneal B cells, while class-switched and control mAb-treated littermates. By contrast, DNP-specific IgG secondary Ab responses were severely inhibited. responses were 20-fold lower in CD20 mAb-treated mice com- The influence of B cell depletion on TI humoral immune re- pared with control mAb-treated littermates on day 28. All mice sponses was assessed in littermates treated with CD20 or control were retreated with CD20 or control mAb on day 21 and were mAb 7 days before TNP-LPS (TI-1 Ag) or DNP-Ficoll (TI-2 Ag) rechallenged with DNP-KLH on day 28. Secondary IgM and IgG immunizations. Following TNP-LPS immunizations, hapten-spe- Ab responses were impaired (15- and 40-fold lower, respectively) cific IgM levels were similar in both CD20 and control mAb- in CD20 mAb-treated mice by day 35, while significant secondary treated littermates, while TNP-specific IgG titers were significantly Ab responses were observed in control mAb-treated littermates lower in CD20 mAb-treated mice (Fig. 2C). With thioglycolate The Journal of Immunology 365

A B220+GL-7hi B220+GL-7- B220+GL-7hi B220+GL-7-

FIGURE 3. Germinal centers in B cell-depleted mice. A, Representative CD20 expression by B cells ϩ with germinal center (B220 GL-7high) and follicular Naive mice Immunized mice ϩ Ϫ B (B220 GL-7 ) phenotypes assessed by three-color im- Control IgG2a MB20-11 munofluorescence staining with flow cytometry analysis. Control IgG2a MB20-11

Mice were immunized with NP18-CGG 8 days before 0.5 0.06 0.9 0.4 splenocytes were isolated and stained using MB20-11 or B220 mAbs (heavy line) or isotype-matched control (light line) mAb with fluorochrome-conjugated IgG2a- specific secondary Ab. B, Representative germinal cen- 55.6 1.57 67.3 1.64 ter B cell depletion. Mice were untreated or immunized with NP -CGG 7 days before being given MB20-11 or 18 C B220+ B220+ B220+ B220+ B220+ control mAb for 7 days. Numbers indicate the relative GL-7hi CD24hiCD21- CD24hiCD21+ CD24+CD21+ CD1dhiCD21hi ϩ high ϩ Ϫ percentages of B220 GL-7 or B220 GL-7 lym- CTL mAb 0.3 4 3 24 3 phocytes within the indicated gates. C, Spleen B cell CD20 mAb Downloaded from 3 subsets. Bar graphs indicate mean (ϮSEM) numbers of 0.2 2 16 2 2 each B cell subset in naive and immunized littermates 7 0.1 1 8 1 days following MB20-11 or isotype control mAb treat- ** 1 0 * 0 ** ** 0 ** ** 0 ** ** 0 ** ** ment as in B. Significant differences between sample Naive Immun. Naive Immun. Naive Immun. Naive Immun. Naive Immun. -p Ͻ 0.01. D, Rep ,ءء ;p Ͻ 0.05 ,ء ;means are indicated resentative spleen germinal center depletion. Mice were Control IgG2a MB20-11

D http://www.jimmunol.org/ immunized with NP-CGG and injected with control or CD20 mAb 7 days later. Seven days after mAb injection, spleens were harvested, sectioned, and stained for TCR␤ chain, B220, and the GL-7 Ag. A–D, All results repre- sent Ն3 independent experiments.

ββ ββ TCR TCR by guest on October 5, 2021 B220 B220 GL-7 GL-7

and CD20 mAb treatment before TNP-LPS immunization, both were also significantly reduced in CD20 mAb-treated mice com- TNP-specific IgM and IgG levels were significantly decreased pared with control mAb-treated littermates (Fig. 2E). Thus, (Fig. 2D). Hapten-specific IgM and IgG responses to DNP-Ficoll CD20ϩ B cell depletion reduced TI-1 IgG and TI-2 IgM and IgG

Table I. Immunization effects on CD20 mAb-induced B cell depletiona

Naive Mice Immunized Mice

Tissue B Subsetb Control mAbc MB20-11 Control mAb MB20-11

Bone marrow Pro/pre 2.7 Ϯ 0.3 3.2 Ϯ 0.5 1.5 Ϯ 0.1 1.7 Ϯ 0.1 Immature 1.9 Ϯ 0.2 1.6 Ϯ 0.2 0.32 Ϯ 0.04 0.19 Ϯ 0.02* Mature 2.6 Ϯ 0.3 0.06 Ϯ 0.01** 0.20 Ϯ 0.04 0.01 Ϯ 0.01**

Blood B220ϩ 4.6 Ϯ 0.4 0.08 Ϯ 0.02** 9.7 Ϯ 0.3 0.05 Ϯ 0.01**

Spleen Mature 32 Ϯ 3 0.7 Ϯ 0.1** 22 Ϯ 3 0.75 Ϯ 0.2** T1 4.9 Ϯ 1.0 0.7 Ϯ 0.1** 4.6 Ϯ 0.9 1.3 Ϯ 0.2** T2 2.5 Ϯ 0.7 0.01 Ϯ 0.01** 5.9 Ϯ 1.2 0.14 Ϯ 0.04** Marginal zone 1.6 Ϯ 0.5 0.01 Ϯ 0.01** 3.5 Ϯ 0.3 0.02 Ϯ 0.01**

Peripheral lymph node B220ϩ 1.1 Ϯ 0.1 0.09 Ϯ 0.02** 2.2 Ϯ 0.3 0.16 Ϯ 0.07**

a Mice were either unimmunized or injected with DNP-KLH on day 0. On day 28, the immunized mice were boosted with DNP-KLH. Naive mice received control or CD20 mAb on day 0, with tissues harvested on day 7. Immunized mice were injected with control or CD20 mAb on day 35, with tissues harvested on day 42. b B cell subsets were: bone marrow pro/pre-B (B220lowIgMϪ), immature B (B220lowIgMϩ), and mature B (B220highIgMϩ); spleen mature (B220ϩCD24ϩCD21ϩ), T1 (B220ϩCD24highCD21Ϫ), T2 (B220ϩCD24highCD21ϩ), and marginal zone (B220ϩCD21highCD1dϩ). c Values (ϮSEM) indicate cell numbers (ϫ10Ϫ6) present in mice 7 days after mAb treatment (n Ն 3): blood shown as cells (ϫ10Ϫ5/ml), lymph nodes as pooled bilateral inguinal and axial lymph nodes, and bone marrow as both femurs pooled. Significant differences between means are indicated: .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء 366 B CELL DEPLETION ABROGATES ADAPTIVE HUMORAL IMMUNITY IN VIVO

responses, while peritoneal B cell depletion was necessary to ab- A IgM rogate TI-1 IgM responses. 1.5 CTL mAb CD20 mAb 1.0 Naive Germinal center B cells are depleted by CD20 mAb treatment

0.5 ** The effect of CD20 mAb treatment on germinal center B cells was ** assessed by immunofluorescence staining. Most B220ϩGL-7high 0 ** 020406080 100 germinal center B cells expressed CD20 at levels 7-fold higher Days ϩ Ϫ mAb boost than those found on B220 GL-7 follicular B cells (Fig. 3A). IgG1 IgG2c However, a small population (1–2%) of germinal center B cells 1.5 1.2 expressed little to no CD20. Since germinal center cells are max- 1.0 0.8 imally established by day 8 following immunizations with NP-

0.5 0.4 ** ** CGG (9), mice were treated with CD20 or isotype control mAb on ** day 7 after immunization. On day 14, B220ϩGL-7high germinal 0 0 center cell numbers were decreased by 99% in naive mice follow- IgG2b IgG3 1.5 1.5 ing CD20 mAb treatment and by 75% in immunized mice (Fig. 3, B and C). The majority of B220ϩ B cells remaining within the 1.0 1.0 * * spleens of immunized mice following 7 days of CD20 mAb treat- ** 0.5 0.5 ment represented recent pre-B and immature B cell emigrants from the bone marrow that had not begun to express CD20 at significant

0 0 Downloaded from 020406080 100 020406080 100 densities, as described previously (32). By contrast, mature bone Days Days mAb boost mAb boost marrow (B220highIgMϩ), blood (B220ϩ), mature spleen (B220ϩ ϩ ϩ ϩ Ϫ B CD24 CD21 ), spleen T1 (B220 CD24highCD21 ), spleen T2 IgM d96 IgG d104 ϩ high ϩ ϩ high 2.0 2.0 (B220 CD24 CD21 ), and marginal zone (B220 CD1d CTL mAb high 1.5 CD20 mAb 1.5 CD21 ) B cells were depleted by 92–99% following CD20 mAb Naive -/- 1.0 Rag 1.0 treatment in naive mice and mice immunized with NP-CGG or DNP-KLH (Fig. 3C and Table I). Histological examination of http://www.jimmunol.org/ 0.5 0.5 spleen sections revealed dramatically reduced follicular and ger- 0 0 10-2 10-3 10-4 10-5 10-2 10-3 10-4 10-5 minal center B cell staining 7 days after CD20 mAb treatment (Fig. Serum Dilution Serum Dilution 3D). Therefore, immunization does not affect the overall efficiency C D of B cell depletion and most germinal center B cells expressed 500 CTL mAb 1.0 CTL mAb CD20 at high levels, thus making them sensitive to CD20 mAb- 400 CD20 mAb 0.8 Naive Naive mediated depletion. 300 0.6 200 0.4 Memory B cells and Ab responses are depleted by CD20 mAb by guest on October 5, 2021 100 0.2 ϩ 0 ** ** 0 The effect of CD20 B cell depletion on memory Ab responses -1 0 2 4 7 10 -1 0 2 4 7 10 was assessed using DNP-KLH-immunized mice that were then treated with CD20 or control mAb 21 days later. Despite effective E Bone Marrow Spleen B cell depletion for 57 days as described (31, 32), significant DNP- 16 800 specific IgM and IgG Ab levels were maintained in CD20 and 12 600 control mAb-treated mice for at least 77 days following primary 8 400 immunizations (Fig. 4A). The mice were then retreated with CD20 or 4 200 control mAb on day 82 and rechallenged with DNP-KLH 7 days later.

0 ** **0 ** ** Secondary Ab responses were elicited in control mAb-treated mice, but IgM levels were 26-fold lower (day 96) and IgG titers were 6-fold lower (day 104) in CD20 mAb-treated mice (Fig. 4B). To compare FIGURE 4. Long-lived Ag-specific Ab levels and memory Ab re- primary and secondary Ab responses, naive mice were immunized sponses in CD20ϩ B cell-depleted mice. A, Ag-specific Ab levels after B with Ag in parallel with the rechallenge of their previously immu- cell depletion. Littermate pairs of mice were immunized (filled arrows) with DNP-KLH on day 0 and treated with MB20-11 (n ϭ 5) or control nized littermates. These primary IgM responses were greater than sec- (n ϭ 5) mAb on days 21 and 82 (shaded arrows). All mice were rechal- ondary responses in previously immunized mice after CD20 mAb lenged with DNP-KLH on day 89. Previously unimmunized littermates treatment, but IgG responses were relatively modest (Fig. 4, A and B). were also immunized with DNP-KLH on day 89 to quantify primary Ab responses. B, Relative titers of DNP-specific IgM (day 96) and IgG (day 104) in pooled sera from immunized mice in A with background measured using Rag1Ϫ/Ϫ mouse serum. C and D, Memory Ab responses after adop- D, High-affinity NP-specific IgG1 levels in individual serum samples from ϩ tive transfer. Purified splenic B220 cells were isolated from NP-CGG- C were measured by ELISA using NP5-BSA and NP25-BSA to indicate the primed mice (42 days after immunization) or from unimmunized litter- relative affinities of the Ab responses. Values represent mean (ϮSEM) mates and adoptively transferred into Rag1Ϫ/Ϫ mice (day Ϫ1) along with ratios of the titers for each serum sample. E, Bone marrow and spleen ASC an equal number of splenic T cells isolated from CGG-primed mice. The responses. Numbers of cells secreting NP25-specific IgG1 were determined mice were then immediately given MB20-11 or control mAb. All mice by ELISPOT. Values indicate mean (ϮSEM) ASC numbers from mice in were boosted with NP-CGG on day 0, with serum harvested on the indi- C on day 10. Less than 0.1 ASC/105 cells were detected in the bone marrow cated days. A–C, Hapten-specific Ab levels in individual serum samples and spleens of mice with primed B cells treated with MB20-11 (primed B were measured by isotype-specific ELISA. Values represent mean ϩ CD20) and naive B cells treated with control mAb (naive B plus IgG2a). (ϮSEM) relative OD units obtained using individual sera (unless indicated Differences between mice with primed B cells treated with control mAb otherwise) from Ն4 mice in each group. Significant differences between CD20 (primed B ϩ IgG2a) and primed B cells ϩ CD20 mAb or naive B cells ϩ .p Ͻ 0.01 ,ءء ;p Ͻ 0.01. IgG2a mAb were significant ,ءء ;p Ͻ 0.05 ,ء ;and control mAb-treated mice are indicated The Journal of Immunology 367

A Blasting Cells CFSElowCD138+ C IgM ASCs IgG ASCs 15 *** 9 Total ** ** CD20+ 10 *** 6 CD20- ** ** 5 3

0 0 CFSE CD20 Bone Spleen Bone Spleen Marrow Marrow CFSElowCD138- CFSEhiCD138- D Bone Marrow Spleen 9 CTL 24 CD20 6 16

3 8

0 0 CD20 CD20 CD138hi CD138hi CD138hi CD138hi BrdU+ BrdU- BrdU+ BrdU- B Spleen Spleen E Bone Marrow IgM Bone Marrow IgG 1.4 5

0.7 2.5

0 0 CD138 CD20 Bone Marrow Spleen IgM Spleen IgG

Bone Marrow Downloaded from 4 ** 8 3 ** 6 * 2 4 1 2 0 0 CD138 CD20 CD20 CD20 Thio - + Thio - ϩ FIGURE 5. CD20 expression by plasmablasts and ASCs. A, Representative CD20 expression by purified B220 cells that were labeled with CFSE and http://www.jimmunol.org/ cultured with LPS for 72 h. CD20 expression by three B cell subpopulations was assessed by immunofluorescence staining with flow cytometry analysis. CD20 expression (heavy lines) and background control anti-mouse IgG2a/c secondary polyclonal Ab (thin lines) staining are indicated for each B cell subset. B, Spleen and bone marrow B220lowCD138high cells from naive mice express lower levels of CD20 than B220ϩCD138Ϫ cells. Dot plots of B220 vs CD138 staining with labeled B220ϩCD138Ϫ and B220lowCD138high populations are shown (left panels). B220ϩCD138Ϫ (thin line) and B220lowCD138high (heavy line) cells were assessed for CD20 expression (right panels). Background control anti-mouse IgG2a/c secondary poly- clonal Ab staining is shown (shaded line). A and B, Results represent those obtained in three independent experiments. C, Bone marrow and spleen plasma cells express CD20. Bone marrow and splenocytes were either unsorted or sorted into CD20ϩ and CD20Ϫ populations using magnetic beads. Values indicate mean (ϮSEM) IgM and IgG ASC numbers per 104 cells as quantified by ELISPOT analysis (n ϭ 3). D, CD138high cells were maintained in the spleen and bone marrow of CD20 (n ϭ 4) or control (n ϭ 4) mAb-treated mice. Mice were fed BrdU for 2 wk before CD20 mAb by guest on October 5, 2021 treatment. Seven days later, splenic and bone marrow CD138high cells were analyzed for BrdU uptake by flow cytometry. Values represent the mean (ϮSEM) total number of cells gated within the indicated population. E, CD20 mAb depletes splenic but not bone marrow ASCs in vivo. Mice were untreated or given thioglycolate (Thio) before receiving CD20 (n ϭ 4) or control (n ϭ 4) mAb. Values indicate mean (ϮSEM) IgM and IgG ASC numbers contained in a whole spleen or two femurs quantified by ELISPOT analysis 7 days after mAb injection. C–E, Differences between sample .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء ;means were significant

Thus, CD20 mAb treatment inhibited memory IgM and IgG B cell 13 Ϯ 4 and 643 Ϯ 196 (per 105 cells), respectively. However, responses, but did not deplete preformed serum Ab. NP-specific ASCs were undetectable in CD20 mAb-treated Adoptive transfer experiments were conducted to verify the mice (Fig. 4E). Rag1Ϫ/Ϫ mice given naive B cells generated effect of CD20 mAb treatment on memory Ab responses and to spleen ASCs at very low numbers, but none were detected in the exclude the induction of plasma cells from primary immune bone marrow. Thus, CD20 mAb treatment effectively depleted ϩ responses. NP-CGG-primed B220 B cells or naive B cells memory B cells and thereby eliminated secondary Ab ϩ were mixed with equal numbers of CGG-primed Thy1.2 T responses. cells and transferred into Rag1Ϫ/Ϫ mice before treatment with CD20 or control mAbs and challenge with NP-CGG. NP-spe- cific IgG1 Ab titers and avidities were determined by ELISA CD20 expression by plasmablasts and plasma cells in vitro and using low-valency NP5-BSA or high-valency NP25-BSA. NP- in vivo specific IgG1 Abs (400 Ϯ 50 ␮g/ml) were easily observed in Ϫ Ϫ Plasmablasts and plasma cells express CD138, a commonly used control mAb-treated Rag1 / mice receiving NP-primed B cells, while Ab levels were not measurable in CD20 mAb- marker for ASCs (41, 42). Plasmablasts may be induced in vitro by treated mice (Fig. 4C). Control mAb-treated Rag1Ϫ/Ϫ mice stimulating B cells with LPS to induce TLR-dependent plasma cell given naive B cells had low NP-specific IgG1 Ab levels. The differentiation or with CD40 mAb in combination with IL-4 to NP-specific Abs produced in control mAb-treated Rag1Ϫ/Ϫ mimic TD plasma cell differentiation (40). Therefore, B cells were ϭ labeled with CFSE, cultured with LPS or CD40 mAb plus IL-4, mice receiving primed B cells were high avidity (NP5:NP25 ϭ and divided into three subpopulations based on their relative CFSE 0.91), while NP5:NP25 ratios were lower (NP5:NP25 0.13) in Rag1Ϫ/Ϫ mice given naive B cells (Fig. 4D). The numbers of intensities and CD138 expression (43). After LPS stimulation, pro- ϩ plasma cells and plasmablasts measured functionally as NP- liferating plasmablasts (CFSElowCD138 ) expressed CD20 at sig- specific IgG1 ASCs in the bone marrow and spleens of control nificant levels (Fig. 5A). CFSElowCD138Ϫ proliferating cells and mAb-treated Rag1Ϫ/Ϫ mice that received primed B cells were CFSEhighCD138Ϫ nonproliferating cells also expressed CD20, but 368 B CELL DEPLETION ABROGATES ADAPTIVE HUMORAL IMMUNITY IN VIVO

ABC 4 CTL mAb 9 1wk 8 1wk LFA/VLA mAbs 4wk * 4wk * 3 10wk * 6 10wk * 6 ** ** 2 4 3 ** ** 1 ** 2 * ** ** * * ** **** ** ** 0 0 0 678910 CD20 CD20 -+ Weeks after immunization -+- + - + LFA/VLA --+ + LFA/VLA --+ + D E 1 1wk 50 1wk 6 1wk 4wk 4wk 4wk 10wk 10wk 10wk 4 ** 0.5 25 2

** ** 0 ** **** 0 **** **** 0 **** ** **** CD20 -+- + CD20 -+- + CD20 -+- + LFA/VLA --+ + LFA/VLA --+ + LFA/VLA --+ + Downloaded from G H 3 1wk 100 CTL/CTL 4wk CTL/CD20 10wk 75 LFA-VLA/CTL LFA-VLA/CD20 1.5 50 ** ** http://www.jimmunol.org/ 25 * * ** * * ** ** * ** * * 0 **** **** 0 ** 6810 12 14 16 CD20 -+- + Weeks after immunization LFA/VLA --+ + FIGURE 6. Bone marrow replenishment after ASC depletion. A, LFA-1 and VLA-4 mAb treatment depletes Ag-specific plasma cells from the bone marrow. LFA-1 and VLA-4-blocking mAbs (n Ն 5) or control rat IgG (n Ն 5) were administered 6 wk after NP-CGG immunizations. Bone marrow was Ϯ harvested at the indicated times, with NP-specific ASCs enumerated using ELISPOT assays. Values represent mean ( SEM) NP25-specific ASC numbers per femur. B and C, CD20ϩ B cell depletion inhibits repopulation of the bone marrow by Ag-specific ASCs. Mice were treated with the indicated mAbs 6 wk

Ն Ն by guest on October 5, 2021 after immunization with NP-CGG. NP33-(B) and NP6-specific (C) bone marrow ASCs were enumerated by ELISPOT assay at week 1 (n 7), week 4 (n 3), ϭ Ϯ and week 10 (n 4) after mAb injection. Values represent mean ( SEM) NP33-orNP6-specific ASC numbers per femur. D–G, B cell subsets within tissues after LFA-1 and VLA-4 mAb treatments. Tissue B cell subsets in immunized mice were analyzed by flow cytometry at week 1 (n Ն 3), week 4 (n Ն 3), and week 10 (n ϭ 4) after mAb injection. Values represent mean (ϮSEM) numbers of B220ϩIgMhigh mature bone marrow B cells (D), B220ϩCD21ϩCD24ϩ mature spleen B cells (E), B220ϩCD21highCD1dϩ spleen marginal zone B cells (F), and B220ϩCD21ϩCD24high spleen T2 B cells (G). H, Depletion of bone marrow plasma cells decreases NP-specific serum IgG levels. Serum was harvested at the indicated times after immunization from mice treated with control (n ϭ 8), CD20 (n ϭ ϭ ϭ 8), LFA-1/VLA-4 (n 8), or both CD20 and LFA-1/VLA-4 (n 7) mAbs. NP6-specific serum IgG titers were determined by ELISA. Values at each time point represent the mean percentage of the initial NP-specific titers before mAb injection at week 6. A–H, Differences between sample means for control mAb-treated .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء ;mice and the indicated samples at the same time point were significant at levels 60% lower than plasmablasts. Similar results were ob- mined. Using CD138 staining with in vivo BrdU-labeling techniques, tained following B cell activation with CD40 mAb plus IL-4 stim- in vivo-generated short-lived plasmablasts (CD138highBrdUϩ) and ulation (data not shown). Thus, plasmablasts generated in vitro long-lived plasma cells (CD138highBrdUϪ) have been phenotypically expressed cell surface CD20. recognized (44). Therefore, mice were fed BrdU for 2 wk before Although CD20 may be expressed on in vitro-derived ASCs, treatment with CD20 or control mAb. Seven days later, spleen and CD20 is down-regulated in human B cells differentiating to plasma bone marrow cells were harvested and assessed for CD138 and BrdU cells (29). To determine whether CD20 is down-regulated in murine staining. Despite effective B cell depletion after CD20 mAb treatment, ASCs in vivo, CD20 expression was assessed by immunofluores- short-lived and long-lived plasma cell numbers were unchanged cence staining of spleen and bone marrow from naive mice. B220low (Fig. 5D). CD138high cells in the bone marrow expressed CD20, but at densities To directly assess the effect of CD20 mAb treatment on ASC ϩ Ϫ 80 and 75% lower than splenic and bone marrow B220 CD138 B numbers in vivo, naive mice were given CD20 or control mAb cells, respectively (Fig. 5B). In fact, when spleen and bone marrow 7 days before spleen and bone marrow cells were harvested, single-cell suspensions from naive mice were sorted with magnetic with ASC numbers determined by ELISPOT. IgM and IgG ASC ϩ Ϫ ϩ beads into CD20 and CD20 populations, the CD20 populations numbers in the bone marrow of mice receiving CD20 and con- of cells contained the majority of ASCs (Fig. 5C). Thus, ASCs ex- trol mAb were comparable (Fig. 5E). However, CD20 mAb pressed CD20 at low, but detectable levels in vivo. treatment significantly decreased the number of splenic IgG and IgM ASCs. Thus, bone marrow ASCs expressed insufficient ASC resist CD20 mAb-mediated depletion in vivo CD20 levels for their depletion in vivo, while some splenic Because CD20 expression is drastically reduced on ASCs in vivo, ASCs were depleted. Administration of thioglycolate induces sys- the effect of CD20 mAb on ASC numbers in naive mice was deter- temic inflammation that enhances B cell depletion by CD20 mAb in The Journal of Immunology 369 vivo (32). However, cotreatment of mice with thioglycolate and marrow with ASCs when the plasma cell niche was depleted. How- CD20 mAb did not enhance the degree of ASC depletion (Fig. 5E). ever, bone marrow repopulation was abrogated by the concurrent de- pletion of mature and memory B cells with CD20 mAb. Long-lived plasma cell depletion Since CD20 mAb did not reduce the bone marrow ASC compart- Discussion ment, an alternative approach was tested to determine whether These studies are the first to demonstrate in mice that most nascent long-lived plasma cells could be depleted. Because bone marrow humoral immune responses were blocked by CD20ϩ B cell deple- plasma cells express the LFA-1 and VLA-4 adhesion molecules tion before immunization, while predeveloped serum Ab levels (26, 45), whether function-blocking LFA-1 and VLA-4 mAbs were not affected by either short- or long-term CD20ϩ B cell de- could purge bone marrow ASCs from their survival niche (22) was pletion (Figs. 1, 2, and 4). Abrogation of primary and secondary assessed. Coinjection of LFA-1 and VLA-4 mAbs reduced NP- Ab responses is explained by the effective depletion of follicular, specific IgG ASC numbers by ϳ75% in the bone marrow of mice marginal zone, B1, germinal center, and memory B cell subsets immunized with NP-CGG 6 wk earlier (Fig. 6A). NP-specific ASC (Figs. 3 and 4 and Table I). Remarkably, long-lived Ab titers per- numbers in the bone marrow were reduced for up to 28 days, after sisted in the absence of memory B cell function and recall re- which NP-specific ASCs returned. Bone marrow ASCs were only sponses to Ag challenge (Figs. 1, 2, and 4). Thus, long-lived Ab depleted when LFA-1 and VLA-4 mAbs were used in combination production does not require persisting Ag to drive the memory B and similar results were obtained for NP-specific spleen ASCs cell pool to chronically generate plasmablasts and short-lived (data not shown). Thereby, coblockade of LFA-1 and VLA-4 plasma cells as previously suggested (23). Likewise, maintenance caused temporary bone marrow ASC depletion. of serum Ab levels does not require Ag-independent memory B Repopulation of the bone marrow with NP-specific ASCs 4 wk cell differentiation into plasma cells, as once suggested (24). In Downloaded from after LFA-1/VLA-4 mAb injection may have been due to mobi- fact, inflammation-induced increases in serum Ig levels and hy- lized bone marrow ASCs surviving by circulating within other pergammaglobulinemia were inhibited by CD20ϩ B cell depletion mouse tissues, followed by ASC relocalization to the bone marrow without an effect on basal serum Ig levels (Fig. 1, B and C). There- once the mAbs were cleared; alternatively, mobilization of ASCs fore, Ag-independent cytokine or TLR signaling may induce mem- from the bone marrow survival niche may have led to ASC death ory or other B cell subsets to differentiate into plasma cells (24, due to loss of survival signals (22), with repopulation of the bone 25), but this is not necessary for the maintenance of long-lived http://www.jimmunol.org/ marrow due to new ASC generation from memory B cells. To serum Ab levels. Thus, long-lived plasma cells or their immediate determine how the bone marrow came to be repopulated with Ag- precursors are indeed intrinsically long-lived and are distinct from specific ASCs, immunized mice were treated with either CD20 or the memory B cell pool. control mAbs in combination with LFA-1/VLA-4-blocking mAbs. Germinal center B cells, in vitro-generated plasmablasts, and Bone marrow cells were harvested 1, 4, and 10 wk later. After 1 some ASCs expressed cell surface CD20 at significant levels (Figs. wk of mAb treatment, NP-specific IgG ASC numbers were re- 3A and 5, A and B), which allowed their removal from B cell duced by ϳ70% ( p Ͻ 0.02) in mice receiving LFA-1/VLA-4 preparations (Fig. 5C). By contrast, in vivo CD20 mAb treatment mAbs or both LFA-1/VLA-4 and CD20 mAbs (Fig. 6, B and C). depleted only ϳ75% of splenic ASCs and had no effect on bone by guest on October 5, 2021

Both total (NP33-specific) and high-affinity (NP6-specific) IgG marrow ASC numbers (Fig. 5E) or established Ag-specific Ab ASCs were reduced similarly by LFA-1/VLA-4 mAb treatment. responses (Figs. 4 and 6). Likewise, CD20 mAb treatment did not By weeks 4 and 10, CD20ϩ B cell depletion prevented the recov- significantly deplete either short-lived CD138highBrdUϩ plasma- ery of NP-specific ASCs in the bone marrow of mice receiving blasts or long-lived CD138highBrdUϪ plasma cells (44) in vivo LFA-1/VLA-4 mAbs, while ASCs repopulated the bone marrow of (Fig. 5D). Spleen ASC depletion despite normal CD138high B cell mice receiving only LFA-1/VLA-4 mAbs. Thus, repopulation of numbers suggests that the use of CD138 as a marker for plasma the bone marrow after ASC purging with LFA-1/VLA-4 mAbs cells may only be appropriate for a mature CD20Ϫ subset of ASCs was due to new ASC production because CD20ϩ B cell depletion (46). Nonetheless, the in vivo decrease in splenic ASC numbers prevented bone marrow ASC repopulation when given in combi- after CD20 mAb treatment is likely due to residual CD20 expres- nation with LFA-1/VLA-4 mAbs. sion. Alternatively, the depletion of marginal zone, germinal cen- To verify that repopulation of the bone marrow ASC pool in ter, and memory B cells may prevent the formation of new plas- LFA-1/VLA-4 mAb-treated mice was not due to failed depletion mablasts, while short-lived plasma cells died off naturally during of the CD20ϩ mature/memory B cell pool, the effects of LFA-1/ the 7 days of CD20 mAb depletion. Even though some spleen VLA-4 plus CD20 mAb treatments on B cell depletion in tissues ASCs were depleted in vivo, they did not appear to contribute was assessed. Mature bone marrow B cells, and splenic marginal significantly to serum Ig levels in comparison to fully mature bone zone and T2 B cells were cleared 1 and 4 wk after treating mice marrow plasma cells. The lack of long-lived bone marrow ASC with CD20 mAb (Fig. 6, D–G), but returned by week 10. By con- depletion was most likely attributable to either their low-density trast, LFA-1/VLA-4 mAb treatment only depleted mature bone CD20 expression or the absence of effector monocytes within their marrow B cells, splenic marginal zone B cells, and ϳ50% of microenvironmental niches. Thus, although CD20ϩ B cell deple- spleen T2 B cells after 1 wk, with full repopulation of these subsets tion was effective at inhibiting primary and secondary humoral by 4 and 10 wk. One week after LFA-1/VLA-4 mAb treatment, the immune responses, serologic “memory” resulting from long-lived number of mature spleen B cells increased, but these numbers plasma cells was unaffected. normalized by 4 wk after mAb administration. Serum from mice Function-blocking mAbs against LFA-1 and VLA-4 purged Ag- receiving LFA-1/VLA-4 mAbs did not contain detectable levels of specific ASCs from the bone marrow (Fig. 6A), potentially induc- rat IgG beyond 1 wk after mAb injection, suggesting that the effect ing their death due to loss of survival niche signals (22). The bone of LFA-1/VLA-4 mAb treatment does not persist after mAb clearance marrow was repopulated with plasma cells as soon as the LFA-1 (data not shown). Finally, consistent with reduced bone marrow and VLA-4 mAbs were cleared from the circulation. However, plasma cell numbers after combined LFA-1/VLA-4 and CD20 mAb coinjection of mice with LFA-1/VLA-4 and CD20 mAbs led to treatment, serum NP-specific IgG levels were also significantly re- prolonged ASC depletion from the bone marrow (Fig. 6, B and C). duced (Fig. 6H). Thus, B cells had the ability to repopulate the bone Therefore, CD20ϩ B cells, and most likely memory B cells, were 370 B CELL DEPLETION ABROGATES ADAPTIVE HUMORAL IMMUNITY IN VIVO required for repopulating empty plasma cell niches in the bone B cells explains the absence of isotype-switched Ab responses in marrow. The prolonged ASC depletion in LFA-1/VLA-4- and CD20 mAb-treated mice. CD20 mAb- cotreated mice was also reflected in serum Ig levels; That CD20 mAb treatment impairs primary and recall Ab re- Ag-specific serum IgG was significantly reduced in mice receiving sponses while maintaining serum Ig levels in mice is consistent both CD20 and LFA-1/VLA-4 mAbs (weeks 7–12) compared with with studies in patients receiving , a chimeric anti-human mice receiving either mAb alone. Others have shown that the mar- CD20 mAb. This mAb can cause decreased Ab responses to some ginal zone B cell subset can be specifically mobilized and depleted recall Ags (49), while total Ig levels in lymphoma and rheumatoid by LFA-1/VLA-4-blocking mAbs (47); however, the current stud- arthritis patients generally remain within the normal range (49– ies now demonstrate that LFA-1 and VLA-4 adhesion molecules 52). Rituximab treatment also decreases primary Ab responses and also regulate mature B cell and plasma cell localization within the class switching in baboons (53). Although antibacterial Ab levels bone marrow. Moreover, this study is the first to show that Ag- are preserved in patients following rituximab- specific long-lived plasma cells can be purged from the bone mar- mediated B cell depletion (52), a positive clinical response is as- row by blocking adhesion receptor function, with memory B cells sociated with a significant fall in autoantibody levels in some pa- required for repopulation of a plasma cell-deficient bone marrow. tients (54–58), but not all (59). However, these conclusions are Therefore, long-lived plasma cells and memory B cells contribute complicated by the fact that most patients also receive supplemen- in interdependent ways to long-lived serological memory; mature tary immunosuppressive therapies and the full extent of B cell and memory B cells are not required for maintaining bone marrow clearance in humans given rituximab is unknown. In autoimmune plasma cell numbers, but are required for repopulation of plasma mice, continuous CD20 mAb treatment does not affect circulating Ig or autoantibody levels (34, 36). Thus, CD20 mAb therapy may cell-deficient bone marrow through Ag-dependent (10) or -inde- Downloaded from pendent (24) mechanisms. Thus, CD20 mAb treatment in combi- not be beneficial if pathogenic autoantibody-secreting cells are nation with LFA-1/VLA-4-blocking mAbs could provide a new long-lived or do not express CD20. However, CD20 mAb treat- therapeutic avenue for bone marrow plasma cell depletion that ment may deplete memory B cells or the precursors of pathogenic may also lead to the identification of additional similar strategies autoantibody-secreting plasma cells, thereby reducing the sources for depleting plasma cells from other tissues. of new Ab-secreting plasma cells in autoimmune disease where ϩ autoreactive memory B cells may be continually “tickled” with

CD20 B cell depletion did not inhibit IgM responses to TNP-LPS http://www.jimmunol.org/ Ag, leading to perpetual plasma cell differentiation. It is important or DNP-KLH immunizations, while IgM responses to DNP-Ficoll to note that the effects of rituximab in human patients may not were significantly inhibited by CD20 mAb treatment (Fig. 2). That completely recapitulate the results detailed in this study. Ritux- CD20 mAb treatment rapidly and effectively depletes marginal zone imab and the anti-mouse CD20 mAb used in this study are vastly B cells but not peritoneal B cells (32) indicates that peritoneal B cells distinct mAbs, with potentially different binding capabilities and may preferentially generate IgM responses to TI-1 and TD Ags. IgM effects in vivo (31–33). Ab generation by peritoneal B cells is further supported by the ob- The current studies demonstrate that depleting the majority of servation that primary IgM Ab responses were not generated in CD20 peripheral B cells by CD20 mAb treatment does not have a dra- mAb-treated mice after rechallenge with DNP-KLH (Fig. 2) since matic negative effect on preexisting Ab levels because plasma cells CD20 mAb eventually reduces peritoneal B cell numbers by the time by guest on October 5, 2021 are long-lived and survive independent of repopulation by the B that these mice were boosted with Ag (32). CD20 mAb plus thiogly- cell compartment. These results contrast with significant reduc- colate treatments also reduced basal serum IgM Ab levels by up to ϩ tions in serum Ab levels within 2 wk of CD19 B cell depletion B half (Fig. 1 ), most likely due to the depletion of peritoneal B cells in mouse models (60), consistent with the measured half-lives of and their natural Ab products (48). Otherwise, CD20 mAb plus thio- serum Abs (61). Thus, serum Ab deficiency may not be a signif- glycolate treatments did not significantly alter bone marrow and icant consequence of long-term B cell depletion using CD20 spleen ASC numbers or basal serum Ig levels (Fig. 5), arguing that mAbs, except when new Ags and pathogens are encountered. peritoneal B cells only produce a portion of serum IgM and relatively However, alternative therapeutic strategies for depleting long-lived little IgG. Thereby augmenting the innate immune response using plasma cells will have to be devised to combat the negative clinical agents like thioglycolate to mediate more effective B cell depletion implications of humoral memory once initiated. Thus, a balance may not augment the reduction of serum Ig levels. Thus, the majority between effective B cell depletion, therapeutic benefit, and immu- of serum Ab appears to be continually produced by long-lived plasma nodeficiency will also need to be established as more potent B cell cells that do not require replenishment once generated. depletion strategies move into clinical practice. The absence of Ag-specific Ab isotype switching and IgG-sub- class Ab responses to TI-1, TI-2, and TD Ags in CD20 mAb- Acknowledgments treated mice is likely due to the depletion of germinal center B We thank Drs. Jonathan Poe and Jean-David Bouaziz for assistance with cells and their precursors (Figs. 2 and 3). Most B cells with a these studies. germinal center phenotype and histologically recognizable germi- nal centers were depleted following 7 days of CD20 mAb treat- Disclosures ment, with at least 70% of germinal center B cells depleted in Thomas F. Tedder is a paid consultant for MedImmune and a consultant immunized mice (Fig. 3, B–D and Ref. 32). Thereby, germinal and a shareholder for Angelica Therapeutics. center-dependent clonal expansion, somatic hypermutation, and isotype switching are unlikely to occur to a significant extent fol- References lowing CD20 mAb treatment. CD20 mAb treatment also prevented 1. Haas, K. M., J. C. Poe, D. A. Steeber, and T. F. Tedder. 2005. B-1a and B-1b cells high-affinity Ag-specific Ab formation and the expansion of ASC exhibit distinct developmental requirements and have unique functional roles in in bone marrow and spleens of recipient mice during adoptive innate and adaptive immunity to S. pneumoniae. Immunity 23: 7–18. 2. Kantor, A. B., and L. A. Herzenberg. 1993. Origin of murine B cell lineages. transfer experiments. Thus, memory B cells expressed sufficient Annu. Rev. Immunol. 11: 501–538. levels of CD20 to be effectively targeted in vivo, with memory Ab 3. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323–335. responses virtually eliminated by CD20 mAb treatments in vivo 4. Kelsoe, G. 1996. Life and death in germinal centers (redux). Immunity 4: (Fig. 4). Thereby, the elimination of germinal center and memory 107–111. The Journal of Immunology 371

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