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The Journal of Immunology, 2007, 178: 897-907.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fc{gamma}RIIB Regulates Autoreactive Primary Antibody-Forming Cell, but Not Germinal Center B Cell, Activity1

Ziaur S. M. Rahman2, Boris Alabyev and Tim Manser

Department of Microbiology and Immunology and Kimmel Cancer Center, Thomas Jefferson Medical College, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The low-affinity FcR for IgG Fc{gamma}RIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created Fc{gamma}RIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of Fc{gamma}RIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic Fc{gamma}RIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that Fc{gamma}RIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Four types of FcRs for IgG have been identified and are widely expressed on hemopoietic cell lineages. These Fc{gamma}Rs play important roles in maintaining the homeostasis and increasing the efficiency of the Ab response by linking IgG Abs with effector responses (1, 2, 3, 4). Mice express Fc{gamma}RI, Fc{gamma}RIIB, Fc{gamma}RIII, and Fc{gamma}RIV (4). Fc{gamma}RI, and Fc{gamma}Rs IIB and III, display high and low affinity for IgGs, respectively, and the low-affinity Fc{gamma}Rs can only bind IgG in the form of immune complexes (IC)3 (5). The recently identified Fc{gamma}RIV binds to mouse IgG2a and IgG2b subclasses with intermediate affinity (6). There are two different signaling classes of FcRs: the activation (Fc{gamma}RI, Fc{gamma}III, and Fc{gamma}RIV) and inhibitory (Fc{gamma}RIIB) receptors. The activation and the inhibitory receptors mediate signals through ITAM in the common {gamma}-chain subunit and ITIM in the cytoplasmic tail, respectively (7, 8, 9). Fc{gamma}RIIB1 and Fc{gamma}RIIB2 are two isoforms of Fc{gamma}RIIB, resulting from the alternative splicing of RNA encoded by a single gene (5, 8, 10, 11). Most murine B cells appear to lack expression of Fc{gamma}RIIB2 and activating Fc{gamma}Rs, but express Fc{gamma}RIIB1 (8, 12).

In a primary T cell-dependent (TD) immune response, Ag-specific B cells differentiate along (short-lived) Ab-forming cell (AFC) and germinal center (GC) B cell pathways (13). GCs are microenvironments formed in the follicles of secondary lymphoid organs by highly proliferative B cells (14). GC B cells are further selected into either the secondary (long-lived) AFC or memory B cell compartments. In addition, GC B cells undergo affinity and specificity maturation. During this process, somatic hypermutation alters the variable regions of BCRs expressed by GC B cells (14, 15, 16) and thus generates a diverse BCR repertoire. Follicular (FO) dendritic cells (FDC) are the major stromal cell type in GCs and can trap Ag on their surface in the form of ICs via complement receptors (17, 18, 19) and FcRs (20, 21, 22). It has been suggested that GC B cells with high affinity and specificity for Ag in ICs on FDCs are selected into either the AFC or the memory B cell compartments and GC B cells with low affinity and autoreactivity are negatively selected (14, 23, 24), although data in support of such models have been elusive (20, 25, 26).

Early models of affinity and specificity maturation were solely based on the role of the BCR in this process. Tarlinton and Smith (27) later proposed an extension of this model by considering an influence of Fc{gamma}RIIB. They suggested that Fc{gamma}RIIB on GC B cells regulates AFC vs memory B cell development. According to their model, early in the response, GC B cells predominantly differentiate into AFCs. Late in the response, when high titers of Ag-specific IgG Abs have been produced, ICs trapped on FDCs become the major source of this Ag in the GC. As a result, GC B cells interact with Ag, the BCR and Fc{gamma}RIIB are co-cross-linked, leading to inhibition of the AFC differentiation pathway and promotion of memory B cell development.

Ravetch and Lanier (7) have proposed that the relative strength of opposing signals from the BCR and Fc{gamma}RIIB on GC B cells determines the net outcome of B cell selection mediated by Ag-containing IC in the GC response. In their model, predominant engagement of ICs by Fc{gamma}RIIB vs BCR on GC B cells with low affinity for Ag or with autoreactivity results in cell death. Conversely, coengagement of the BCR and Fc{gamma}RIIB by ICs on GC B cells with high affinity for Ag mediates signaling events that inhibit proliferation and apoptosis, leading to survival and progression to memory B cells (28).

These models did not address how modulation of Fc{gamma}RIIB surface levels on GC B cells might influence their positive and negative selection. We and others have shown that Fc{gamma}RIIB expression is up-regulated on activated (29, 30) and GC B cells (31, 32, 33) in nonautoimmune mice. In contrast, Fc{gamma}RIIB expression is down-regulated or fails to be up-regulated on GC B cells in many autoimmune-prone strains of mice (31, 33, 34). This reduced expression was suggested to have the potential to accelerate autoimmune disease, consistent with the Ravetch and Lanier model for negative selection of autoreactive GC B cells discussed above (31, 34). Fc{gamma}RIIB-deficient C57BL/6 (B6.RIIB–/–) mice have been shown to spontaneously develop high titers of antinuclear autoantibodies (ANAs) and ultimately fulminant autoimmune disease (35). Taken together, these data raise the possibility that down-regulation or failed up-regulation of Fc{gamma}RIIB on GC B cells in autoimmune strains may be functionally equivalent to an Fc{gamma}RIIB deficiency.

We have previously described two lines of VH chain knockin mice termed HKI65 and HKIR (36, 37). These mice differ only in the absence (HKI65) or presence (HKIR) of a single mutation to arginine (R) at position 55 in the CDR2 region of the VH gene. Both VH transgenes, in combination with a single endogenous {kappa} L chain gene, encode BCR that we term "canonical." Canonical BCRs have specificity for both the hapten arsonate (p-azophenylarsonate (Ars)) and nuclear autoantigens. The HKIR version of canonical Abs displays substantial reactivity against chromatin and dsDNA and can cause kidney dysfunction by glomerular deposition in vivo. Despite their weak and strong autoreactivity, respectively, both HKI65 and HKIR canonical BCRs promote efficient development of B cells to mature FO phenotype and these B cells are not short-lived and do not display features of anergy in vitro (37, 38).

However, peripheral B cells expressing the canonical HKIR BCR have reduced levels of surface IgM and IgD as compared with B cells expressing the HKI65 BCR, apparently due to chronic engagement of autoantigen. In addition, both HKI65 and HKIR B cells can participate in the Ag-driven GC response but HKIR GC B cells do not efficiently seed the memory B cell compartment, suggesting negative selection of these cells in the GC/memory pathway. To determine whether Fc{gamma}RIIB played a role in this negative selection, we generated Fc{gamma}RIIB-deficient HKI65 and HKIR mice and analyzed the primary and Ars-driven development of canonical clonotypes in these mice.


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

C57BL/6 (B6) and C57BL/6.SJL (B6.CD45.1) mice were purchased from The Jackson Laboratory. Ig VH chain knockin mice (termed HKI65 and HKIR) were generated in our laboratory and described previously (37, 38). Fc{gamma}RIIB-deficient (B6.RIIB–/–) mice were supplied by Dr. J. Ravetch (Rockefeller University, New York, NY). All mice were maintained in a pathogen-free barrier facility and were 8–13 wk old when used in experiments.

Adoptive transfer and immunizations

Two adoptive transfer protocols were used. One was similar to those previously described (39, 40). In this protocol, C57BL/6 (CD45.2+) or B6.SJL (CD45.1+) recipient mice were immunized i.p. with 100 µg of Ars-keyhole limpet hemocyanin (KLH) (in alum) 1 wk before transfer (i.v., in PBS) of 5 x 106 splenocytes (105 E4+ cells) or 2.5–5 x 104 FACS purified B220+, IgD+, and E4+ (CD45.2+) cells, respectively. Mice were then injected i.p. with 50 µg of Ars-KLH in PBS immediately after cell transfer. We termed this protocol A. For protocol B, mice were immunized with 150 µg of Ars-KLH in alum i.p. 12–24 h after cell transfer. For secondary immune responses, mice were boosted 7–8 wk after cell transfer and primary immunizations following protocol B.

Abs and other reagents

Abs and other reagents used for flow cytometry and immunohistology included: FITC-GL7; PE, FITC, and PE-Texas Red-anti-B220 (clone RA3-6B2); PE-anti-Fc{gamma}RII/Fc{gamma}RIII (clone 2.4G2); PE-anti-IgD (clone 11-26); streptavidin-CyChrome; FITC-anti-CD21/35(7G6); PE-anti-CD23 (B3B4); PE-anti-CD69 (H1.2F3); PE-anti-CD80 (16-10A1); FITC-anti-CD86 (GL-1); FITC-goat anti-mouse IgG (BD Pharmingen); FITC-metallophillic macrophage-1 (MOMA-1; Serotec); biotin-anti-IgD (clone 11-26; Southern Biotechnology Associates); PE and biotin-anti-mouse CD45.2 (clone 104) and PE-anti-mouse C1qRp (AA4.1; eBioscience); streptavidin-PE (Molecular Probes); FITC-peanut lectin (agglutinin) (PNA); FITC-donkey anti-mouse IgM (Jackson ImmunoResearch Laboratories) and a biotinylated form of the anti-idiotypic mAb E4 (prepared in-house).

Immunohistology

Spleen cryostat sections (5–6 µm) were prepared as described (41). Immunohistology was performed using the Abs listed above and the stained sections were analyzed using a fluorescence microscopy (Leitz Diaplan) and images were captured as described (42).

Flow cytometry

Three- and four-color flow cytometric analysis was done on cell suspensions prepared from spleens of naive and immunized mice stained with multiple combinations of Abs listed above. Biotinylated Abs were detected with streptavidin-CyChrome. Stained cells were analyzed using a Coulter Epics XL/MCL analyzer. Data were analyzed using FlowJo software (Tree Star).

ELISPOT assays for primary and secondary AFCs

Splenocyte suspensions from chimeric mice were plated at 1 x 106 cells/well and diluted serially (1/2) in multiscreen 96-well filtration plates (Millipore) coated with goat anti-mouse IgM (µ-specific) or goat anti-mouse IgG ({gamma}-specific; Caltag Laboratories) for 6 h at 37°C. Transgene-expressing canonical IgM and IgG Abs produced by AFCs were detected by biotinylated anti-clonotypic mAb E4 (prepared in-house) and streptavidin-alkaline phosphatase (Vector Laboratories). E4+ IgM and IgG Abs were developed using the Vector Blue Alkaline-Phosphatase Substrate kit III (Vector Laboratories). ELISPOTs were counted using a computerized imaging video system (Cellular Technology).

ELISA

Ag (anti-Ars) and clonotype-specific (E4) total serum Igs were measured by ELISA on 96-well plates (Immulon-4; Thermo Electron) as previously described (43).

Laser capture microdissection of AFC clusters and mutation analysis

Frozen spleen sections (5–6 µm) from chimeric mice were placed on polyethylenenaphthalate membrane-coated slides (Leica Microsystems) and stained with biotinylated-E4 and PNA-HRP (Vector Laboratories) as described (41). Biotinylated Ab was detected as described above. E4+ AFC clusters were then laser capture microdissected using the laser microdissection microscope (Leica Microsystems) and collected directly into PCR tubes containing 5 µl of 1x PBS, 15 µl of nuclease-free water, and 5 µl of proteinase-K (2 µg/ml). Proteinase-K digestion was performed at 55°C for an hour. Using these samples, PCR amplifications of VH chain knockin (HKI65 and HKIR) Tgs were achieved via nested PCR using the following primer pairs: round 1, 5'-CAACCTATGATCAGTGTCCTC-3' (5' primer, hybridizing 5' of the leader exon) and 5'-GAAATGCAAATTACCCAGGTG-3' (3' primer, hybridizing to the JH2-JH3 intron) and round 2, 5'-CAGGTGTCCACTCTGAGGTTC-3' (5' primer, hybridizing to the end of the leader exon and the beginning of the VH gene) and 5'-GTGTCCCTAGTCCTTCATGACC-3' (3' primer, hybridizing to the sequence upstream of the first round reverse primer in the JH2-JH3 intron). PCR product purification, cloning, sequencing, and mutation analyses were done as described (44).

GC B cell sorting, DNA extraction, and mutation analysis

B220+, E4+, and PNA+ GC B cells were purified using a MoFlo fluorescent activated high-speed sorter (DakoCytomation). Genomic DNA was prepared from these cells using the DNeasy tissue kit (Qiagen) following the manufacturer’s instructions. PCR amplification of VH Tgs, PCR product purification, cloning, sequencing, and mutation analyses were done as described above.

GC B cell sorting, RNA extraction, and real-time RT-PCR

B220+, E4+, and PNA+ GC B cells were sorted using a MoFlo FACS as described above. In vitro LPS-stimulated MACS (Miltenyi Biotec) purified E4+ canonical B cells were used as control. RNA purification, reverse transcription of RNA, real-time RT-PCR and generating raw relative quantification (RQ) values for B lymphocyte-induced maturation protein-1 (Blimp-1) and Bcl-6 gene expression were performed as described (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Normal primary B cell development in Fc{gamma}RIIB–/– canonical VH knock-in mice

We generated HKI65.Fc{gamma}RIIB–/– (termed 65.RIIB–/–) and HKIR.Fc{gamma}RIIB–/– (termed R.RIIB–/–) mice by crossing Fc{gamma}RIIB–/– C57BL/6 congenic mice (B6.RIIB–/–) with HKI65 and HKIR mice, respectively. The HKI65 and HKIR Tgs had been backcrossed to the C657BL/6 background for at least 15 generations. Although both HKI65 and HKIR BCRs promote efficient development of canonical B cells to mature FO phenotype, this does not extend to marginal zone (MZ) B cells, especially in HKIR mice (37). An anti-clonotypic mAb (E4), specific for the BCR expressed by both HKI65 and HKIR canonical B cells, was used to identify such B cells. As shown in Table I, the total number of B220+ cells in 65.RIIB–/– and R.RIIB–/– was comparable to HKI65 and HKIR control mice, respectively. The total number and frequency of E4+ cells in 65.RIIB–/– and R.RIIB–/– mice was also similar as compared with their control counterparts.


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  		Table I. Absolute number of B220+ and E4+ and frequency of E4+ cells in Fc{gamma}RIIB-sufficient and -deficient HKI65 and HKIR mice

 
Parallel spleen sections obtained from naive mice were stained with anti-B220 (red) and MOMA-1 (green) (Fig. 1A, upper panels) and E4 (red) and MOMA-1 (green) (Fig. 1A, lower panels). MOMA-1 stains for metallophillic macrophages that usually line the FO border of the MZ. As shown in Fig. 1A, lower panels, we found E4+ cells were confined to B cell areas in the white pulp in 65.RIIB–/– (second column) and R.RIIB–/– (fourth column) mice with few E4+ cells in the MZ area, identical with the situation observed in Fc{gamma}RIIB-sufficient control mice (first and third columns). These results were consistent with flow cytometry data showing that most E4+ cells (99%) from mice of all four genotypes were IgMlowIgD+ (Fig. 1B, second row) and CD21lowCD23+ (Fig. 1B, fourth row) FO B cells. The distribution of bulk B220+ gated IgMhighIgDlow cells containing transitional and MZ B and IgMlowIgDhigh FO B cells in 65.RIIB–/– and R.RIIB–/– mice was similar to HKI65 and HKIR controls, respectively (Fig. 1B, first row). The slightly lower percentage of IgMhighIgDlow T plus MZ and higher percentage of IgMlowIgDhigh FO B cells observed in HKIR and R.RIIB–/– mice probably resulted from the reduction of the CD21highCD23low MZ B population in these mice (Fig. 1B, third row, right two panels, see below). We have also previously reported the somewhat and substantially reduced levels of surface BCR on HKI65 and HKIR E4+ B cells, respectively (37, 38). To test whether the Fc{gamma}RIIB deficiency had any effect on BCR levels, we examined surface IgM and IgD distributions in 65.RIIB–/– and R.RIIB–/– E4+ cells and found these to be similar to those found on HKI65 and HKIR E4+ cells, respectively (Fig. 1B, second row).


Figure 1
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  		FIGURE 1. Primary development of HKI65 and HKIR canonical B cells is not influenced by an Fc{gamma}RIIB deficiency. A, Adjacent spleen sections obtained from the naive mice of the indicated genotypes were stained with B220 (red) and Moma-1 (green), and E4 (red) and Moma-1 (green). Representative images of B cell FO areas (B220, upper panels) and location of E4+ cells (E4+, lower panels) in those areas are shown. B, Flow cytometric analysis of total (B220 gated) and canonical (E4 gated) B cells for surface IgM and IgD (upper two rows) and CD21/35 and CD23 (lower two rows). C, Splenocytes from naive mice of the indicated genotypes were stained for B220 and B cell immaturity marker AA4.1 (anti-C1qRp). AA4.1+ and AA4.1– gates are shown. D, Levels of activation/costimulatory markers CD69, CD80, and CD86 were measured by flow cytometry on FACS-purified B220+, E4+ canonical B cells (left two columns) and in vitro LPS-activated E4+ cells (rightmost column) obtained from mice of the indicated genotypes. E, Levels of B220 (right column) and CD45 (left column) on CD19-gated total B cells were measured by flow cytometry analysis of splenocyte suspensions from pooled samples of two naive mice of the indicated genotypes. All data are representative of one of three independent experiments.

 
Additional flow cytometric analysis on splenocytes using the AA4.1 mAb (specific for the C1qRp Ag, a marker for immature and transitional B cells) revealed no significant difference in the ratio of bulk B220+ (Fig. 1C) or E4+ cells (data not shown) expressing and lacking this marker in 65.RIIB–/– and R.RIIB–/– strains as compared with HKI65 and HKIR controls. In fact, the percentage of AA4.1+ cells in all lines of knockin mice was at least 2-fold lower than that observed in transgene-negative controls (data not shown), indicating that the canonical VH Tgs efficiently promote B cell development to a mature phenotype. We further examined the levels of the activation/costimulatory markers CD69, CD80, and CD86 to determine whether 65.RIIB–/– and R.RIIB–/– B cells were preactivated in the absence of Fc{gamma}RIIB. Although these markers were up-regulated on B cells from mice of all genotypes in response to LPS in vitro (Fig. 1D, rightmost column), the levels of these markers on ex vivo 65.RIIB–/– (Fig. 1D, blue line, leftmost column) and R.RIIB–/– E4+ (Fig. 1D, blue line, middle column) cells were comparable to controls (Fig. 1D, red lines, left and middle columns). Unexpectedly, as shown in Fig. 1E, we found B220 (right column) and CD45 (left column) levels on Fc{gamma}RIIB-deficient (B6.RIIB–/–, 65.RIIB–/–, and R.RIIB–/–, blue lines, all panels) B cells were slightly and >2-fold lower, respectively than those on their Fc{gamma}RIIB-sufficient counterparts (red lines, all panels). Although the significance of this observation is presently unclear, these reduced levels do not seem to influence primary B cell development or tolerance. Together, these data suggest that primary B cell development and tolerance in HKI65 and HKIR mice, including canonical clonotypes are overtly unaffected by the absence of Fc{gamma}RIIB.

The early stages of Ag-driven proliferation in vivo are not influenced by Fc{gamma}RIIB

To investigate whether the early stages of the B cell proliferative response in vivo were influenced by Fc{gamma}RIIB, a synchronized anti-Ars response was induced using an adoptive transfer and immunization protocol similar to those previously published (39, 40). This allowed a detailed examination of the kinetics of the early stages of donor B cell proliferation and differentiation in vivo. CFSE-labeled splenocytes (1 x 107) from 65.RIIB–/– and R.RIIB–/– mice were adoptively transferred into nonirradiated syngeneic mice that had been immunized with Ars-KLH (in alum) 1 wk earlier followed by boosting with Ars-KLH (in PBS) i.p., immediately after cell transfer (termed protocol A). HKI65 and HKIR splenocytes were used as controls. On day 3 posttransfer and postimmunization, the percentage of donor B cells that diluted CFSE in recipient mice to various extents was evaluated by flow cytometry. As shown in Fig. 2, activated total (B220high, GL7+, upper panels) as well as canonical B cells (B220high, E4+, lower panels) from 65.RIIB–/– (second column) and R.RIIB–/– (last column) mice had undergone similar numbers (5, 6, 7) of divisions. This proliferative behavior was analogous to that observed for HKI65 (first column) and HKIR (third column) B cells.


Figure 2
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  		FIGURE 2. Fc{gamma}RIIB-sufficient and -deficient HKIR B cells proliferate to the same extent during the initial phases of the immune response to Ars. Chimeric mice were created by transferring CFSE-labeled 65.RIIB–/– (second column), R.RIIB–/– (last column), HKI65 (first column), and HKIR (third column) control splenocytes into syngeneic C57BL/6 recipients according to protocol A, as described in the text and Materials and Methods. At day 3 after transfer, chimeric mice were sacrificed and splenocytes were analyzed via flow cytometry. B220high cells were gated and total (B220high, GL7+, upper panels) and canonical (B220high, E4+, lower panels) B cell proliferation was assessed by dilution of CFSE. A total of 3 x 105 lymphocyte-gated events were analyzed. Dashed lines demark unit division intervals, beginning with the unactivated, nondivided population (rightmost interval).

 
Enhanced primary AFC and serum Ab responses in the absence of Fc{gamma}RIIB

To study the behavior of canonical 65.RIIB–/– and R.RIIB–/– cells in the AFC and GC responses, anti-Ars immune responses were induced by using transfer protocol A described above. Five x 106 splenocytes (105 E4+ cells) from 65.RIIB–/–, R.RIIB–/–, and control mice were transferred to syngeneic C57BL/6 recipients. C57BL/6 mice lack the VH gene necessary to encode canonical, E4+ Abs (our unpublished data). Donor cell-derived E4+ AFCs were quantified by ELISPOT assay on day 5 (day 12 after initial immunization) posttransfer. IgG-producing E4+ AFCs in mice receiving 65.RIIB–/– and R.RIIB–/– splenocytes were over 3-fold more frequent on average as compared with controls (Fig. 3A, top panel). No significant difference was found between mice receiving HKI65 and HKIR splenocytes and their Fc{gamma}RIIB deficiency counterparts, respectively.


Figure 3
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  		FIGURE 3. An Fc{gamma}RIIB deficiency augments primary AFC and Ab responses of HKI65 and HKIR canonical B cells. Fc{gamma}RIIB-sufficient and -deficient HKI65 and HKIR canonical B cells were transferred to B6 recipients as described in Materials and Methods. A, Recipient B6 mice had been either preimmunized with Ars-KLH before transfer (upper panel, protocol A), or immunized posttransfer (lower panel, protocol B). The number of E4+ IgG (upper two panels) and IgM (lower panel) secreting AFCs were measured by ELISPOT assay. Each circle represents the number of E4+ AFCs per 1 x 106 splenocytes obtained from an individual recipient mouse. {circ} and •, Data from control and Fc{gamma}RIIB-deficient mice, respectively. Horizontal bars represent the average number of E4+ AFCs. Statistical analysis was performed by Student’s t test. The data for IgG-producing AFCs were obtained from four independent experiments each for protocol A and for protocol B, and the data for IgM-producing AFCs were obtained from two independent experiments using protocol B. B, Adjacent spleen sections, obtained from immunized B6 mice (protocol A) receiving splenocytes from 65.RIIB–/–, R.RIIB–/–, and control mice, were stained with E4 (red) and Moma-1 (green), upper panels, and E4 (red) and anti-IgG (green), lower panels. These data are representative of five mice of each genotype. C, Anti-Ars (upper panel) and E4 (lower panel) total Igs were measured by ELISA, as described in Materials and Methods. Each circle represents the dilution factor at an OD value of 1.0 (405 nm) obtained from the serum of a single mouse. The horizontal bars indicate the average value of dilution factors. Statistical analysis was performed by Student’s t test.

 
To test the possibility that the enhanced AFC responses of 65.RIIB–/– and R.RIIB–/– E4+ cells were unique to the protocol used, another protocol (termed protocol B) was introduced in which we immunized chimeric mice 12–24 h after cell transfer and quantified IgG-producing E4+ AFCs by ELISPOT on day 8 postimmunization. Analogous results were obtained with this protocol (Fig. 3A, middle panel). Using this same protocol, we also quantified IgM-producing E4+ AFCs. We found a 2-fold increase on average in the number of E4+ AFCs in mice receiving both 65.RIIB–/– and R.RIIB–/– splenocytes as compared with HKI65 and HKIR control splenocytes (Fig. 3A, bottom panel). Thus, augmented AFC responses are a characteristic of canonical B cells deficient in Fc{gamma}RIIB, consistent with previously published data (45).

As described above, canonical B cells in all four strains were mostly of mature FO phenotype (Fig. 1). However, to eliminate any possible direct or indirect contribution of Fc{gamma}RIIB–/– MZ or immature B cells to the AFC response, we performed similar immunization and transfer (protocol A) experiments with FACS-purified B220+CD23+CD21low FO B cells and obtained results analogous to those illustrated in Fig. 3A (data not shown).

We also performed histological analysis on day 5 of the anti-Ars responses using protocol A. Parallel spleen sections, obtained from chimeric mice injected with splenocytes (5 x 106) from donor mice of all genotypes, were stained with E4 (red) and MOMA-1 (green) (Fig. 3B, upper panels), and E4 (red) and anti-IgG (green) (Fig. 3B, lower panels). Clusters of E4+ cells, located in the bridging channels (defined by MOMA-1 staining), a typical location for AFCs, were only present in 65.RIIB–/–->B6 (upper row, second panel) and R.RIIB–/–->B6 (upper row, rightmost panel) chimeric mice. Fig. 3B also shows that while IgG+ B cells were confined to E4+ GCs in HKI65->B6 and HKIR->B6 mice, such cells (judged by colocalization of E4 and anti-IgG staining) were also apparent in E4+ AFC clusters in 65.RIIB–/–->B6 (lower row, second panel) and R.RIIB–/–->B6 (lower row, rightmost panel) mice, in addition to GCs. Histological analysis of spleen sections obtained from naive mice (<13 wk) of all genotypes revealed no pre-existing E4+ AFCs in the absence of Fc{gamma}RIIB (data not shown).

Finally, to test whether the elevated AFC responses observed in 65.RIIB–/–->B6 and R.RIIB–/–->B6 mice had any effect on serum Ig levels, Ag (anti-Ars) and clonotype-specific (E4) total Igs were measured by ELISA at various times postimmunization (using protocol B) in chimeric mice receiving Fc{gamma}RIIB-sufficient and -deficient splenocytes. Consistent with the ELISPOT data, elevated serum Ag-specific (anti-Ars, upper panel) and E4+ (lower panel) total Ig levels were observed in 65.RIIB–/–->B6 and R.RIIB–/–->B6 mice as compared with controls on days 7 and 14 of the response (Fig. 3C). At later time points, these serum Ab titers dropped in all four types of chimeric mice to levels that precluded their informative comparison.

Reduced participation of canonical HKIR B cells in the GC response

Our previous data have suggested that canonical R55 E4+ cells can participate in the GC response (36) but not as efficiently as HKI65 E4+ cells (B. Alabyev, Z. S. M. Rahman, and T. Manser, unpublished data). To measure quantitative differences in this regard, we followed the adoptive transfer and immunization protocol A described above. In the first set of experiments, spleen sections, obtained from Ars-KLH-immunized B6 mice receiving FACS-purified E4+ B cells from HKI65 or HKIR mice, were stained with E4 and GL7. Consistent with our previous findings, Fig. 4A illustrates that in HKIR->B6 chimeras, E4+ donor B cells clearly could enter GCs. In addition, the data in Fig. 2 demonstrate that in immunized HKIR->B6 chimeras many proliferating donor E4+ B cells have acquired the GL7 GC B cell marker. Taken together, these data rule out exclusion from the GC as an explanation for the reduced participation of these HKIR anti-Ars, anti-DNA B cells in the GC, and memory responses.


Figure 4
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  		FIGURE 4. Anti-Ars GC responses and Fc{gamma}RIIB expression by canonical HKI65 and HKIR GC B cells. Spleen sections, obtained from B6 mice receiving HKI65 or HKIR splenocytes, 4 or 5 days after cell transfer, were stained with E4 and for the GC marker GL7. A, The results obtained for three representative GCs from mice of each type. B, E4+ cells in GCs were counted from eight randomly chosen small- and medium-sized E4+ GCs per mouse. Each circle indicates the number of E4+ cells counted from a particular GC. {circ} and • represent data from HKI65->B6 and HKIR->B6 E4+ GCs, respectively. The horizontal bars indicate the average number of E4+ cells per GC. These data were obtained from two to three mice at each time point. C, Upper panels, Three-color (B220, E4, and PNA) flow cytometry analysis was performed on splenocytes obtained from adoptively transferred and immunized HKI65->B6 and HKIR->B6 mice on day 5 posttransfer. The percentage of B220high E4+PNA+ GC B cells is shown in rectangular gates. These data represent pooled samples of two to three mice of each genotype. C, Lower panels, Levels of 2.4G2 (staining for Fc{gamma}RIIB) on B220high E4+PNA+ GC B cells (blue) vs B220highE4–PNA– non-GC B cells (red) in the two types of mice are shown.

 
Differences in the participation of B cells in the GC response can be evaluated by changes in GC size, frequency, and the number of GL7+ cells per GC (44, 46, 47). Therefore, as previously described (42, 46), we performed semiquantitative studies on the numbers of E4+ B cells in splenic GCs in HKIR->B6 and HKI65->B6 chimeras 4 and 5 days after injection of donor splenocytes (protocol A). We categorized GCs in three different sizes (small, medium, and large) and E4+ cells in these classes of GCs were counted. The frequency of HKIR->B6 E4+ GCs was similar to HKI65->B6 E4+ GCs (data not shown) but the number of E4+ cells per GC differed. Because there were a few large E4+ GCs in either case, we counted E4+ cells in randomly chosen small and medium GCs. The number of E4+ cells per HKIR->B6 E4+ GC was reduced by almost 2-fold on day 4 and 3-fold on day 5 posttransfer and boosting (Fig. 4B) as compared with that found in HKI65->B6 E4+ GCs. Flow cytometry analysis of splenocytes obtained on day 5 of the GC response revealed similar results (Fig. 4C, upper panels).

To determine whether Fc{gamma}RIIB expression on HKI65 and HKIR canonical GC B cells was altered due to the autoreactivity of these cells, we performed adoptive transfer experiments using protocol A. Splenocytes obtained from immunized recipients, at day 5 posttransfer and boosting, were stained with B220, E4, PNA, and 2.4G2 (anti-Fc{gamma}RII/III). Flow cytometric analysis showed (Fig. 4C, lower panels) that the levels of Fc{gamma}RIIB on E4+ GC B cells (B220highE4+PNA+, in the blue histogram) were comparably higher, in either HKI65->B6 or HKIR->B6 chimeras as compared with levels on non-GC B cells (B220highE4–PNA–, in the red histogram).

No influence of Fc{gamma}RIIB on anti-Ars GC responses by canonical B cells

To test a role for Fc{gamma}RIIB in the GC response, 65.RIIB–/– and R.RIIB–/– E4+ B cells were adoptively transferred to C57BL/6 recipients and GC responses were induced using protocol A. HKI65 and HKIR E4+ donor cells served as controls. Spleen sections, obtained on day 5 posttransfer, were stained with E4 and GL7 and E4+ cells in GCs were counted as described above. No significant differences were found in the frequency of E4+ GCs (data not shown) and the number of E4+ cells per 65.RIIB–/–->B6 (Fig. 5A) or R.RIIB–/–->B6 E4+ GC as compared with HKI65->B6 and HKIR->B6 controls, respectively (Fig. 5A). Flow cytometric analysis (using anti-B220, E4, and PNA) gave rise to similar results (Fig. 5B).


Figure 5
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  		FIGURE 5. Anti-Ars GC responses of HKI65 and HKIR canonical B cells are not altered by an Fc{gamma}RIIB deficiency. A, E4+ cells in GCs, formed by HKI65 and HKIR B cells sufficient ({circ}) or deficient (•) for Fc{gamma}RIIB, were detected and counted as described in the legend to Fig. 4. Each circle indicates the number of E4+ cells counted from a particular GC. The horizontal bars indicate the average number of E4+ cells per GC. These data were obtained from three to five mice of each genotype using immunization and transfer protocol B and sampling 5 days after cell transfer. B, Flow cytometric analysis of E4+ canonical GC B cells from mice generated and sampled as described in A. C, CD45.2+ canonical donor cells in GCs, formed by HKI65 and HKIR B cells sufficient ({circ}) or deficient (•) in Fc{gamma}RIIB, were detected and counted as described in the legend to Fig. 4.

 
Because BCR levels on primary canonical HKIR B cells are low, and reduced BCR levels are a general characteristic of GC B cells (14, 33, 48, 49), we considered the possibility that the diminished number of E4+ B cells observed in HKIR->B6 and R.RIIB–/–->B6 GCs might be due to an inability to detect a large percentage of such cells using the E4 anti-clonotypic mAb. As such, we repeated the experiments described above using canonical splenic B cells (B220+, E4+, CD45.2+) purified via FACS that were transferred into C57BL/6.SJL (B6.CD45.1) congenic recipients. These studies also revealed a 2-fold lower number of CD45.2+ (donor) GC B cells in mice receiving canonical HKIR, as compared with canonical HKI65 B cells, in the presence or absence of Fc{gamma}RIIB (Fig. 5C).

V regions of Fc{gamma}RIIB–/– canonical B cells in primary AFC clusters lack somatic mutations

We next examined whether the E4+ AFC clusters observed in anti-Ars responses in 65.RIIB–/–->B6 and R.RIIB–/–->B6 chimeric mice might be derived from GC B cells that had undergone V region somatic hypermutation. Previous studies including our own have shown that Abs expressed by primary AFCs that arise in normal TD immune responses lack somatic mutations, but in secondary TD responses, AFCs express heavily somatically mutated Abs (50), presumably because they are derived from precursors that have participated in the GC reaction. It was also recently shown that AFCs that arise spontaneously in autoimmune-prone mice may express hypermutated Ab V regions (51).

E4+ AFC clusters in 65.RIIB–/–->B6 and R.RIIB–/–->B6 mice were laser capture microdissected from spleen sections obtained on day 5 of the anti-Ars response using protocol A. VH chain knockin transgenes were amplified, cloned, and sequenced as previously described (44). A very low frequency of mutations, at the background PCR error level (data not shown), was found in both 65.RIIB–/–->B6 (0.06%) and R.RIIB–/–->B6 (0.07%) E4+ AFCs (Table II).


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  		Table II. Somatic hypermutation frequency in Ig H chain knockin transgenes in E4+ AFCs in chimeric micea

 
Somatic hypermutation is not altered in Fc{gamma}RIIB–/– canonical GC B cells

The above data suggest that primary Fc{gamma}RIIB–/– E4+ AFC clusters are not derived from GCs. However, it was possible that Fc{gamma}RIIB–/– canonical GC B cells exited the cell cycle shortly after entering GCs and differentiated into AFCs in extra-GC sites, thus precluding accumulation of mutations in their VH genes. In this case, the frequency of V gene mutations in canonical Fc{gamma}RIIB–/– GC B cells would be predicted to be lower than that in canonical Fc{gamma}RIIB+/+ GC B cells. To test this idea, protocol A was used and on day 5 posttransfer and Ars-KLH boosting, E4+ GC B cells (B220high, E4+, PNA+) were purified by FACS. The VH chain knockin transgenes in these cells were amplified, cloned, and sequenced as previously described (44). We found no significant difference in somatic hypermutation frequencies in E4+ GC B cells derived from any donor B cells of any strains of mice (Table III), further supporting the conclusion that primary E4+ clusters nucleated by 65.RIIB–/– and R.RIIB–/– canonical B cells were not derived from GCs.


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  		Table III. Somatic hypermutation frequency in Ig H chain knockin transgenes in E4+ GC B cells in chimeric micea

 
Fc{gamma}RIIB–/– canonical GC B cells do not express increased levels of Blimp-1

GC B cells express low levels of Blimp-1 and high levels of Bcl-6 (52, 53, 54). Conversely, AFCs have low levels of Bcl-6 and high levels of Blimp-1 expression (55, 56). We examined whether the enhanced E4+ AFC responses observed in 65.RIIB–/–->B6 and R.RIIB–/–->B6 mice was due to up-regulation of Blimp-1 and down-regulation of Bcl-6 in Fc{gamma}RIIB–/– E4+ GC B cells, which might have led these cells to the AFC pathway. 65.RIIB–/–, R.RIIB–/–, and control splenocytes (5 x 106) were used in protocol A to induce Ars-specific GC responses. On day 5 posttransfer, E4+ GC B cells (B220highE4+PNA+) were purified by FACS and RNA was extracted to perform QPCR for Blimp-1 and Bcl-6 RNA. Although Blimp-1 RNA was up-regulated in LPS-stimulated B cells in vitro, the levels of this RNA were much lower in both Fc{gamma}RIIB–/– and Fc{gamma}RIIB+/+ E4+ GC B cells (Fig. 6, upper panel, Blimp-1) and no significant difference was found among GC B cells of all genotypes. Reciprocal results were obtained for Bcl-6 RNA (Fig. 6, lower panel, Bcl-6). Together, these data indicate that enhancement of the anti-Ars AFC responses from HKI65 and HKIR canonical B cells due to an Fc{gamma}RIIB deficiency is independent of GC responses.


Figure 6
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  		FIGURE 6. Blimp-1 and Bcl-6 RNA levels in canonical GC B cells. RNA was extracted from FACS-purified B220highE4+PNA+GC B cells, obtained on day 5 posttransfer and immunization, from chimeric mice receiving B cells of the indicated genotypes using protocol A. RNA extracted from in vitro LPS-activated (72 h) MACS-purified E4+ cells was used as a control. Raw RQ values of Blimp-1 (upper panel) and Bcl-6 (lower panel) RNA expression in GC B cells and LPS-activated HKI65, 65.RIIB–/–, HKIR, and R.RIIB–/– canonical B cells were obtained via real-time RT-PCR. Amplification was done in triplicate. These data are representative of four independent experiments.

 
The impaired memory response of canonical HKIR B cells is not reversed in the absence of Fc{gamma}RIIB

We previously have shown that despite participation of canonical B cell clonotypes with the arginine (R) mutation at position 55 in the GC reaction, such cells do not efficiently enter the memory compartment as measured by an anamnestic serum Ab response (36). Therefore, we examined the influence of Fc{gamma}RIIB on anti-Ars memory responses following adoptive transfer protocol B. Mice were rested for 2 mo post-Ars-KLH immunization before boosting. IgG producing E4+ AFCs were quantified by ELISPOT assay on day 4 postsecondary immunization. The number of secondary E4+ AFCs in 65.RIIB–/–->B6 chimeric mice (Fig. 7, left panel) was 2-fold higher than that of HKI65->B6 controls, indicating the regulation of secondary AFCs by Fc{gamma}RIIB. However, there was a 4-fold reduction in E4+ AFCs in mice receiving R.RIIB–/– splenocytes (Fig. 7) as compared with mice receiving 65.RIIB–/– splenocytes. In addition, a comparably reduced number of E4+ AFCs was observed in R.RIIB–/–->B6 and HKIR->B6 mice (Fig. 7, right panel). These data reinforce the conclusion that Fc{gamma}RIIB does not play a prominent role in the negative selection of autoreactive HKIR B cells in the GC/memory pathway.


Figure 7
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  		FIGURE 7. Memory responses of canonical Fc{gamma}RIIB-sufficient or -deficient B cells. The number of donor cell-derived E4+ IgG secreting secondary AFCs per 1 x 106 was measured by ELISPOT assay on day 4 postsecondary immunization using protocol B. Each circle represents the number of E4+ AFCs per 1 x 106 splenocytes obtained from an individual immunized recipient. {circ} and •, Data from mice receiving Fc{gamma}RIIB-sufficient and -deficient donor cells, respectively. Horizontal bars represent the average number of E4+ AFCs. Statistical analysis was performed by Student’s t test.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In total, the data presented here support previous conclusions that Fc{gamma}RIIB plays a pivotal, B cell autonomous role in regulating the magnitude of the AFC response. These data do not, however, suggest that this inhibitory receptor is directly involved in the action of B cell tolerance "checkpoints" in the GC/memory pathway. An Fc{gamma}RIIB deficiency did not noticeably alter the primary development or activation state of canonical HKIR B cells, including the substantially reduced BCR levels characteristic of these antinuclear Ag-reactive clonotypes. Moreover, Ars-induced recruitment into, and initial participation of, highly (HKIR) and weakly (HKI65) autoreactive canonical clonotypes in the TD immune response and GC reactions were indistinguishable between versions of these clonotypes expressing or lacking Fc{gamma}RIIB. The degree of augmentation of the Ars-induced primary AFC response due to an Fc{gamma}RIIB deficiency was similar between canonical HKIR and HKI65 clonotypes. Finally, the anamnestic anti-Ars AFC response of canonical HKI65, but not HKIR, B cells was enhanced by an Fc{gamma}RIIB deficiency, indicating that the substantially reduced population of the memory B cell compartment of canonical clonotypes containing, as compared with lacking, the R55 mutation was not overtly influenced by the absence of Fc{gamma}RIIB.

The experiments of Ravetch and colleague (57) have demonstrated that an Fc{gamma}RIIB deficiency predisposes mice of a C57BL/6, but not a BALB/c, genetic background to the spontaneous development of high autoantibody titers, including ANAs, and ultimately fulminant autoimmune disease by 6–8 mo of age. This same group has studied the influence of genetic background on the development of transgenic B cells expressing the 3H9/56R ANA autoantibody, whose autoreactivity was previously reported to be regulated by L chain receptor editing (58). They found that this editing was intact on a BALB/c background, but was perturbed on a C57BL/6 background, and the latter mice developed serum titers of 3H9/56R ANA Abs, mainly of the IgM isotype (45). However, when an Fc{gamma}RIIB deficiency was introduced into the B6.3H9/56R strain, the resulting mice developed high titers of IgG ANA Abs by 3 mo of age. Taken together, these results were interpreted to indicate that Fc{gamma}RIIB acts as a modifier of autoimmunity, as opposed to being directly involved in the induction of B cell tolerance, as previously proposed (7). That is, once tolerance is broken in the B cell compartment, an Fc{gamma}RIIB deficiency functions to allow IgG-expressing autoreactive B cells to progress to secretory phenotype.

Our experiments were analogous to these past studies in that we examined the influence of an Fc{gamma}RIIB deficiency on the regulation of ANA B cell activity on the C57BL/6 background. They differed in that regulation of canonical HKIR ANA clonotype autoreactivity does not take place by receptor editing, but apparently via BCR down-regulation (37, 38). Also, we provided foreign Ag drive to these clonotypes in the form of Ars-KLH, whereas how 3H9/56R clonotypes were recruited into the immune response in the experiments of Ravetch et al. (45) is not clear. In this regard, our experiments did not indicate spontaneous activation of HKIR or HKI65 clonotypes due to an Fc{gamma}RIIB deficiency. Nonetheless, our data concur with the conclusions of Ravetch and colleagues in suggesting that a major effect of the Fc{gamma}RIIB deficiency on autoreactive clonotype behavior is to promote the development of IgG-producing AFCs. We are currently determining whether and to what extent an Fc{gamma}RIIB deficiency predisposes canonical HKIR chimeric mice to spontaneously develop autoimmune disease later in life and, if so, whether the onset of disease is accelerated or delayed by Ars-KLH immunization.

In the context of the above discussion, it is important to note that the balance between activating and inhibitory signaling in primary autoreactive B cells may be substantially altered when cross-reactive foreign Ag is provided to this compartment. B cells that internalize, process and present this Ag are likely to garner high levels of cognate, self-tolerant T cell help. This, in turn, could result in negative signaling pathways in these B cells being suppressed or overridden by costimulatory pathways. For example, IL-4 has been shown to abrogate the inhibitory effects of Fc{gamma}RIIB on B cell activation induced by intact anti-BCR Ig reagents in vitro (59). In contrast, under conditions where cognate T cell help is limiting or unavailable, "spontaneous" activation of members of the autoreactive B cell compartment via other costimulatory pathways (e.g., TLRs) may be more readily suppressed by negative regulators such as Fc{gamma}RIIB. Thus, in this latter situation the protective effects of Fc{gamma}RIIB may be manifested at both early and late stages of autoantigen-driven AFC proliferation and differentiation.

Our data have important implications for evaluation of previously published models that ascribe a role for Fc{gamma}RIIB in the regulation of positive and negative selection of B cell clonotypes and the AFC/memory B cell differentiation pathways in the GC. These results do not support the Tarlinton and Smith (27) model inasmuch as they fail to indicate that the dramatic enhancement of the primary canonical HKI65 and HKIR AFC responses resulting from an Fc{gamma}RIIB deficiency is due to a "shunting" of these clonotypes from the GC to the AFC pathway. GC B cells derived from Fc{gamma}RIIB-deficient and -sufficient HKIR and HKI65 B cells all express low and equivalent levels of Blimp-1 RNA and high levels of Bcl-6 RNA. Also, an Fc{gamma}RIIB deficiency did not decrease the quantity of HKIR or HKI65 clonotypes present in GCs. Moreover, V gene somatic hypermutation frequencies were comparable and negligible in GC B cells and primary AFCs, respectively, derived from all four of these B cell clonotypes. Taken together, these data support a regulatory function for Fc{gamma}RIIB in the AFC pathway that acts independently of events occurring in the GC.

Moreover, as discussed above, our data do not indicate a major role for Fc{gamma}RIIB in the negative selection of autoreactive B cells during the GC/memory pathway (7). Further studies will be required to determine whether this receptor plays any role in the positive selection of GC B cells. Nonetheless, these considerations beg the question of why levels of Fc{gamma}RIIB are up-regulated on GC B cells and whether lack of this up-regulation, observed in many autoimmune-prone strains of mice, contributes to the etiology of autoimmunity. Although we (20) and others (60, 61) have obtained data indicating that expression and up-regulation of Fc{gamma}RIIB on FDCs may be important for promoting various aspects of B cell selection and differentiation in the GC, the role of Fc{gamma}RIIB on GC B cells remains unresolved.

Numerous studies have shown that the autoantibodies produced in systemic autoimmune disease states, particular to nuclear autoantigens, bear the hallmarks of transit through the GC/memory pathway: heavily somatically mutated V regions and recurrent types of mutations that increase affinity for cognate autoantigen (e.g., resulting in the introduction of arginines in CDRs) (62, 63). If Fc{gamma}RIIB does not directly participate in the negative selection of autoreactive B cells during the GC/memory B cell pathway how, then, can the pivotal contribution of a deficiency of this receptor to the development of autoimmune disease on certain mouse and human genetic backgrounds be explained? Our results, and the data of Ravetch and colleagues, suggest that at early stages in the multifactorial march toward development of fulminant autoimmune disease, the increased production of stable serum IgG autoantibodies due to an Fc{gamma}RIIB deficiency could well represent a major step. Such autoantibodies could readily form autoantigen-containing ICs, that could stimulate inflammatory and APCs via the activating Fc{gamma}Rs. This, in turn, could lead to the increased production of costimulatory factors and the presentation of the autoantigens in these ICs to rare, autoreactive T cells, contributing to the breakdown of peripheral T cell tolerance. A "vicious cycle" of autoreactive B and T cell activation perhaps leading to the development of autoantigen-driven GC, AFC, and memory B cell responses might be the result.


   Acknowledgments
 
We thank Scot Fenn and Richard Ila for technical assistance and all members of the Manser laboratory for their indirect contributions. We also thank Dr. Brian O’Hara (Department of Pathology, Thomas Jefferson University, Philadelphia, PA) for assistance with the laser capture microdissection.


   Disclosures
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 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 National Institutes of Health Grant RO1 AI 46806 (to T.M.). Back

2 Address correspondence and reprint requests to Dr. Ziaur S. M. Rahman, Department of Microbiology and Immunology and Kimmel Cancer Center, Jefferson Medical College, Bluemle Life Sciences Building 708, 233 South 10th Street, Philadelphia, PA 19107-5541. E-mail address: zrahman{at}mail.jci.tju.edu Back

3 Abbreviations used in this paper: IC, immune complex; TD, T cell dependent; AFC, Ab-forming cell; GC, germinal center; FO, follicular; FDC, FO dendritic cell; KLH, keyhole limpet hemocyanin; Ars, p-azophenylarsonate; PNA, peanut lectin (agglutinin); MZ, marginal zone; ANA, antinuclear autoantibody; RQ, relative quantification; MOMA-1, metallophillic macrophage-1; Blimp-1, B lymphocyte-induced maturation protein-1. Back

Received for publication August 15, 2006. Accepted for publication November 2, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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