B cell clones that sustain long-term plasmablastgrowth in T-independent extrafollicularantibody responsesMei-Chi Hsu, Kai-Michael Toellner, Carola G. Vinuesa, and Ian C. M. MacLennan*
Medical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham B15 2TT, United Kingdom
Communicated by Michael Potter, National Institutes of Health, Bethesda, MD, February 22, 2006 (received for review September 8, 2005)
Some antigens induce Ab responses without T lymphocyte help.Among these, many polysaccharide-based antigens cause marginalzone B cells to proliferate and differentiate into plasma cells. B1cells also respond to some of these antigens. In this article, wereport that antigen-specific B1b cells, in response to the T-independent antigen (4-hydroxy-3-nitrophenyl)-acetyl (NP)-Ficoll,develop into clones that sustain Ab production for months withcontinued production of plasma cells and the accumulation ofantigen-specific B cells in follicles. The persistence of this T-independent plasmablast response contrasts with the short-termplasmablast growth associated with T-dependent extrafollicularresponses. The nature of the cells responding to NP-Ficoll wasprobed by using chimeras that have B1 cells but lack primary Blymphopoietic capacity and have very few B2 cells or T cells. Thechimeras were constructed by transferring 105 IgM� IgD� perito-neal exudate cells into mice unable to produce their own T and Bcells because of deficiency in recombinase-activating gene 1(RAG-1). The chimeras mounted sustained IgM and IgG3 anti-NP Abresponses to NP-Ficoll. This finding was associated with continuedNP-specific extrafollicular plasmablast growth and the accumula-tion of NP-specific B cells in follicles. B cells were not found in themarginal zones of chimeras, and they also lacked recirculating IgD�
cells and CD3� cells. The absence of B2 and T cells confirms thathemopoietic cell chimerism leading to primary lymphopoiesis hadnot been established.
B1 cells � memory � plasmablasts
A group of thymus-independent (TI) antigens, known as TI-2antigens, are linked by their inability to induce responses in
Bruton tyrosine kinase-deficient mice (1, 2). Many of thenaturally occurring antigens in this group are bacterial capsularand cell-wall polysaccharides. Typically, responsiveness to TI-2antigens is acquired later in ontogeny than that to other TIantigens or thymus-dependent (TD) antigens (3). This delay hasmassive consequences for infection from bacteria with densepolysaccharide capsules such as the pneumococci, which causelarge numbers of infant deaths annually from meningitis andpneumonia (4, 5). Characteristically TI-2 antigens induce splenicmarginal zone B cells to grow as plasmablasts in extrafollicularfoci of the spleen (6–8). Kearney et al. (7) have studied responsesto phosphorylcholine that forms part of bacterial cell-wall poly-saccharide. Natural Ab exists that binds this determinant, whichis recognized both in the marginal zone and B1 repertoires.Using transgenic B cells specific for phosphorylcholine with Igvariable-region idiotypes that are associated with entry to eitherthe B1 or marginal zone repertoires, these authors have shownthat both types of B cells can be involved in the response to thisantigen.
Classically, B cell memory is associated with TD Ab responses,and these memory cells are derived from B cells that haveproliferated and undergone affinity maturation and selection ingerminal centers (9). Although in certain circumstances TI-2antigens are also able to induce germinal center formation, theseinstances are abortive and generate neither plasma cells nor
memory B cells (10, 11). Memory B cells can be defined as cellsthat have undergone antigen-driven proliferation and then be-come nonproliferating cells that can be induced, on reexposureto antigen, to proliferate and secrete Ab. Evidence for theproduction of cells that fulfill these criteria from B1 cellsresponding to a TI-2 antigen is presented in this article.
Multiple copies of haptens such as (4-hydroxy-3-nitrophenyl)-acetyl (NP) conjugated to the neutral polysaccharides, Ficoll orhydroxyethyl starch, are frequently used as test TI-2 antigens (8,10, 12–16). NP-Ficoll induces extrafollicular responses from bothnaıve and memory marginal zone B cells (8, 17). By contrast,IgDhigh CD23� recirculating B cells with specificity for NP do notmount an Ab response to this antigen in vivo (6, 17). BackgroundIgM, but not IgG Ab, that binds NP is found in nonimmunizedmice, indicating that these specificities are included in the B1repertoire. Furthermore, Pyk-2-deficient mice, which are re-ported to lack marginal zone B cells but have B1 cells, produceAb responses, albeit diminished, to the related hapten 2-4-trinitro phenyl conjugated to Ficoll (18).
B cells recruited into extrafollicular Ab responses to the TDantigen NP-chicken �-globulin grow as plasmablasts (prolifer-ating Ab-secreting cells) for �4 days before terminally differ-entiating into plasma cells (nonproliferating Ab-secreting cells)(19). By contrast, plasmablasts can be found for months inextrafollicular Ab responses induced by immunization withNP-Ficoll (20). In this article, we address the basis for thispersistence of plasmablasts in the response to NP-Ficoll. At first,the most obvious explanation seemed to be that naıve B cellsrecently produced in the marrow continue to be recruited intothe response by persistent antigen. NP-Ficoll is resistant todegradation in the body and is taken up and retained byspecialized macrophages in the splenic marginal zone (21); it canbe held as an immune complex on the surface of folliculardendritic cells (20). Experiments reported here indicate thatsustained responses to NP-Ficoll can be maintained withoutcontinued recruitment of naıve B cells. This article goes on tocharacterize mature B cell clones that can maintain a pool ofNP-specific plasmablasts in response to NP-Ficoll.
ResultsSelf-Sustaining B Cell Clones Maintain Long-Term Plasmablast Re-sponses to NP-Ficoll. In response to i.p. immunization with NP-Ficoll, WT mice produced persistent NP-specific plasmacytoidcells (plasmablasts, plasma cells, or both) in splenic extrafollicu-lar foci (Fig. 1A). Even 3 months after immunization, �10% ofthe plasmacytoid cells were in cell cycle, as assessed by Ki-67nuclear staining. Comparable proportions of plasmacytoid cells
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
were found to be in cell cycle in our previous studies of responsesto NP-Ficoll in WT mice, as assessed by the uptake of thethymidine analogue 5-bromo-2�-deoxyuridine (20). To testwhether this persistence of plasmablasts in responses to NP-Ficoll requires continued recruitment of naıve B cells, chimericmice were constructed that had mature B cells but no capacityfor primary B lymphopoiesis. The chimeras were created bytransferring 107 lymph-node cells containing mature B and Tcells into congenic recombinase-activating gene 1 (RAG-1)-deficient mice, which lack the capacity to produce T and B cellsfrom hemopoietic progenitors (22) (Fig. 1B). Studies show thatby 14 days after cell transfer such chimeras have reconstitutedboth follicular and marginal zone B cell subsets and produce Abin response to immunization with NP-Ficoll (17). In this article,we tested whether the supply of mature NP-specific B cells wouldbe exhausted through differentiation into short-lived Ab-producing cells. The chimeras were immunized i.p. with NP-Ficoll on days 14, 49, and 72 after cell transfer. Against expec-tation, plasmacytoid cells containing large amounts of anti-NPAb were seen in splenic extrafollicular foci after each immuni-zation (Fig. 1 C–E). As in WT mice, a proportion of these cellswere plasmablasts, as assessed by expression of Ki-67 (Fig. 1 Dand E). The additional immunizations had little obvious effecton the response apart from a possible trend for these immuni-zations to be followed by a temporary increase in the proportion
of NP-specific plasmablasts (Fig. 1E). Ab titers achieved afterthe first immunization were maintained or somewhat increasedafter secondary and tertiary immunizations with NP-Ficoll.Typical Ab responses of chimeras are shown in the experimentdescribed below and depicted in Fig. 2.
NP-Specific B Cells Accumulate in B Cell Follicles but Not MarginalZones During Responses to NP-Ficoll. Immunohistology of thespleens of chimeras that had received two or three immuniza-tions with NP-Ficoll show that NP-specific B cells as well asplasmablasts and plasma cells were present (Fig. 1 C and D). TheNP-specific B cells induced by NP-Ficoll were located mainly infollicles, both primary follicles, which lack germinal centers (Fig.1C), and secondary follicles, which have germinal centers (Fig.1D). Some NP-specific B cells were also located in the outer Tzone, where their size and frequent expression of Ki-67 indicatesthese cells were mostly B blasts. The combined numbers ofNP-specific B cells in the follicles and T zone and the proportionof these in cell cycle, as assessed by Ki-67 expression, are shownin Fig. 1F. The presence of B cells in follicles is consistent withour earlier finding of persistent and mainly nonproliferatingNP-specific B cells in the follicles of WT mice after primaryimmunization with NP-Ficoll (20). Critically, there was a virtualabsence of NP-specific follicular B cells before immunization inboth WT mice and chimeras (Fig. 1F). This finding implies that
Fig. 1. Evidence for persistent B cell clones that sustain extrafollicular responses to NP-Ficoll. (A) Immunohistological analysis of the splenic-specific Ab responseof WT mice to NP-Ficoll. Blue triangles represent the number of NP-specific plasmablasts per mm2 of section at intervals after immunization; red triangles depictthe total number of NP-specific plasmacytoid cells per mm2 (the sum of the numbers of NP-specific plasmablasts and NP-specific plasma cells). Triangles representvalues from individual mice; continuous red and dashed blue lines connect median values. The number next to the day-0 red triangle indicates the number ofsuperimposed points. (B) Construction of chimeras that have mature B cells but no capacity for B lymphopoiesis. Flow cytometry shows IgM and IgD expressionof donor B220� peripheral lymph-node cells (Left); 107 lymph-node cells were transferred into each RAG-1-deficient recipient. IgM and IgD expression of splenicB cells 54 days after lymph-node cell transfer is shown (Right); the red circle highlights an emergent IgMhigh IgDlow population not obvious in the donorpopulation. (C) A spleen section from a chimera immunized on days 14 and 49 after cell transfer with NP-Ficoll and analyzed 6 days after the second immunization.NP-specific plasmacytoid cells (with strong blue cytoplasmic staining) are seen in an extrafollicular focus (ExF), whereas NP-specific B cells (with less strong bluesurface staining) are seen among the orange-stained IgD� recirculating B cells in a primary follicle (PF). (D) A similar section, this time identifying plasmablastswith cytoplasmic NP-binding (blue) and nuclear Ki-67 expression (brown) in an ExF. Some NP-specific B cells are seen in a secondary follicle (SF), and a proportionof these are Ki-67�; the other Ki-67� cells in the follicle are germinal center B cells. (E) Plots similar to those in A, except that the responses of chimeras are followed.Groups of chimeras were immunized 14, 14 and 49, or 14, 49, and 72 days after construction by lymph-node transfer. Red symbols show the total numbers ofNP-specific plasmacytoid cells per mm2, and blue symbols show the numbers of NP-specific plasmablasts per mm2. Red lines and dashed blue lines connect medianvalues. The number next to the day-0 red circle indicates the number of superimposed points. (F) Similar plots to those in E, depicting data from the same chimerasbut showing the numbers, in follicles and the T zone, of NP-specific B cells (red circles) and B blasts (blue circles) per mm2 of section. Red lines and dashed bluelines connect median values.
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the NP-specific B cells in the follicles of immunized chimerashave arisen after immunization by proliferation and that manyhave now come out of cell cycle. Despite the follicular locationof the NP-specific B cells of chimeras immunized with NP-Ficoll,their phenotype was not that of germinal center B cells. First, asshown above, many of the NP-specific B cells were present infollicles that lack germinal centers, and even when they were infollicles with germinal centers, many were not in cell cycle (Fig.1F). On the basis of immunohistology, these cells lack thegerminal center B cell-associated features of Bcl6 expression andpeanut agglutinin binding (data not shown). It is a theoreticalpossibility that hemopoietic cell chimerism was established bylymph-node lymphocyte transfer, although this hypothesis isunlikely because no host stem-cell depletion was undertakenbefore cell transfer. If this phenomenon had occurred, it couldhave led to B lymphopoiesis and the recruitment of naıve B cellsinto response. This possibility is addressed and excluded as anexplanation for the persistence of plasmablasts in the responseto NP-Ficoll in experiments described in the last section ofResults.
There Are Sufficient B1b Cells in Lymph Nodes to Reconstitute the B1bCell Compartment of Chimeras. The experiments described aboveraise the suspicion that the long-term response of chimeras mightbe sustained by B1 cells because these, unlike B2 cells, are knownto have the capacity for self-renewal in the periphery (23). Arecent article has highlighted the ability of B1b cell clones toprovide potent long-lasting TI-protective Ab responses against arelapsing fever bacterium, Borrelia hermsii (24). Furthermore,this study showed the expansion of antigen-specific clones of B1cells. At first, it seemed that our choice of peripheral lymph-nodecells as a source of donor cells for chimera construction did notfavor this conclusion; for, as expected, the B cells in peripherallymph-node suspensions were overwhelmingly IgDhigh IgMvariable
recirculating B cells (Fig. 1B); such IgDhigh cells are known notto respond to NP-Ficoll in vivo (6, 17), although they can give riseto marginal zone cells (8, 25–27) that can respond to this antigen(6, 8, 28). Flow cytometry of the donor B220� lymphocytesshowed that �2% of B cells have an IgMhigh IgDlow phenotypethat characterizes B1 cells and marginal zone B cells (Fig. 1B).Despite this result, analysis of peritoneal exudate cell (PEC)suspensions from chimeras showed that an IgM�, IgDlow/�,CD11b�, CD23low B1 population emerges over time in RAG-1�/� mice that have received peripheral lymph-node cells (see
Fig. 4A, which is published as supporting information on thePNAS web site). In comparison to PEC from WT mice, a lowerproportion of the IgM� PEC in these chimeras expressed CD5(see Fig. 4B), indicating that lymph-node cells predominantlyreconstituted the B1b compartment. The small number of B1cells transferred compared to the numbers of B1 cells recoveredin the PEC of chimeras seems likely to reflect proliferation aftertransfer.
Small Numbers of B1 Cells Endow RAG-1�/� Mice With the Ability toMount Long-Term Ab Responses to NP-Ficoll Without Reconstitutingthe Marginal Zone or Recirculating B Cell Pools. To investigate thepossible contribution of B1 cells to the persistent response toNP-Ficoll, a new series of chimeras were constructed by trans-ferring into RAG-1�/� mice small numbers of B1 cells withminimal numbers of B2 cells (Fig. 2 A). The aim was to create aB1 cell pool but not recirculating and marginal zone B2 cellpools. The chimeras constructed with PEC enriched for B1 cellsand depleted of B2 cells received 105 IgM� PEC depleted ofIgD-expressing cells. A second group of chimeras received 105
IgM� PEC depleted of CD5-expressing cells. This group wasused to test whether B1b cells were able to support long-termresponses to NP-Ficoll. Positive control groups were includedthat received either 106 total PECs (a mixture of B1 andrecirculating B cells) or, as in the first experiments, 107 periph-eral lymph-node cells (mainly recirculating B cells but also, asshown above, some B1 cells). A final group of chimeras received105 IgDhigh PECs (recirculating cells depleted of B1 cells). Thephenotypes of representative donor populations are shown inFig. 2A. Chimeras were immunized i.p. with NP-Ficoll 14 and 42days after cell transfer and were finally assessed on day 48.
Serological analysis of the response of the different chimerasto NP-Ficoll is shown in Fig. 2B. The chimeras constructed withIgD� IgM� PEC produced IgM and IgG3 Ab after immunizationwith NP-Ficoll (Fig. 2B). A similar serum Ab response wasdetected in the other groups of chimeras, except those con-structed by transfer of 105 IgD� PEC, which failed to mount anNP-specific Ab response to NP-Ficoll. In addition to the specificAb response to NP-Ficoll, it can be seen that 14 days after celltransfer and before primary immunization natural IgM, but notIgG3, anti-NP Ab is present in the serum of all chimeras exceptthose constructed with IgD� cells.
The spleens of the chimeras reconstituted with IgD� IgM�
PEC had NP-specific plasmacytoid cells in extrafollicular foci
Fig. 2. The relative ability of different B cell subsets to render RAG-1-defficient mice responsive to NP-Ficoll. (A) Flow cytometry plots of the unsorted and sorteddonor populations used to construct chimeras in RAG-1�/� mice (a plot of donor peripheral lymph-node cells is shown in Fig. 1B). (B) NP-specific serum IgM andIgG3 Ab titers of chimeras immunized with NP-Ficoll on days 14 and 42 after cell transfer. The titers were determined on blood samples taken immediately beforethe first immunization, just before the second immunization, and 6 days after the second immunization. The symbols correspond to the donor cells used toconstruct the chimeras shown in A.
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(Fig. 3A). Between 39% and 64% of these cells had switched toIgG3 (see Table 1, which is published as supporting informationon the PNAS web site) and consistently �10% of these wereKi-67�. In addition, NP-specific B cells were found in splenic Bcell follicles of these chimeras (Fig. 3A). These cells are probablycorrespond to B220� NP-binding B cells identified by flowcytometric analysis of these spleens (Fig. 3A), which are amixture of IgM� and, presumably switched, IgM� cells. No cellsexpressed IgD, nor the germinal center B cell marker GL7,whereas some cells expressed intermediate levels of syndecanconsistent with them being plasmablasts.
Analysis of the B cell compartments in these chimeras con-structed with 105 PEC depleted of IgD� B cells shows that theyhave small numbers of IgD� IgM� B cells in the follicles (Fig. 3Band see Table 1). IgD� cells were not found in their follicles, andthis accords with flow cytometry of these spleens, which revealedthe presence of IgM�- but not IgD-expressing B cells (Fig. 3Band see Table 1). Despite the presence of IgD� IgM� B cells infollicles, no B cells were found in the marginal zone. This findingcontrasts markedly with chimeras constructed by transfer of 107
peripheral lymph-node cells. These chimeras have follicles withmany IgD� B cells and well developed IgDlow/�, IgM� marginalzone B cell populations (Fig. 3C). The absence of IgD� B cellsor B cells in the marginal zone of the chimeras constructed withIgD� IgM� PEC indicates that hemopoietic chimerism leadingto primary B lymphopoiesis had not been established.
Analysis of the spleens from the other groups of chimerasshows that all of the chimeras that produced an Ab response toNP-Ficoll had NP-specific plasmacytoid cells in their spleen (Fig.3A and see Table 1). Many of these cells had switched to produceIgG3. These chimeras also had NP-specific B cells in their splenicfollicles. On the basis of f low cytometry, these cells were mainly
IgM� IgD�. Although NP-binding cells from chimeras con-structed with IgD-depleted PEC or CD5-depleted PEC did notexpress surface IgD, a significant minority of the NP-binding Bcells from chimeras constructed by transfer of 107 lymph-nodecells or 106 PEC expressed IgD (Table 1).
Taken together, these transfer experiments indicate that B1bcells can give rise to persistent extrafollicular responses toNP-Ficoll. At this stage, the participation of B1a cells, i.e., thoseB1 cells that express CD5 are not excluded. Recirculating IgDhigh
B cells seem unlikely to contribute to these long-term responses,although as mentioned above, large numbers of recirculatingcells will reconstitute a marginal zone population, which gives aresponse to NP-Ficoll (17); however, this response is likely to beshort-term.
DiscussionAb responses to NP-Ficoll are associated with plasmablasts thatpersist for months in extrafollicular foci (20). This finding differsstrikingly from conventional TD extrafollicular Ab responses inwhich plasmablasts are only present for a few days (19). Thechimeras constructed with small numbers of IgD� PEC lackedB2 cells, indicating that primary B lymphopoietic capacity hadnot been established. Consequently, the persistence of NP-specific plasmablasts after immunization could not have beenattributable to continued recruitment of naıve B cells. At thisstage, it is not possible to exclude whether plasmablast persis-tence is attributable to long-term self-renewal. An alternativepossibility is that the NP-specific B cells that accumulate infollicles in these responses are involved in maintaining the poolof plasmablasts. These two possibilities are not mutually exclu-sive. The location of these NP-specific B cells distinguishes themfrom conventional memory B cells generated in response to
Fig. 3. Transfer of 105 PEC depleted of IgD� cells enables RAG-1�/� mice to sustain long-term responses to NP-Ficoll without restoring the recirculating andmarginal zone pools of B2 cells. (A) Analysis of NP-specific cells in the spleen of a chimera constructed 48 days before with 105 IgD-depleted PEC. Thephotomicrograph shows IgM� plasmacytoid cells (large arrow). Stromal elements in a follicle (F) and marginal sinus (M) are stained blue for mucosal addressincell-adhesion molecule expression; There is a cluster of NP-specific B cells in the follicle (small arrow), but none of these cells are located in the marginal zone,which lies outside the marginal sinus. Flow cytometry shows analysis of IgM, IgD, GL7, and CD138 expression by NP-binding cells gated into B220high and B220low
subsets. The spleen was taken 48 days after cell transfer with immunizations with NP-Ficoll on days 14 and 42. (B) IgM and IgD expression in the spleen of a chimeraconstructed with 105 IgD-depleted PEC. (C) IgM and IgD expression in the spleen of a chimera constructed with 107 peripheral lymph-node (LN) cells. (B and C)The chimeras were also immunized with NP-Ficoll on days 14 and 42 after cell transfer and analyzed on day 48; The arrow points to clustered IgM� plasmacytoidcells in the red pulp. T, T zone.
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NP-protein conjugates that typically localize to the marginalzone (29) and to the blood and bone marrow (30). Follicularlocalization opens the possibility that these TI-2 antigen-inducedB cells might be recruited back into the response by NP-Ficollheld as immune complex on follicular dendritic cells (20).
Recently, Haas et al. (31) have proposed distinct roles for B1aand B1b cells on the basis of different selection requirements forthese two populations during ontogeny. They found that miceoverexpressing CD19 produced B1a cells with few B1b cells andnoted that these mice had high levels of natural Ab. By contrast,CD19-deficient mice were found to lack B1a cells and natural Abbut had B1b cells and mounted Ab responses against the TI-2antigen type-3 pneumococcal capsular polysaccharide. In ourstudies, the excellent response to NP-Ficoll by chimeras recon-stituted with 105 CD5� peritoneal exudate B cells is consistentwith this conclusion. Nevertheless, these chimeras did produce asmuch background (natural) anti-NP IgM as chimeras reconsti-tuted with whole-PEC preparations. It may be that very smallnumbers of contaminating CD5� B cells were able to expand toproduce normal levels of Ab in these chimeras. An alternativepossible explanation is that WT B1b cells did produce natural Abbut that this function was impaired if the B1b cells lacked CD19.
The B1 cell clones involved in long-term Ab responses mightgive rise to pathology. For example, the protracted antigen-driven TI Ab responses may provide the cellular basis forauto-Ab production in diseases such as Guillain–Barre syn-drome. Nonmutated IgM and IgG Abs (IgM and IgG3 in mice)against a glycolipid motif found on Campylobacter jejuni, whichcrossreacts with neural gangliosides, mediate this relativelycommon self-limiting paralytic disease (32). These Abs persistfor longer than would be expected from a short-lived TI-extrafollicular Ab response. Another situation in which theseresponses by B1b cells might give rise to disease lies in thecontinued production of plasmablasts by clones respondingNP-Ficoll or similar persistent antigens. This sort of responsepresents a potential danger for the development of B cellneoplasms, which are associated with serial acquisition of geneticchanges. This theory is well exemplified in the plasmacytomas ofBALB�c mice, which only develop after protracted exposure tomineral oil given into the peritoneal cavity (33). The role ofantigen in the development of these plasmacytomas can beinferred from the relative protection of mice rendered pathogen-free from plasmcytoma development (34). These neoplasmsoften show signs of receptor editing but only approximately halfshow Ig V-region gene mutations (35). At least those neoplasmswithout these mutations might have been generated during Abresponses not involving a germinal center reaction. The perito-neal location of these plasmacytomas has led to the suggestionthat these neoplasms may arise from B1 cells. The continuedproduction of plasmablasts by B1b cell clones described in thepresent article appears to provide a situation in which plasma-blasts could acquire the sequential genetic changes associatedwith plasmacytoma development.
Materials and MethodsAnimals and Immunizations. RAG1�/� mice 6 to 8 weeks of age ona C57BL�6 background were obtained from the Biomedical
Services Unit (University of Birmingham) and were kept underspecific pathogen-free conditions at all times. Rag1�/� micewere reconstituted, as indicated in the text, with congenic WTcells from C57BL�6 mice (Harlan Olac, Bicester, U.K.) by i.v.injection; 2 weeks later the chimeras received their first dose ofNP-Ficoll (Biosearch Technologies, Novato, CA; 30 �g in sterilePBS) i.p. They were challenged again with NP-Ficoll at timesspecified in the text. All experiments involving animals werecarried out under license from the British Home Office, whichwas granted after local animal ethics committee approval.
Cell Purification and Adoptive Transfer. Total PECs were obtainedfrom washes of the peritoneal cavity with cold RPMI medium1640. Cells were stained with IgD and IgM fluorochrome-conjugated Ab and positively selected for IgDhigh IgMvariable andIgDlow/� IgMhigh with a MoFlow f low cytometric sorter(DAKO). CD5� cells were negatively selected from total PECsuspension by magnetic bead separation (Miltenyi Biotec, Bisley,U.K.).
Flow Cytometry Reagents. After isolation, single-cell suspensionsfrom donor mice or chimeras were stained with the following Abconjugates: B220-biotin, CD11b-FITC, CD138-phycoerythrin,CD5-biotin, IgD-phycoerythrin, IgM-FITC (all substances wereobtained from BD Biosciences, San Diego, CA). The monoclo-nal Ab GL-7, which binds to a molecule expressed by germinalcenter B cells (36), was a kind gift of G. Kelsoe (Duke University,Durham, NC) and was used as a biotin conjugate. Biotin wasdetected by using streptavidin-CyChrome (BD Biosciences).NP-binding cells were identified by using a locally preparedNP-phycoerythrin conjugate. The cells were analyzed by using aFACScalibur flow cytometer (BD Biosciences).
ELISA. For testing NP-specific Abs, NP15-BSA-coated 96-wellplates were used. The serum Abs from chimeras were detectedby alkaline phosphatase-conjugated goat anti-mouse IgM orIgG3 (Southern Biotech, Birmingham, AL), and the enzymebound to plates was developed with p-nitrophenylphosphatesubstrate (Sigma–Aldrich). For standardization, a positive con-trol was run along with test samples in all plates. The titers forserum samples were calculated as the log serum concentrationrequired to achieve 30% maximum OD.
Immunohistochemistry Staining. This process was carried out asdescribed in refs. 20 and 37. Briefly, 5-�m, acetone-fixed frozensections were stained for IgD, mucosal addressin cell-adhesionmolecule (38), NP-binding, and expression of the proliferation-associated nuclear antigen Ki-67 (39). NP-binding cells were de-tected by using NP-conjugated sheep IgG and biotinylated donkeyanti-sheep secondary Ab. The detection of NP-specific cells wasrevealed in a streptavidin-alkaline phosphatase system. Mucosaladdressin cell-adhesion molecule was also stained by using strepta-vidin-alkaline phosphatase. IgD and Ki-67 were detected by usingimmunoperoxidase. The stained sections were viewed under �20objective, and the number of positively stained cells per mm2 ofsection was determined by using a 10-mm graticule.
This work was funded by grants from the British Medical ResearchCouncil.
1. Amsbaugh, D. F., Hansen, C. T., Prescott, B., Stashak, P. W., Barthold, D. R.& Baker, P. J. (1972) J. Exp. Med. 136, 931–949.
2. Mond, J. J., Scher, I., Mosier, D. E., Baese, M. & Paul, W. E. (1978) Eur.J. Immunol. 8, 459–463.
3. Hazlewood, M., Nusrat, R., Kumararatne, D. S., Goodall, M., Raykundalia, C.,Wang, D. G., Joyce, H. J., Milford-Ward, A., Forte, M. & Pahor, A. (1993) Clin.Exp. Immunol. 93, 157–164.
4. Butler, J. C., Breiman, R. F., Lipman, H. B., Hofmann, J. & Facklam, R. R.(1995) J. Infect. Dis. 171, 885–889.
5. Kaczmarski, E. B. (1997) Commun. Dis. Rep. CDR Rev. 7, R55–R59.
6. Lane, P. J., Gray, D., Oldfield, S. & MacLennan, I. C. (1986) Eur. J. Immunol.16, 1569–1575.
7. Martin, F., Oliver, A. M. & Kearney, J. F. (2001) Immunity 14, 617–629.8. Vinuesa, C. G., Sunners, Y., Pongracz, J., Ball, J., Toellner, K.-M., Taylor,
D., MacLennan, I. C. & Cook, M. C. (2001) Eur. J. Immunol. 31, 1340–1350.
9. Coico, R. F., Bhogal, B. S. & Thorbecke, G. J. (1983) J. Immunol. 131,2254–2257.
10. Shih, T. A., Meffre, E., Roederer, M. & Nussenzweig, M. C. (2002) Nat.Immunol. 3, 570–575.
Hsu et al. PNAS � April 11, 2006 � vol. 103 � no. 15 � 5909
IMM
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11. Vinuesa, C. G., Cook, M. C., Ball, J., Drew, M., Sunners, Y., Cascalho, M.,Wabl, M., Klaus, G. G. & MacLennan, I. C. (2000) J. Exp. Med. 191,485–494.
12. Amlot, P. L., Grennan, D. & Humphrey, J. H. (1985) Eur. J. Immunol. 15,508–512.
13. Nishimura, H., Minato, N., Nakano, T. & Honjo, T. (1998) Int. Immunol. 10,1563–1572.
14. Ohnishi, K., Melchers, F. & Shimizu, T. (2005) Eur. J. Immunol. 35, 957–963.15. Sato, S., Steeber, D. A. & Tedder, T. F. (1995) Proc. Natl. Acad. Sci. USA 92,
11558–11562.16. Tsitsikov, E. N., Gutierrez-Ramos, J. C. & Geha, R. S. (1997) Proc. Natl. Acad.
Sci. USA 94, 10844–10849.17. Vinuesa, C. G., Sze, D. M.-Y., Cook, M. C., Toellner, K.-M., Klaus, G. G. B.,
Ball, J. & MacLennan, I. C. M. (2003) Eur. J. Immunol. 32, 297–305.18. Guinamard, R., Okigaki, M., Schlessinger, J. & Ravetch, J. V. (2000) Nat.
Immunol. 1, 31–36.19. Sze, D. M., Toellner, K.-M., Garcia de Vinuesa, C., Taylor, D. R. & MacLen-
nan, I. C. (2000) J. Exp. Med. 192, 813–821.20. Vinuesa, C. G., O’Leary, P., Sze, D. M., Toellner, K.-M. & MacLennan, I. C. M.
(1999) Eur. J. Immunol. 29, 1314–1323.21. Humphrey, J. H. (1985) Immunol. Lett. 11, 149–152.22. Taccioli, G. E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P. A. & Alt, F. W.
(1993) Science 260, 207–210.23. Herzenberg, L. A. (2000) Immunol. Rev. 175, 9–22.24. Alugupalli, K. R., Leong, J. M., Woodland, R. T., Muramatsu, M., Honjo, T.
& Gerstein, R. M. (2004) Immunity 21, 379–390.
25. Dammers, P. M., de Boer, N. K., Deenen, G. J., Nieuwenhuis, P. & Kroese,F. G. (1999) Eur. J. Immunol. 29, 1522–1531.
26. Kumararatne, D. S. & MacLennan, I. C. (1981) Eur. J. Immunol. 11, 865–869.27. Srivastava, B., Quinn, W. J., III, Hazard, K., Erikson, J. & Allman, D. (2005)
J. Exp. Med. 202, 1225–1234.28. Balazs, M., Martin, F., Zhou, T. & Kearney, J. (2002) Immunity 17, 341–352.29. Liu, Y. J., Zhang, J., Lane, P. J., Chan, E. Y. & MacLennan, I. C. (1991) Eur.
J. Immunol. 21, 2951–2962.30. McHeyzer-Williams, L. J. & McHeyzer-Williams, M. G. (2005) Annu. Rev.
Immunol. 23, 487–513.31. Haas, K. M., Poe, J. C., Steeber, D. A. & Tedder, T. F. (2005) Immunity 23,
7–18.32. Boffey, J., Nicholl, D., Wagner, E. R., Townson, K., Goodyear, C., Furukawa,
K., Conner, J. & Willison, H. J. (2004) J. Neuroimmunol. 152, 98–111.33. Potter, M. & Wiener, F. (1992) Carcinogenesis 13, 1681–1697.34. Byrd, L. G., McDonald, A. H., Gold, L. G. & Potter, M. (1991) J. Immunol. 147,
3632–3637.35. Diaw, L., Siwarski, D., Coleman, A., Kim, J., Jones, G. M., Dighiero, G. &
Huppi, K. (1999) J. Exp. Med. 190, 1405–1416.36. Takahashi, Y., Dutta, P. R., Cerasoli, D. M. & Kelsoe, G. (1998) J. Exp. Med.
187, 885–895.37. Cunningham, A. F., Serre, K., Toellner, K.-M., Khan, M., Alexander, J.,
Brombacher, F. & MacLennan, I. C. (2004) Eur. J. Immunol. 34, 686–694.38. Briskin, M. J., McEvoy, L. M. & Butcher, E. C. (1993) Nature 363, 461–464.39. Gerdes, J., Dallenbach, F., Lennert, K., Lemke, H. & Stein, H. (1984) Hematol.
Oncol. 2, 365–371.
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