Viral Particles Drive Rapid Differentiation of Memory B Cells into Secondary Plasma Cells Producing Increased Levels of

This information is current as Franziska Zabel, Deepa Mohanan, Juliana Bessa, Alexander of September 24, 2021. Link, Antonia Fettelschoss, Philippe Saudan, Thomas M. Kündig and Martin F. Bachmann J Immunol 2014; 192:5499-5508; Prepublished online 12 May 2014;

doi: 10.4049/jimmunol.1400065 Downloaded from http://www.jimmunol.org/content/192/12/5499

Supplementary http://www.jimmunol.org/content/suppl/2014/05/11/jimmunol.140006 Material 5.DCSupplemental http://www.jimmunol.org/ References This article cites 39 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/192/12/5499.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Viral Particles Drive Rapid Differentiation of Memory B Cells into Secondary Plasma Cells Producing Increased Levels of Antibodies

Franziska Zabel,* Deepa Mohanan,* Juliana Bessa,† Alexander Link,† Antonia Fettelschoss,* Philippe Saudan,† Thomas M. Kundig,*€ and Martin F. Bachmann*,‡

Extensive studies have been undertaken to describe naive B cells differentiating into memory B cells at a cellular and molecular level. However, relatively little is known about the fate of memory B cells upon Ag re-encounter. We have previously established a system based on virus-like particles (VLPs), which allows tracking of VLP-specific B cells by flow cytometry as well as histology. Using markers, it is possible to adoptively transfer memory B cells into a naive mouse and track responses of naive and

memory B cells in the same mouse under physiological conditions. We have observed that VLP-specific memory B cells quickly Downloaded from differentiated into plasma cells that drove the early onset of a strong humoral IgG response. However, neither IgM+ nor IgG+ memory B cells proliferated extensively or entered germinal centers. Remarkably, plasma cells derived from memory B cells preferentially homed to the bone marrow earlier and secreted increased levels of Abs when compared with primary plasma cells derived from naive B cells. Hence, memory B cells have the unique phenotype to differentiate into highly effective secondary plasma cells. The Journal of Immunology, 2014, 192: 5499–5508. http://www.jimmunol.org/ or induction of long-lived humoral memory, interaction of splenic follicles close to GCs (17, 18). In contrast, PCs are mostly B cells with their cognate Ag alone is not sufficient, be- found in lymphoid organs where they have been induced as well F cause this process is dependent (1). CD4+ T cells as in the bone marrow (BM), which serves as the primary survival help activation through cell-bound molecules, such as, for niche up to several years (16, 19, 20). They can usually only be example, CD40L (2) and secreted molecules such as IL-4, IFN-g, detected in the for a short duration early after or IL-21 (3, 4), which results in switching, B cell prolif- (9, 21). In general, GC-derived memory B cells as well as PCs eration, and differentiation into memory B as well as plasma cells persist independently of Ag presence (16, 21–23). (PCs) (5, 6). Upon secondary , the humoral response is usually The long-lived humoral is mainly driven by the faster, stronger, and of the IgG isotype. The cellular basis of this by guest on September 24, 2021 (GC) response. Upon activation by cognate Ag, memory response is only starting to emerge. Recently, functional follicular B cells move to the edge of B cell follicles for interaction differences between IgM+ and IgG+ memory B cells have been with specific CD4+ Th cells. Subsequently, some of the activated described upon Ag recall (24). Dogan et al. (24) used a transgenic B cells move back into the B cell follicles and initiate the GC mouse model that irreversibly marked B cells GFP positive when response, which results in mutated and affinity-matured memory activation-induced deaminase (AID) has been expressed. AID is B cells and Ab-forming cells (AFCs) (7–10). Typically, memory involved in somatic hypermutations and class switching of the B cells express isotype-switched IgG or IgA Igs on their surface. BCR, which normally occurs in GCs. Upon immunization with However, immunization with protein or polysaccharide Ags can SRBCs, they observed GFP+ memory B cells that expressed either also result in a pool of IgM+ memory B cells (11–15). Memory IgM or IgG on their surface. After antigenic re-exposure, the B cells appear to efficiently recirculate throughout the lymphatic majority of the GFP+ IgM+ memory B cells initiated GC reactions system and are found at comparable frequencies in all lymphoid and gave rise to a IgG1+ memory pool. In contrast, GFP+ IgG+ organs as well as in the blood (16). The majority of memory B memory B cells failed to efficiently proliferate or enter GCs but cells reside in the marginal zone and at a lower frequency in the rapidly differentiated into PCs. Different observations were made with a soluble protein Ag (PE) after secondary immunizations. The generated IgG1+ memory B cells expanded and a proportion *Department of Dermatology, Zurich University Hospital, 8091 Zurich, Switzerland; differentiated into PCs. However, unlike the previous study, IgM+ † ‡ Cytos Biotechnology AG, CH-8952 Zurich-Schlieren, Switzerland; and Jenner Insti- memory B cells were not observed to enter GCs, which was tute, University of Oxford, Oxford OX3 7DQ, United Kingdom probably due to the presence of IgG Abs (13, 25). Additional Received for publication January 13, 2014. Accepted for publication April 15, 2014. studies after secondary tetanus toxoid immunizations in humans This work was supported by Swiss National Science Foundation Grant 31003A_149925. showed a peak of PCs at ∼day 6 in the blood (26, 27), followed by Address correspondence and reprint requests to Franziska Zabel, Dermatology De- a small increase of memory B cells at day 14 (27). Analysis of the partment, University Hospital Zurich, Gloriastrasse 31, CH-8091 Zurich, Switzer- land. E-mail address: [email protected] BCRs showed increased affinities due to further increased muta- The online version of this article contains supplemental material. tions after secondary immunizations without distinct dissection of Abbreviations used in this article: AFC, Ab-forming cell; AID, activation-induced the isotypes. This observation suggests that memory B cells went deaminase; BM, bone marrow; CS, class-switched; FCM, flow cytometry; GC, ger- into a second GC reaction upon Ag rechallenge. minal center; IF, immunofluorescence; ms, mouse; PC, ; PNA, peanut To be able to follow naive and memory B cell responses in the agglutinin; VLP, virus-like particle. same animal, we established an adoptive transfer system using Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 memory B cells specific for virus-like particles (VLPs). Studies of www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400065 5500 MEMORY B CELLS FORM POTENT SECONDARY PCs memory B cell responses using prime-boost regimens in the same follows: ∼0.5 3 107 cells for B220+ MACS-purified memory B cells, 5 + 5 + host have the disadvantage that specific IgG Abs are present, which ∼1 3 10 cells for IgG memory B cells, and ∼3 3 10 cells for the IgM generally suppress B cell responses. Therefore, we used an adoptive memory B cells. Control mice received one-tenth of whole of naive Ly5.1 or naive IgHa mice, respectively. transfer model to study the cellular response preventing the bias of the cellular response if specific IgG would be present. We used ELISA VLPs derived from the bacteriophage Qb as model Ag, which Blood was taken at indicated time points. For determination of Qb-VLP– induce strong and long-lasting GC-derived memory B cell, PC, as specific Ab titers, ELISA plates (Nunc Immuno MaxiSorp, Rochester, NY) well as Ab responses. Both the repetitive surface of the VLPs as were coated overnight with 100 mlQb-VLPs (1 mg/ml), and binding of well as the RNA that is spontaneously packaged during production serum Abs was detected by HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Pairs of allotype-specific markers were contribute to the magnitude of the response (28–31). Using this a used to determine Abs produced either by B cells of the IgH strain (biotin strategy, we were able to follow naive and memory B cell re- mouse (ms) anti-ms IgG1[a] [clone 10.9], biotin ms anti-ms IgG2a[a] sponses under physiological conditions without the need of ad- [clone 8.3]) or IgHb strain (biotin ms anti-ms IgG1[b] [clone B68-2], biotin ditional tools and treatments. We found that these VLPs induced ms anti-ms IgG2a[b] [clone 5.7]). Ab binding was detected by HRP- IgM+ as well as IgG+ memory B cells. Adoptive transfer experi- labeled streptavidin (BD Biosciences). Absorbance readings at 450 nm of the 1,2-phenylenediamine dihydrochloride color reaction were ana- ments demonstrated that, in contrast to previous reports, both sub- lyzed. sets failed to efficiently proliferate or enter GCs. Unexpectedly, both populations rapidly differentiated into unique secondary PCs, ELISPOT which homed to the BM and produced superior amounts of Abs Qb-VLP–specific AFC frequencies were determined, as described (33). compared with their primary counterparts derived from naive Briefly, 24-well plates were coated with 10 mg/ml Qb-VLPs. Single-cell Downloaded from B cells. suspensions of BM cells were added in DMEM containing 2% FCS and incubated for 5 h at 37˚C. Cells were washed off and plates were incubated with goat anti-mouse IgG (EY Labs), followed by alkaline phosphatase– Materials and Methods conjugated donkey anti-goat IgG Ab (Jackson ImmunoResearch Labora- Mice tories) before development of alkaline phosphatase color reactions. Wild-type C57BL/6 mice were purchased from Harlan (Horst, The Neth- Flow cytometry http://www.jimmunol.org/ erlands). A breeding pair of Ly5.1 [B6.SJL-Ptprc(a) Pepc(b)/BoyJ(002014)] mice were obtained from The Jackson Laboratory and further bred at the Single-cell suspensions of splenocytes and BM cells were prepared. Prior germfree BZL mouse facility at the University Hospital (Zurich, Swit- staining, RBC lysis of samples was performed. If not specifically indicated, zerland). IgHa mice were bred at Biosupport (Schlieren, Switzerland). All all Abs for flow cytometry (FCM) and immunofluorescence (IF) were mice experiments were conducted in accordance with ethical principles purchased from BD Biosciences and eBioscience. FcRs were generally and guidelines of the Cantonal Veterinary Office. blocked with an anti-CD16/32 Ab. To detect specific memory B cells, splenocytes were stained for differential surface expression and were characterized as positive for B220 (PE-Cy7); negative for IgD, CD4, CD8, CD11b, CD11c, and Gr-1 (all PE, PE-Cy7, or allophycocyanin-Cy7); and Qb-VLPs are derived from the bacteriophage Qb. The VLP self-assembles positive for binding to fluorescence-labeled Qb-VLP (Alexa 488). GC during the production process in Escherichia coli. Along the way, E. coli- B cells were further analyzed for binding to biotinylated peanut agglutinin by guest on September 24, 2021 derived mRNA (ssRNA) is packaged within the particle. The Qb-VLP (PNA; Vector Laboratories) and subsequent detection with streptavidin- consists of 180 subunits and forms a particulate and repetitive structure. labeled allophycocyanin-Cy7. To be able to distinguish subtypes of spe- The purification process is described elsewhere (32). cific memory B cells, B were stained for binding to PE anti- IgM or PE anti-IgG. Specific PC numbers were obtained by staining Immunization splenocytes and BM cells that were characterized as PE Cy7-B220low and Mice were immunized with 50 mgQb-VLP i.v. to generate memory B cells negative for IgD, CD4, CD8, CD11b, CD11c, and Gr-1 (all PE). Cells were that were isolated from the spleen and adoptively transferred. To challenge permeabilized using FACS lysing solution (BD Biosciences) and stained mice, 20 mgQb-VLP was administered i.v. For immunization, the VLPs for intracellular binding to labeled Qb-VLP (Alexa 488), PE anti-IgM, or were diluted with sterile PBS and injected in a volume of 150 ml into the PE anti-IgG. Surface binding was blocked by unlabeled Qb-VLP, anti- tail vein. IgM, or anti-IgG. Transferred B cells were analyzed for allotype marker (Ly5.1) expression and stained with anti-Ly5.1 allophycocyanin Ab. BrdU BrdU labeling staining was performed with the BrdU staining kit for FCM allophyco- cyanin provided by eBioscience. Qb-VLPs were labeled by fluorochrome BrdU (Sigma-Aldrich) was administered as a 0.8 mg/ml solution in the Alexa 488 according to the producer’s instructions (Invitrogen). The cell drinking water (light protected and changed every second day) for 9 d from acquisition was measured by the FACS Canto II (BD Biosciences), and the day of immunization. data were analyzed by Flow Jo version 7.6.4 (Tree Star software).

Adoptive transfer of memory B cells Cell purification and sorting a were collected from congenic Qb-VLP–immunized Ly5.1 or IgH To transfer distinct memory B cell populations, splenocytes of memory mice in DMEM supplemented with 2% FCS, antibiotics, and 10 mM mice were either purified by B220+ MACS beads (Miltenyi Biotec) or HEPES. A single-cell suspension of the splenocytes was prepared. RBCs sorted as IgM+ or IgG+ memory B cells. B220+ MACS-purified B cells were lysed by ACK buffer (0.15 M ammonium chloride, 0.01 M potassium from immunized mice were further sorted positive for B220 PE-Cy7 (as hydrogen carbonate [pH 7.2–7.4]). In general, Ly5.1+ splenocytes con- + control for MACS purification) and negative for IgM, IgD, Gr-1, CD11b, taining memory B cells were transferred into Ly5.2 C57BL/6 recipient CD11c, CD4, and CD8 (all PE) expression. This population represented mice, which allowed us to track the memory and the endogenous B cell a the CS specific and unspecific B cells. A second population that was also response. Memory B cells generated in IgH mice were transferred into + b B220 MACS purified was further sorted positive for B220 PE-Cy7 (as C57BL/6, which expresses the IgH allotype. Hence, we were able to control for MACS purification) and negative for IgG, IgD, Gr-1, CD11b, distinguish Abs resulting from the transferred memory B cells (IgHa b CD11c, CD4, and CD8 (all PE) expression. This population represented positive) or from newly activated naive B cells of the host (IgH positive). + b the specific and nonspecific IgM memory B cells. Samples were sorted Recipient mice (Ly5.2 or IgH ) received one-tenth of a donor mouse with FACS Aria. Purities of 95–98% were yielded. (Ly5.1 or IgHa)-derived memory spleen. Splenocytes were either directly transferred into recipient mice or memory B cells were further purified by Immunofluorescence MACS and/or cell sort. A number of ∼1 3 107 cells of total splenocytes containing ∼0.05–0.10% (5–10 3 103) VLP-specific class-switched (CS) IF staining was performed, as previously described (34). In short, 6- to memory B cells or ∼0.005–0.01% (500–1000) VLP-specific IgM+ memory 8-mm cryostat sections on Superfrost Plus glass slides (Fisher Scientific) B cells were transferred. The corresponding purified populations were were air-dried rehydrated in ice-cold acetone and blocked with 1% (w/v) transferred with the respective numbers found in total splenocytes, as BSA (Sigma-Aldrich) and 1% (v/v) normal mouse serum (Jackson The Journal of Immunology 5501

ImmunoResearch Laboratories). Sections were then incubated as indicated splenocytes containing memory B cells were isolated and adop- with Abs in PBS containing 0.1% (w/v) BSA and 1% (v/v) normal mouse tively transferred into naive mice expressing IgHb. As expected, serum. B cell follicles were identified with Alexa 647–labeled anti-B220. the Ab response was dominated by the transferred memory B cells GC B cells were visualized by binding to biotinylated PNA (Vector Lab- oratories). Specific B cells were detected for binding to Alexa 488–labeled (Fig. 1B). Qb. Transfer-derived B cells were stained with biotinylated CD45.1. Only small numbers of specific memory B cells are found in Biotinylated Abs were detected with Alexa 546–labeled streptavidin (Molecular Probes). Images were acquired on an Axioplan microscope recipient mice after antigenic challenge with an AxioCam MRm (Zeiss) camera using Open laboratory software The magnitude of the memory B cell response at the cellular level (Improvision) and edited with Adobe Photoshop software. was assessed next and compared with responses of naive mice. Statistics Congenic Ly5.1+ mice were immunized. After 4–6 wk, spleno- + Statistical analysis of two groups was performed with a double Student’s cytes were isolated and either directly or by B220 MACS beads + t test with Welsh’s correction. For the comparison of three groups or the purified and transferred into recipient mice (Ly5.2 ). Twenty-four kinetic analysis of two groups, the statistical analysis was performed with hours later, the mice were challenged with Qb-VLP. After indi- an F-test (ANOVA). Statistical significance is defined as p , 0.05. cated time points, we checked for the population of CS specific B cells (percentage of VLP-specific B cells within B220+, IgMlow, Results IgDlow,Gr12,CD42,CD82,CD11b2, CD11c2) within the spleen. Accelerated Ab response in the presence of memory B cells As expected, after immunization of naive mice (28, 29), up to To be able to selectively trace the response of memory B cells 25% of B cells with a CS phenotype (B220+,IgMlow,IgDlow, 2 2 2 2 2 to VLP challenge, we established an adoptive transfer model Gr1 ,CD4 ,CD8 , CD11b ,CD11c )werespecificforQb at Downloaded from (Supplemental Fig. 1). To this end, Ly5.1 congenic mice were the peak of the response (Fig. 2A, 2B). Surprisingly, the overall immunized with Qb-VLP, and 4–6 wk later either total spleno- Qb-specific B cell response was strongly reduced (∼103)inthe cytes or B220+ MACS-purified B cells were isolated and trans- presence of transferred splenocytes containing memory B cells ferred into Ly5.2+ recipient mice. After 24 h, the mice were as well as B220+ purified B cells (Fig. 2B). Reduced expansion challenged with Qb-VLP. To minimally perturb the memory B of specific host B cells (Fig. 2A) correlated with the observation

cells, we tried to limit the experimental handling of the cells as that host B cells mounted a relatively poor Ab response when http://www.jimmunol.org/ much as possible. Thus, recipient mice received either nonpuri- memory B cells were present (Fig. 1B). fied splenocytes or B220+ MACS-purified B cells. As control, To dissect the memory versus response, we analyzed splenocytes from naive Ly5.1 mice were transferred into Ly5.2 the expression of the allotype marker Ly5.1 within the specific CS 2 2 recipients. Twenty-four hours after the transfer, mice were chal- B cell population (Qb-VLP+ B220+, IgMlow, IgDlow, Gr1 , CD4 , 2 2 2 lenged with 20 mgQb-VLP. The hallmark of memory B cell CD8 ,CD11b ,CD11c ). Unexpectedly, between 70 and 90% of responses is an accelerated Ab response. Indeed, we observed Qb-specific B cells were host (Ly5.2+) and not donor (Ly 5.1+) that the humoral response at early time points was higher in derived in mice receiving splenocytes from immunized mice memory B cell (memory transfer and B220+ purified transfer) (Fig. 2C). This indicates that memory B cells do not proliferate recipient mice compared with control mice, which only received nearly as well as naive B cells and even inhibit the host primary by guest on September 24, 2021 naive splenocytes (Fig. 1A). However, the Ab titers at later time B cell response. This suppression is in agreement with a study by points (t . day 12) were comparable between mice receiving Jenkins and colleagues (13), which reported some suppression of primed or naive B cells. Memory B cells therefore accelerated the the host response in the presence of switched Ig memory B cells. early Ab response, whereas they made a minor contribution at To further analyze the proliferative capacity of memory B cells, later time points (Fig. 1A). By using congenic mice expressing we performed an in vivo pulse-chase experiment with BrdU allotype a (IgHa) instead of b (IgHb), we were able to distinguish (Fig. 2D). Mice received an adoptive transfer of either memory or the Ab production resulting from transferred memory B cells or naive splenocytes and were challenged with Qb-VLP 24 h later. from de novo activated naive B cells within the recipients. Hence, BrdU was supplied in drinking water for 9 d starting from the day mice of the IgHa strain were immunized, and, after 4–6 wk, of immunization. On day 5 after immunization, we analyzed

FIGURE 1. Transferred memory B cells dominate the early humoral response. Qb-positive memory B cells derived from immunized congenic mice (Ly5.1 or IgHa) were transferred into recipient mice (Ly5.2 or IgHb) and challenged with Qb-VLP. Mice that received an adoptive transfer of naive splenocytes served as a control and presented a primary response. (A)Qb-VLP–specific total IgG Ab titer of mice that received undepleted memory, B220+ MACS purified, or naive splenocytes. (B)Qb-VLP–specific IgG1 and IgG2a Ab titer originated of allotype a or b of mice that had received splenocytes from immunized mice containing memory B cells. Mean with SEM. Mice per group, n = 3. Data are representative of at least three independent experiments. The p values in (A) represent the significance of memory and B220-purified memory transfer to naive transfer at indicated time points. 5502 MEMORY B CELLS FORM POTENT SECONDARY PCs Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. Memory B cells do not efficiently proliferate after Ag re-encounter. Memory B cells derived from congenic immunized mice (Ly5.1+) were transferred into recipient mice (Ly5.2+) either undepleted or B220+ MACS purified and challenged with Qb-VLP. Mice that received an adoptive transfer of naive splenocytes served as a control and presented a primary response. (A) Gating strategy of FCM analysis on day 13 after immunization upon adoptive transfer. (B) Frequencies of Qb-specific B cells within the CS B cell population (B220+, IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, Gr-12). (C) Distribution of responding transferred memory B cells (donor, Ly5.1) within the CS-specific B cell population (Qb+B220+, IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, Gr-12). (D) Experimental design of the proliferation assay. Mice received an adoptive transfer on day 21 and were immunized with 20 mgQb-VLP 24 h later. BrdU labeling was performed for 9 d by providing 0.8 mg/ml BrdU in the drinking water from the day of immunization. Specific B cell responses were analyzed on day 5, a time point during the pulse phase of BrdU, and day 13, a time point of the chase phase of BrdU. (E) Percentage of Qb+ CS B cells (B220+, CD42, CD82, CD11b2, Cd11c2, Gr12) that were positive for BrdU is shown. Mean with SEM. Mice per group, n = 3. Data are representative of at least three independent experiments. The p value represents the significance of memory and B220-purified memory transfer to naive transfer. specific CS B cells (Qb+,B220+,CD42,CD82,CD11b2, Cd11b2, Whether transferred memory B cells homed to other organs Gr12) for their BrdU incorporation. The majority (∼80%) of the than the spleen, which could also contribute to secondary specific CS B cell population was positive for BrdU in both B cell responses, was addressed next. To this end, we quantified groups, indicating that most cells had duplicated in the last 5 d the number of transferred lymphocytes and specific B cells (Fig. 2E). We also analyzed the BrdU incorporation on day 13; (Supplemental Fig. 2A) in different lymphoid and nonlymph- after 4 d, BrdU labeling had been stopped. At this time point, oid organs. No transferred lymphocytes were found in the kid- ∼35% of specific CS B cells (Qb+, B220+, CD42, CD82, ney and liver (data not shown) and only a small number in CD11b2, Cd11b2, Gr12) of mice that had received an adoptive blood, BM, and lung. However, the majority of transferred transfer of memory splenocytes were still positive for BrdU. In nonspecific lymphocytes were detected in lymph nodes and the comparison, hardly any specific CS B cells of mice that had re- spleen (Supplemental Fig. 2). A small number was also found in ceived only naive splenocytes were positive for BrdU. These the lung. In contrast, the majority of specific B cells was only results clearly show that B cells in naive hosts efficiently prolif- detected in spleen (Supplemental Fig. 2C). Hence, secondary B cell erate between days 9 and 12 upon Ag encounter, whereas the responses did not occur besides the BM or secondary lymphoid number of nonproliferating cells was much higher in the presence organs and in particular the spleen, confirming that memory B cells of memory B cells. only inefficiently expanded upon antigenic challenge. The Journal of Immunology 5503 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Memory B cells inefficiently form GCs. An adoptive transfer experiment was performed to assess the ability of memory B cells entering GCs. PNA expression was assessed by FCM (A–C) and histology (D–I). (A) Gating strategy of FCM analysis of Qb-specific CS B cells expressing PNA and Ly5.1 on day 12 after challenge. (B) Distribution of PNA expression gated on Qb+B220+, IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, and Gr-12 B cells. (C) Distribution of Ly5.1 expression gated on PNA+Qb+B220+, IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, and Gr-12 B cells. (D–G) IF staining. Green: staining for Qb-VLP binding (stained with Qb-VLP–labeled Alexa 488); blue: staining with anti-B220 Ab; red: indicated at either PNA or anti- Ly5.1. (D) GC formation during a primary response on day 26 after immunization. Scale bar, 100 mm. (E and F) Simultaneous immune fluorescence staining of spleen sections for detection of donor-derived GCs. (E) Example of a host-derived GC (no signal for Ly5.1) on day 9 after Ag rechallenge. Scale bar, 100 mm. (F) Example of a unique donor-derived GC (positive signal for Ly5.1) on day 26 after Ag rechallenge. Scale bar, 100 mm. (G) Spleen sections have been stained for PNA expression indicating GC formation. Scale bar, 1000 mm. (H and I) Quantitative analysis of numbers of GCs by their PNA expression per section of spleen. (H) Quantitative analysis of numbers of GCs per section of spleen by staining for PNA expression of mice that received naive splenocytes. The total number of GCs is displayed along with the number of those GCs that were positive for Ly5.1 (donor derived). (I) Quantitative analysis of numbers of GCs per section of spleen by staining for PNA expression of mice that received memory splenocytes. The total number of GCs is displayed along with the number of those GCs that were positive for Ly5.1 (donor derived). Mice per group, n = 2 [only for (G)–(I)] or 3 of at least three independent experiments.

Induction of GC formation was assessed next by FCM and Qb+, B220+, IgMlow, IgDlow, Gr12, CD42, CD82, CD11b2, histology (Fig. 3). One half of the spleen was used for FCM and CD11c2) in the spleen. Although a significant population of PNA+ the other half for histological analysis. Congenic (Ly5.1+) mice B cells could be observed in the presence of transferred spleno- were immunized, and spleens 4–6 wk later were isolated and ei- cytes containing memory B cells, the majority of these cells were ther unpurified or B220+ MACS beads purified were transferred host derived (Fig. 3A–C). Differences were even more pronounced into recipient mice (Ly5.2+). After 24 h, the mice were challenged if GC formation was assessed by histology (Fig. 3D–I). We with Qb-VLP. The PNA expression was assessed within the spe- identified B cell follicles by B220 expression and stained for PNA cific CS B cell population (percentage of PNA expression within and Qb-VLP binding. Congenic B cells were visualized by Ly5.1 5504 MEMORY B CELLS FORM POTENT SECONDARY PCs staining (Fig. 3D–F). The onset of the GC response was delayed in mice, a sizeable population of specific PCs could be detected in mice that had received memory B cells because very few GCs are mice that received splenocytes from immunized mice when found on day 6 post-Ag challenge when compared with mice that compared with control mice (naive splenocyte transfer) (Fig. 4B). had received only naive splenocytes (Fig. 3G). The number of A large fraction of these PCs was donor (Ly5.1+) and therefore established GCs was more similar at a later time point (day 12, memory B cell derived (Fig. 4C). This indicated that transferred Fig. 3G). Noteworthily, in both experimental groups none of the memory B cells could quickly differentiate into PCs. To exclude GCs were positive for Ly5.1 expression, indicating that none of that the PC population was cotransferred during the adoptive the transferred derived B cells entered a GC reaction (Fig. 3H, 3I). transfer of unpurified splenocytes, a group of mice was left Fig. 3F shows a rare exception of a memory B cell–derived GC, unchallenged. Neither a significant specific PC population nor which was positive for the congenic Ly5.1 marker. Similar find- a humoral response could be observed in these mice, indicating ings were made by Pape et al. (13). They could observe sup- that PCs were induced in the host after immunization (data not pression in GC formation when switched Ig memory B cells were shown). present. Memory B cell–derived secondary PCs rapidly migrate Rapid differentiation of memory B cells into secondary PCs to the BM PC numbers in the spleen were enumerated by FCM in a next set of The BM is known to serve as an important survival niche for long- experiments (Fig. 4A). PCs were defined as positive for intracel- lived PCs. We therefore analyzed the BM for specific AFC numbers lular Ab specific for Qb-VLP, and negative for B220, CD4, CD8, by ELISPOT. To this end, Ly5.1+ mice were immunized and + CD11b, CD11c, and Gr1. Congenic (Ly5.1 ) mice were immu- spleens isolated after 4–6 wk. Twenty-four hours after the transfer, Downloaded from nized, spleens 4–6 wk later were isolated, and unpurified were the mice were challenged with Qb-VLP. There were strongly in- transferred into recipient mice (Ly5.2+). After 24 h, the mice were creased numbers of specific PCs (Fig. 4D) in the presence of challenged with Qb-VLP. Despite the smaller number of Qb- transferred splenocytes containing memory B cells early after specific CS B cells in the presence of splenocytes from immunized challenge, which correlated with the increased Ab titer on early http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Memory B cells preferentially differentiate into secondary PCs. Memory B cells derived from congenic immunized mice (Ly5.1+) were transferred into recipient mice (Ly5.2+) and challenged with Qb-VLP. Mice that received an adoptive transfer of naive splenocytes served as a control and presented a primary response. (A) Gating strategy of FCM analysis of Qb+ PCs on day 5 after immunization upon adoptive transfer. (B) Frequencies of Qb- specific PCs (B2202, CD42, CD82, CD11c2, CD11b2, Gr-12). (C) Distribution of responding transfer-derived PCs (donor, Ly5.1) within the specific B cell population (Qb+B2202, CD42, CD82, CD11c2, CD11b2, Gr-12). Mean with SEM. Mice per group, n = 3. Data are representative of at least three independent experiments. (D) Meta-analysis of specific PC numbers in BM by ELISPOT assay of five independent experiments. The analyzed time points vary among the five experiments, as follows: n = 6 for days 3, 5, and 9; n = 9 for day 6; n = 15 for days 12 and 20. Mean with SEM, p values derived by Mann–Whitney U test. The Journal of Immunology 5505 time points (Fig. 1). Frequencies of BM PCs early after challenge both memory B cell populations inefficiently proliferated but dif- were 5- to 20-fold increased in the presence of memory B cells, ferentiated into PCs upon challenge with viral particles. The differ- indicating that 80–95% of PCs are memory B cell derived under entiation into PCs of purified memory B cell populations was less these conditions. Thus, memory B cells rapidly differentiated into efficient compared with nonpurified memory B cells. This sug- PCs and migrated to the BM. gests that the purification procedure hampers the differentiation capacity of memory B cells. IgM+ as well as IgG+ memory B cells fail to efficiently proliferate The secondary memory B cell–derived PCs secrete more Abs It has been reported that IgM+ memory B cells preferentially As described above, memory B cells rapidly differentiated into proliferate upon antigenic challenge, whereas IgG+ memory B PCs, which could be detected in the spleen by FCM and BM by cells differentiate into PCs (24). Would that also be the case for ELISPOT. Interestingly, spot sizes of ELISPOT experiments were VLP-specific memory B cells? To address this question, we neg- strongly increased in the presence of splenocytes from immunized atively purified IgM+ and IgG+ memory B cell populations from mice containing memory B cell–derived PCs (Fig. 6A–C). This spleens of congenic immunized mice and transferred them into suggested that memory B cell–derived PCs produce increased Ly5-disparate recipient mice. Mice were challenged after 24-h amounts of Ab. To address this point quantitatively, we performed posttransfer with Qb-VLP. During primary responses, we found FCM experiments to detect intracellular binding of labeled Qb- that VLPs induced IgM+ (5–10% of total Qb+ memory B cells) VLP in PCs. Consistent with the hypothesis of higher intracel- as well as IgG+ (90–95% of total Qb+ memory B cells) memory lular Ab levels, we observed increased binding of Qb-VLP in + + B cells (data not shown). Neither IgM nor IgG memory B cells memory B cell–derived PCs compared with PCs derived from naive Downloaded from efficiently proliferated and differentiated into a sizeable pop- B cells (Fig. 6D, 6E) in both spleen and BM. To directly measure ulation of specific B cells with a CS phenotype (Fig. 5A) when levels of intracellular Abs, we performed intracellular IgG staining compared with primary responses (transfer of naive splenocytes). (Fig. 6F). These experiments confirmed increased amounts of As we already observed during transfer experiments of unpurified Ab within secondary PCs derived from memory B cells. Hence, memory splenocytes (Fig. 2C), the majority of the specific CS secondary memory B cell–derived PCs produced increased levels

B cell population upon memory B cell transfer was host derived of specific Abs compared with primary PCs derived from naive http://www.jimmunol.org/ (Fig. 5B). In addition, we observed a similar PC population be- B cells. tween the three analyzed groups (Fig. 5C). However, in contrast Taken together, we have shown that VLP+ IgM+ and VLP+ IgG+ to findings with unpurified memory splenocytes, we only found up memory B cells inefficiently expanded upon cognate Ag restim- to 25% of PCs donor derived (Fig. 5D). This indicates that ulation. Qb-specific memory B cells did not re-enter GCs, but by guest on September 24, 2021

FIGURE 5. Transfer of purified memory subtypes shows a similar proliferation and differentiation profile upon Ag challenge as unpurified memory splenocytes. Memory B cells derived from congenic immunized mice (Ly5.1+) were negatively purified and transferred as IgM+ or IgG+ memory B cells into recipient mice (Ly5.2+) and challenged with Qb-VLP. Mice that received an adoptive transfer of naive splenocytes served as a control and presented a primary response. (A) Frequencies of Qb-specific B cells within the CS B cell population (B220+ IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, Gr-12). The p value derived from two-way ANOVA analysis. (B) Distribution of responding transferred memory B cells (donor, Ly5.1) within the CS-specific B cell population (Qb+B220+, IgM2, IgD2, CD42, CD82, CD11c2, CD11b2, Gr-12). (C) Frequencies of Qb-specific PCs (B2202, CD42, CD82, CD11c2, CD11b2, Gr-12). (D) Distribution of responding transfer-derived PCs (donor, Ly5.1) within the specific PC population (Qb+B2202, CD42, CD82, CD11c2, CD11b2, Gr-12). Mean with SEM. Mice per group, n = 3. Data are representative of at least three independent experiments. 5506 MEMORY B CELLS FORM POTENT SECONDARY PCs

FIGURE 6. PCs derived from mem- ory B cells are highly potent for Qb. Congenic mice (Ly5.1+) were immu- nized, and unpurified whole splenocytes were transferred into recipient mice (Ly5.2+). Transfer of naive splenocytes served as a control. BM was analyzed for specific PCs. (A) Example of ELISPOTs on day 6 after immunization. (B) Over- all and (C) individual analysis of ELI- SPOT sizes. (D) Dot plot and histogram of intracellular binding of Qb-VLP

of PCs of mice that received memory Downloaded from splenocytes. (E) Mean fluorescence inten- sity of intracellular Qb-VLP binding of PCs in recipient mice that are either donor (Ly5.1+)orhostderived(Ly5.2+). (F) Mean fluorescence intensity of simulta- neous intracellular staining of PCs with increased binding for Qb-VLP as well http://www.jimmunol.org/ as anti-IgG. Mean with SEM. Mice per group, n = 3 or 4 [only (F)], respectively. Data are representative of three inde- pendent experiments. by guest on September 24, 2021

quickly differentiated into secondary PCs, which preferentially in which minimally manipulated specific memory B cells were homed to the BM and secreted higher levels of Abs. transferred. A number of control experiments demonstrated that results were qualitatively the same if nonpurified splenocytes, Discussion B220+ purified B cells, or isotype-switched purified memory In contrast to primary B cell responses, which are well described at B cells were transferred. Using this system, we found that mem- the cellular and molecular level, relatively little is known about ory B cells failed to efficiently proliferate or initiate GC formation. memory B cell responses. Several factors are responsible for the In contrast, to our knowledge, we describe in this study for the first problems in studying memory B cell responses. These include time the differentiation of highly effective secondary PCs derived difficulties in distinguishing primary from secondary B cell from memory B cells. responses in the same host and the presence of specific Abs that Different memory B cell subsets have been described. Classical may interfere with secondary B cell responses. The latter problem memory B cells may be best described as surface IgG+ B cells that is particularly obvious for immunization with live , in do not secrete Abs. This is in contrast to PCs that secrete Abs and which specific Abs may block replication and hence antigenic are IgG+ in the cytoplasm but not on the cell surface. Both sub- exposure. Specific Abs may, however, also inhibit secondary re- types are GC derived (35, 36) and mediate host protection for sponses by blocking access of B cells to specific Ag. To be able to a long period of time. In addition, there exist surface IgM+ track previously activated B cells, knockin mice have been gen- memory B cells exhibiting hypermutated BCRs that also persist erated that allow specific tracking of B cells that have previously over extended periods of time (11, 12). Recently, it has been expressed AID (24). Adoptive transfer of memory B cells into shown that these latter memory B cells constitute a proliferation- allotype-disparate hosts also allows selective characterization of competent pool of memory B cells that may differentiate into memory B cell responses even in the absence of specific Abs. IgG+ memory B cells upon antigenic challenge. In contrast, IgG+ This latter approach is complicated by the fact that positive selec- memory B cells rapidly differentiated into PCs upon challenge tion and in particular Ag-specific selection of B cells blocks re- (24). In an additional study, secondary B cell responses were sponsiveness of transferred B cells and that extensive manipula- primarily mediated by IgG+ memory B cells because they were tion of memory B cells may in general interfere with B cell re- better able to respond to antigenic challenge in the presence of sponsiveness. This issue is particularly worrisome because trans- specific Abs (13). Only a poor IgM+ memory B cell–mediated ferred purified memory B cells fail to extensively proliferate. To response was detected, which was attributed to the presence of avoid these problems, we established an adoptive transfer system specific Abs. After the adoptive transfer of IgM+ memory B cell The Journal of Immunology 5507 population into naive recipient mice, Pape et al. (13) could ob- Disclosures serve that those memory B cells proliferated and re-entered GCs. F.Z., J.B., A.L., P.S., and M.F.B. have options and/or stock of Cytos Bio- More than 50% of this population class-switched from the IgM to technology AG. D.M., A.F., and T.M.K. have no financial conflicts of in- the IgG subtype. In none of the studies was a quantitative cellular terest. analysis performed. With regard to the highly immunogenic model Ag Qb-VLP, we were able to track and quantify naive and memory B cell responses within one animal. In contrast to recent References findings, our adoptive transfer experiments did not reveal such 1. Parker, D. C. 1993. T cell-dependent B cell activation. Annu. Rev. Immunol. 11: multiple layers of B cell memory. In our experimental system, 331–360. 2. Banchereau, J., F. Bazan, D. Blanchard, F. Brie`re, J. P. Galizzi, C. van Kooten, both memory B cell compartments failed to extensively expand Y. J. Liu, F. Rousset, and S. Saeland. 1994. The CD40 antigen and its ligand. upon challenge with VLPs but similarly differentiated into sec- Annu. Rev. Immunol. 12: 881–922. 3. Rudge, E. U., A. J. Cutler, N. R. Pritchard, and K. G. Smith. 2002. Interleukin 4 ondary PCs. The fact that neither unmanipulated memory B cells reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc (as found in whole splenocyte populations) nor purified memory gamma RII-mediated B cell suppression. J. Exp. Med. 195: 1079–1085. B cell populations efficiently proliferated renders it unlikely that 4. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, et al. 2000. Interleukin 21 manipulation of cells was responsible for the failure of memory and its receptor are involved in NK cell expansion and regulation of B cells to divide and expand. As a control, we chose to transfer function. Nature 408: 57–63. naive Ly5.1 splenocytes into the Ly5.2-positive recipient. No 5. McHeyzer-Williams, L. J., and M. G. McHeyzer-Williams. 2004. Develop- mentally distinct Th cells control plasma cell production in vivo. 20: similar suppression of the host B cell response or, in other words, 231–242. enhanced PC and Ab response was observed when compared with 6. Rajewsky, K. 1996. and learning in the system. Nature Downloaded from 381: 751–758. the host response of recipient mice after memory B cell transfer. 7. Jacob, J., and G. Kelsoe. 1992. In situ studies of the primary immune response to A possible explanation for the discrepancies is that we used (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar highly repetitive VLPs of 30 nm diameter loaded with the TLR7/8 lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176: 679– 687. ligand RNA in this study. Such viral particles are known to drive 8. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Fo¨rster, and J. G. Cyster. T cell–independent IgM responses and are able to cause strong 2002. Balanced responsiveness to chemoattractants from adjacent zones deter- mines B-cell position. Nature 416: 94–99. and long-lasting IgG responses in the absence of adjuvant (29, 37). http://www.jimmunol.org/ 9. Blink, E. J., A. Light, A. Kallies, S. L. Nutt, P. D. Hodgkin, and D. M. Tarlinton. Hence, it is likely that viral particles are the stronger stimulus for 2005. Early appearance of germinal center-derived memory B cells and plasma memory B cells than SRBCs or soluble protein Ags used in the cells in blood after primary immunization. J. Exp. Med. 201: 545–554. 10. Allen, C. D., T. Okada, and J. G. Cyster. 2007. Germinal-center organization and previous studies. From a physiological point of view, presence of cellular dynamics. Immunity 27: 190–202. viral particles in lymphoid organs constitutes a situation that asks 11. Schittek, B., and K. Rajewsky. 1992. Natural occurrence and origin of somati- for immediate Ab production rather than extensive expansion of cally mutated memory B cells in mice. J. Exp. Med. 176: 427–438. 12. Tangye, S. G., and K. L. Good. 2007. Human IgM+CD27+ B cells: memory the memory B cell pool. This may explain why memory B cells B cells or “memory” B cells? J. Immunol. 179: 13–19. rapidly differentiate into PCs in the absence of major proliferative 13. Pape, K. A., J. J. Taylor, R. W. Maul, P. J. Gearhart, and M. K. Jenkins. 2011. events. It will be interesting to directly compare responses to re- Different B cell populations mediate early and late memory during an endoge- nous immune response. Science 331: 1203–1207. by guest on September 24, 2021 petitive Ags with those to soluble proteins. 14. Foote, J. B., T. I. Mahmoud, A. M. Vale, and J. F. Kearney. 2012. Long-term The present data indicate that, during secondary B cell responses, maintenance of polysaccharide-specific antibodies by IgM-secreting cells. J. Immunol. 188: 57–67. secondary PCs are generated, whereas naive B cells are recruited into 15. Klein, U., R. Kuppers,€ and K. Rajewsky. 1997. Evidence for a large compart- a parallel primary B cell response resulting in a new wave of memory ment of IgM-expressing memory B cells in humans. Blood 89: 1288–1298. B cells. Hence, each antigenic re-exposure may trigger both secondary 16. Bachmann, M. F., T. M. Kundig,€ B. Odermatt, H. Hengartner, and R. M. Zinkernagel. 1994. Free recirculation of memory B cells versus antigen- and primary B cell responses. In this model, the memory B cell pool dependent differentiation to antibody-forming cells. J. Immunol. 153: 3386– induced in the primary response essentially represents the Ab rep- 3397. ertoire produced in the secondary response (38). In contrast, the 17. Anderson, S. M., M. M. Tomayko, A. Ahuja, A. M. Haberman, and M. J. Shlomchik. 2007. New markers for murine memory B cells that define memory B cells generated during the secondary response are largely mutated and unmutated subsets. J. Exp. Med. 204: 2103–2114. derived from naive B cells and may therefore harbor slightly different 18. Aiba, Y., K. Kometani, M. Hamadate, S. Moriyama, A. Sakaue-Sawano, M. Tomura, H. Luche, H. J. Fehling, R. Casellas, O. Kanagawa, et al. 2010. specificities than the concomitantly produced Abs. As a consequence, Preferential localization of IgG memory B cells adjacent to contracted germinal the B cell response remains dynamic, and antigenic subspecificities centers. Proc. Natl. Acad. Sci. USA 107: 12192–12197. encountered during the primary response are not endlessly carried 19. Slifka, M. K., M. Matloubian, and R. Ahmed. 1995. Bone marrow is a major site of long-term antibody production after acute viral infection. J. Virol. 69: 1895– forward, preventing adaptation of the B cell responses to newly 1902. emerging variants. , in which the originally 20. Sze, D. M.-Y., K.-M. Toellner, C. Garcı´adeVinuesa,D.R.Taylor,and encountered influenza virus strain dominates subsequent responses I. C. M. MacLennan. 2000. Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival. J. Exp. Med. 192: 813–821. to new viral strains, would be such an example of a constraint B cell 21. Manz, R. A., M. Lo¨hning, G. Cassese, A. Thiel, and A. Radbruch. 1998. Survival response. However, although this phenomenon may be induced ex- of long-lived plasma cells is independent of antigen. Int. Immunol. 10: 1703– 1711. perimentally in animals, during natural infection of humans this does 22. Schittek, B., and K. Rajewsky. 1990. Maintenance of B-cell memory by long- not appear to be the case, and, in line with our data, the B cell response lived cells generated from proliferating precursors. Nature 346: 749–751. is always well adapted to currently circulating viral strains (39). 23. Maruyama, M., K. P. Lam, and K. Rajewsky. 2000. Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407: 636–642. Taken together, our data demonstrate rapid differentiation of 24. Dogan, I., B. Bertocci, V. Vilmont, F. Delbos, J. Me´gret, S. Storck, C. -A. Reynaud, memory B cells to powerful secondary PCs, whereas the sec- and J.-C. Weill. 2009. Multiple layers of B cell memory with different effector ondary pool of memory B cells is to a large extent derived from functions. Nat. Immunol. 10: 1292–1299. 25. Benson, M. J., R. Elgueta, W. Schpero, M. Molloy, W. Zhang, E. Usherwood, naive B cells, allowing plasticity of the memory B cell repertoire and R. J. Noelle. 2009. Distinction of the memory B cell response to cognate upon multiple antigenic exposures. antigen versus bystander inflammatory signals. J. Exp. Med. 206: 2013–2025. 26. Odendahl, M., H. Mei, B. F. Hoyer, A. M. Jacobi, A. Hansen, G. Muehlinghaus, C. Berek, F. Hiepe, R. Manz, A. Radbruch, and T. Do¨rner. 2005. Generation of Acknowledgments migratory antigen-specific plasma blasts and mobilization of resident plasma We thank A. Link, J. Bessa, and Thomas Kundig€ for carefully reading the cells in a secondary immune response. Blood 105: 1614–1621. 27. Fro¨lich, D., C. Giesecke, H. E. Mei, K. Reiter, C. Daridon, P. E. Lipsky, and manuscript; Prof. M. Groettrup and Prof. T. Brunner for helpful discus- T. Do¨rner. 2010. Secondary immunization generates clonally related antigen- sions; and G. Weiss for help with statistics. specific plasma cells and memory B cells. J. Immunol. 185: 3103–3110. 5508 MEMORY B CELLS FORM POTENT SECONDARY PCs

28. Gatto, D., S. W. Martin, J. Bessa, E. Pellicioli, P. Saudan, H. J. Hinton, and 34. Link, A., F. Zabel, Y. Schnetzler, A. Titz, F. Brombacher, and M. F. Bachmann. M. F. Bachmann. 2007. Regulation of memory antibody levels: the role of 2012. Innate immunity mediates follicular transport of particulate but not soluble persisting antigen versus plasma cell life span. J. Immunol. 178: 67–76. protein antigen. J. Immunol. 188: 3724–3733. 29. Jegerlehner, A., P. Maurer, J. Bessa, H. J. Hinton, M. Kopf, and M. F. Bachmann. 35.Good-Jacobson,K.L.,andM.J.Shlomchik. 2010. Plasticity and heteroge- 2007. TLR9 signaling in B cells determines class switch recombination to neity in the generation of memory B cells and long-lived plasma cells: the IgG2a. J. Immunol. 178: 2415–2420. influence of germinal center interactions and dynamics. J. Immunol. 185: 30. Hou, B., P. Saudan, G. Ott, M. L. Wheeler, M. Ji, L. Kuzmich, L. M. Lee, 3117–3125. R. L. Coffman, M. F. Bachmann, and A. L. DeFranco. 2011. Selective utilization 36. Shlomchik, M. J., and F. Weisel. 2012. Germinal center selection and the de- of Toll-like receptor and MyD88 signaling in B cells for enhancement of the velopment of memory B and plasma cells. Immunol. Rev. 247: 52–63. antiviral germinal center response. Immunity 34: 375–384. 37. Jegerlehner, A., T. Storni, G. Lipowsky, M. Schmid, P. Pumpens, and 31. Bessa, J., A. Jegerlehner, H. J. Hinton, P. Pumpens, P. Saudan, P. Schneider, and M. F. Bachmann. 2002. Regulation of IgG antibody responses by density M. F. Bachmann. 2009. Alveolar and lung dendritic cells sense and CD21-mediated costimulation. Eur. J. Immunol. 32: 3305–3314. RNA and drive mucosal IgA responses. J. Immunol. 183: 3788–3799. 38. Weiss, U., and K. Rajewsky. 1990. The repertoire of somatic antibody mutants 32. Cielens, I., V. Ose, I. Petrovskis, A. Strelnikova, R. Renhofa, T. Kozlovska, and accumulating in the memory compartment after primary immunization is re- P. Pumpens. 2000. Mutilation of RNA phage Qbeta virus-like particles: from stricted through affinity maturation and mirrors that expressed in the secondary icosahedrons to rods. FEBS Lett. 482: 261–264. response. J. Exp. Med. 172: 1681–1689. 33. Bessa, J., N. Schmitz, H. J. Hinton, K. Schwarz, A. Jegerlehner, and 39. Wrammert, J., K. Smith, J. Miller, W. A. Langley, K. Kokko, C. Larsen, M. F. Bachmann. 2008. Efficient induction of mucosal and systemic immune N. Y. Zheng, I. Mays, L. Garman, C. Helms, et al. 2008. Rapid cloning of high- responses by virus-like particles administered intranasally: implications for affinity human monoclonal antibodies against influenza virus. Nature 453: 667– design. Eur. J. Immunol. 38: 114–126. 671. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021