B cell homeostasis and plasma cell homing controlledby Krüppel-like factor 2Rebecca Winkelmanna, Lena Sandrocka, Martina Porstnera, Edith Rotha, Martina Mathewsa, Elias Hobeikab,Michael Rethb, Mark L. Kahnc, Wolfgang Schuha,1, and Hans-Martin Jäcka,1,2

aDivision of Molecular Immunology, Nikolaus-Fiebiger-Center, Department of Internal Medicine III, Friedrich-Alexander-University Erlangen-Nürnberg,D-91054 Erlangen, Germany; bCentre for Biological Signalling Studies (BIOSS), University of Freiburg, Department of Molecular Immunology, Faculty ofBiology, University of Freiburg and Max-Planck Institute for Immunobiology, D-79108 Freiburg, Germany; and cDivision of Cardiovascular Medicine,Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6069

Edited* by Michael J. Bevan, University of Washington, Seattle, WA, and approved November 30, 2010 (received for review September 2, 2010)

Krüppel-like factor 2 (KLF2) controls T lymphocyte egress fromlymphoid organs by regulating sphingosin-1 phosphate receptor1 (S1Pr1). Here we show that this is not the case for B cells. Instead,KLF2 controls homeostasis of B cells in peripheral lymphatic organsand homing of plasma cells to the bone marrow, presumably bycontrolling the expression of β7-integrin. Inmice with a B cell-specificdeletion of KLF2, S1Pr1 expression on B cells was only slightly af-fected. Accordingly, all splenic B cell subsets including B1 cells werepresent, but their numbers were increased with a clear bias for mar-ginal zone (MZ) B cells. In contrast, fewer peyers patches harboringfewer B cells were found, and fewer B1 cells in the peritoneal cavityas well as recirculating B cells in the bone marrow were detected.Upon thymus-dependent immunization, IgG titers were diminished,and antigen-specific plasma cells were absent in the bone marrow,although numbers of antigen-specific splenic plasmablasts were nor-mal. KLF2 plays also a role in determining the identity of follicularB cells, as KLF2-deficient follicular B cells showed calcium responsessimilar to those of MZ B cells and failed to down-regulate MZ B cellsignature genes, such as CD21 and CXCR7.

cell trafficking | S1P1 | LKLF | B cell development | knockout mouse

Maturation of lymphocytes in primary lymphoid organs, con-trolled egress into the periphery, and proper positioning in

lymphoid tissues are critical for efficient adaptive immune re-sponses. Differential expression of Krüppel-like factor 2 (KLF2)promotes egress of T cells from lymphoid organs into the bloodand T-cell migration to lymph nodes (1, 2). Accordingly, KLF2 isexpressed in naive T cells, down-regulated upon activation, andreexpressed in memory T cells (1, 3–5). KLF2-deficient T cellsinefficiently exit from the thymus and thus accumulate there; this isthought to be a consequence of a decrease in sphingosin-1 phos-phate receptor 1 (S1Pr1) expression, which is regulated by KLF2(2, 6). In addition, KLF2 increases the expression of β7-integrin(gene symbol: Itgb7) and CD62L (L-selectin) on T cells (1, 2). How-ever, other downstream effects of KLF2 expression are less clear,although they very likely contribute to the migratory behavior ofT cells. For example, vav-cre- and lck-cre-mediated deletion ofKLF2 in T cells resulted in the up-regulation of the inflammatorychemokine receptors CCR3 andCCR5 (7) on thymocytes, whereasCD4-cre–mediated deletion led to the up-regulation of onlyCXCR3 and spontaneous IL4 production in naive T cells (8).Although the role of KLF2 in T-cell migration has been studied

extensively, its function in B cells is not fully understood. KLF2expression at the RNA and protein level is induced by pre-B cellreceptor (pre-BCR) signals (9). Analogous to T cells, KLF2 tran-scripts are abundant in resting mature B cells, down-regulatedupon mitogenic activation, and reexpressed in plasma and mem-ory B cells (10–12). Because KLF2 controls the expression of β7-integrin and CD62L, and because these adhesion molecules playalso an important role in B cell trafficking (13–15), we thoughtthat KLF2 controls homeostasis and trafficking of B lineage cells.

To address this question, we investigated the function of KLF2 ina mouse model with a B cell-specific deletion of KLF2.

ResultsKLF2 Expression Profiling in Mature B Cell Subsets. Microarrayanalyses showed that KLF2 is expressed in pre-B cells, naive Bcells, and plasma cells (9–12). We confirmed KLF2 mRNA ex-pression in isolated plasma cells from bone marrow and restingB cells by Taqman RT-PCR (Fig. S1). In addition, we founda down-regulation of KLF2 transcripts upon stimulation witheither LPS or a mixture of anti-CD40/IL4/anti-IgM (αBCR) (Fig.S1). Western blot analysis using rabbit serum raised against aKLF2 peptide (9) confirmed that KLF2 protein was expressed infreshly isolated CD43-negative splenic B cells and was down-regulated after treatment with an αBCR mixture (Fig. 1A). Wealsomade two additional observations. In resting B cells, we foundtwo forms of KLF2, suggesting that the protein is posttransla-tionally modified. Upon activation, the high molecular form dis-appeared faster than the low molecular form. More surprisingly,although abundant in isolated follicular (FO) B cells, KLF2 pro-tein was barely detectable inmarginal zone (MZ) B cells fromWTmice (Fig. 1B).

Normal B Cell Development in the Bone Marrow of KLF2-DeficientAnimals. One function of KLF2 is to keep cells in a quiescent state(16–18). Applied to B lymphoid cells, an increase of KLF2 ex-pression upon pre-BCR induction should terminate the cell cycleof proliferating pre-B cells (9, 19). If so, KLF2 deficiency shouldresult in hyperproliferation of pre-B cells, and thus, in an increaseof the pre-B cell pool. To test this hypothesis, we established B cell-specific KLF2-knockout mice by crossing mice carrying a floxedKLF2 (KLF2flox) allele (20) with the B cell-specific deleter strainmb1-cre (21) (Fig. S2 A and B). In these mice, deletion of KLF2occurs in early B cell precursors, as demonstrated by Western blotanalyses of CD19+ B lymphoid cells from bonemarrow and spleen(Fig. S2C). In flow cytometric analyses, the frequencies andnumbers of pro-B (CD19+/c-kit+), pre-B (CD19+/CD25+) andimmature (B220low/IgM+; IgM+/IgD−/CD93+) B cells (Fig. S3)did not differ between KLF2-deficient and KLF2-sufficient mice.This excludes KLF2 as the quiescence factor to terminate pre-BCR–mediated proliferative expansion of functional pre-B cells(19). However, frequencies and numbers of recirculating mature

Author contributions: R.W., W.S., and H.-M.J. designed research; R.W., L.S., M.P., E.R.,M.M., and W.S. performed research; E.H., M.R., and M.L.K. contributed new reagents/analytic tools; R.W., W.S., and H.-M.J. analyzed data; and R.W., W.S., and H.-M.J. wrotethe paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1W.S. and H.-M.J. contributed equally to this work.2To whom correspondence should be addressed. E-mail: hjaeck@molmed.uni-erlangen.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012858108/-/DCSupplemental.

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B cells (IgD+/IgM+ and B220hi/IgM+) are decreased by half (Fig.2A and Fig. S3C, respectively), indicating that KLF2-deficientmature B cells might have a defect in their homing capacity to thebone marrow.

Splenomegaly and Increased Numbers of Splenic B Cell Subsets inKLF2-Deficient Mice. Immature bone marrow B cells enter theblood stream and migrate to the spleen, where they develop intotransitional B cells (22) and differentiate into mature FO orMZBcells (23, 24).When we compared B cell subsets in the spleen fromKLF2-deficient and WT mice by flow cytometry, we observeda clear effect of KLF2 deletion on B cell homeostasis (Fig. 2B).KLF2-deficient mice have enlarged spleens (Fig. S4A) with a two-to threefold increase in the total number of B220+/IgM+ splenicB cells, an increase in all transitional B cell stages (T1–T3), a two-to threefold increase in numbers of FO B (B220+/CD21lo/CD23hi), and a four- to fivefold increase of MZ B cells (B220+/CD21hi/CD23lo/neg) (Fig. 2B). In addition, slightly but not signif-icantly increased numbers of B1a cells (IgM+/CD5+/CD43+)were detected in the spleen (Fig. 2B and Fig. S4B).Next, we examined the splenic architecture. Lymphoid follicles

in the spleen of WT mice consist of a core with IgDhi/IgMlo FO Bcells, separated by a ring of MOMA+ metallophilic macrophagesfrom the marginal zone harboring IgDlo/IgMhi MZ B cells (Fig.S5). In the spleen of KLF2-deficient mice, follicles with FO andMZ B cells are present. However, follicles are larger in size, andthe separation between MZ and FO B cells is not as clear as inKLF2-deficient mice (Fig. 2C and Fig. S5).

B Cell Homeostasis Is Disturbed in Other Peripheral Lymphatic Com-partments of KLF2-Deficient Mice. Mature FO B cells leave thespleen, circulate in the bloodstream and lymphstream, and enterother peripheral lymphatic organs (25). To determine whetherKLF2 controls migration and homing of mature peripheral B cellsubsets, we analyzed other lymphoid compartments for the pres-ence of B cells. By flow-cytometric analysis, we found no differ-ences in the numbers and frequencies of CD19+/IgM+ B cells ininguinal lymph nodes (Fig. 2D). In contrast, in the blood of KLF2-deficient animals, frequencies of B cells were decreased two-to threefold (Fig. 2E). Similarly, the peyers patches in KLF2-deficient animals were smaller, their numbers were reduced (Fig.S6A) and they contained two- to threefold fewer CD19+/IgM+ Bcells (Fig. 2F). Consistent with the reduced number of peyerspatches and B cells, serum titers of “natural” IgA were reduced asdetermined by ELISA (Fig. 3A, Right).The effect of KLF2 deletion on B1 cells in the peritoneal cavity

was even more severe, with a clear reduction in B1a (IgM+/CD5+) and B1b (IgM+/CD5−/CD11b+) cells (Fig. 2G and Fig.S6B). Because B1 cells are the major source for production of“natural” IgM in the serum (26), we determined the amount ofserum Ig titers by ELISA but could not detect any differences in“natural” IgM or IgG serum levels (Fig. 3A, Left and Center).

Reduced Numbers of Plasma Cells Observed in Bone Marrow AfterBoost Immunization with Thmyus-Dependent Antigen. To investigatewhether KLF2-deficient B cells could participate in humoral im-mune responses, we immunized WT as well as KLF2-deficient ani-mals with either thymus-independent (TI) or thymus-dependent(TD) antigens (Figs. 3 and 4). Despite an increase in numbers ofFO and MZ B cells in the spleen, antigen-specific antibody IgMand IgG titers in response to TI-1 (TNP-LPS, Fig. 3B) and TI-2 (TNP-ficoll, Fig. 3C) antigens were fairly normal in KLF2-deficient animals, with the exception of a slight but significant in-crease in IgG titers against TNP-LPS at day 14 after immunization(Fig. 3B). Similarly, IgMand IgG responses against theTDantigenTNP-coupled keyhole limbet hemocyanin (TNP-KLH) did notdiffer significantly between WT and KLF2-deficient mice 14 d af-ter primary immunization (Fig. 4A). However, 35 d after the pri-mary TNP-KLH immunization, antigen-specific total IgG (Fig.4A) and 42 d after immunization also antigen-specific IgG1 titerswere significantly reduced in KLF2-deficient mice (Fig. S7B).The secondary immune response against TNP-KLH (Fig. 4B)

mirrored the primary response. Over a period of 7 days, the ki-netics of antigen-specific IgG and IgM titers did not differ betweenWT and knockout mice. However, total IgG and IgG1 titers, butnot IgM titers, were significantly reduced 14 d after boost immu-nization (Fig. 4B and Fig. S7B). This was not due to a drop in thenumber of antibody-secreting (CD138hi/κhiλhi) plasma cells in theblood and the spleen, as their numbers were similar 5 and 14 d afterboost immunization in the blood and spleen, respectively (Fig. 4C).In contrast, the number and frequency of plasma cells in the bonemarrow were two- to threefold decreased 14 d after boost immu-nization with TNP-KLH (Fig. 4C, Lower). To confirm this findingfor antigen-specific plasma cells, we enumerated TNP-specificIgG-secreting cells by ELISPOT assays in spleen and bonemarrowfrom mice 14 d after a secondary TNP-KLH immunization (Fig.4D). We found similar numbers of antigen-specific IgG-secretingcells in the spleen of both WT and knockout mice. However, incontrast to WT mice, TNP-IgG-secreting cells were absent in thebonemarrow of KLF2-deficient mice. Hence, KLF2 contributes tothe homing of plasma cells to survival niches in the bone marrow.

KLF2 Controls Expression of CD62L and β7-Integrin but Not S1Pr1.Because KLF2-deficient mice have more B cells in the spleen andfewer B cells in most other peripheral organs, KLF2 could playa role in the egress, migration, and homing of peripheral B cellsubsets by controlling the expression of genes encoding migrationfactors and adhesion molecules. For example, previous studiesidentified the chemoattractant receptor S1Pr1 and the adhesionmolecules CD62L and β7-integrin as KLF2 target genes (1, 2, 7, 27).Egress of thymocytes and B cells from the thymus and the bone

marrow, respectively, to the blood is controlled by expression ofS1Pr1, a direct KLF2 target gene (2, 6, 28–30). In addition, S1Pr1 isrequired for efficient egress of plasma cells from the spleen into theblood (12). Because KLF2-deficient B cells egress from the bonemarrow, and because KLF2-deficient plasma cells enter the blood-stream,wewould expect thatKLF2 deficiency, in contrast to T cells,would not significantly affect S1Pr1 expression in B cells. Indeed,this was the case. In microarray and flow-cytometric analyses, wedetected, in splenic FOB andMZB cells as well as in blood B cells,very similar levels of S1Pr1 (Fig. 5 A and B, Upper Row). We con-firmed the presence of functional S1Pr1 on KLF2-deficient MZ Bcells by injecting mice with the S1Pr1-agonist FTY720 (Fig. 5C).FTY720 decreases S1Pr1 on the cell surface, and, as a result, MZ Bcells are attracted by the chemokine CXCL13 to the follicular zone(31). As documented in Fig. 5C, FTY720 abolishes the clear sepa-ration of FO B and MZ B cells.There are conflicting reports about the role of KLF2 in con-

trolling the expression of chemokine receptors in T cells (7, 8). Inour microarray and TaqMan RT-PCR analyses, the expression ofmost chemokine receptors was not affected in KLF2-deficient

0 24 48 72 h αBCRkDa

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Fig. 1. Expression analysis of KLF2 in B cells. (A and B) Western blot analysesof KLF2 abundance in (A) CD43-depleted splenic B cells before and aftermitogenic activation with αBCR mix and (B) purified splenic FO and MZ Bcells from WT mice. Membranes were first stained with an anti-KLF2 serumand then with an antiactin serum. Actin abundance served as a control forthe amount of loaded protein.

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FO and MZ B cells (Fig. S8). Only transcripts for CXCR7 areup-regulated in KLF2-deficient FO B cells on microarrays andTaqMan RT-PCR assays (Fig. 5A and Fig. S8). An almost iden-tical profile of chemokine receptor expression in KLF2-deficientB cells is reported in the accompanying manuscript by Hart et al.In contrast to S1Pr1, the abundance of the α4β7-integrin het-

erodimer was reduced in KLF2-deficient mice on recirculatingbone marrow B (Fig. S9A), blood B (Fig. 5B), and FO B cells(Fig. 5 A and B and Fig. S9B). Here, we measured expression ofβ7-integrin (gene symbol: Itgb7) mRNA by Affymetrix microarrayand RT-PCR, and the amount of α4β7-integrin on protein levelby flow cytometry. In contrast, α4β7-integrin is hardly expressedon WT and KLF2-deficient MZ B cells (Fig. 5B). This was notsurprising, as WT MZ B cells barely produce KLF2 (Fig. 1B).CD62L, an adhesion molecule critical for lymphocytes to home

to peripheral lymph nodes (13, 15), can still be detected atmRNA and protein levels on KLF2-deficient FO B and blood Bcells. However, compared with the corresponding WT B cellsubset, CD62L expression was reduced in KLF2-deficient B cells(Fig. 5B, Lower Row). Because MZ B cells from WT mice hardlyexpress KLF2, it was not surprising that MZ B cells showedlower CD62L expression than WT FO B cells. In summary,KLF2-deficieny results in a strong reduction of β7-integrin anda weaker down-regulation of CD62L on FO B cells.

KLF2 Deficiency Results in an MZ-Like Phenotype in FO B Cells. Be-cause KLF2 is barely detected in WT MZ B cells (Fig. 1B), wespeculated that KLF2-deficient FO B cells might show somemolecular and functional hallmarks of MZ B cells. This is indeedthe case. As shown by flow cytometry (Figs. 2B and 6A) and on

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Fig. 2. Disturbed B cell homeostasis in peripheral lymphatic organs. Cell suspensions from different organs of WT (FACS dot plots to the left) and KLF2-deficient (FACS dot plots to the right) mice (6–10 wk old) were surface stained with indicated antibodies. Frequencies (Left and Center) and absolute cellnumbers (Right) of (A) recirculating B cells (IgD+/IgM+) from bone marrow, (B) total B cells (B220+/IgM+), MZ B cells (B220+/CD23−/CD21hi), FO B cells (B220+/CD23+/CD21low), transitional B cells (T1: B220+/AA4.1+/IgMhi/CD23−, T2: B220+/AA4.1+/IgMhi/CD23+ and T3: B220+/AA4.1+/IgMlow/CD23+), as well as B1a (IgM+/CD5+/CD43+) and B1b cells (IgM+/CD5−/CD43+) from spleen are shown. (C) Immunofluorescence of acetone-fixed tissue cryosections (color code of antibodiesshown to the right). Frequencies of total CD19+/IgM+ B cells in (D) inguinal lymph node, (E) blood, and (F) peyers patches as well as (G) frequencies of B1a (IgM+/CD5+/CD11b+), B1b (IgM+/CD5−/CD11b+), and B2 (IgM+/CD5−/CD11b−) cells in peritoneal lavages. Results of all analyzed littermates are summarized in thediagrams shown to the right, with one dot representing one mouse.

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splenic cryosections (Fig. 6B), KLF2-deficient FO B cells fail todown-regulate complement receptors 1/2 (CD21/35) and expressCD21/CD35 at levels similar to those on WT MZ B cells (Fig.6A). In addition, CXCR7, a chemokine receptor expressed onMZ B cells (32), is up-regulated on KLF2-deficient FO B cells,as shown by microarray and TaqMan RT-PCR analyses (Fig. 5Aand Fig. S8).Furthermore, compared with WT FO B cells, KLF2-deficient

FO B cells showed a slight but statistically significant increase inthe baseline of intracellular Ca2+, similar to that found in WTMZ B cells (Fig. 6C and Fig. S10). In contrast, KLF deficiencydid not affect either intracellular or extracellular Ca2+ flux inMZ B cells (Fig. 6C and gating in Fig. S10A). Finally, MZ-like Bcells with higher expression of CD21/35 could also be detected inthe inguinal lymph nodes by flow cytometry (Fig. S11). Thesefindings point to a function of KLF2 in determining the identityof FO B cells.

DiscussionIn this study, we investigated the effect of KLF2 deficiency onplasma cell homing to the bone marrow and B cell homeostasis.In T cells, KLF2 controls the exit of thymocytes into the blood byregulating the expression of the chemoattractant receptor S1Pr1(1, 2, 23). In contrast to T cells, we show here that KLF2 is notrequired to maintain surface expression of S1Pr1 on B lineagecells. Consistent with this, WT MZ B cells, which produce onlysmall amounts of KLF2, show high levels of S1Pr1 (31). We alsoshow here that S1Pr1 on KLF2-deficient B cells is functional. Weconclude this from three findings: (i) KLF2-deficient B cells aredisplaced from the marginal zone upon FTY720 treatment invivo; (ii) immature KLF2-deficient B cells egress from the bonemarrow and are positioned to the correct anatomical location insplenic follicles; and (iii) KLF2-deficient plasma cells reach the

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Fig. 4. Impaired thymus-dependent immune response in KLF2-deficientmice. Sera of WT mice (blue open triangles) and KLF2-deficient mice (redfilled triangles) were collected after (A) primary and (B) boost TNP-KLH im-munization and analyzed by ELISA for antigen-specific antibodies. (C) Flow-cytometric analyses of frequencies (Left and Center) and absolute numbers(Right) of CD138hi/κhi/λhi plasma cells in spleen, blood, and bone marrow ofWT and KLF2-deficient mice at indicated time points after boost TNP-KLHimmunization. (D) ELISPOT assay of TNP-specific IgG-secreting cells in bonemarrow and spleen 14 d after boost immunization with TNP-KLH. and D)Triplicate of one representative experiment (Left), and summary of resultsof all analyzed littermates, with one dot representing the mean value ofELISPOT triplicates of one mouse (Right).

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bloodstream. The fact that all three events in B cell maturationdepend on the expression of S1Pr1 (28, 30, 31) strongly supportsour conclusion.It is known that plasma cells require CXCR4 and CXCR3 to

reach the bone marrow (33–35) but not how they enter the bonemarrow and which survival signals are required there (36, 37).KLF2-deficient B cells respond to TD antigens and develop inthe spleen into IgG-secreting plasma cells. However, these cellsdo not reach the bone marrow. Our finding that KLF2-deficientB cells down-regulate β7-integrin on transcript level and the α4β7-

heterodimer on protein level could add another player to themechanism that controls the homing of plasma cells to the bonemarrow. α4β7-Integrin is critical for IgA-secreting plasma cells tohome to the gut (14, 38, 39), which could explain the decrease innatural serum IgA in KLF2-deficient mice. Furthermore, vas-cular cell adhesion molecule-1 (VCAM-1), a ligand for α4β7-integrin, is expressed in the bone marrow in combination withmucosal addressin cell adhesion molecule-1 (MAdCAM-1) (38).Therefore, it is tempting to speculate that α4β7-integrin is criticaleither for entry of plasma cells into the bone marrow or for theirlocalization to survival niches in the bone marrow. However, someCD138hi/IgL-chainhi plasma cells were still present in the bonemarrow of our KLF2-deficient mice. Therefore, KLF2 deficiencycould either affect the trafficking of only a subset of plasma cells(e.g., from the spleen) or delay plasma cell homing to the bonemarrow. Further characterization of KLF2-deficient plasma cellsand transfer experiments with GFP-labeled plasma cells fromspleen and other secondary organs will address this issue.The down-regulation of α4β7-integrin in KLF2-deficient mice

could also explain most of the defects in B cell homeostasis, as thephenotype is mirrored in mice with defective α4β7-integrin func-tion. For example, treatment with blocking antibodies againstα4β7-integrin (13), or germline deletion of β7-integrin (15), resultsin an accumulation of B cells in the spleen and a reduced capacityof B cells to enter peyers patches and mesenteric lymph nodes butnot peripheral lymph nodes. Unfortunately, none of these studiesexamined the effect of defective β7-integrin function on B1 cellhomeostasis in the peritoneal cavity or the presence of antigen-specific plasma cells in the bone marrow.Surprisingly, the strong reduction in peritoneal B1 cells in our

KLF2-KO mice was not accompanied by decreased serum IgMlevels (26). The four- to fivefold increase in the MZ B cell com-partmentmight compensate for the loss of theB1 cell compartmentas a producer of “natural” IgM (24). Alternatively, splenic CD5-positive B1a cells, which were slightly increased in KLF2-KOmice,could be another source for most of the natural serum IgM (40).During the preparation of our manuscript, Hoek et al. also

reported the effect of conditionalKLF2 deletion (viaCD19-cre) onB cells (41). Although some of theirfindings, such as the increase inFO and MZ B cells and the reduction in peritoneal B1 cells, areconsistent with our results, there are notable differences. For ex-ample, Hoek et al. did not analyze secondary immune responses

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and plasma cell homing. In addition, they propose that S1Pr1 andCXCR5 are strongly down-regulated in MZ B cells and up-regu-lated in FO B cells and conclude that this explains why FO B cellsinvade the marginal zone in the spleen. In contrast, we did not findsignificant changes in S1Pr1 expression at the RNA and proteinlevel in KLF2-deficient MZ and FO B cells, and CXCR5 expres-sion remained unchanged in both populations. The drastic down-regulation of S1Pr1 inKLF2-deficientMZB cells in theHoek et al.paper is surprising, as MZ B cells still disappear in that study afterblockage of S1Pr1 function through FTY720 treatment.We do notknow the reason for the differences between the results of Hoeket al. and our own. However, an accompanying paper by Hart et al.describes studies in a mouse with CD19-cre mediated KLF2 de-letion with essentially the same results as reported in our paper.In summary, KLF2 controls homeostasis of mature B cell

subsets in peripheral lymphatic organs and homing of antibody-secreting plasma cells to the bone marrow, presumably by con-trolling the expression of the adhesion molecule β7-integrin.

Materials and MethodsProcedures. Standard procedures and methods such as animal handling, flowcytometry, isolation and activation of B cells, histology, immunizations andantigen-specific ELISA, ELISPOT assays, RNA and Western blot analyses, andstatistical analyses are described in SI Appendix.

Analysis of Intracellular Calcium Flux. Calcium flux measurements wereperformed as described (42, 43). Briefly, 5 × 106 total spleen cells wereincubated with the Ca2+ dye Indo-1 AM and fluorochrome-conjugatedantibodies against CD21 and CD23 to distinguish between FO (CD23hi/CD21lo) and MZ (CD23lo/neg/CD21hi) B cells. To measure changes in in-tracellular Ca2+, stained cells were suspended in Ca2+-free Krebs–Ringersolution containing 0.5 mM EGTA and stimulated with 10 μg/mL anti-μHCF(ab’)2 (Jackson Laboratories). To measure extracellular Ca2+ influx, Ca2+

was restored after 240 s. Changes in Indo-1 AM fluorescence ratios wereanalyzed in gated FO and MZ B cell populations with a LSRII flow cytom-eter (BD Biosciences) using FlowJo software (Tree Star). Equal Indo-1loading was assessed by ionomycin stimulation.

FTY720 Treatment. For FTY treatment, 6- to 10-wk-old animals were injectedi.p. with either 20 μg FTY720 (Cayman Chemicals) or an equivalent volume ofsaline. Four hours after injection, animals were killed, and spleens were re-moved and processed for cryosections.

ACKNOWLEDGMENTS.We thank M. Wabl for discussion and Uwe Appelt forcell sorting. This research was supported in part by the InterdisciplinaryCenter for Clinical Research Erlangen (IZKF), the Deutsche Forschungsge-meinschaft (Training Program GK592, Research Grant FOR 832 JA 968; toH.M.J.), an intramural grant from Erlanger Leistungsbezogene Anschubfi-nanzierung und Nachwuchsförderung (to W.S.), Deutsche Forschungsge-meinschaft Center Grant SFB620, and the German Excellence Initiative(EXC294; to M.R.).

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