antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin

0014-2980/00/0808-2226$17.50+.50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

Antigen persistence is required for somaticmutation and affinity maturation of immunoglobulin

Yang Wang1, Guangming Huang2, Jing Wang1, Hector Molina2, David D. Chaplin2 andYang-Xin Fu1

1 Department of Pathology, The University of Chicago, Chicago, USA2 Department of Internal Medicine, Howard Hughes Medical Institute, Washington University

School of Medicine, St Louis, USA

Whether germinal centers (GC) with follicular dendritic cell (FDC) clusters are the essentialsites for affinity maturation of immunoglobulin is still controversial. To re-evaluate the roleof GC/FDC in affinity maturation and somatic mutation in a defined antigen system,lymphotoxin- § –/– and TNF receptor I–/– mice, lacking GC/FDC, were immunized with (4-hydroxy-3-nitrophenyl) acetyl-sheep RBC (NP-SRBC). In contrast to soluble hapten-carriersystems, NP-SRBC allows us to compare affinity maturation in the presence or absence ofadjuvant. These mice showed a dramatically impaired ability to generate high-affinity IgG toNP, but retained the ability to produce low-affinity anti-NP IgG when NP-SRBC was used inthe absence of adjuvant. In contrast to wild-type mice, somatic mutation of the expressedIgG heavy chain gene was rarely detected in these GC/FDC-deficient mice. This suggeststhat GC/FDC are essential for affinity maturation. Trapping antigen-specific B cells inside theT cell zone of TNFRI–/– mice may prolong the interaction between T and B cells, which allowsclass switching but no further affinity maturation of IgG. Interestingly, GC/FDC-deficientmice could be induced to generate high-affinity, somatically mutated IgG antibodies byimmunization with the same amount of NP-SRBC antigen emulsified in incomplete Freund’sadjuvant or repeated immunization with the antigen alone. Thus, these data support a modelin which prolonged availability of antigen is required for somatic mutation and affinity matu-ration, and FDC or adjuvants facilitate such processes by slowly releasing antigens.

Key words: Affinity maturation / Follicular dendritic cell / Germinal center / Lymphotoxin / Tumor

Received 30/12/99Revised 17/4/00Accepted 2/5/00

[I 20436]

Abbreviations: FDC: Follicular dendritic cells GC: Germi-nal centers LT: Lympotoxin wt: Wild type

1 Introduction

Germinal centers (GC) are activated antigen-specific Bcell clusters that develop around follicular dendritic cells(FDC) inside primary B cell follicles upon antigen chal-lenge [1–5]. Several lines of indirect evidence suggestthat the GC/FDC complex provides an essential micro-environment for effective class switching and somaticmutation of the expressed immunoglobulin (Ig) genes: (1)the generation of memory B cells and production of high-affinity antibody is temporally associated with the forma-tion of GC [1–5]; (2) B cells with somatically mutated Iggenes were originally identified within GC [1, 4]; and (3)isotype switching of Ig genes was primarily detectedwithin GC after the onset of somatic mutation, suggest-ing that GC may facilitate the process of class switching[6]. It is thought that antigenic and non-antigenic stimula-

tion by FDC clusters to B cells is essential to maintainGC [3, 7, 8]. However, the exact role of the GC/FDCcomplex in somatic mutation and class switching of Ighas not been conclusively defined.

The dominant role of the GC has been undermined byrecent studies of antibody responses in mutant mousestrains that lack FDC and GC. Matsumoto et al. [9]clearly demonstrated that somatic mutation and affinitymaturation could be induced in lymphotoxin (LT)a–/– micefollowing immunization with (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) in alum even though thesemice lack FDC clusters and morphologically defined GC[9]. Subsequent independent studies using other GC/FDC-deficient mice, including strains with targetedmutations of either the receptor or ligands of the TNFand LT families, generally demonstrated similar resultsusing different antigens in adjuvant [7, 10–13]. In con-trast to earlier views that GC/FDC are the essential sitefor affinity maturation, these recent studies stronglyargue that GC/FDC are not essential for affinity matura-tion. However, it was later found that LT § –/– mice failed to

2226 Y. Wang et al. Eur. J. Immunol. 2000. 30: 2226–2234

Fig. 1. Mice deficient in FDC and GC retain Ig class switch-ing but fail to generate high-affinity antibody. wt (+/+), LT § -deficient (−/−), or TNFR-I-deficient (−/−) mice were immu-nized i.p. with NP-SRBC (108) in PBS and boosted 4 weekslater with 108 NP-SRBC. Sera were collected 7 days after thesecondary immunization. Anti-NP antibodies were mea-sured by ELISA using either NP45-BSA (empty bars), NP30-BSA (filled bars), NP13-BSA (vertical hatching), or NP2.5-BSA(horizontal hatching) as the plate-bound capture antigens.Only high-affinity antibodies are captured by NP2.5-BSA.Antibody levels are expressed as relative units (RU) by com-parison to a standard serum. wt mice are compared toTNFR-I–/– mice (A); or compared to TNFR-I–/– mice (B). Dataare expressed as means ± SD from three to four mice/group. One of three experiments is presented.

generate a detectable IgG response to anti-sheep redblood cells (SRBC) [14] and other antigens in theabsence of adjuvants [15]. In contrast to some GC-deficient mice which display an intrinsic defect in theinteraction between T and B cells [7, 8, 16], there were nointrinsic defects in either T or B cells from LT § –/– mice[14]. The lack of antibody response may be due to thelack of GC/FDC. Similarly, several FDC-deficient strains,including TNF–/– mice and TNF receptor-I–/– (TNFR-I–/–)mice, also showed no defect in B cell response in vitro,but demonstrated an impaired GC formation and anti-SRBC IgG response in vivo. Interestingly, those miceretain IgG responses to soluble antigens in adjuvant [13,17–19]. Therefore, the role of GC/FDC in affinity matura-tion and Ig class switching remain unclear.

There have been no experiment designs to reconcilethese contradictory results. The discrepancies amongprevious studies may be caused by different experimen-tal design, in at least three potentially important ways.First, the antigens used were different: a complex partic-ulate antigen in the case of SRBC versus a single solubleepitope of hapten. Second, adjuvants were used in someexperiments but not the others. Third, various dosagesof antigens were used. It is difficult to study affinity matu-ration using the classic hapten-carrier system withoutadjuvant. To better define the role of the GC/FDC com-plex and the nature of antigen in affinity maturation, weconjugated a well-defined hapten, NP, to SRBC so thataffinity maturation to a single epitope could be comparedat a given dose in the presence or absence of adjuvants.Here, we report that the degree of antigen (NP) availabil-ity determines whether a somatically mutated, high-affinity antibody response is formed. Persistence of anti-gen by adjuvant or repeated immunization rescues thesomatic mutation and affinity maturation disability in themice lacking GC/FDC.

2 Results and discussion

2.1 Mice genetically deficient in FDC and GC failto generate somatically mutated high-affinityantibodies but retain the ability to undergoisotype class switching

It has been recently reported that LT § –/– mice respondedto NP-OVA in alum but failed to respond to SRBC [9, 12].It is difficult to compare antibody response using NP-OVA with SRBC immunization. Soluble antigens have tobe mixed with adjuvant, while SRBC alone could inducea strong IgG responses in wild-type (wt) mice. To gain abetter understanding of the role of GC/FDC in IgGresponse to a single defined antigen, SRBC were conju-gated with NP to form NP-haptenated SRBC. The

advantage of our approach is that the response to sucha single epitope (NP) in a given dose can be directlycompared in the presence or absence of adjuvant. Fur-thermore, the nature of somatic mutation/affinity matu-ration can be readily defined in this NP system: (1) affinitymaturation can be estimated using ELISA in which thetarget antigen is either sparsely haptenated with NP(NP2.5-BSA, detecting only relatively high-affinity anti-bodies) or densely haptenated (NP13- or NP30-BSA,detecting both high- and low-affinity anti-NP antibodies);(2) C57BL/6 mice preferentially utilize the VH186.2 genein response to NP, and specific somatic mutations in thisgene correlate with acquisition of high-affinity antibodyto NP [1, 5, 9, 20]; and (3) the IgG response to the sameamount of NP-SRBC can be directly compared in thepresence and absence of adjuvant.

Eur. J. Immunol. 2000. 30: 2226–2234 Antigen persistence for somatic mutation 2227

Fig. 2. TNFR-I–/– mice immunized with NP-SRBC lack somatic mutation of the expressed VH186.2 gene. RNA was recoveredfrom the spleens of mice immunized twice i.p. with NP-SRBC in PBS as in Fig. 1. The expressed VH186.2 and closely relatedmRNA were recovered by RT-PCR and sequenced. Among 35 closely related genes, the VH 186.2 gene has unique sequences.For example, the codons at position 75 and 76 are CCC and TCC. Selected codons of the CDR1 and CDR2 domains of the germ-line VH186.2 gene are shown on the top lines. Nucleotides identical to the germ-line sequence are indicated by dashes. Codonscontaining sequences different from the germ-line sequence are shown. Without immunization with NP-SRBC, VH186.2 tran-scripts could not be amplified from either wt or TNFR-I–/– mice (data not shown).

Wild-type, LT § –/–, and TNFR-I–/– mice were immunizedi.p. with NP-SRBC in PBS and boosted 4 weeks later. Allsera collected 7 days after the secondary immunizationcontained high levels of low-affinity anti-NP IgG astested by ELISA (Fig. 1 A, B). In contrast to wt mice,LT § –/– mice and TNFR–/– mice produce almost no high-affinity IgG to NP (Fig. 1 A, B). The data suggests thatGC/FDC complexes may play a key role in supportingthe generation of high-affinity antibodies but is notessential for effective class switching of Ig.

It is established that affinity maturation is primarily theresult of selection of high-affinity antibodies from the ini-tial pool of responding B cells and the introduction ofnew sequence variants into the antibody V regions bysomatic hypermutation [1, 2]. Analysis of the anti-NPresponse in the C57BL/6 background is simplifiedbecause the primary VH gene segment selected in this

NP response is VH186.2 in this strain of mouse. To char-acterize the molecular element of affinity maturation ofanti-NP response in GC/FDC-deficient mice, we choseC57BL/6 TNFR-I–/– mice (N12) to analyze the selection ofthe VH186.2 gene and its mutation.

At day 7 after the second immunization with NP-SRBC,the VH186.2 gene and closely related genes from thesame VH family in the spleen were amplified by reversetranscription-PCR, and then cloned and sequenced [9].The independent derivation of each analyzed VH186.2sequence was verified by the uniqueness of somaticmutation in complementarity determining (CDR) regions.The ratios of the usage of the VH186.2 gene and itsrelated family members were determined and comparedFig. 2). High ratios of the usage of the VH186.2 gene andits related family members suggest that a proper NP-specific B cells are selected. Therefore, 16 of 16 ampli-


Fig. 3. Antigen persistence restores affinity maturation inGC/FDC-deficient mice. (A) Production of high-affinity anti-body is restored in GC/FDC-deficient mice by immunizationwith adjuvant. Both wt and TNFR-I–/– mice (3–4 mice/group)were immunized with NP-SRBC (108) emulsified with IFA andan identical booster immunization was administered 4weeks later. Sera were collected 7 days after the boosterimmunization. (B) Restored production of high-affinity anti-body in LT § –/– mice repeated immunization. LT § –/– mice wereprimed with 108 NP-SRBC and then immunized every 7 dayswith 107 NP-SRBC i.p. in PBS for 4 weeks. Sera from eachgroup (3–4 mice/group) were collected 7 days after the fifthimmunization. Anti-NP IgG was measured by ELISA usingeither NP45-BSA (empty bars), NP13-BSA (vertical hatching),or NP2.5-BSA (horizontal hatching) as the plate-bound cap-tured antigens. The data from one of three experiments ispresented as means ± SD.

fied sequences from wt mice and 15 of 16 amplifiedmRNA from TNFR-I–/– mice, were authentic VH186.2,indicating that the TNFR-I–/– mice had retained their abil-ity to select B cells expressing NP-specific Ig, suggest-ing that this step of selection can occur outside the GC/FDC complex.

The pattern and frequency of somatic mutations withinthe VH186.2 gene were also analyzed. Seven days afterthe booster immunization, wt mice showed an averageof 11.6 mutations per amplified VH 186.2 sequence in theregion between the beginning of CDR1 and the start ofthe V-D-J junction of IgG (Fig. 2). Mutation of Trp to Leuat amino acid position 33 in the CDR1 of VH186.2 ishighly correlated with acquisition of higher affinity for NP[1, 5, 9]. For sequences amplified from immunized wtmice, codon 33 in the CDR1 region showed mutationfrom Trp to Leu in 12 of 16 independent sequenes. Thiswas consistent with the data demonstrating affinity mat-uration by ELISA (Fig. 1), and suggested that somatichypermutation played a substantial role in the affinitymaturation process. In contrast, almost no mutations(0.5 mutation events/amplified sequence) were detectedin immunized TNFR-I–/– mice, and no codon 33 muta-tions were identified in the 15 cloned VH186.2 sequences(Fig. 2). This suggests that following immunization withNP-SRBC the efficiency of somatic mutation is dramati-cally reduced in the absence of GC/FDC, leading tomore than one log reduction (tenfold) in high-affinity anti-NP IgG antibody in the TNFR-I–/– mice (Fig. 1).

2.2 Production of high-affinity antibodies in GC/FDC-deficient mice can be restored bymaintaining antigen persistence

However, several recent studies are contradictory to thisstudy [9]. For example, immunization of LT § –/– mice withNP-OVA adsorbed to alum induced high-affinity anti-NPantibodies with abundant somatic mutations in theVH186.2 sequences [9]. To test whether contradictoryresults between this study and previous studies are dueto different doses or type of antigens, or to adjuvanteffect, we immunized wt and TNFR-I–/– mice twice withequivalent amounts of NP-SRBC emulsified in IFA (nobacteria product). At 7 days after the secondary immuni-zation, sera from TNFR-I–/– mice contained high levels ofboth low- and high-affinity IgG, similar to the levels seenin similarly immunized wt mice (Fig. 3 A). Furthermore,immunization of TNFR-I–/– mice with NP-SRBC in adju-vant elicited the formation of antibodies using VH186.2carrying substantial numbers of somatic mutations(Fig. 4). For both wt and TNFR-I–/– mice, 15 of 15 ampli-fied sequences were VH186.2. All but one of thesequences amplified from the TNFR-I–/– mice showed

somatic mutations in the region between the first codonof the mature IgG1 and the start of the V-D-J junction(average number of mutations 6.7 compared to 17.4 inthe sequences amplified from wt mice). Of 15 indepen-dent amplified sequences from the TNFR-I–/– mice, 13showed the key Trp to Leu mutation at codon 33 in theCDR1 region. Therefore, adjuvant plays an essential rolein the restoration of somatic mutation and selection ofhigh-affinity variants even in the absence of FDC and GC.

The pesistence of antigens may be the key for the main-tenance of B cell memory response [21]. It is possiblethat persistence of antigen caused by adjuvant or FDCallows prolonged B cell stimulation for somatic mutation,and affinity maturation to occur. Repeated immunizationmay compensate for the loss of FDC. To test this possi-bility, LT § –/– mice were immunized weekly with NP-SRBC


Fig. 4. Immunization with adjuvant restores somatic mutations of the VH186.2 gene in NP-SRBC immunized TNFR-I–/– mice. Asin Fig. 2, selected codons of the CDR1 and CDR2 domains of the germ-line VH186.2 gene joined to DFL16.1 are shown on thetop lines. Nucleotides identical to the germ-line sequence are indicated by dashes. Multiple somatic mutations are found in adju-vant immunized TNFR-I–/– mice, including the affinity-specifying Trp to Leu mutation at codon 33. RNA was collected from thespleen of immunized mice (3 mice/group).

in PBS. After five immunizations, the sera were collectedand affinity maturation was determined. The amount ofhigh-affinity antibody was restored to the level observedin wt mice (Fig. 3 B). The role of antigen dose in the selec-tion of B cells seems less critical in this setting since LT § –/–

mice fail to generate effective IgG production in responseto a wide range of antigens, from 5 × 105 to 5 × 108 NP-SRBC or SRBC. Therefore, antigen persistency is the keyto select B cells with higher affinity to antigens. The datafurther suggests that one role of GC/FDC or adjuvant insomatic mutation is to retain antigen within the microenvi-ronment of responding B cells, especially when the anti-gen may not otherwise be persistently available.

2.3 Trapping antibody-producing cells insideT cell zones in GC/FDC-deficient mice

Although GC or FDC clusters can be reduced by block-ing TNF, these structures can be rescued by introducinga stronger antigenic stimulation [22]. To rule out the

potential of restored FDC or GC formation in TNFR-I–/–

mice after strong immunization, the spleens were col-lected after immunization with NP-SRBC, a strong anti-gen to assess formation of GC with or without adjuvant.GC/FDC were formed within most splenic follicles of wtmice, but no splenic GC/FDC were detected in theknockout strain (Fig. 5 A). No FDC were detected in othertissues in those immunized mice.

GC/FDC-deficient mice could produce high level of low-affinity antibody. Where are antibody-producing cellsgenerated and how does effective class switching occurin the absence of GC? To identify where antibody-producing cells are located in these GC/FDC-deficientmice after antigenic challenge, the spleen sections werestained with anti-IgG or anti-IgM antibodies. In contrastto other B cells, antibody-producing B cells show strongstaining for both IgG and IgM within the cell and on thecell membrane. Ten days after the second immunizationwith SRBC in IFA, the spleens of TNFR-I–/– mice showedmany more IgM- and IgG-producing cells within the T


Fig. 5. Trapping of antibody-producing cells inside of T cell zones in TNFR-I–/– mice in the absence of GC/FDC. (A) No GC weredetected in TNFR-I–/– mice even after immunization of 108 SRBC in adjuvant. The spleen sections were stained with PNA (blue)and anti-B 220 antibody (brown). No FDC clusters were detected by anti-FDCM-1 antibody (data not shown). (B) Increasedantibody-producing cells inside of T cell zone of TNFR-I–/– mice. Spleen sections from three mice/group were stained with anti-IgM (blue) and anti-IgD (brown) or with anti-IgG (blue) and anti-IgD (brown) to visualize the location of IgG and IgM producingcells in the splenic sections from the mice immunized with SRBC in IFA.

cell zones of the white pulp compared to wt animals(Fig. 5 B). Similar results were obtained using spleen sec-tions of TNFR-I–/– mice immunized with NP-SRBC orSRBC wthout adjuvant. Antibody-producing cells couldbe readily visualized 4–20 days after rechallenge. There-fore, trapping of antigen-specific B cells in T cell zone ofTNFR-I–/– mice may allow prolonged interaction betweenT and B cells, leading to further Ig class switching andsometimes production of higher level of low-affinity anti-body (Fig. 3). However, such interaction fails to generateeffective affinity maturation if antigen is not persistentlyavailable.

GC/FDC-deficient mice can produce high-affinity anti-body in the presence of adjuvant if GC/FDC exist inectopic sites, such as lung and liver. LT § –/– mice have nolymph nodes (LN) and Peyer’s patches but do haveaccumulation of T and B cells in non-lymphoid tissues[23]. We could not detect any GC/FDC or antibody-producing cells in those tissues (data not shown). Theformation of GC-like structures in the absence of FDCclusters may not be sufficient to provide a microenviron-ment for affinity maturation. GC-like structures consist-ing of clusters of peanut agglutinin (PNA)+ cells are read-ily visualized in mesenteric LN (MLN) of TNFR-I–/– miceafter immunization. These scattered PNA+ clusters arepresent without evidence of FDC clusters in any lym-phoid tissue [23]. Similar GC-like structures were alsofound in MLN of RAG-1–/– mice reconstituted with LT § -deficient splenocytes or bone marrow. Without adjuvant,very low levels of high-affinity Ig to NP are detected in

these mice, suggesting such GC structures are not func-tional for affinity maturation. Koni et al. [12] confirmedour results showing that GC were present in MLN of LT g –/–

mice in the absence of FDC clusters. They proposed thatthese PNA+ cells were associated with affinity maturationin the lymphoid tissues since these mice produced high-affinity antibody in response to NP-conjugated chicken+ -globulin in alum. It remains possible, however, thataffinity maturation in the LT g –/– mice was a consequenceof the use of alum during immunization by their protocolrather than the presence of the PNA+ clusters.

It is still under debate whether B cells are activated in Tcell zones or B cell follicles. Earlier studies indicated thatB cells are activated in the T cell zone and move to the Bcell folicles to form GC where they undergo further selec-tion for the generation of high-affinity antibody [1–4]. Incontrast, a recent study analyzing the interactionsbetween antigen-specific T and B cells from transgenicmice has shown that antigen-specific B cells inside Bcell follicles do not travel into the T cell zones before theformation of GC [24]. Our finding in immunized TNFR-I–/–

mice that antibody-producing B cells are localized insidesplenic T cell zones may imply that activated B cells arepresent in T cell zones due to the lack of chemokine fromactivated FDC, but fail to travel back to B cell follicles(Fig. 5). Migration of B cells into the B cell zones appearsto be impaired in TNF–/– mice [8, 25]. TNF-activated FDCmay produce chemokines to attract activated B cellsback to the follicles [26]. The “trapping” of antigen-specific B cells in this study is consistent with such


notion. Such reduction of chemokines has not beenrecovered by stronger immunization with adjuvant. It ispossible that antigen persistence by adjuvant is the keyto allow Ig affinity maturation of B cells outside B cell fol-licle. It has been demonstrated that low antigen dose inadjuvant favors selection of somatic mutants with hall-marks of antibody affinity maturation [27]. Although therole of adjuvant may be complex, our data suggest thatfor affinity maturation, a key function of adjuvant is toslowly release low doses of antigen, facilitating theselection of B cell with high-affinity receptors.

Memory B cells are thought to be generated in GC andthey often express higher affinity of Ig to antigen [5, 20].Neither LT § –/– mice nor TNFRI–/– mice produce memorycells since the transfer of the primed cells from theseimmunized knockout mice (NP-SRBC or NP-KLH) intoirradiated wt mice failed to generate memory IgGresponse [28]. Interestingly, Karrer et al. [29] recentlyreported memory IgG responses were detected inTNFRI–/– mice infected with live LCMV but no IgGresponse with virus-derived antigens alone [29]. In con-trast to NP-SRBC, live virus provides prolonged antigenexposure for the generation of memory B cells. Together,these results are consistent with the nation that antigenpersistency may be critical for the generation of strong Igresponse and memory IgG response. When antigens arelimited, it is likely that GC/FDC is also essential for thegeneration of memory B cells. It will be of interest toaddress whether FDC is also essential for the mainte-nance of memory B cells.

In some FDC-deficient mice, ectopic PNA clusters cansometimes be found in T cell zone of organized lymphoidtissues, LN in particular [7, 8, 12, 23]. In our study, LT § –/–

mice showed no significant PNA clusters in the spleenafter immunization and no more then 2 % of LT § –/– miceretain one LN-like structure in the mesenteric area [23].We have found no GC-like structures in non-lymphoidtissues in these mice. Furthermore, we found no LN-likestructure in any of the 15 LT § –/– mice who showed high-affinity IgG to NP after immunization with NP-SRBC (withadjuvant or NP-OVA in alum. In the absence of adjuvant,those mice fail to generate high-affinity antibody. Inter-estingly, we found that TNFRI–/– mice often have multiplePNA clusters in lymphoid tissues, MLN in particular [23].These mice still failed to generate high-affinity IgG ifadjuvant was not used for immunization. It is conceiv-able that affinity maturation occurs is closely associatedwith the status of antigen persistence but not associatedwith the formation of PNA clusters.

LT § –/– mice failed to generate IgG in response to SRBC(without adjuvant), but they generate high level of lowaffinity IgG to NP in response to NP-SRBC in this study.

Several possibilities may account for the differences.First, immune response to NP-SRBC and SRBC may bedifferent. However, we found that LT § –/– mice failed togenerate detectable IgG to SRBC with or with NP conju-gation (data not shown), but produced high levels of lowaffinity IgG to NP (Fig. 1); second, the SRBC systemdoes not allow us to distinguish between low- and high-affinity Ig to SRBC. It is possible that only high affinityantibody can efficiently bind to SRBC coated on theplate and determined by ELISA. Using the NP systemenables us to further study somatic mutation and affinitymaturation.

3 Concluding remarks

Our study indicates that the GC/FDC complex actsimportantly to support affinity maturation when the anti-gen is present only transiently. When antigen exposure isprolonged, either by repeated immunization with antigenalone or using antigen with a depot adjuvant, efficientaffinity maturation with somatic mutation can occur. Incontrast, the GC/FDC complex is not essential for classswitching. FDC-supported GC may provide a functionalmicroenvironment for several other activities by express-ing soluble cytokines and surface ligands [3, 7]. Takentogether with the recent discovery of secondary Ig generearrangements in GC, GC may also be the key periph-eral structures for the generation of B cell diversity[30–32]. Factors that affect the development and main-tenance of FDC and GC will be important modulators ofsystemic B cell immune responses. Our study furthersupports a model that the geographical localization ofantigens to secondary lymphoid tissuess and the doseand time of antigen exposure are key variables determin-ing whether or not an effective immune response occurs[21, 33]. Such study will help us to understand the role ofantigens and GC/FDC complex in systemic immuneresponse and various immunization protocols for basicand clinical studies as well as vaccinations.

4 Materials and methods

4.1 Mice and immunization

C57BL/6J and TNFR-I–/– mice also called P55–/– mice (no.002818) were obtained from The Jackson Laboratory (BarHarbor, ME). LT § –/– mice were generated in our laboratory[15] on a mixed 129Sv × C57BL/6 background and werebred under specific pathogen-free conditions. The micewere immunized i.p. with 1 × 108 SRBC or NP-SRBC with orwithout IFA and then received a booster immunization 4weeks after the initial immunization. Spleens and sera werecollected 7 days after the second immunization as previ-


ously described [14]. In some cases, the mice were immu-nized with NP-SRBC weekly for 5 weeks and 5 weeks lateraffinity maturation of Ig was determined by ELISA.

4.2 Measurement of antigen-specific Ig

Specific antibodies were measured and analyzed as previ-ously described [9, 14]. For measurement of anti-SRBC anti-bodies, 96-well Falcon plates (Becton Dickinson, LincolnPark, NJ) were coated with SRBC [9, 14]. For anti-NP anti-bodies, 96-well Immulon 4 plates (Dynatech Laboratories,Chantilly, VA) were coated with NP-BSA (1–3 ? g/ml, Bio-search Tech., San Rafael, CA) for 2 h. Different degrees ofNP substitution were used as indicated, ranging from NP2-BSA to NP45-BSA. Unbound antigens were washed awaywith PBS. Diluted mouse sera were then added and incu-bated at 4 °C for 1 h. Bound antibodies were detected using100 ? l of 1:2,000 diluted alkaline phosphatase-conjugatedgoat anti-mouse IgG-antibody (Southern Biotechnology, Bir-mingham, AL), followed by addition of the alkaline phospha-tase substrate p-nitrophenyl phosphate (Sigma, St. Louis,MO) at 1 mg/ml.

4.3 Evaluation of spleen follicle structure byimmunohistochemical staining

Spleens from the mice 10 days after primary or 7 days afterbooster immunization were harvested, embedded in O.C.T.compound (Miles, Elkhart, IN), and frozen in liquid nitrogen.Frozen sections (6–10 ? m thick) were fixed, quenched, andstained as previously described [14].

4.4 Sequence analysis of the expressed VH186.2 genes

The sequences of expressed VH186.2 and highly relatedgenes were determined by minor modifications of previouslydescribed methods. At the indicated times after secondaryimmunization, spleens from three mice/group were col-lected and total RNA was extracted using the RNAzolreagent (Biotecx Lab, Houston, TX). cDNA was synthesizedfrom 0.2 ? g of total RNA using oligo(dT) primed reverse tran-scription, and subjected to nested PCR to recover VH186.2and highly related gene sequences joined to the IgG1 con-stant region. In the first reaction, the sense primer was 5'-CATGCTCTTCTTGGCAGCAACAGC-3' (for VH186.2), andthe antisense primer was 5'-GTGCACACCGCTGGACAGGGATCC-3' (for C + 1). The second reaction use senseprimer 5'-CAGGTCCAACTGCAGCAG-3' and antisenseprimer 5'-AGTTTGGGCAGCAGA-3'. Amplified fragmentswere cloned into pCRII (Invitrogen), and randomly pickedtransformants were sequenced.

Acknowledgements: This work was supported in part bya NIH grant to Y.-X. Fu (R01 HD37104). Y.-X. Fu is a recipient

of clinical investigator award from NIAID, NIH (AI 01410).D. D. Chaplin is an investigator of the Howard Hughes Medi-cal Institute. The authors would like to thanks Drs. UrsulaStorb, Marcus Clark, and Shuhua Han for their critical com-ments and advice.

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Correspondence: Yang-Xin Fu, Department of Pathology,MC6027, The University of Chicago, 5841 S. Maryland Ave,Chicago, IL 60637, USAFax: +1-773-702-6260e-mail: yfu — midway.uchicago.eduor: David D. Chaplin, HHMI, Washington University Schoolof Medicine, 660 S. Euclid, St. Louis, MO 63110, USAe-mail: chaplin — im.wustl.edu


antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin

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