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The Journal of Immunology, 2007, 179: 2270-2281.
Copyright © 2007 by The American Association of Immunologists, Inc.
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The Recirculating B Cell Pool Contains Two Functionally Distinct, Long-Lived, Posttransitional, Follicular B Cell Populations1

Annaiah Cariappa, Cristian Boboila, Stewart T. Moran, Haoyuan Liu, Hai Ning Shi and Shiv Pillai2

Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Disparate models for the development of peripheral B cells may reflect significant heterogeneity in recirculating long-lived B cells that have not been previously accounted for. We show in this study that the murine recirculating B cell pool contains two distinct, long-lived, posttransitional, follicular B cell populations. Follicular Type I IgMlow B cells require Ag-derived and Btk-dependent signals for their development and make up the majority of cells in the recirculating follicular B cell pool. Follicular type II B cells do not require Btk- or Notch-2-derived signals, make up about a third of the long-lived recirculating B cell pool, and can develop in the absence of Ag. These two follicular populations exhibit differences in basal tyrosine phosphorylation and in BCR-induced proliferation, suggesting that they may represent functionally distinct populations of long-lived recirculating B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During binary cell fate decisions, distinct signaling receptors ensure that daughter cells representing each of two distinct fates are generated, and that development is not completely skewed in favor of one type of daughter population. During the generation of B and T cells, for instance, the Flk2 receptor tyrosine kinase, in conjunction with IL-7-derived signals, may drive the commitment of progenitors toward the B lineage, while signals from Notch1 ensure the simultaneous commitment of progenitors to a T cell fate (1, 2, 3). Broadly similar mechanisms involving reciprocal pairs or sets of pairs of signaling receptors may operate during other commitment events that appear to involve binary cell fate decisions. One such binary cell fate decision involves the commitment of peripheral B cells to a follicular or a marginal zone B cell fate.

The development of naive peripheral B cells depends on the signals delivered by the BCR, as well as on survival and differentiation signals derived from other receptors and transcriptional regulators, including B cell activating factor receptor (BAFF-R), Notch2, and NF{kappa}B1 (4, 5, 6, 7, 8, 9, 10). Developmental signals that originate from the BCR are postulated to include both constitutive ligand-independent signals, as well as signals that depend on interactions between distinct self-Ags and their cognate BCRs, apparently below the avidity threshold required to trigger receptor editing and clonal deletion (11, 12).

Immature B cells in the bone marrow represent the first defined stage of B cell development in which the BCR is expressed on the cell surface. These cells also express high levels of CD24 and CD93 (13, 14, 15). IgMhighIgDlow/–CD93+ B cells that express low levels of CD21/CR2 and CD23 (16) represent newly formed or transitional type 1 (NF/T1)3 B cells. They may reside in an extra-follicular location in the spleen before their entry into B lymphoid follicles. These NF/T1 B cells have a short half-life, and they mature after a brief delay into transitional type 2 (T2) cells. T2 cells still express high levels of CD24 and CD93, reflecting their relatively recent generation in the bone marrow, express high levels of IgM, and are CD23+ (13, 17, 18). A population of CD93+ B cells transitional type 3 (T3) cellsthat are IgMlow has also been described (18). T2 and T3 B cells are believed to represent transient intermediates on their way to becoming long-lived recirculating follicular B cells that remain CD23+, but no longer express CD93 (Fig. 1A). In a recent study, it has been suggested that T3 B cells represent anergic B cells (19). In a different classification, nonrecirculating splenic IgMhighIgDhigh CD23+CD21high cells were also designated as T2 cells (16). We and others have suggested that these latter cells represent specialized splenic precursors of marginal zone (MZ) B cells called MZ precursor, or MZP, B cells (7, 9, 10, 20, 21, 22, 23). MZP B cells reside only in splenic follicles in rodents and are nonrecirculating B cells.


Figure 1
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  		FIGURE 1. Models for transitional B cells and the role of Ag in splenic B cell differentiation. A, Sequential progression through transitional T1, T2, and T3 stages leads to the generation of mature B cells. T1 cells are AA4.1+CD23–CD45+ B cells, T2 B cells are AA4.1+CD23+IgMhigh B cells, and T3 B cells are AA4.1+CD23+IgMlow B cells. Mature B cells are AA4.1–CD23+ IgM+ B cells (after Allman et al., Ref. 18 ). B, A signal strength model for peripheral B cell development. NF/T1 B cells are IgMhighIgDlowCD21low cells. T2/FP cells are IgMhighIgDhighCD21int B cells. Relatively strong BCR signals and Btk are required for the generation of IgDhighIgMlow FO B cells, while relatively weak signals collaborate with Notch2 and NF-{kappa}B1 to yield MZ B cells (after Pillai et al.; Ref. 10 ). C, Ag mediated MZ B cell development. Ag mediates MZ B cell development while CD24lowCD23+ follicular B cells develop in the absence of Ag (after Wen et al.; Ref. 27 ).

 
A number of mutations that result in striking defects in MZ B cell development are accompanied by normal or enhanced follicular B cell numbers. MZ B cell development is defective in the absence of negative regulators of BCR signaling, such as Aiolos and CD22, but mature follicular B cell numbers are enhanced in these mice (20, 24, 25). The loss of MZ and MZP B cells in Aiolos–/– mice was reversed in mice harboring defects in both Aiolos and Btk, suggesting that enhanced Btk signaling compromises MZ B cell development (20), but contributes to normal or increased numbers of IgDhighIgMlow follicular B cells. Although this signal strength model (Fig. 1B) is consistent with the normal development of MZ B cells observed in the absence of Btk in replete mice, other non-BCR-linked genes, such as Notch2, RBP-J{kappa}, and NF{kappa}B1 are also required for MZ B cell development (6, 7, 8). We have therefore suggested that developing B cells that receive weaker BCR signals may be receptive to inductive signals delivered by intracellular Notch2 and by NF{kappa}B1 to differentiate first into MZP B cells and to then migrate into and be retained in the marginal zone area as MZ B cells (10). NF{kappa}B activation occurs downstream of the BAFF receptor, and Notch2 and NF{kappa}B1 function synergistically during MZ B cell development (26). Another study using BCR transgenic mice has suggested that weaker BCR signals contribute to follicular B cell development rather than MZ B cell development (27 ; Fig. 1C), and this view will be discussed below in the context of our current findings.

In contrast to defects in the Btk/PLC{gamma}2 pathway that compromise follicular but not MZ B cell development, a number of other mutations in genes encoding signaling proteins result in a marked reduction of MZ B cells. Mutations in CD19 (28), the p50{alpha}-p55{alpha}-p85{alpha} regulatory subunits of PI3K (29), and the p110 {delta} subunit of PI3K (30, 31) all present defects in BCR/Btk/Plc{gamma}2 activation, but also have defects in the development of both B-1 and MZ B cells. It is recognized that PI3 kinase not only contributes to the activation of Btk, but also is required for the activation of Akt and the subsequent inhibition of Forkhead family proteins. It has been suggested that the peripheral B cell developmental defects seen in the absence of PI3K signaling (but not in the absence of Btk and PLC{gamma}2) may reflect the failure to activate the Akt pathway in PI3K mutant mice (29). CD19 functions as an adaptor for PI3K activation, and it is possible that, during B cell development, these molecules contribute to the migration of B cells to the marginal zone and/or the peritoneal cavity, as well as to the retention of cells in those locations. Indeed, mutations in Pyk2, a tyrosine kinase linked to integrin signaling (32), the Rho guanine nucleotide exchange factor Lsc, (33), the adaptor DOCK-2 (34), the Rac2 Rho family GTPase (35), and integrins (36) exhibit defective MZ B cell development because these genes presumably contribute to the migration or retention of MZ B cells in the marginal zone.

Most of the evidence for presumed stages of peripheral B cell development is inferential and has been obtained from the analysis of mutants or the study of the sequential emergence of B cell populations following nonablative irradiation. Direct evidence for precursor-product type relationships of specific populations during B lymphocyte development is generally lacking. A major confounding factor in the study of peripheral B cell development is the phenomenon of homeostatic proliferation. Transfer of purified B cell populations into nonirradiated replete mice results in very poor subsequent recovery of the transferred cells, making precursor-product type studies on cellular populations by cell transfer virtually impossible. Transfer of B cell populations into mice that lack lymphocytes as a consequence of immunodeficiency or irradiation results in homeostatic proliferation, the expression of high levels of CD21, and the generation preferentially of MZ B cells (37, 38, 39, 40). In these circumstances, following transfer into lymphopenic hosts, transitional and follicular B cells primarily give rise to MZ B cells. Lymphocyte development appears to proceed in a distinct manner in replete mice. Some genes that are required for homeostatic proliferation in lymphopenic mice are not required for normal MZ B cell development in replete mice. As an example, homeostatic B cell proliferation in lymphopenic recipients is impaired in the absence of Btk, but MZ B cell development proceeds normally in replete mice that lack Btk (39). Similarly, although NF{kappa}B1 contributes to MZ B cell development (8), it is not required for the homeostatic proliferation of B cells (39).

Our studies, described below, indicate that two functionally distinct, long-lived, recirculating, posttransitional, follicular B cell populations can be distinguished, one of which represents a pool of B cells that develops in the apparent absence of self-Ag-mediated BCR ligation and does not require Btk, and is also preserved in the absence of Notch2-derived signals. Therefore, this long-lived population could represent the cell type that undergoes a cell fate decision, giving rise to either MZ B cells or IgMlow follicular B cells. The latter IgMlow B cell population requires Ag and Btk signals for development, presumably resulting from the relatively avid interaction of developing B cells in vivo with cognate self-structures. Possible reasons for the existence of these two distinct follicular B cell populations will be discussed below.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The HyHEL10 IgH and IgL knock-in mice have been described previously (41, 42) and were kindly made available by Dr. J. Cyster (University of California, San Francisco, CA) and Dr. M. Nussenzweig (Rockefeller University, New York). Notch2+/– mice (7) were made available by Dr. Y. Hamada (University of Tokyo, Tokyo, Japan). C57BL/6 mice were purchased from The Jackson Laboratories and housed in a pathogen-free facility. All mice used for experiments were between 8 and 12 wk of age. Animal procedures were cleared by the Subcommittee on Research Animal Care at Massachusetts General Hospital.

Flow cytometric analysis and fluorescent-activated cell sorting

Flow cytometry and flow sorting were performed as previously described (43, 44). The following murine mAb conjugates were used: allophycocyanin (APC)- and r-PE-Cy7-1B4B1 (anti-IgM, rat IgG), and R-PE-, biotinylated-, and Cy5-11-26 (anti-IgD, rat IgG2a, {kappa}, all from Southern Biotechnology Associates, Birmingham, AL), and purified 2.4G2 (anti-CD16/CD32 {Fc{gamma} III/II receptor}, rat IgG2b, {kappa}, culture supernatant), APC- and Pacific Blue-RA3-6B2 (anti-CD45R/B220, rat IgG2a, {kappa}), PE-Cy7-RA3-6B2, PE-Cy7-R6-60.2 (anti-IgM, rat {LOU}, rat IgG2a, {kappa}), FITC- and PE-7G6, (anti-CD21/CD35, rat IgG2b), PE-M1/69 (anti-CD24, Heat Stable Ag, rat {DA} IgG2b, {kappa}), PE-S7 (anti-CD43, Leukosialin, rat IgG2a, {kappa}), PE-B3B4 (anti-CD23, rat IgG2b, {kappa}), and PE-AA4.1 (Early B Lineage, rat {Harlan Sprague Dawley} IgG2b, {kappa}) all from BD Pharmingen, and APC- and PE-AA4.1 (eBioscience). Hen egg lysozyme (HEL; Sigma-Aldrich) was biotinylated, and was revealed by streptavidin (SA)-PE-Texas Red or PerCP-Cy5.5, and biotinylated IgD was revealed by SA-PE-Cy5.5 or SA-PerCP (all from BD Pharmingen). For intracellular cytokine staining, the following R-PE-conjugated Abs were used: XMG1.2 (anti-IFN-{gamma}, rat IgG1), MP6-XT22 (anti-TNF, rat IgG1), JES6-5H4 (anti-IL-2, rat IgG2b), 11B11 (anti-IL-4, rat IgG1), MP5-20F3 (anti-IL-6, rat IgG1), and C15.6 (anti-IL-12, rat IgG1), all from BD Pharmingen. Intracellular cytokine staining was performed after surface staining, following treatment with 0.1% saponin (Sigma-Aldrich), and GolgiPlug 1 µl/ml (brefeldin A; BD Pharmingen). Intracellular phosphotyrosine residues were stained with FITC-4G10 (anti-phosphotyrosine mAb; Upstate Biotechnology) following surface staining and treatment with 0.1% saponin. Staining at 4°C or after fixation with paraformaldehyde gave similar results.

Flow cytometric analysis was performed on a dual laser FC500 (Beckman Coulter), and sorting was performed on a MoFlo sorter (DakoCytomation), a FACSAria (BD Biosciences), and an Epics Altra hypersort system (Beckman Coulter). The purity of sorted samples always exceeded 96%. Processed sample data was analyzed using FloJo v8.1.1 (Tree Star) software. IgM/IgD gates in the spleen were set according to Hardy et al. (45).

Bromodeoxyuridine (BrdU) labeling

Continuous labeling with BrdU (Sigma Aldrich) was performed as described earlier (43). In brief, 0.25 mg/ml BrdU and 2 mg/ml glucose were administered in drinking water for 22 days, and the decay in labeling was followed for 4 wk after cessation of BrdU administration. The BrdU decay data curves were generated by the least squares nonlinear regression analysis method.

Lymphocyte proliferation assay

The method described previously (24) was followed with some modifications. In brief, sorted cell fractions were plated at 1 x 105 cells/well in triplicate in 96-well flat-bottom microtiter plates with medium alone or with medium and one of the following: goat anti-mouse IgM (Fab’)2 (µ-chain specific), 10 µg/ml (Jackson ImmunoResearch Laboratories), polyclonal goat anti-mouse IgD antiserum (activating), 10 µg/ml (eBioscience), purified hamster anti-mouse CD40, 1 µg/ml (BD Pharmingen), LPS from Escherichia coli serotype O55:B5, 10 µg/ml (Sigma Aldrich). The cells were cultured for 48 h, then pulsed with 2 µCi/well of [3H]thymidine (PerkinElmer) for an additional 18 h, and harvested and read in a betaplate reader.

Affymetrix DNA GeneChip probe array assay

IgMhighIgDhighCD21int follicular type II (FO II) B cells and IgMlowIgDhighCD21int follicular type I (FO I) B cells were sorted directly into RNAlater (Qiagen). Total cellular RNA was prepared using RNeasy columns (Qiagen). RNA was amplified twice using RiboAmp RNA Amplification Kit (Arcturus) and single-stranded biotinylated cRNA probes were generated using the cDNA as template, and the BioArray RNA Transcript Labeling Kit (Enzo Biochem), which uses T7 RNA polymerase, and biotin-labeled nucleotides. The probes were purified using Qiagen columns, fragmented, and hybridized to the murine genome MOE430A set GeneChip (Affymetrix) at the Harvard Center for Genomic Research (Cambridge, MA). Hybridization signals were detected by a GeneChip Scanner 2500 (Affymetrix) and checked for uniformity. Probe sets were all scaled to the same target intensity of 500 using global scaling, and the data were analyzed using the Affymetrix Microarray Analysis Suite (MAS 5.0). The samples were validated using two sets of internal controls: Affymetrix internal control genes, and a known set of genes that should be up- or down-regulated in the two follicular B cell samples.

In vitro differentiation

Flow-purified splenocytes were plated in 48-well tissue culture plates at a density of 0.5–1 x 105 cells/well in triplicate in 300 µl of complete {alpha}-DMEM with or without HEL, and cultured in a CO2 incubator at 37°C and 5% CO2. Two days later, each of three wells were pooled, filtered, and stained for flow cytometry using the same fluorochrome combination as used for the sort.

Statistical analysis

The p values for differences between groups were calculated by the Mann-Whitney U test using StatView (version 5.0.1). BrdU decay curves were generated by the least squares nonlinear regression analysis method using MacCurveFit 1.5 (Kevin Raner Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In these studies, we sought to test the hypothesis that there are two distinct, long-lived, recirculating, posttransitional follicular B cell populations, one of which owes its origins to relatively strong Btk-dependent BCR signals (and may be consequently prohibited from differentiation into the MZ B cell lineage), whereas the other requires weaker BCR signals and might represent a distinct type of mature follicular B cell that develops in the absence of cognate Ag in a "default" manner, dependent perhaps on constitutive BCR signaling and exposure to BAFF. Such a population of follicular B cells would theoretically retain the ability to be drawn into the MZ B cell pool when required.

IgMhighIgDhigh B cells in the spleen can be separated into two distinguishable B cell populations

Hardy et al. (45) described two categories of IgDhigh B cells distinguishable by their different levels of surface IgM, and by the fact that one category was markedly diminished in Xid mice. Xid mice carry a mutant version of the TEC kinase, Btk. IgDhighIgMlow (Fraction I) B cells are lost in the absence of Btk-derived signals, whereas IgDhighIgMhigh (Fraction II) B cells were maintained in Xid mice. It has sometimes been assumed that IgDhighIgMhigh Fraction II B cells are also transitional T2 B cells (16), but this has never been rigorously established or disproved, and this is an issue that will be addressed below.

As seen in Fig. 2, IgDhighIgMhigh Fraction II B cells can be separated into two distinct populations. Both are CD23+. One population expresses high levels of CD21. These cells also express high levels of CD1d and CD9 (46, 47 ; AC and SP, unpublished observations) and correspond to the cells that we and others have previously called MZP B cells (7, 9, 10, 20, 21, 22, 23). A distinct subpopulation of Fraction II B cells expresses intermediate levels of CD21 (20), and also lower levels of CD1d and CD9 than MZP cells (47). We refer to these Btk-independent IgMhighIgDhighCD21int B cells as FO II or FO II B cells to differentiate these cells from the Btk-dependent Fraction I IgDhighIgMlow population, which we categorize as FO I or FO I B cells.


Figure 2
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  		FIGURE 2. Splenic IgMhighIgDhigh B cells can be separated into two distinct populations. IgMhighIgDhighCD21high B cells are MZP cells. IgDhighIgMhighCD21int cells represent FO II B cells. FO II and MZP B cells were separated by flow sorting and recharacterized as shown.

 
There were a number of issues regarding FO II B cells that we sought answers to. Are FO II B cells long-lived B cells or are they part of the transitional pool? Although Fraction II cells survive in the absence of Btk, it is unclear whether the surviving cells are FO II B cells or MZP B cells. Do FO II B cells develop in a Btk-independent manner, and are they preserved in mice in which B cells develop in the apparent absence of a cognate Ag? Are cells of the Btk-dependent FO I B cell population lost in the absence of a cognate Ag?

The majority of FO II B cells are posttransitional B cells

The elegant studies of Allman et al. (17, 18) have revealed the presence of a population of AA4.1/CD93+ B cells that express CD23 and are referred to as T2 cells. We have attempted to delineate the relationship between all peripheral B cell populations, as categorized by different groups, using six-color flow cytometry. The vast majority of AA4.1+ B cells reside in the newly formed/T1 B cell pool, and in the T2 pool. As shown in Fig. 3A, a large proportion (~80%) of the AA4.1+ CD23+ T2 population is made up of IgMhighIgDlow B cells that do not overlap with the FO II pool. A proportion of the IgMhighIgDhigh CD23+ FO II pool may be made up of relatively recent emigrants from the bone marrow that have just entered the follicular B cell compartment, and we wished to determine what proportion of FO II B cells express markers of the transitional/T2 population. We used AA4.1, as well as the widely used CD24 marker, to identify the relative proportions of transitional B cells in the FO II B cell pool in adult mice. A small proportion of FO II B cells (~10%) expressed AA4.1 or were CD24high (Fig. 3B). These data indicate that the vast majority of FO II B cells are AA4.1neg posttransitional B cells, and that these cells are therefore potentially long-lived B lymphocytes.


Figure 3
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  		FIGURE 3. A small proportion of FO II B cells express transitional markers. A, Most AA4.1+CD23+IgMhigh T2 cells are IgDlow B cells. Six-color flow cytometric analysis was performed using Abs against CD45R, CD23, CD21, IgM, IgD, and CD93. B, A small proportion of IgMhighIgDhighCD21int FO II B cells express transitional markers such as AA4.1 (i) or CD24 (ii); n = 3 mice.

 
FO II B lymphocytes are long-lived cells

To determine whether FO II B cells are long-lived or short-lived B cells, we performed continuous BrdU labeling studies. Labeling studies have been reported on unfractionated Fraction II B cells (48), but we wished to separate these IgMhighIgDhigh B cells into MZP and FO II B cell pools. Mice were fed BrdU continuously for 3 wk, labeling was stopped abruptly, and BrdU-positive cells in different B cell populations in the spleen were enumerated for the following 4 wk as the label decayed and were plotted using regression analysis. As can be seen from Fig. 4, FO I, MZP, and FO II B cells are long-lived splenic follicular B cell populations, whereas NF/T1 cells, as is well established, represent a short-lived B cell population. MZ B cells accumulate higher levels of BrdU initially, but are also long-lived B cells. Because FO II cells are long-lived B cells, can be identified in lymph nodes and the bone marrow, and show clear evidence of recirculation when assessed using a parabiosis approach (44), they represent a distinct pool of long-lived recirculating B cells, easily distinguishable phenotypically from FO I B cells. Because these B cells have a long half-life similar to those of FO I B cells, the vast majority of the FO II cells are clearly posttransitional B cells, in keeping with the data presented in Fig. 3. BrdU labeling studies were also performed on bone marrow B cells (44) and FO II B cells identified in the bone marrow were as long lived as FO I B cells (data not shown).


Figure 4
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  		FIGURE 4. FO II B cells are long-lived cells. Following continuous BrdU labeling for 22 days, labeling was abruptly stopped and allowed to decay during a 4 wk period. Regression analysis of the data is shown. Error bars, SEM. NF, Newly formed B cells. n = 3 mice per time point.

 
Alteration of Igµ mRNA levels in FO I vs FO II B cells

The specific reduction in cell surface IgM levels in FO I vs FO II cells may reflect an alteration of gene expression at a transcriptional or posttranscriptional level, or might reflect the posttranslational down-regulation specifically of IgM BCRs. We compared the expression, at the level of mRNA accumulation, of selected specific B cell genes in purified FO I and FO II B cells using a microarray approach. As can be seen in Fig. 5, although mRNA accumulation of CD21, CD1d, and CD24 are comparable between FO I and FO II B cells, there is a striking reduction of µ-chain mRNA accumulation noted in FO I B cells. Although there might or might not be a posttranslational component to surface IgM regulation in FO I B cells, a major differentiation-related alteration between FO I and FO II B cells occurs at the level of the regulation of µ-mRNA levels by transcriptional or posttranscriptional means.


Figure 5
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  		FIGURE 5. FO I and FO II B cells can be distinguished at the level of IgM mRNA accumulation. mRNA levels of CD1d, CD21, CD22, CD23, and IgM were compared in purified FO I and FO II cells, on an Affymetrix microarray. GAPDH expression was similar in both samples in each of two separate analyses.

 
FO I and FO II B cells exhibit differences in basal tyrosine phosphorylation, basal cytokine synthesis, and proliferation following BCR ligation

Basal tyrosine phosphorylation of FO II B cells is set at a slightly higher level than that of FO I B cells, suggesting intrinsic differences between these two populations. Basal tyrosine phosphorylation levels of FO II B cells resemble the levels observed in NF/T1 B cells, and the MZ and MZP B cell populations (Fig. 6A). However, mobilization of calcium following BCR crosslinking was similar in FO I and FO II B cells (data not shown). Sorted FO I cells proliferated less than sorted FO II cells in response to BCR ligation with either anti-IgD or anti-IgM (Fig. 6B). FO I cells also proliferated less well in response to CD40 ligation, but these cells do not have a global impairment in proliferation because the response to LPS was robust (Fig. 6B). A relatively small proportion of activated B cells synthesizes cytokines (49), and we have noted basal synthesis of cytokines in a small number of ex vivo follicular B cells. However, the proportion of cytokine-synthesizing cells is consistently greater in the FO II population than in FO I B cells (Fig. 7). The proportions of FO II B cells that synthesize cytokines under basal conditions more closely resemble the proportions seen in newly formed B cells, and in MZP and MZ B cells (data not shown), broadly mirroring the results of studies on basal tyrosine phosphorylation. Taken together, the differences in basal tyrosine phosphorylation, the in vitro proliferation data, and the comparison of cytokine synthesis in unstimulated cells suggest that FO I and FO II B cells may represent functionally distinct populations.


Figure 6
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  		FIGURE 6. FO I cells proliferate less than FO II cells following BCR stimulation and CD40 ligation. A, Basal tyrosine phosphorylation was compared by flow cytometry in FO I and FO II B cells. B, Proliferation was examined in the context of anti-IgD-mediated activation (left), anti-CD40 triggering (left), anti-IgM-mediated activation (right), and LPS-mediated activation (right). n = 3 mice per group. Error bars, SEM.

 

Figure 7
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  		FIGURE 7. Under basal conditions, cytokine synthesis is more frequently observed in the FO II population of follicular B cells. The synthesis of TNF, IFN-{gamma}, IL-2, IL-4, IL-6, and IL-12 was compared in ex vivo unstimulated FO I and FO II B cells using intracellular staining and flow cytometry. Representative results are shown, but similar results were noted in six mice.

 
FO II B cells are preserved in mice heterozygous for Notch2 and in the absence of Btk-derived signals

MZ B cells are lost in mice in which the recombination signal binding protein RBPJ{kappa} or Notch2 has been conditionally deleted in the B lineage, and are significantly diminished in mice that are heterozygous for Notch2 (6, 7, 50). We wished to establish whether the long-lived FO II B cell population is generated independently of both Btk-derived and Notch2-dependent differentiation signals. We examined FO II B cells and other splenic B cell populations in wild-type mice and in Notch2 heterozygotes. As can be observed in Fig. 8, A and B, although there is a significant decrease in MZ B cells in Notch2 heterozygotes, and a significant increase in FO I B cells, the relative absence of Notch2 does not significantly influence the FO II B cell population, which is slightly more abundant in Notch2 heterozygotes. As seen in Fig. 8C, it is clear that at steady-state, FO II B cells are present in normal numbers in Xid mice, although, as has long been established, Btk-derived signals are required for the preservation of FO I B cells (45). These data suggest that although the generation or survival of FO I B cells depends on Btk, and MZ B cells represent a Notch2-dependent population, long-lived FO II B cells do not require Btk or Notch2 for their generation or maintenance.


Figure 8
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  		FIGURE 8. FO II cells develop independently of Notch2 and Btk. A, Peripheral splenic B cells were analyzed by flow cytometry in wild-type mice and Notch2 heterozygotes. The FO II B cell population was preserved. n = three mice per group. B, Quantitation of FO I, FO II, MZP, and MZ B cell populations in wild-type and Notch2 heterozygotes, based on mice analyzed in A. Error bars, SEM. C, FO II B cells are preserved in Xid mice.

 
In replete as well as lymphopenic BCR knock-in mice lacking cognate Ag, the FO II B cell population is preserved, whereas FO I B cells are markedly diminished

A number of BCR transgenic mice have been identified that exhibit a marginal zone B cell bias with a relative decrease in follicular B cells. One possible explanation for this phenomenon is that these mice are relatively lymphopenic and that homeostatic proliferation drives the generation of MZ B cells (40). We decided to examine mice with an Ag-specific BCR in a nonlymphopenic or a relatively lymphopenic setting. Anti-HEL transgenic BCR expressing B cells have been extensively studied (41, 42, 51). We examined the distribution of splenic peripheral B cell populations in an anti-HEL H and L chain BCR knock-in mouse in the absence of HEL. Heterozygous BCR knock-in mice essentially express "physiological" levels of the BCR, and knock-in Ig genes are subject to somatic alterations that may occur during an immune response (52). Our expectations were that, in the absence of any known cognate Ag, the FO I population would be markedly diminished (as has been noted previously in anti-HEL transgenic mice), but that FO II, MZP, and MZ B cells would be preserved.

As seen in Fig. 9A, in nonlymphopenic mice heterozygous for the anti-HEL BCR knock-in H and L chain genes, only a proportion of all B cells were HEL specific. One explanation for this phenomenon is the presence of ongoing receptor editing and V gene replacement (data not shown). HEL-binding B cells were largely excluded from the FO I compartment, suggesting that endogenous Ags that cross-react sufficiently with HEL and potentially drive FO I B cell development may be of very low abundance. The IgD/IgM profile of HEL-binding B cells resembles that described for Xid mice, in keeping with the view that Ag and Btk-derived BCR signals may be required for the generation of the FO I phenotype. In mice homozygous for anti-HEL H and L chain genes, the IgD/IgM profiles of HEL-binding cells were virtually identical to those of heterozygous knock-in mice (Fig. 9A). These mice exhibit a marked reduction of all IgDhighIgMlow FO I B cells, presumably because there are fewer non-HEL binding B cells in the homozygous mice and HEL-specific B cells are largely excluded from the FO I fraction.


Figure 9
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  		FIGURE 9. Differentiation of newly formed B cells into IgMhighIgDhigh B cells occurs in the absence of Ag. A, Analysis of IgM- and IgD-expressing HEL-binding B cells in spleens of anti-HEL BCR knock-in heterozygous and homozygous mice. FO I, FO II, and MZ B cell populations are quantitatively represented on the right. B, Newly formed B cells differentiate into IgMhighIgDhigh B cells in the absence of Ag. CD43negCD21negCD23neg splenic B cells from homozygous knock-in mice were purified using flow cytometry, reanalyzed after sorting, and incubated for 48 h in the absence or presence of HEL. Cells were analyzed for the expression of IgM and IgD using fluorochromes not used during the initial sort. C, IgMhighIgDhighCD21int FO II B cells were flow purified from homozygous anti-HEL BCR knock-in mice and incubated in the absence or presence of HEL. Cells were analyzed after 48 h using the same colors for anti-IgM and anti-IgD as used initially for purification. D, IgMhighIgDlowCD21low newly formed B cells were purified using flow sorting from homozygous anti-HEL BCR knock-in mice and incubated in the absence or presence of HEL. Cells were analyzed after 48 h using the same colors for anti-IgM and anti-IgD as used initially for purification. E, IgMhighIgDlowCD21low newly formed B cells and IgMhighIgDhighCD21int FO II B cells were flow purified from wild-type mice and individually incubated with or without HEL for 48 h. Cells were analyzed after 48 h using the same colors for anti-IgM and anti-IgD as used initially for purification.

 
We tested the assumption that the few, but clearly identifiable, non-HEL-binding B cells in homozygous knock-in mice may have arisen because of "spontaneous" receptor editing and V gene replacement events in the absence of Ag. All {kappa}L chain positive non-HEL-binding B cells in the FO I compartment in homozygous anti-HEL knock-in mice had undergone VH gene replacement (data not shown; Ref. 53). This phenomenon may explain, in part, why only a fraction of the B cells in heterozygous BCR knock-in mice express anti-HEL BCRs. Although FO I HEL-binding B cells were diminished in homozygous knock-in mice, immunohistochemical studies on homozygous knock-in mice revealed the presence of HEL-binding B cells in splenic follicles, and these presumably represent primarily FO II and MZP B cells (data not shown). Although the absence of specific Ag leads to a marked reduction in HEL-specific FO I B cells, FO II B cell numbers were preserved in both relatively normopenic heterozygous knock-in mice and in lymphopenic homozygous mice, suggesting that this long-lived follicular B cell population, in contrast to FO I B cells, develops and is maintained in the absence of any known self Ag.

Spontaneous development of FO II B cells in the absence of Ag

The expression of cell-surface CD93 decreases soon after peripheral B cells mature, whereas CD23 levels and IgD levels increase during the process of maturation. T1 cells presumably develop into T2 cells that are initially IgDlow, which go on to mature into IgDhigh T2 cells, which might then in turn mature into the cells with the phenotype of AA4.1– long-lived FO II B cells. We sought to determine whether the IgMhighIgDhigh phenotype may be acquired in vitro, in the absence of any cognate Ag, by newly formed/T1 cells. We also wished to examine whether FO II B cells have the potential to assume a FO I-like phenotype in vitro following exposure to cognate Ag. There were many reasons to choose an in vitro approach. The differentiation potential of specific B cell populations cannot be ascertained in vivo by cell transfer because of the confounding effect of homeostatic proliferation, and cell transfer studies in replete mice are not readily interpretable. Clearly, exposure of certain BCR transgene-expressing B cells to self-Ags leads to the induction of tolerance in vivo. An in vitro approach permits the easy titration of cognate Ag and the use of specific B cell populations, but focuses specifically on differentiation potential, without addressing issues that relate to tolerance.

We purified newly formed B cells by an immunodepletion approach, selecting cells that were CD43–CD23–CD21– by flow cytometry from a homozygous anti-HEL BCR knock-in mouse (Fig. 9B). This negative selection approach was initially used to avoid any potential activation of B cells by Abs against IgM and IgD. When incubated for 48 h in the absence of Ag, these newly formed B cells spontaneously assumed an IgMhighIgDhigh phenotype resembling that of Fraction II B cells. However, in the presence of low concentrations of HEL (10 ng/ml), these cells assumed a FO I B cell-like IgDhighIgMlow phenotype after 48 h (Fig. 9B). This negative selection approach for cell sorting was initially undertaken because there is no method available to purify FO II B cells without using Abs against surface Igs. In separate experiments, we also purified FO II B cells from homozygous anti-HEL knock-in mice using what are described as nonactivating Abs to IgM, IgD, and CD21, and exposed these cells to the same concentrations of Ag used in the experiments with newly formed B cells. As seen in Fig. 9C, incubation of FO II B cells in the absence of Ag for 48 h caused no discernible change in the IgMhighIgDhigh phenotype of FO II B cells. However, incubation of these cells with low concentrations of HEL in vitro resulted in most of these cells assuming an FO I-like phenotype after 48 h. Differentiation is linked to the specific Ag and not any potential contaminant (such as LPS, for instance), because the same HEL preparation did not induce the differentiation of Ag-nonspecific B cells (Fig. 9E), nor did it induce an alteration in the size of HEL-specific cells as assessed by forward scatter (data not shown). Purification of NF/T1 cells from homozygous anti-HEL knock-in mice (Fig. 9D) or from wild-type mice (Fig. 9E, left) using "nonactivating" Abs against IgM, IgD, and CD21 also led to the in vitro assumption of an FO II-like phenotype by NF/T1 B cells similar to the results observed in Fig. 9B. These studies suggest that both the depletion and positive selection approaches to purification of T1 and FO II B cells are effective and equally valid, and that newly formed cells are already programmed to differentiate spontaneously and quantitatively into cells that express high levels of IgM and IgD. Long-lived IgMhighIgDhigh CD21int B cells might retain the FO II-like phenotype in vivo because they are not tickled by cognate self-Ags. These data are consistent with the view that one subset of long-lived follicular B cells develops in a "default", Ag-independent manner, while the other requires cognate Ag-derived and Btk-dependent signals. The absence of a known cognate Ag in a transgenic animal does not rule out the presence of some unidentified cross-reacting self-Ag in the host animal that is recognized by the transgenic BCR. However, in the HEL knock-in model, if such a postulated self-Ag exists, it is incapable of allowing differentiation of FO II B cells into FO I B cells in vivo.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our studies demonstrate the existence of two distinguishable long-lived recirculating follicular B cell populations. FO II B cells are posttransitional cells that develop in a Btk-independent manner. FO I B cells require Btk, which in turn is presumably activated by self-Ags, for their development. Relatively strong BCR signals are already known to facilitate FO I B cell development, but are not required for FO II and MZ B cell development. Although the latter populations can both develop in the absence of Btk-derived signals, MZ B cells require Notch2, while FO II B cells do not require Notch2 for development. It appears therefore that FO II B cells represents a "default" follicular B cell population that requires BAFF, but does not require Btk activation or Notch2 derived signals for development. It is likely that a "default" follicular B cell population noted in a previous study, but not further characterized, is actually the FO II B cell pool (27). A model for B cell differentiation, building in part on the models for transitional B cell development generated by Allman et al. (17, 18), is provided in Fig. 10. It is emphasized that this model, although supported by the analysis of loss of function mutant mice, remains speculative. T1 cells progress to the T2 stage and then express higher levels of surface IgD to enter the FO II B cell pool. We speculate that if a specific B cell clone encounters a self-Ag of the appropriate avidity it may be selected at the T2-IgDhigh stage and differentiate further into an FO I cell possibly passing through an intermediate T3 stage; alternatively, such a clone may be selected by self-Ag at the FO II stage and mature into an FO I B cell. IgDhigh B cells of the FO II type can develop in an Ag-independent manner. Ag-independence is always inferred, because it is impossible to rule out the presence of some cross-reactive, unidentified, self Ag for any BCR. Studies showing the retention of FO II cells in the absence of MZP and MZ B cells in certain mutant mice strains suggest, but do not prove, that FO II B cells may represent long-lived follicular precursors of MZ B cells.


Figure 10
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  		FIGURE 10. A schematic model for peripheral B cell development in the absence of Ag. See text for details.

 
Our in vitro differentiation studies are limited in scope and have focused on the crucial alterations in surface IgM and IgD that accompany differentiation. They were initiated primarily because of the lack of suitable in vivo approaches. Although it is unlikely that all the phenotypic changes that occur in the specialized follicular environment in vivo can be recreated in vitro, the quantitative shifts in the phenotypes of purified B cell populations that were observed speak to the validity of using these surface markers to obtain information regarding the differentiation process.

FO I and FO II B cells are present in all follicular compartments as well as in the bone marrow perisinusoidal niche (44). It is likely that these two distinguishable types of cells respond to immunogens in a broadly similar fashion, given the ability of mice, such as the MD4 anti-HEL transgenic mice, that have very few FO I B cells, to readily make Abs in response to immunization with HEL. Purified follicular B cells (deficient in FO I cells) from anti-HEL transgenic mice have been shown to be functional (54), and it is therefore likely that FO II B cells are functional, competent B lymphocytes. A likely explanation for why two distinct follicular B cell populations have evolved is that Btk-dependent BCR signals and Notch2 represent opposing inducers that respectively drive FO I B cell and MZ B cell generation, ensuring the reciprocal generation of these two distinct terminally differentiated sublineages of B-2 B cells. FO I B cells are rarely depleted in a dramatic fashion during the course of an infection, but blood-borne pathogens can cause a drastic activation and depletion of the MZ B cell pool (55, 56). FO II B cells may constitute a long-lived cellular reservoir that can potentially be recruited as it transits the spleen to replenish the MZ B cell pool following a severe and acute challenge by a blood-borne pathogen.

The Ag/BCR/Btk dependent generation of FO I B cells in turn presumably ensures that not all developing B cells are driven by Notch2 ligand-dependent signals into the MZ B cell pool. Strong BCR signals may ensure that a subset of B cells is driven irreversibly to a follicular (FO I) fate and thus may be actively inhibited from committing to entry into the MZ B cell pool. It is this requirement for reciprocal inhibitory events during development that might have driven the evolution of two distinguishable and developmentally distinct long-lived follicular B cell populations. Further studies are called for to examine how strong BCR signals may antagonize inductive signals for MZ B cell development, and to determine how apparently weaker BCR signals may also be required, as clearly suggested in the work of Wen et al. (27), during the program of MZ B cell development.


   Acknowledgments
 
We thank Michael Carroll, Michel Nussenzweig, Jason Cyster, Yoshio Hamada, and Christopher Klug for making mice available. We thank Joanne Yetz-Aldape, John Daley, Suzan Lazo-Kallanian, and Michelle Connole for help with flow cytometry. Sharon Germana is thanked for help with the proliferation assays. Jennifer Couget is thanked for help with microarrays.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AI06493 and CA102793 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Shiv Pillai, MGH Cancer Center, Building 149, 13th Street, Charlestown, MA 02129. E-mail address: pillai{at}helix.mgh.harvard.edu Back

3 Abbreviations used in this paper: NF/T1, newly formed or transitional type 1; T2, transitional type 2; T3, transitional type 3; MZ, marginal zone; MZP, MZ precursor; APC, allophycocyanin; HEL, hen egg lysozyme; SA, streptavidin; BrdU, bromodeoxyuridine; FO II, follicular type II; FO I, follicular type I. Back

Received for publication November 9, 2006. Accepted for publication June 1, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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