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-Bearing Follicular Dendritic Cells Deliver a Late Antigenic Signal That Promotes Somatic Hypermutation This information is current as of September 23, 2021. Yongzhong Wu, Selvakumar Sukumar, Mohey Eldin El Shikh, Al M. Best, Andras K. Szakal and John G. Tew J Immunol 2008; 180:281-290; ; doi: 10.4049/jimmunol.180.1.281 http://www.jimmunol.org/content/180/1/281 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Immune Complex-Bearing Follicular Dendritic Cells Deliver a Late Antigenic Signal That Promotes Somatic Hypermutation1

Yongzhong Wu,2* Selvakumar Sukumar,2* Mohey Eldin El Shikh,* Al M. Best,† Andras K. Szakal,‡ and John G. Tew3*

We reasoned that immune complex (IC)-bearing follicular dendritic cells (FDCs) promote somatic hypermutation (SHM). This hypothesis was tested in murine reactions induced in vitro by coculturing 6-day (4-hydroxy-3-nitrophenyl) acetyl- primed but unmutated ␭؉ B cells, chicken ␥-globulin (CGG) memory T cells, FDCs, and ICs (anti-CGG plus NP-CGG). in primed ␭؉ B cells were obtained only when both FDCs and were present. FDCs alone promoted B survival and Ab production but there were no mutations without more immunogen. Moreover, the rate was enhanced when FDCs were activated. Trapped ICs ranged from 200 to 500 Å apart on FDC membranes and this correlated with the periodicity known to optimally signal BCRs. FDCs are unique in their ability to retain ICs for months and a second signal mediated by FDC-ICs Downloaded from appeared to be needed a week or more after immunization by immunogen persisting on FDCs. However, the time needed to detect extensive SHM could be reduced to 7 days if ICs were injected together with memory T cells in vivo. In marked contrast, no mutations were apparent after 7 days in vivo if ICs were replaced by free Ag that would not load on FDCs until Ab was produced. The data suggest that specific Ab production leads to the following events: Ab encounters Ag and ICs are formed, ICs are trapped by FDCs, B cells are stimulated by periodically arranged Ag in ICs on FDCs, and this late antigenic signal promotes SHM. The Journal of Immunology, 2008, 180: 281–290. http://www.jimmunol.org/

erminal centers (GCs)4 are a distinctive architectural mice where Ig secretion, and thus the amount of ICs trapping on feature of secondary lymphoid tissues that become ap- FDCs is markedly reduced (16), calling into question the need G parent upon appropriate stimulation with Ag. The GC for ICs (at least at high levels) in promoting SHM. If IC-bearing reaction is characterized by rapidly proliferating specific B cells FDCs participate in SHM, we reasoned that it should be pos- adjacent to GC Th cells and follicular dendritic cells (FDCs) sible to directly demonstrate this in vitro where T cells, B cells, bearing immune complexes (ICs) with intact specific Ag (1, 2). ICs, and FDCs can be manipulated at will. Major events known to occur in GCs include Ig class switching, -protein conjugates are frequently used as by guest on September 23, 2021 somatic hypermutation (SHM), and selection of somatically because provide a single defined for study of SHM mutated B cells with high-affinity receptors (3–7). A variety of in a protein-specific T-dependant fashion. The hapten (4-hydroxy- studies provide rationale and support for the concept that IC- 3-nitrophenyl) acetyl (NP) conjugated to a carrier protein has been bearing FDCs promote SHM (8–14). However, more direct ev- extensively used in SHM studies and the information available on idence defining the role of FDCs in SHM is needed given that the anti-NP response is abundant. The following are characteristic some studies do not support this concept. Specifically, mature of B cells responding to NP in C57BL/6 mice: 1) they generally ␣ FDC reticula do not develop in lymphotoxin knockout mice, express the ␭ 1 L chain (17, 18), 2) typically, their BCRs are yet if the Ag dose is high enough, SHM is readily apparent in encoded by the VH186.2/DFL16.1/JH2 gene rearrangement (19– these FDC reticula-defective animals (15). The authors ac- 23), 3) Tyr96 in the BCR appears to be involved in NP binding knowledge that FDCs may be present but in the absence of (20), 4) in CDR3 tends to be limited (21, 23), reticula they were not obvious (15). Moreover, SHM occurs in and 5) replacement of Trp (W) with a Leu (L) in position 33 in the

VH region increases the affinity of BCRs expressing VH186.2/ † Ϫ ϳ *Departments of Microbiology and Immunology, Department of Biostatistics, and DFL16.1/JH2 (V␭1) by 10-fold (24). ‡ Department of Anatomy and Neurobiology, and Immunology Group, Virginia Com- To test the hypothesis that SHM is promoted by IC-bearing monwealth University, Richmond, VA 23298 FDCs, GC reactions were established in vitro by coculturing Received for publication January 26, 2007. Accepted for publication October ␭ϩ ␥ 16, 2007. NP-primed, but unmutated, day 6 B cells, chicken -glob- The costs of publication of this article were defrayed in part by the payment of page ulin (CGG)-primed memory T(CGG) cells and FDCs bearing NP- charges. This article must therefore be hereby marked advertisement in accordance CGG. After 7 days, RNA was isolated and mutations in the with 18 U.S.C. Section 1734 solely to indicate this fact. ␥ VH186.2 gene switched to C were analyzed. SHM was appar- 1 This work was supported by National Institutes of Health Grant AI-17142. ent only when B cells were stimulated with both immunogen 2 Y.W. and S.S. contributed equally to the article. and FDCs, and ICs were more stimulatory than free immuno- 3 Address correspondence and reprint requests to Dr. John G. Tew, Department of gen. We find these data exciting, because the use of IC-bearing Microbiology and Immunology, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298-0678. E-mail address: [email protected] FDCs allowed study of SHM in GC reactions in vitro with normal B cells in an Ag-specific system for the first time. In 4 Abbreviations used in this paper: GC, germinal center; FDC, follicular ; IC, immune complex; SHM, somatic hypermutation; NP, (4-hydroxy-3-nitro- vivo studies confirmed the importance of ICs in that SHM could phenyl) acetyl; CGG, chicken ␥-globulin; EM, electron microscopy; NIP, (4-hydroxy- be detected in just 7 days but only when FDCs were preloaded 3-iodo-5-nitrophenyl) acetyl. with ICs. The results suggest that the initial exposure to Ag Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 primes T cells and stimulates specific B cells to begin making www.jimmunol.org 282 FDCs AND SHM

Ab. Specific Ab binds immunogen and forms ICs and ICs are trapped by FDCs that promote the GC reaction. The results suggest that about a week after primary immunization, a second Ag signal is needed to induce SHM and this late signal appears to be delivered to GC B cells by periodically arranged ICs per- sisting on FDCs. Materials and Methods Mice, immunization, and irradiation Six- to 8-wk-old C57BL/6 mice were purchased from the National Cancer Institute, were housed in standard shoebox cages, and given food and water ad libitum. Mice were immunized with CGG 1 mo before the isolation of

T cells that would include memory T(CGG) cells as described previously (25–27). To prime B cells, 50 ␮lof200␮g/ml alum precipitated NP-CGG to- gether with heat-killed Bordetella pertussis were injected s.c. in each front leg and hind foot to stimulate and expand NP-specific B cells, which were isolated 6 days later. To activate FDCs, 25 ␮g of LPS were injected into each front leg and hind foot and mice were given 3 days for the FDCs in the draining lymph nodes to be activated. FDCs were isolated 24 h after irradiating mice with FIGURE 1. FDCs enhanced NIP-specific IgG production by B cells Downloaded from 1000 rad in a 137Cesium irradiator to kill most and enrich for isolated 6 days after primary immunization but an additional encounter FDCs (26). Animals were handled in compliance with Virginia Common- with immunogen and FDCs was required for SHM. GC reactions were wealth University Institutional Animal Care and Use Committee initiated by culturing 1 ϫ 106 unmutated but 6 day-primed B cells, guidelines. 0.5 ϫ 106 T cells, and 0.5 ϫ 106 FDCs in the presence of 100 ng of NP Abs and reagents (36)-CGG as free Ag or in ICs. The contents in each culture are indi- cated across the bottom and after 7 days of culture, supernatant fluids Rat anti-mouse FDC (FDC-M1), biotin mouse anti-rat ␬ (MRK-1), anti- were collected for NIP-specific IgG assays and cell pellets were col- http://www.jimmunol.org/ mouse CD21/CD35 (clone 7G6), and anti-mouse CD32/CD16 ( lected for RNA extraction. A, NIP-specific IgG production; B, muta- 2.4G2) were purchased from BD Biosciences. Mouse CD45R (B220) tions per 1000 bases of the V 186.2 clones recovered from the same MicroBeads, mouse CD90 (Thy1.2) MicroBeads, anti-biotin MicroBeads, H cultures as in A. The rate of mutations per 1000 bases in each of the six and MACS LS columns were purchased from Miltenyi Biotec. Biotin- labeled rat anti-mouse ␬ was purchased from Zymed Laboratories. Al- conditions illustrated in this figure was calculated after analyzing: 10, kaline phosphatase-labeled goat anti-mouse IgG (HϩL) and P-nitro- 13, 20, 10, 14, and 14 VH186.2 clones, respectively. The data from phenylphosphate substrate were obtained from Kirkegaard & Perry these clones representing each of the six conditions were used to look Laboratories. OVA conjugated with (4-hydroxy-3-iodo-5-nitrophenyl) for significant ␹2 values. The statistical analysis indicated that FDCs acetyl (NIP-OVA) and NP(36)-CGG were obtained from Biosearch plus Ag or FDCs plus IC with primed B cells and memory T cells (final

Technologies. Anti-CGG was obtained from hyperimmunized mice two conditions) induced more mutations than found in any other con- by guest on September 23, 2021 with serum anti-CGG IgG levels in excess of 1 mg/ml. dition (p Ͻ 0.01). Other controls included replacing CGG-primed T Isolation of FDCs cells with irrelevant T cells and NP-CGG-anti-CGG with OVA-anti- OVA ICs. The SHM that is so readily apparent with NP-CGG-anti-CGG FDCs were isolated as previously described (28). Briefly, lymph nodes and CGG-primed T cells was not obtained in these negative controls. from lethally irradiated mice were gently digested and the cell suspension The data in this figure were from a single experiment but the results are ␬ incubated with rat anti-mouse FDC-M1, biotin-mouse anti-rat (clone representative of three experiments of this type. MRK-1), and anti-biotin magnetic microbeads sequentially. Finally, the FDCs were positively selected using a magnetic column (MACS). Approx- imately 85–95% of these cells expressed the FDC phenotype, FDC-M1ϩ, CD32ϩ, CD21/35ϩ, CD40ϩ, and ICAMϩ (28). ␭ Analysis of mutations in the V region Isolation of B cells and T(CGG) cells The anti-NP response predominantly uses the VH186.2 gene (20–23) and it Single-cell suspensions were prepared from lymph nodes of mice primed was analyzed in this study to assess . Cells were collected for 6 days with NP-CGG. After depleting ␬ϩ cells with biotin-anti ␬ and ␭ϩ on day 7 of culture and RNA was isolated using TRIzol (Invitrogen Life anti-biotin microbeads using a MACS column, the B cells were isolated Technologies). The RNA was then reverse transcribed to cDNA with with anti-B220 microbeads using MACS. To obtain memory T(CGG) cells, GeneAmp Gold RNA PCR Core kit (Applied Biosystems) using oligo dt. lymphocytes from mice immunized with CGG were incubated with 40 ␮l The VH186.2 gene followed by D and J regions and a proximal segment of of anti-CD90.2 microbeads and T cells were selected using MACS. For the C ␥ gene segment was amplified by two rounds of nested PCR with experiments in Fig. 5, negative selection was used to avoid tagging the B high-fidelity Pfu-Ultra DNA polymerase (Stratagene). The forward and cells or T cells with Ab that might promote phagocytosis or complement- ϩ reverse primers for the first-round PCR, were 5Ј-catgctcttcttggcagcaa mediated lysis in vivo. The ␭ B cells from naive mice were isolated by Ј Ј Ј Ј ␬ϩ ␬ cagc-3 and 5 -gtgcacaccgctggacagggatcc-3 , for second-round PCR, 5 - depleting cells with biotin-anti and anti-biotin microbeads and T cells caggtccaactgcagccag-3Ј and 5Ј-agtttgggcagcaga-3. The PCR was con- with anti-CD90.2 microbeads. The T(CGG) cells were obtained by depleting ducted as follows: 95°C 1 min, 56°C 1 min, 72°C 1 min for 30 cycles B cells with anti-B220 microbeads. followed by 72°C for 10 min. The expected 400-bp PCR product was In vitro GC reactions and the anti-NIP Ab response then electrophoresed on 1.2% agarose gel, purified by gel extraction using QIAQuick kit (Qiagen) and cloned using the Zero Blunt TOPO In vitro GC reactions were initiated by culturing 1 ϫ 106 unmutated but PCR Cloning kit (Invitrogen Life Technologies). The plasmids were 6-day primed B cells, 0.5 ϫ 106 T cells, and 0.5 ϫ 106 FDCs in the transformed into one-shot TOP10 chemically competent cells (Invitro- presence of 100 ng of NP (36)-CGG as free Ag or ICs (NP-CGG plus gen Life Technologies) and plated onto Luria-Bertani medium contain- anti-CGG). Mutations in NP-stimulated B cells occur after day 6 (10, 23, ing 50 ␮g/ml ampicillin. Multiple discrete colonies were picked the 29) and we confirmed this in multiple experiments. The ICs were made by following day and grown for 24 h in Luria-Bertani broth containing 50 incubating NP-CGG with anti-CGG at a ratio of 6 ng/ml anti-CGG to 1 ng ␮g/ml ampicillin in a 37°C shaker. The plasmids were isolated using a of NP-CGG for 2 h and then adding IC containing 100 ng of NP-CGG to QIAprep Spin miniprep kit (Qiagen) and the insert was sequenced using appropriate wells in 24-well culture plates. Supernatant fluids were har- an ABI sequencer. The sequences were analyzed using the GCG Wis- vested 7 days later and were assayed for NIP-specific IgG Ab by ELISA consin Package (Accelrys), ClustalW (30, 31), and BioEDIT (www. using NIP-OVA. mbio.ncsu.edu/BioEdit/bioedit.html) software packages. The Journal of Immunology 283

␭ϩ Table I. Sequences from B cells stimulated with Ag or IC and normal FDCs (N-FDCs) with T(CGG) cells (data corresponds with Fig. 1) Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

Redundant sequences were excluded from our calculation of mutations 0.1 M cacodylate buffer for 90 min, dehydrated in a graded series of eth- per 1000 bases to avoid multiplying the effect of a single mutation. To anols, infiltrated, and embedded in PolyBed 812 epoxy resin. Sections were avoid including N-terminal additions, changes within five bases of the V-D cut using a LKB 2128 ultramicrotome to a thickness of ϳ800 Å, and junction were also excluded from our calculations of mutations per 1000 mounted on slotted grids. Sections were left unstained with uranyl acetate bp. The background rate of mutation detected using this system was low and lead citrate for better detection of the HRP labeling. Thin sections were Ͻ ( 0.1 mutations per 1000 bases in over 100 control sequences analyzed). studied with a JOEL electron microscope at 50 kV for cytochemical la- In addition, the C region of 24 sequences with mutated VH186.2 genes beling to increase contrast on unstained sections. were examined and no mutations were found. In short, error attributable to random mutation or polymerase error was very low. Adoptive transfer experiment Immunohistochemistry Recipients were irradiated with 600 rad under the maximum scatter con- Draining lymph nodes (popliteal, brachial, and axillary) from mice adop- 137 ␭ ditions by using a Cs source (Mark I, Model 68-0146; J. L. Shepherd and tively transferred with naive B cells and T(CGG) cells and stimulated with Associates). This dose is sufficient to arrest populations but Ag or ICs were harvested and flash frozen in Optimum Temperature Com- leave functional populations of radio-resistant FDCs (32). Twenty-four pound (Tissue-Tec). Serial 10-␮m-thin sections were cut from the frozen hours after irradiation, the mice were reconstituted by injecting 2.5 ϫ 106 blocks using a Jung Frigocut 2800E cryostat and fixed in acetone. The ␭ϩ ϫ 6 naive B cells and 2.5 10 memory T(CGG) cells in each hind foot or sections were then rehydrated and incubated with IgM rat anti-GL-7 Ab ϩ front leg. The ␭ B cells for these experiments were obtained by negative (eBioscience), followed after washing by HRP-conjugated mouse anti-rat ϩ selection by removing T cells and ␬ B cells. These mice were divided into IgM (Southern Biotechnology Associates). The HRP was then developed two groups with one group of three mice receiving 5 ␮g of NP-CGG in using a diaminobenzidine substrate (Biomeda). Digital pictures were taken preformed ICs in the hind feet and front legs (ICs were made with 20 ␮g using an Olympus BH-2 microscope with an Optronics image acquisition ␮ of NP-CGG plus 120 g of anti-CGG incubated in vitro for 2 h). The device. Morphometric analysis was performed using BioQuant Nova as ␮ control group of three mice received 5 g of NP-CGG in each hind foot described previously (33). and front leg. One week later, serum and draining lymph nodes were har-

vested and serum Ab levels and mutations in the VH186.2 gene segment in the ␭ϩ B cells were determined. Statistical analysis Electron microscopy The number of mutations per sequence was compared using a Poisson Purified FDCs labeled with HRP anti-HRP ICs were fixed with 1% para- regression analysis (SAS Proc Genmod version 9.1; SAS Institute) An formaldehyde plus 0.87–0.9% glutaraldehyde (electron microscopy grade; offset of log306 was used to normalize the fitted group means to the number Electron Microscopy Sciences) in 0.1 M cacodylate buffer then the labeling of nucleotides. Significant ␹2 values were determined at p Ͻ 0.05. Analysis was developed for peroxidatic activity using a Diaminobenzidine Cobalt of GC numbers, area, and IgG in Fig. 5 were done using a two-tailed Ͻ Chromogen kit (Biomeda). The specimens were postfixed in 1% OsO4 in Student t test and significance was accepted at p 0.05. 284 FDCs AND SHM

␭ϩ Table II. Amino acid sequences from B cells stimulated with Ag or IC and normal FDCs (N-FDCs) with T(CGG) cells (data corresponds with Fig. 1) Downloaded from http://www.jimmunol.org/

Results SHM in the presence of FDCs and ICs in vitro

SHM in vivo To begin testing the hypothesis that IC-bearing FDCs promote by guest on September 23, 2021 The V 186.2 gene segment switched to Ig␥ was analyzed using SHM, GC reactions were elicited in vitro using FDCs, CGG- H ␭ϩ ␭ϩ our reagents to confirm that mutations are not apparent 7 days after primed memory T(CGG) cells, and B cells. The B cells were immunizing mice with NP-CGG but that most segments are mu- tated 14 days after in vivo challenge (data not shown) (10, 23, 29). A rate of 9 mutations/1000 bases was found at day 14 and this is consistent with published results (8.4–15 mutations/1000 bases) (23, 34).

FIGURE 3. Activation of FDCs enhanced SHM. FDCs express TLR-4 and 3 days after injection of LPS, the levels of molecules known to pro- mote FDC accessory activity including ICAM-1, VCAM-1, and Fc␥RIIB were significantly elevated (37). GC reactions were set up as described in Fig. 1 except the FDCs were activated. The contents in each culture are indicated at the bottom of the figure and LPS was used as a control in the second set of cultures at 25 ␮g/ml. The rate of mutations per 1000 bases in FIGURE 2. Clonal relatedness of sequences suggesting ongoing muta- each of the five conditions was calculated after analyzing: 10, 7, 18, 20, and tion in in vitro GC reactions. A phylogram was deduced from independent 14 clones, respectively. The data from these clones were used to look for sequences derived from cultures set up with B cells, T cells, FDCs, and ICs significant ␹2 values in statistical analysis. The analysis indicated that both (BTFDCIC) using the ClustalW program from the European Molecular Ag and IC stimulated more mutations with LPS-activated FDCs than nor- Biology Laboratory. The first 14 sequences are listed in Table I and addi- mal FDCs (p Ͻ 0.01). It was also clear that ICs, which would load on FDCs tional clones were sequenced yielding 7 more sequences to extend this immediately, stimulated better than Ag that would be converted into ICs analysis to 21. over time as Ab was produced (p Ͻ 0.01). The Journal of Immunology 285

␭ϩ Table III. Sequences from B cells stimulated with Ag or IC and activated FDCs (A-FDCs) with T(CGG) cells (data corresponds with Fig. 3) Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

obtained from mice immunized 6 days earlier with NP-CGG. hapten was interacting with the newly produced anti-NIP. The 6 days allowed NP-specific ␭ϩ B cells to clonally expand, Moreover, in marked contrast with all previous conditions, nu- class switch, and to begin producing IgG anti-NIP under phys- merous mutations were obtained upon second exposure to im- iological conditions in vivo. However, as shown by others (10, munogen (Fig. 1B, columns 5 and 6). All sequences sampled 23, 29) and confirmed here, these ␭ϩ B cells taken 6 days after were germline until immunogen was added with the FDCs and primary immunization are not mutated. However, somatic mu- the 3–5 mutations per 1000 bases with immunogen was signif- tation would be one of the next major events in a GC reaction. icantly different from total lack of mutations in all previous These 6-day B cells were cultured for 7 additional days in vitro conditions ( p Ͻ 0.01; by ␹2). The numbers of clones sequenced and the production of specific anti-NIP Ab and mutations in the for each culture condition and negative controls including FDCs

VH186.2 gene segment were determined (Fig. 1). In the absence with irrelevant ICs and irrelevant T cells are described in the of FDCs, the anti-NIP IgG levels were low (20–25 ng/ml; Fig. legend to Fig. 1. 1A) and sequences were germline after the 6 days in vivo and 7 B cells do not survive well in the absence of FDCs and FDCs days in vitro (Fig. 1B, columns 1–3). Addition of FDCs to ac- produce molecules including activating factor belonging to tivated B cells and memory T(CGG) cells dramatically increased the TNF family (BAFF) that promote B cell survival (35). Ap- anti-NIP production (Fig. 1A, column 4), but did not promote proximately 80% of our B cells survive for a week in the presence SHM as indicated by a lack of mutations (Fig. 1B, column 4). of FDCs compared with only 20% in the absence of FDCs (36). In vivo, specific Ab production leads to IC formation followed We reason the low levels of Ab in cultures lacking FDCs is, at least by IC trapping and retention on FDCs in GC light zones. To in part, a reflection of a loss in B cells. Nevertheless, the critical model this in vitro, Ag (NP (36)-CGG) or ICs were added to the comparison is with cultures containing FDCs in the absence of T and B cells as a source of immunogen and this addition cor- specific immunogen and cultures with FDCs plus immunogen. The related with a reduction in free anti-NIP levels persisting in addition of FDCs gave a major boost in Ab production that correlated cultures with FDCs (Fig. 1A, columns 5 and 6) suggesting the with increased B cell survival but no mutations. In marked contrast, 286 FDCs AND SHM

␭ϩ Table IV. Amino acid sequences from B cells stimulated with Ag or IC and activated FDCs (A-FDCs) with T(CGG) cells (data corresponds with Fig. 3) Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 the addition of FDCs plus immunogen resulted in SHM. We exam- lates with increased accessory activity as indicated by a 2- and ined B cell survival in the presence of FDCs with specific and non- 10-fold increase in specific IgG production (37). If FDC molecules specific ICs and without immunogen and were unable to detect any promote SHM, then activated FDCs should have enhanced ability difference in B cell number or viability as long as FDCs were present. to promote SHM. To test this prediction, LPS-injected mice were

The specific nucleotide changes in the mutated VH186.2 gene given 3 days for FDCs in the draining lymph nodes to be activated segment presented in Fig. 1 are indicated in Table I and the and then FDCs were isolated, which involves multiple washes resulting changes in amino acid sequences are indicated in Ta- which should remove any persisting LPS. Nevertheless, to control ble II. In cultures stimulated with FDC plus IC, there were 14 for carryover, we added LPS to selected cultures to determine independent clones analyzed and these clones contained 21 mu- whether we could find an LPS effect (Fig. 3, column 2). LPS has tations. Among the 21 mutations, 17 were unique mutations not been found to promote SHM (38) and that was confirmed in distributed over 16 different positions in the V region and all these studies. Like normal FDCs (Fig. 1), activated FDCs failed to mutations were substitutions and not the consequence of inser- promote SHM in the absence of second exposure of B cells to tion or deletion. As expected the mutations show a bias for immunogen (Fig. 3, column 3). However, addition of Ag doubled transition (76%) over transversion (24%), and AGT/C (this usu- the frequency of mutations found with normal FDCs ( p Ͻ 0.01) ally represented as RGYW) intrinsic hot spots for somatic mu- and preformed ICs yielded ϳ12 mutations/1000 bases at day 13 (6 tations were found 10 times. Moreover, these point mutations days in vivo and 7 days in vitro) which compared favorably with were concentrated in CDRs (62%) rather than framework re- the 9 mutations/1000 bases obtained in vivo at day 14. Tables III gions (38%). As expected, changes in amino acid sequence and Table IV illustrate the alterations in the VH186.2 gene segment were apparent in framework regions in Table II but changes and alterations in amino acids, which are similar to those shown in were most frequent in CDRs 1 and 2. Clone N-FDCIC 8 has Tables I and II except changes are more frequent. tryptophan replaced with leucine in position 33 and that shift is known to be associated with higher affinity anti-NP (24). In ICs on FDCs are periodically arranged addition, a phylogram was deduced from sequences obtained Immunogens in ICs on FDCs induce more potent Ab responses from the cultures with FDCs and ICs (Fig. 2). Note the clonal than free Ag plus FDCs (39, 40) and ICs with FDCs were more relatedness of some sequences suggesting ongoing mutation in potent stimulators of SHM than free Ag as illustrated in Figs. 1 and the cultures. 3. Repeating are known to simultaneously engage multi- ple BCRs and promote B cell activation (41). Moreover, - Activated FDCs are potent inducers of SHM independent GCs that include low levels of hypermutation can be We recently reported that FDCs express TLR4 and that LPS up- obtained when multiple NP epitopes are conjugated to Ficoll regulates many FDC molecules. Moreover, FDC activation corre- (42). Studies of DNP on polymers indicate that epitopes spaced The Journal of Immunology 287 Downloaded from

FIGURE 5. Comparison of Ag with ICs on induction of SHM in vivo. FIGURE 4. Periodicity of IC trapping by FDCs in vitro. FDCs were Mice were irradiated with 600 rad and reconstituted with negatively se- ␭ϩ lected naive B cells and memory T(CGG) cells. These mice were divided

incubated overnight with HRP ICs, washed, and prepared for transmission http://www.jimmunol.org/ EM. A, An FDC not stained with uranyl acetate and lead citrate to better into two groups with one receiving 5 ␮g of NP-CGG in preformed ICs in illustrate the labeling with HRP around the entire cell. The area in the the hind feet and front legs. The control group received 5 ␮g of NP-CGG ϩ rectangle is shown at higher power in B illustrating the periodic distribution in each hind foot or front leg. After 7 days, ␭ B cells were isolated from of the HRP ICs. This is unique to ICs as HRP labeling of FDCs with lymph nodes and analyzed for VH186.2 mutations. A, NIP-specific IgG FDC-M1, ICAM-1, or VCAM-1 may be patchy but a constant periodicity measured by ELISA in three mice per group and the error bar represents has not been noted. SD and the differences are statistically significant (p Ͻ 0.01) and the data are representative of two experiments. B, The number of mutations per ␭ϩ 1000 bases of VH186.2 clones sequenced. The B cells from the draining 120–670 Å apart are optimal for BCR cross-linking and B cell acti- lymph nodes of the three mice were pooled and RNA extracted. We ana- vation (43). Studies with scanning and transmission electron mi- lyzed 10 clones in each condition and the difference in mutation frequency by guest on September 23, 2021 croscopy (EM) indicate that ICs trapped in vivo are organized on was statistically significant (p Ͻ 0.01). C and D, Representative illustra- ␮ FDC dendrites with an orderly spiraling periodicity of 440–490 Å tions of 10- m-thin sections of draining lymph nodes from the two groups of mice labeled with anti-GL7 to identify GC B cells. E and F, Cumulative (44). To determine whether ICs trapped by FDCs in vitro are pe- data representing total number of GCs and area of GCs per midsagittal riodically arranged, FDCs were incubated overnight with ICs using section of all six mice challenged with Ag or IC. HRP as Ag, washed, and prepared for transmission EM (Fig. 4). Note that the HRP IC labeling of the plasma membrane is easily seen because the cell was not stained with uranyl acetate and lead mice produced more NIP-specific Ab than mice injected with

citrate that would have made HRP more difficult to see because free Ag (Fig. 5A). Moreover, mutations in the VH186.2 gene these stains increase the electron density of cell structures (Fig. segment from the IC-injected mice were at a frequency of 12 4A). The area in the rectangle in A is shown at higher power in Fig. mutations/1000 bases (Fig. 5B). In marked contrast, no muta- 4B and the results revealed a periodic arrangement of Ag trapped tions were found after 7 days in clones from control mice that in vitro by FDCs in a range between ϳ200 and 500 Å. This pe- were given memory T cells and free Ag (Fig. 5B). This fre- riodic distribution of epitopes is well within the 120–670 Å known quency of 12 mutations/1000 bases in just 7 days is comparable to optimally stimulate B cells (43). with results obtained 14 days after a typical primary immuni- zation (23, 34). Furthermore, immunohistochemistry revealed ICs promote SHM in vivo robust GCs in IC-injected mice as evidenced by GL7 labeling of Our in vitro data (Figs. 1 and 3) indicated that IC-bearing FDCs GC B cells (Fig. 5, C and D). Morphometric analysis indicated promoted SHM better than free immunogen ( p Ͻ 0.05 for both that both the number and area of GL7-positive GCs were higher experiments with the additional sequences used in Fig. 2 in- in draining lymph nodes from sites injected with ICs (Fig. 5, E cluded in the data in Table I) and we sought to confirm the and F). Mice injected with Ag would be expected to have mu- importance of ICs in vivo. We reasoned that several days would tations by 14 days but they were not apparent by 7 days. In be required to induce specific Ab and produce ICs for FDCs to short, the time needed for extensive SHM was reduced by about ϩ trap and that IC-bearing FDCs would be the rate-limiting factor a week when naive ␭ B cells were challenged in vivo with Ag for SHM in vivo. To test this hypothesis, irradiated mice were in ICs that would immediately localize on FDCs. reconstituted with naive ␭ϩ B cells together with memory

T(CGG) cells and given either preformed ICs or NP-CGG alone Discussion for a control. Serum and ␭ϩ B cells were harvested 7 days after The concept that IC-bearing FDCs promote GC reactions and play immunogen challenge in vivo and NIP-specific serum Ab levels an important role in promoting SHM is supported by a number of

and mutations in the VH186.2 gene segment were determined. studies (8–14) but not all (15, 16). Demonstrating SHM with nor- As expected on the basis of previous work (39), IC-injected mal B cells in vitro, where cell types can be readily manipulated, 288 FDCs AND SHM

FIGURE 6. Kinetics of events that promote SHM in GC reactions. Events in the first panel occur in the first 4 days after Ag challenge. Dendritic cells

(DCs) trap Ag and transport it into the deep cortex, where they complete Ag processing and prime naive T cells emerging from the high endothelial venules Downloaded from (HEVs). Specific B cells in afferent lymph and emerging from HEVs also interact with Ag. As soon as specific T cells are primed, they provide help for specific B cells to begin making Ab (AbY; see arrows) detectible 3–4 days after Ag challenge. This specific Ab begins to form ICs which fix complement and these ICs are readily cleared by (M␾) particularly in the medullary sinuses (MS) and subcapsular sinuses (SS). In addition, some ICs are trapped by FDCs that attract B and T cells into the follicles. In the 4- to 7-day period (second panel), IgG production expands, illustrated by plasma cells migrating toward medullary cords, and clearance of ICs or “immune elimination” continues. FDCs trap more ICs including IgG-ICs and the FDC associated ICs promote GC reactions with dark and light zones (light green in panel 2). The GC reactions lead to enhanced Ig class switching and more IgG production.

By day 7, soluble Ag has flowed through the node or has been cleared by except for Ag persisting in ICs trapped on FDCs in GC light zones. http://www.jimmunol.org/ In the 7- to 14-day period (panel 3), a strong Ag signal is delivered to GC B cells by periodically arranged ICs on FDCs that promotes SHM leading to cells with higher affinity BCRs that compete for limited Ag persisting on FDCs. These mutated cells are induced to produce high-affinity IgG important in affinity maturation. has been challenging. In the present study, we sought to set up GC cells internalize, degrade, and clear ICs with a T1/2 in macro- reactions in vitro where the hypothesis that FDCs promote SHM phages of ϳ30 min (45), ICs trapped by FDCs are retained for could be tested under defined conditions. We recently refined months to years (45). This unique ability to retain intact surface- methods for FDC isolation (28) and found that FDCs bearing the bound Ag (in the form of ICs) in GC light zones makes FDCs an by guest on September 23, 2021 appropriate ICs promote activation-induced attractive candidate to deliver an antigenic signal to GC B cells a production, Ig class switching, and affinity maturation by NP- week or more after the initial immunization. Moreover, ICs on specific B cells (25). These results prompted this study to deter- FDCs were arranged periodically between ϳ200 and 500 Å apart mine whether FDCs promote SHM in GC reactions in vitro. The and were more potent than free Ag in their ability to stimulate results reported here indicate that, over a 13-day period, B cells SHM (Figs. 1 and 3). primed in vivo can be stimulated with activated IC-bearing FDCs Mutations in the in vitro GC reactions had numerous similarities (37) in vitro and exhibit SHM at rates comparable with those seen with mutations observed in vivo. Specifically, sequences from cul- in vivo. In brief, the data suggest that the initial Ag exposure tures containing IC-bearing FDCs accumulated numerous muta- primes T cells and stimulates specific B cells to begin making tions in the VH186.2 region while the IgG C region remained un- specific Ab that interacts with Ag to produce ICs. Most ICs will be changed (data not shown). An increased rate of mutation in CDR efficiently trapped and eliminated by phagocytic cells but some are 1 and 2 is typical of in vivo data (46) and suggests a selective trapped by FDCs in GC light zones and SHM is promoted in GC pressure and the same trend is apparent in the in vitro data reported B cells restimulated by Ag in FDC-ICs after day 6. The sequence here. Evidence for selection in the in vitro GC reactions is also of these events is illustrated and summarized in Fig 6. suggested by the observation that changes at the VD junction in Passive transfer studies indicate that it is important for ICs to be control sequences were never found (B cells alone, B and T cells made using IgG to promote SHM (13). SHM is typically not ap- plus Ag or ICs, B and T cells plus FDCs without Ag). However, parent in the first week after primary immunization (10, 23, 29) changes at the VD junction in cultures with IC-bearing FDCs were and we reason that IgG Ab production, IgG-IC formation leading found, especially with activated FDCs (see Tables I and III). to trapping by FDCs, takes time and helps explain the delay. As Changes at the VD junction included multiple bases in sequence illustrated in Fig 5, SHM can be shifted into the first week if and these alterations likely reflect rare B cells with end-region specific ICs and specific T cells are injected into normal mice but changes that were selected and expanded by Ag on FDCs. Tyr95 not by specific T cells alone. Thus, the rate-limiting step appears to appears to be important for NP binding (20); tyrosine was main- be IgG production. We think it is important to note that, in the tained or replaced by phenylalanine which is a conservative absence of a second exposure to Ag, no mutations were apparent change that has been found in anti-NP responses (29). We also in B cells (after 6 days in vivo and 7 days in vitro) even though Ab reasoned that leucine at position 33 would be selected in the in production was abundant in the presence of FDCs. Taken together, vitro GC reaction but it was rarely observed and this is not under- the data suggest that class switch recombination leads to IgG pro- stood. However, a lack of leucine at position 33 was obtained in duction and IC formation and that IgG-ICs trapped by FDCs de- vivo when SCID mice were injected with T and B cells and given liver a follow-up antigenic signal that promotes SHM in conven- ICs (47) and the same result was obtained when we injected ICs in tional immune responses. Although macrophages and dendritic our in vivo study (data not shown). Remarkably, the tryptophan to The Journal of Immunology 289 leucine replacement at position 33 is not found during the late periodically between ϳ200 and 500 Å apart on FDC membranes stages of immune responses when the selection process should be that have trapped ICs in vitro. Repeating epitopes are known to virtually complete and Ab affinity is highest (29). ICs given at the simultaneously engage multiple BCRs and promote B cell activa- beginning of an experiment may result in levels of ICs on FDCs tion (41). Studies of DNP on polymers indicated that epitopes that are comparable with FDC-ICs at later stages of a normal im- spaced 120–670 Å apart are optimal (43) and the range of 200– mune response. This raises the possibility that sequences with tryp- 500 Å found with ICs trapped by FDCs in this in vitro study fits tophan at 33 may be favored by mature levels of FDC-ICs result- that periodicity. Moreover, T cell-independent GCs can be ob- ing in a rapid transition through the leucine stage. The observation tained when NP is conjugated to Ficoll which should allow simul- that leucine at position 33 was only occasionally found in the taneous engagement of multiple BCRs and that arrangement in- present study when ICs developed rapidly in culture or preformed cludes low levels of hypermutation (42). Similarly, SHM could be ICs were given would be consistent with this interpretation. demonstrated in an in vitro culture system by cross-linking BCRs A number of FDC molecules are thought to be involved in GC and providing T cell help (38). The periodic organization of ICs on reactions, including CXCL-12 and CXCL-13 that attract GC T and FDCs should also allow immunogenic epitopes to be arranged in a B cells, IL-6 that promotes terminal B cell differentiation, CD21L repeating pattern much like a T-independent Ag and facilitate and CD320 that promote Ab production, Fc␥RIIB that minimizes cross-linking of BCRs. In short, extensive interaction between the ITIM signaling in B cells, and ICAM and VCAM that promote B cell membrane and long FDC dendrites bearing epitopes that adhesion between FDCs and B cells (26, 39, 48–50). How signif- simultaneously engage numerous BCRs likely provides a potent icant these molecules are in promoting SHM remains to be deter- signal that would not be delivered by free Ag. This periodic ar- mined. However, the in vitro model where GC reactions are easily rangement of epitopes on FDCs may explain why ICs were more Downloaded from manipulated should be useful for such studies. Given that blocking potent immunogens than free Ag. Adoptive transfer experiments FDC-CD21L-B cell CD21 interactions inhibit Ab and activation- confirmed that IC-bearing FDCs were important for optimal GC induced cytidine deaminase production (25), we thought that FDC- reactions and SHM was obtained in just a week. In short, use of CD21L would likely be important for SHM, and that was tested by FDCs to model GC reactions in vitro made it possible to demon- blocking FDC-CD21L-CD21 interactions but blocking did not strate SHM in Ag-stimulated B cells in vitro. The results suggest

have a significant effect (data not shown). Nevertheless, lack of that induction of specific Ab leads to IC formation, ICs being http://www.jimmunol.org/ inhibition in this study corresponds with reports of SHM occurring trapped by FDCs, GC B cells being stimulated by Ag in these normally in CD21 knockout mice that also have reduced Ab re- FDC-ICs after about a week in vivo, and this late second Ag signal sponses (51, 52). It is also known that FDCs are activated as a leads to the promotion of SHM. consequence of interacting with ICs (53) and the need for ICs could reflect the need for FDC-Fc␥RII occupancy to produce fac- Acknowledgment tors needed to sustain mutating B cells that have been adequately We thank Judy Williamson for preparing EM specimens, cutting sections signaled in vivo. However, if SHM has initiated in vivo and the for EM, and assistance in transmission EM. need for ICs is to properly activate FDCs via FcR engagement, then irrelevant ICs should work as well as relevant ICs. However, Disclosures by guest on September 23, 2021 cultures with FDCs stimulated with either irrelevant ICs (OVA The authors have no financial conflict of interest. anti-OVA) or containing irrelevant T cells did not induce the SHM that is so readily apparent with relevant ICs and relevant T cells. References The data reported here are most compatible with the interpretation 1. Szakal, A. K., M. H. Kosco, and J. G. Tew. 1989. Microanatomy of lymphoid tissue during the induction and maintenance of humoral immune responses: struc- that the B cell population needs stimulation by specific Ag and ture function relationships. Annu. Rev. Immunol. 7: 91–109. specific T cells late in the primary response for optimal SHM. The 2. Tew, J. G., T. E. Mandel, and A. W. Burgess. 1979. 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