HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rao, S. P. Articles by Manser, T. Search for Related Content PubMed PubMed Citation Articles by Rao, S. P. Articles by Manser, T.

Differential Expression of the Inhibitory IgG Fc Receptor FcRIIB on Germinal Center Cells: Implications for Selection of High-Affinity B Cells¹

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The murine low-affinity receptor for IgG, FcRIIB, mediates inhibition of B cell receptor-triggered events in primary B cells. We investigated the expression of FcRIIB on germinal center (GC) cells to better understand its role in memory B cell development. Immunohistological analyses demonstrated differential regulation of FcRIIB on GC cells. Its levels are markedly down-regulated on GC B cells and up-regulated on follicular dendritic cells (FDC) at all times during the GC response. Analyses of surface expression of FcRIIB by flow cytometry and FcRIIB mRNA levels by RT-PCR analysis confirmed that this FcR is down-regulated in GC B cells. In mice lacking FcRIIB, the development of the secondary FDC reticulum in GCs is substantially delayed, although the overall kinetics of the GC response are unaltered. These findings have direct implications for models proposed to account for the selection of high-affinity B cells in the GC and suggest a role for FcRIIB in promoting the maturation of the FDC reticulum.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptors for the Fc portion of IgG (FcR) are widely distributed on hemopoietic cells and play an important role in immune regulation by linking Ab-mediated responses with effector functions. The murine high-affinity FcRI and the low-affinity FcRII and FcRIII (1) are distinguished by their structure, function, distribution, and affinities for IgG (1, 2). These FcR contain homologous extracellular domains; however, they vary considerably in their transmembrane and cytoplasmic regions. FcRI and FcRIII are associated with a common -chain, which contains the immunoreceptor tyrosine-based activation motif in its cytoplasmic tail. In contrast, FcRII contains in its cytoplasmic tail an immunoreceptor tyrosine-based inhibitory motif (3). FcRIIB is encoded by a single gene in the mouse. However, alternative mRNA splicing results in two FcRII isoforms, FcRIIb1 and FcRIIb2 (4, 5). Murine B cells exclusively express FcRIIb1 and lack expression of the activation FcRs (6). Coaggregation of the B cell Ag receptor (BCR)⁵ and FcRIIb1 on B cells results in the inhibition of Ag internalization, activation, proliferation, and Ab secretion (7, 8, 9, 10). Previous in vitro studies have also shown that FcRIIB is capable of independently generating an apoptotic signal upon clustering (10, 11, 12).

Germinal centers (GC) are inducible lymphoid microenvironments that arise primarily in response to T-dependent Ags. During the GC response, a high frequency of mutations is introduced in the variable regions of the BCRs expressed by GC B cells. Ag present in the form of immune complexes (IC) retained on follicular dendritic cells (FDC) within the GC is thought to drive the selection of high-affinity B cell mutants, ultimately resulting in the formation of the memory B cell compartment. During this process of interaction between ICs and GC B cells, it is believed that B cells bearing high-affinity BCR are preferentially stimulated, resulting in the uptake of Ag and its subsequent presentation to Th lymphocytes (13, 14, 15). This model considers only the role of BCR but ignores the role of FcRIIB in B cell selection upon interaction with ICs. Given the general inhibitory role of FcRIIB, it is unclear how GC B cells, on interaction with ICs, could be activated and internalize the Ag for presentation to Ag-specific Th cells.

In this context, it has been suggested that the consequence of coengagement of BCR and FcRIIB by ICs retained on FDC in GC is dependent on the balance between concurrent activation and inhibitory signals, leading to stimulatory, inhibitory, or apoptotic responses (10). FcRIIB expression is also induced on FDC within the GC, and it has been proposed that this high level expression allows FDCs to convert ICs to a highly immunogenic form for B cell activation (16). Interestingly, none of the studies leading to these models has examined the expression levels of FcRIIB on GC B cells. Recent studies have indicated that the surface expression of FcRIIB is reduced on GC B cells in autoimmune strains of mice (17, 18). In contrast, in vitro experiments have indicated that activated B cell blasts differ from resting B cells in that they express significantly elevated levels of FcRIIB, as well as its downstream signaling molecules Src homology domain 2-containing inositol 5-phosphatase and Src homology domain 2-containing inositol 5-phosphatase-2 (19). These data highlight the importance of elucidating the role of FcRIIB in memory B cell development and the GC reaction.

In the present study, we examined whether expression of FcRIIB is modulated in GC B cells during a T-dependent immune response and have characterized the GC response in mice lacking this receptor. Our results indicate that both FcRIIB mRNA and protein expression are significantly down-regulated in GC B cells as compared with non-GC B cells. In addition, whereas the kinetics and magnitude of the GC response appear unaltered in FcRIIB-deficient mice, the development of FDC reticula in GCs is delayed, supporting a role for this receptor in FDC maturation. The implications of these results are discussed in the context of models proposed to account for selection of high-affinity B cells in the GC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and immunizations

FcRIIB^-/- mice on a C57BL/6 background (20) and C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). The generation and characterization of common -chain-deficient mice have been described previously (21). These mice, obtained from Dr. J. Ravetch (Rockefeller University, New York, NY), had been backcrossed to C57BL/6 mice for eight generations. All mice were maintained in pathogen-free conditions. Mice (10–12 wk old) were immunized i.p. with 3 x 10⁸ SRBC (Lampire Biological Labs, Pipersville, PA) in PBS or 100 µg 4-hydroxy-3-nitrophenyl acetyl-chicken -globulin (NP₁₃-CGG) in alum and were sacrificed at different time intervals for analysis.

Antibodies

The following Abs were used for immunohistology or flow cytometric analysis: PE-Texas Red-conjugated anti-B220 (clone RA3-6B2; Caltag Laboratories, Burlingame, CA); FITC-labeled GL7; PE-labeled anti-FcRII/FcRIII (clone 2.4G2); PE-labeled anti-B220 (clone RA3-6B2); biotin-labeled anti-CD35 (clone 8C12) (all purchased from BD PharMingen, San Diego, CA); unconjugated rat IgG Ab to mouse FDCs (FDC-M1 and FDC-M2, gifts from Dr. M. Kosco-Vilbois, Serono Pharmaceutical Research Institute, Plan-les-Quates, Switzerland); FITC-labeled MOMA-2 (Serotec, Oxford, U.K.); biotin-labeled (Fab')₂ mouse anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); biotin-labeled peanut agglutinin (PNA; Sigma-Aldrich, St. Louis, MO); PE or biotin-labeled anti-IgD (clone 11-26; Southern Biotechnology Associates, Birmingham, AL) and streptavidin (SA)-PE (Molecular Probes, Eugene, OR). The K9.361 hybridoma was a kind gift from Dr. U. Hammerling (Sloan Kettering Memorial Hospital, New York, NY).

Histology

Spleens from immunized mice were obtained at various times after immunization and processed for histological analysis as previously described (22). Briefly, spleens were embedded in Tissue-Tek OCT compound (Fisher Scientific, Bridgewater, NJ) by freezing in a 2-methylbutane bath cooled with liquid nitrogen. Frozen spleens were stored at -70°C until sectioned. Sections of 6 µm were cut on a cryostat microtome and mounted onto 0.01% poly-L-lysine (Sigma-Aldrich)-coated slides. The sections were air dried for 1 h, fixed in acetone for 15 min, air dried again for at least 2 h, and stored at -70°C until further analysis.

Frozen sections were rehydrated in TBS for 20 min followed by blocking in TBS, 5% BSA, 0.05% Tween 20 for another 20 min. Double immunohistology was then performed by staining with fluorescent Abs described above for 30 min. For unconjugated Abs, as a secondary step, sections were stained for 30 min with biotinylated mouse anti-rat Ab. The slides were then washed three times in TBS, 5% BSA, 0.05% Tween 20, and the biotinylated Abs were revealed by SA-PE. The slides were washed and stored in PBS in the dark. The stained sections were analyzed using a fluorescence microscope (Leitz Diaplan, Leitz, Wetzlar, West Germany), and digital images were captured using a Kodak DC290 Zoom digital camera and MDS-290 software (Eastman Kodak, Rochester, NY).

Flow cytometry and cell sorting

Cell suspensions prepared from spleens excised from mice on day 8 post-SRBC immunization were depleted of erythrocytes by ammonium chloride Tris lysis (23). For four-color surface staining, 2 x 10⁶ cells resuspended in PBS containing 2% BSA were incubated with pretitered dilutions of GL7-FITC, biotinylated anti-IgD, anti-B220-PET-Texas Red, and 2.4G2-PE for 30 min at 4°C. SA Red 670 was used as a second-step reagent. Cells were analyzed using a Coulter Epics XL/MCL analyzer, and the data were analyzed by WinMDI software (The Scripps Research Institute, La Jolla, CA). For sorting, spleen cells prepared from C57BL/6 or Fc chain^-/- mice on day 8 post-SRBC immunization were stained with GL7-FITC, anti-B220-PE and anti-IgD-biotin followed by SA Red 670. GC and non-GC B cells were separated on a Coulter Epics Elite flow cytometric cell sorter.

RT-PCR assay

mRNA was extracted from sorted GC and non-GC B cells using the RNeasy Miniprep kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. Using 0.2 or 0.5 µg of RNA from each sample, cDNA synthesis and FcRIIB-specific PCR amplification was performed in a single step using the Superscript one-step RT-PCR system (Life Technologies, Gaithersburg, MD). FcRIIB cDNA was amplified using the 5' primer 5'-AAGTCTAGGAAGGACACTGC-3' and the 3' primer 5'-ATCCTGGCCTTCTGGCTTGC-3'. As an internal control, -actin mRNA was amplified using primers purchased from Clontech Laboratories (Palo Alto, CA). RT-PCR was conducted in 50-µl volumes with one cycle programmed to perform cDNA synthesis at 50°C for 30 min and 94°C for 2 min, followed immediately by PCR amplification for 30 cycles at 95°C for 1 min, 55°C for 45 s, and 72°C for 1 min. A final extension was done at 72°C for 5 min. One-fifth volume of the PCR was electrophoresed on a 1.5% agarose gel, and the bands were visualized under UV light.

Gel hybridization

Agarose gels were dried at 65°C for 2 h under vacuum, denatured in 0.5 M NaOH, 1.5 M NaCl for 1 h, and neutralized for 1 h in 0.5 M Tris, 3 M NaCl. The gels were then prehybridized at 42°C in prehybridization buffer solution containing 6x 0.6 M NaCl, 0.15 M Tris (pH 8.0), 6 mM EDTA; 0.05 M Denhardt’s solution; 0.1% SDS; and 250 µg of herring sperm DNA. Probes were prepared from PCR products by ³²P labeling using a random primed DNA labeling kit (Roche Molecular Biochemicals, Mannheim, Germany), denatured, and added to the prehybridization buffer, and the gels were hybridized overnight at 42°C. The gels were washed at low stringency in 5x SSC, 5% SDS for 1 h followed by two 30-min washes at 43°C. Subsequently, a high stringency wash in 0.1x SSC, 0.1% SDS at 60°C was performed. The gels were scanned on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after overnight exposure. The bands were quantitated after subtracting the background signal using Image Quant software (Molecular Dynamics). The integrated intensity of each band representing individual PCR products was measured and normalized using the -actin band intensity for each individual sample.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kinetics of the GC response to SRBC immunization

SRBC have been previously shown to induce robust GC responses in an adjuvant-independent manner (24, 25). However, only limited information was available regarding the magnitude and kinetics of this response. Therefore, we first examined the number and sizes of GC in the spleens of SRBC immunized C57BL/6 mice. Spleen sections obtained from days 4–40 after immunization were stained with GL7. The total number of GCs in each section were counted and categorized into three different sizes by counting GL7⁺ cells as previously described (26). A summary of the results is presented in Fig. 1. On day 4 after SRBC immunization, the majority of GL7⁺ cells were scattered in follicles. Small foci of GL7⁺ cells localized within the FDC network as well as a few well-developed small GCs were detectable (data not shown). By day 6, there was a large expansion in the number of GC comprising all sizes. This large number of GCs was maintained until day 12, after which the numbers declined. Large GCs were present from day 6, reached a peak by day 10, and thereafter declined with a corresponding increase in medium and small GCs. Although the number of medium sized GCs started to decline after day 12, the small GCs continued to increase, perhaps due to the dissolution of some of the large and medium sized GCs. The numbers of small GC gradually declined from day 16 onward, and by day 40 only a few GCs were present, almost all of which were small.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Kinetics of the GC response in C57BL/6 mice after immunization with SRBC. C57BL/6 mice were immunized with SRBC, and spleens removed at the indicated time points. Sections were stained with GL7-FITC. The total number of GL7+ cell clusters per section was counted for each time point. Error bars indicate the SD from three independent experiments. Following the convention previously described (26 ), the GCs were categorized into three sizes. GC clusters with >500-cell diameters were categorized as large. Medium GCs ranged from 250- to <500-cell diameters, and the small GCs were 50- to 150-cell diameters. Data are representative of at least three sections and two mice at each time point.

Although a strong correlation exists between the patterns of PNA and GL7 staining, earlier studies reported the presence of both PNA⁺GL7⁺ as well as PNA⁺GL7^- cell populations in spleen (27, 28). Immunohistological analysis of spleen sections from C57BL/6 mice derived 8 days after primary SRBC immunization was performed by staining with GL7 and PNA. As shown in the inset in Fig. 2, most PNA⁺ cells are GL7⁺ (and thus appear yellow), although some areas stained positive for either GL7 only or PNA only, suggesting heterogeneity in the expression of their respective ligands. Further, a rim of PNA⁺ cells that were GL7^- (Fig. 2, arrow) was also detected surrounding the GC. Although the significance of these differences in the staining pattern of GL7 and PNA is presently unclear, the results show that there is a more restricted pattern of expression of the ligands for GL7 than of the ligands for PNA in GCs. Therefore, we used GL7 as a marker to define GC cells in our subsequent analyses.

View larger version (68K): [in this window] [in a new window]   FIGURE 2. FcRIIB expression is differentially regulated in GCs. Fc chain-/- and C57BL/6 mice were immunized with SRBC, and spleens were removed on day 8. Additionally, Fc chain-/- mice were immunized with NP-CGG, and spleens were removed on day 10. Parallel sections (A–C; D and E; F and G) were stained with GL7-FITC or PNA-biotin followed by SA-FITC. For FcRIIB staining, either 2.4G2-PE for sections from Fc chain-/- mice or K9.361-biotin for sections from C57BL/6 mice was used in combination with anti-B220 or GL7. The areas shown by arrows in C, E, and G correspond to GL7+ regions within GCs in B, D, and F, respectively. These areas show low to undetectable levels of 2.4G2 staining. Original magnification, x200. Boxed inset, Representative histological image from a spleen section derived from C57BL/6 mice 8 days after immunization with SRBC. The section was stained with GL7-FITC and PNA-biotin which was revealed with SA-FITC. Arrow, The rim of PNA+ and GL7- cells surrounding the main GL7+ cluster. Original magnification, x200.

Differential regulation of FcRIIB expression on GC cells

We next examined whether there was any difference in the level of FcRIIB expression on GC and follicular B cells. In contrast to macrophage/monocyte lineage cells, B cells exclusively express the b1 isoform of FcRII and have been reported to lack expression of other Fc family receptors such as FcRI, FcRIII, and the b2 isoform of FcRII (1, 3). The mAb 2.4G2 recognizes the extracellular domains of FcRII and FcRIII. To avoid FcRIII staining with 2.4G2, we immunized Fc chain^-/- mice with SRBC. These mice express neither FcRI nor FcRIII due to the absence of the common -chain, required for assembly and membrane transport of these receptors. However, FcRIIB expression is not perturbed in these mice. Immunohistological analysis of spleen sections from Fc chain^-/- mice that were immunized with SRBC 8 days earlier was performed using 2.4G2 in combination with GL7 and anti-B220. Representative images are shown in Fig. 2. As shown in Fig. 2B, follicular mantle B cells clearly stain with 2.4G2, and the marginal zone stains even more strongly with 2.4G2, suggesting relatively high levels of FcRIIB expression. GL7⁺ GCs revealed an interesting pattern of 2.4G2 staining. Although FcRIIB expression was undetectable on B cells toward one side of the GC, intense 2.4G2 staining was observed polarized to the other side of the GC, suggesting up-regulated FcRIIB expression in this area. Similar results were obtained when sections were stained with PNA and 2.4G2 (Fig. 2A). This sectored pattern of FcRIIB expression was consistently observed. Parallel sections stained with Ab to B220 and 2.4G2 were then analyzed. As shown in Fig. 2C, B220⁺ marginal zone cells exhibited relatively high levels of FcRIIB (and thus appear orange in Fig. 2C), and the B220⁺ follicular mantle cells also coexpressed FcRIIB (and thus appear yellow in Fig. 2C). In contrast, the B220⁺ areas corresponding to GC exhibited sectoring, with undetectable levels of FcRIIB in one area (thus appearing green; Fig. 2C, arrow) and high levels of FcRIIB in the other area.

We then examined whether the observed differential regulation of FcRIIB was also a feature of GC responses to the well-characterized model Ag, NP. Day 10 spleen sections from NP₁₃-CGG-immunized Fc chain^-/- mice were stained with 2.4G2 and GL7 or anti-B220. These experiments also showed sectoring, with high levels of FcRIIB in one region and low to undetectable levels in the other region of GCs (Fig. 2, D and E). The analysis was further extended by using an FcRIIB-specific mAb, K9.361 (29, 30, 31), that was made available to us at a later time during the study. K9.361 exclusively recognizes FcRIIB (Ly17.2 allotype of Ly17.1/Ly17.2 alloantigen system) in C57BL/6 mice and does not bind to FcRIII (31). Immunohistological analysis of day 8 spleen sections from SRBC-immunized C57BL/6 mice with K9.361 and GL7 or anti-B220 revealed exactly the same sectored pattern of GC staining observed with 2.4G2 (Fig. 2, F and G). These data collectively indicate that the expression of FcRIIB is differentially modulated in these two areas of GCs.

FcRIIB expression is markedly reduced on GC B cells and up-regulated on FDC

We previously demonstrated that FcRIIB is up-regulated on FDC within the GC (16). In the experiments described above (Fig. 2), we could not evaluate whether the GC B cells present in the FDC-rich regions of the GC also expressed FcRIIB. To investigate this issue, chimeric mice were created by lethally irradiating FcRIIB^-/- mice and reconstituting them with bone marrow from FcRIIB^+/+ C57BL/6 mice. Because FDCs are resistant to radiation (32), they are derived from the host in these chimeras and therefore cannot express FcRIIB. The donor cells, including B cells, however, can express the FcRIIB receptor. Day 9 spleen sections from NP-CGG-immunized chimeric mice were examined for expression of FcRIIB in GCs. In the absence of FcRIIB expression on FDC, any staining of GC cells with the 2.4G2 Ab should result from FcR expression on B cells. As shown in Fig. 3, although FDCs were present in the GC, there was a lower level of FDC-M1 staining in general (Fig. 3A) and as expected, 2.4G2 staining on FDC was absent. B cells in these GCs, including those present in the FDC reticulum; exhibited markedly reduced levels of 2.4G2 staining compared with mantle zone B cells (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   FIGURE 3. FcRIIB is down-regulated on all GC B cells including those in FDC-rich areas. Chimeric mice created by lethally irradiating FcRIIB-/- mice and reconstituting them with bone marrow from FcRIIB+/+ C57BL/6 mice were immunized with NP-CGG. Sections prepared from spleens derived on day 9 postimmunization were examined for expression of FcRIIB in GCs. Two-color staining was performed on parallel sections with GL7-FITC and FDC-M1, or GL7-FITC and 2.4G2-PE. FDC-M1 was revealed with secondary biotin-labeled mouse F(ab')2 anti-rat IgG followed by SA-PE. Shown are representative images of GCs in parallel sections. The FDC reticulum (A) is not as well developed as in normal mice, and 2.4G2 staining on B cells in FDC-rich regions is very low to undetectable (B). Original magnification, x 200.

GC B cells express low to undetectable levels of FcRIIB at all stages of the GC response

We further investigated whether reduced expression of FcRIIB on GC B cells is characteristic of all stages of the GC response. FcRIIB expression was evaluated on SRBC-immunized C57BL/6 mice from day 4 to day 30 and day 4 to day 16 in Fc chain^-/- mice. Spleen sections were stained with GL7 in combination with 2.4G2, FDC-M1, or FDC-M2. Representative images are shown in Fig. 4. The results of this analysis revealed "sectoring" of GC due to up-regulated FcRIIB levels in the region rich in FDCs and low to undetectable levels of FcRIIB expression on GC B cells where the FDC network was absent. These observations were made at all time points of the GC response starting from day 4 when GC clusters first appeared (Fig. 4A). Furthermore, this was consistent for all GC sizes.

View larger version (67K): [in this window] [in a new window]   FIGURE 4. FcRIIB expression is low to undetectable on GC B cells in FDC poor regions throughout the GC response. Day 4 to day 30 parallel spleen sections derived from C57BL/6 (A) and day 4 to day 16 parallel spleen sections from Fc chain-/- mice (B) after SRBC immunization were stained with GL7-FITC in combination with 2.4G2-PE, FDC-M1, or FDC-M2. FDC-M1 and FDC-M2 were revealed with secondary biotin-labeled mouse F(ab')2 anti-rat IgG followed by SA-PE. Note that the pattern of FcRIIB and FDC-M1 staining results in sectoring the GC into two discrete regions at all time points of the response. Original magnification, x 200.

Flow cytometric and RT-PCR analysis of FcRIIB expression in GC B cells

As an alternative assay for FcRIIB expression on splenic GC B cells, we used flow cytometry. The mAbs 2.4G2 and K9.361 were used to determine the levels of FcRIIB on B cells from Fc chain^-/- and C57BL/6 mice, respectively. Four-color immunofluorescence analysis was performed, and surface expression of FcRIIB was analyzed on B220⁺ gated GL7⁺IgD^- (GC) and GL7^-IgD⁺ (non-GC) B cells. As shown in Fig. 5, compared with the GL7^-IgD⁺ non-GC B cell population, there was a substantial decrease of FcRIIB expression on GL7⁺IgD^- GC B cells. The extent of surface FcRIIB down-regulation on GC B cells detected with 2.4G2 (Fig. 5A) and K9.361 (Fig. 5B) was similar, thus confirming our immunohistology observations (Fig. 2). It is possible that due to the differences in the sensitivities of flow cytometry and fluorescence microscopy techniques, low level expression of FcRIIB on GC B cells was not detectable in immunohistological analyses. However, the unimodal distribution of FcRIIB staining indicates that all GC B cells exhibit this low expression level with no evidence for the presence of any minor population of GC B cells that express FcRIIB at levels similar to GL7^-IgD⁺ B cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Comparison of FcRIIB expression between splenic GL7-IgD+ non-GC and GL7+IgD- GC B cells. Fc chain-/- and C57BL/6 mice were immunized with SRBC, and spleen cells were obtained on day 8 after immunization. Four color flow cytometric analyses was performed after the cells were stained with GL7-FITC and anti-B220-PE-Texas Red in combination either with anti-IgD-biotin and 2.4G2-PE or with anti-IgD-PE and K9.361-biotin. The biotin was revealed with SA-Red 670. The histograms show FcRIIB expression levels on B220+GL7-IgD+ non-GC (– – – –) and B220+GL7+ IgD- GC B cells () in Fc chain-/- mice (A) and C57BL/6 mice (B). The shaded portions represent the staining obtained with a rat isotype control (A) and a mouse isotype control (B).

View larger version (36K): [in this window] [in a new window]   FIGURE 6. FcRIIB mRNA levels are 6-fold lower in GC B cells compared with non-GC B cells. C57BL/6 mice were immunized with SRBC. On day 8 postimmunization, mRNA was extracted from sorted GC and non-GC B cells followed by cDNA synthesis and FcRIIB-specific PCR amplification. As an internal control, -actin mRNA was amplified (not shown). PCR products removed after different numbers of PCR cycles were subjected to agarose gel electrophoresis. The gel was hybridized with an FcRIIB-specific probe and analyzed by PhosphorImager (see Materials and Methods). The relative intensity of each band representing individual PCR products was measured and expressed as units of integrated band intensity. , GC B cells; , non-GC B cells.

FDC exhibit maturational defects in FcRIIB-deficient mice

The differential regulation of FcRIIB on GC B cells and FDC prompted us to examine the GC response in mice lacking this receptor. Histological analysis of spleen sections from day 6 to day 22 post-SRBC immunization was performed on FcRIIB^-/- and C57BL/6 mice with GL7, FDC-M1, and FDC-M2. In C57BL/6 mice, the GC FDC reticulum was seen as fine processes extending throughout one side of the GC (see Fig. 4). This was true at all time points after immunization. In contrast, as shown in Fig. 7, this reticular pattern of staining was very weak in FcRIIB-deficient GCs at early and intermediate times in the GC response. In these GCs, strong FDC-M1 and FDC-M2 staining exhibited a largely punctate pattern until day 16. Thereafter, FDC staining was similar to that seen in C57BL/6 mice. The primary FDC reticula in FcRIIB^-/- mice, however, appeared normal at all stages of the GC response as judged by staining with the anti-CD35 Ab-8C12. Interestingly, even the punctate pattern of FDC staining observed during the early GC response of FcRIIB^-/- mice was largely restricted to one side of the GC. To investigate the cellular origin of this punctate staining, parallel sections from the experiment illustrated in Fig. 7 were stained with GL7, FDC-M1, and the MOMA-2 mAb. MOMA-2 has been previously reported to detect monocytes and tingible body macrophages (33). Representative images from this analysis are shown in Fig. 8. At early time points in FcRIIB^-/- mice (day 8 is illustrated in Fig. 8), MOMA-2 and FDC-M1 staining were largely colocalized to the punctate bodies, and these were rare in C57BL/6 GCs. At later times (day 20 is illustrated), whereas the reticular pattern of FDC-M1 staining in FcRIIB^-/- GCs appeared similar to C57BL/6 GCs, Moma-2/FDC-M1 double-positive cells were still clearly visible but remained rare in C57BL/6 GCs. Interestingly, we also observed MOMA-2 positive, FDC-M1 negative cells in the GCs of FcRIIB^-/- mice at these later time points.

View larger version (91K): [in this window] [in a new window]   FIGURE 7. Delayed development of the secondary FDC reticulum in SRBC-immunized FcRIIB-/- mice. Parallel sections of spleens from day 6 to day 22 post-SRBC immunization were stained with GL7-FITC in combination with FDC-M1, FDC-M2, or biotin-labeled anti-CD35 (clone 8C12). The FDC-M1 or FDC-M2 Abs were revealed with biotin-labeled mouse F(ab')2 anti-rat IgG. The biotin was revealed by SA-PE. Data are shown only until day 18. The staining pattern on days 20 and 22 was similar to day 18. Note the relatively low level of FDC-M1 and FDC-M2 expression in general, and the largely punctate pattern of FDC staining with both FDC-M1 and FDC-M2 until day 16 postimmunization. Original magnification, x200.

View larger version (66K): [in this window] [in a new window]   FIGURE 8. FDC-M1 staining colocalizes in a punctate pattern with MOMA-2 staining in GC during the early to intermediate stages of the response in FcRIIB-/- mice. Parallel sections of day 8 and day 20 spleens from FcRIIB-/- and C57BL/6 mice post-SRBC immunization were stained with combinations of GL7-FITC and FDC-M1 or MOMA-2-FITC and FDC-M1 or MOMA-2-FITC alone. The FDC-M1 was revealed with biotin-labeled mouse F(ab')2 anti-rat IgG followed by SA-PE. Original magnification, x200

View larger version (24K): [in this window] [in a new window]   FIGURE 9. The kinetics and magnitude of the GC response to SRBC immunization in FcRIIB-/- mice are similar to those in C57BL/6 mice. FcRIIB-/- mice were immunized with SRBC, and spleens were removed at the indicated time points. Sections were stained with GL7-FITC. The total number and the different sizes of GCs were determined as described in the legend to Fig. 1. Error bars represent SD. Data are representative of at least three sections and two mice at each time point.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Early models (13, 14, 35) explaining the mechanisms involved in memory B cell development proposed that B cells with somatically mutated Ag receptors are selected based on their capacity to be activated by Ag held as ICs on FDC. These models, however, did not consider the role of FcRIIB. Later models that implicated a role for FcRIIB in the selection of GC B cells were primarily based on data obtained from in vitro studies of primary B cells. Ravetch and Lanier (36) proposed that negative or positive selection of GC B cells depends on whether FcRIIB alone is engaged or coengaged with the BCR, respectively. Interaction of ICs on FDC with B cells through FcRIIB alone results in apoptosis, whereas coengagement with the BCR results in survival of B cells. Because our results demonstrate that FcRIIB is substantially down-regulated on GC B cells, negative selection due to an apoptotic signal generated through FcRIIB seems unlikely.

A model proposed by Tarlinton and Smith (37) suggested that early during the GC response, centrocytes usually initiate differentiation to Ab-forming cells (AFC). Ab produced by these AFC competes with centrocytes for the Ag present in the form of ICs on FDC in GC. Late in the primary response, when the Ab titer or affinity reaches a maximum, AFC differentiation diminishes, and a switch to memory B cell production takes place. This switch requires coengagement of the BCR and FcRIIB. This model implies that FcRIIB is not involved in the differentiation to AFC early in the GC response but is a requirement for memory formation later in the GC response. However, our data on the time course expression of FcRIIB on GC B cells (Fig. 4) do not support this model. However, we did not examine whether FcRIIB expression is reduced on GC B cells present in FDC-rich regions at all times during the GC response. Further studies will be required to determine whether a small subpopulation of GC B cells expresses normal levels of FcRIIB at a specific time in the GC response.

In contrast to those described above, Tew et al. (38) proposed a model that takes into account the significance of FcRIIB on FDC in selection of high-affinity GC B cells. According to this model, the high density of FcRIIB on FDC allows them to bind most of the Fc regions in the ICs on their surface. This minimizes the interaction of these Fc regions with FcRIIB on GC B cells, thus preventing the inhibition of B cell activity. The studies that led to the proposal of this model, however, did not examine the expression of FcRIIB on GC B cells. Based on our findings, it appears that the primary mechanism by which GC B cells escape the inhibitory FcRIIB signal is by down-regulating the expression of this receptor. Our flow cytometric and RT-PCR analyses (Figs. 5 and 6), however, did reveal low level expression of FcRIIB on GC B cells. Flow cytometric analysis performed on day 4 and later time points (day 16 and day 22) showed similar results (S. P. Rao and T. Manser, unpublished observations). Therefore, it is possible that both Fc blocking by FcRIIB on FDC and the down-regulation of expression of FcRIIB on B cells synergize to allow B cell stimulation by ICs. In any case, the functional relevance of such low FcRIIB levels is not clear and will be the subject of future investigations.

Recent studies have suggested a role for FcRIIB down-regulation in the development of autoimmunity. One study reported a 10-fold down-regulation on GC B cells in autoimmune-prone New Zealand Black mice and a 4-fold reduction on GC B cells in New Zealand White mice (17). In another study by the same group (18), FcRIIB expression on the GC B cells from nonautoimmune mice, including C57BL/6, was up-regulated. The reasons for the discrepancy of our results and the results of this previous study are presently unclear. Factors that may account for this discrepancy are use of keyhole limpet hemocyanin as an immunogen and examining the expression of FcRIIB on GC B cells after secondary challenge in their study. More likely, the differences are due to the criteria used for defining GC B cells. Although in our study FcRIIB expression was analyzed on GC B cells that were defined as GL7⁺IgD^-, in the previous study all PNA^high cells were considered GC B cells. It was recently demonstrated that no single marker can unambiguously distinguish GC B cells from other subsets, and flow cytometric analyses showed that PNA^high cells include IgD⁺ cells (27). Therefore, analyses of surface expression of FcRIIB on GC B cells defined on the basis of PNA binding alone may be misleading. These data emphasize the need to more thoroughly investigate the phenotypic criteria used in defining GC B cells.

It was also previously reported that FcRIIB deficiency on a C57BL/6 background results in the development of severe autoimmune glomerulonephritis in old mice (39). These findings formed the basis for the suggestion (36) that absence or abnormal down-regulation of FcRIIB is a mechanism that allows autoreactive IgG autoantibodies to be produced, predisposing to autoimmune disease. However, because our data show that down-regulation of FcRIIB on GC B cells is characteristic of mice that are not autoimmune prone, the mechanistic role for FcRIIB in the development of autoimmune conditions needs further evaluation. For example, there is no formal evidence demonstrating that autoreactive B cells in the GC, which in normal situations are believed to be negatively selected (13, 14), can be recruited into the memory pool due to down-regulation or absence of FcRIIB.

Our data also provide new insights into the role of FcRIIB in the "maturation" of FDC. We have reported that FDC-specific Abs, especially FDC-M1, recognize Ags that are induced on FDC in secondary but not primary follicles (16), suggesting that the expression of these Ags is associated with the maturation of FDC. Our data indicate that the gross GC response in FcRIIB^-/- mice is comparable in kinetics and magnitude with that of normal mice. However, the time course histological analysis with FDC-specific Abs revealed a significant delay in the normally strong reticular expression pattern of the FDC-M1 and FDC-M2 Ags, indicating a defect in the timing of maturation of FDC in the GC. Moreover, during the early phases of the GC response in FcRIIB^-/- mice, strong FDC-M1 staining colocalizes in a punctate pattern with MOMA-2 staining. Further studies are required to determine the nature of the cells that give rise to this staining pattern. They may be a subset of tingible body macrophages that express the FDC-M1 Ag, but it is also interesting to speculate that they are FDC precursors (40) that have yet to develop dendritic morphology.

B cell recall responses are thought to develop in GCs in response to ICs trapped on FDC. In this context, a recent study (16) investigated the significance of FcRIIB on the accessory activity of FDC. In contrast to normal FDCs, those from FcRIIB-deficient mice were incapable of augmenting IC-mediated B cell recall responses in vitro. Given our findings, it is possible that this lack of accessory activity of FDCs may be due not only to absence of FcRIIB but to a defect in FDC maturation as well. More generally, our data suggest that the function of FcRIIB on FDC is not merely to trap ICs but also to promote FDC maturation and expression of hitherto unknown molecules that may be involved in the regulation of GC B cell selection, memory development, and recall responses. Based on the correlation between the appearance of the FDC-M1 and FDC-M2 Ags and FcRIIB up-regulation on FDC during immune responses, it is tempting to speculate that the pathways involved in the regulation of expression of these molecules are one and the same. Clearly, more studies are required to elucidate the role of FcRIIB and mechanisms regulating the expression of this receptor on GC B cells and FDCs.

Another observation made in our study was the sectoring of GCs into two clearly discernible compartments when stained with Abs specific for FDC or FcRIIB. This sectoring was observed at all times during the GC response and raises the possibility that these areas in murine GC may correspond to the light zone composed of centrocytes (area in which the FDC network is organized) and the dark zone composed of centroblasts (area where the FDC network is absent) reported in earlier studies on human tonsillar GCs (41, 42). If so, the delay in FDC maturation in FcRIIB deficient mice may perturb the selection, affinity maturation and memory B cell development of centrocytes in the light zone. However, in the mouse, formal evidence that GC B cells in these areas actually represent centroblasts and centrocytes is currently lacking.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI 46806 (to T.M.) and by National Institutes of Health Training Grant 5-T32-AI07492 (to S.P.R.).

² Current address: Department of Immunology and Inflammation, Biogen, Cambridge, MA 02142.

³ Current address: Merck & Co., 126 East Lincoln Avenue, Rahway, NJ 07065.

⁴ Address correspondence and reprint requests to Dr. Tim Manser, Kimmel Cancer Institute, Bluemle Life Sciences Building, 708, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: manser{at}lac.jci.tju.edu

⁵ Abbreviations used in this paper: BCR, B cell Ag receptor; GC, germinal center; FDC, follicular dendritic cell; IC, immune complex; NP, 4-hydroxy-3-nitrophenyl acetyl; CGG, chicken -globulin; PNA, peanut agglutinin; AFC, Ab-forming cell; SA, streptavidin.

Received for publication February 22, 2002. Accepted for publication June 5, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ravetch, J. V., J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[Medline]
2. Hulett, M. D., P. M. Hogarth. 1994. Molecular basis of Fc receptor function. Adv. Immunol. 57:1.[Medline]
3. Daeron, M.. 1997. Fc receptor biology. Annu. Rev. Immunol. 15:203.[Medline]
4. Lewis, V. A., T. Koch, H. Plutner, I. Mellman. 1986. A complementary DNA clone for a macrophage-lymphocyte Fc receptor. Nature 324:372.[Medline]
5. Ravetch, J. V., A. D. Luster, R. Weinshank, J. Kochan, A. Pavlovec, D. A. Portnoy, J. Hulmes, Y. C. Pan, J. C. Unkeless. 1986. Structural heterogeneity and functional domains of murine immunoglobulin G Fc receptors. Science 234:718.[Abstract/Free Full Text]
6. Bolland, S., J. V. Ravetch. 1999. Inhibitory pathways triggered by ITIM-containing receptors. Adv. Immunol. 72:149.[Medline]
7. Phillips, N. E., D. C. Parker. 1984. Cross-linking of B lymphocyte Fc receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis. J. Immunol. 132:627.[Abstract]
8. Phillips, N. E., D. C. Parker. 1985. Subclass specificity of Fc receptor-mediated inhibition of mouse B cell activation. J. Immunol. 134:2835.[Abstract]
9. Coggeshall, K. M.. 1998. Inhibitory signaling by B cell FcRIIb. Curr. Opin. Immunol. 10:306.[Medline]
10. Pearse, R. N., T. Kawabe, S. Bolland, R. Guinamard, T. Kurosaki, J. V. Ravetch. 1999. SHIP recruitment attenuates FcRIIB-induced B cell apoptosis. Immunity 10:753.[Medline]
11. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, J. V. Ravetch. 1997. Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling. Cell 90:293.[Medline]
12. Ashman, R. F., D. Peckham, L. L. Stunz. 1996. Fc receptor off-signal in the B cell involves apoptosis. J. Immunol. 157:5.[Abstract]
13. Nossal, G. J.. 1994. Negative selection of lymphocytes. Cell 76:229.[Medline]
14. Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature 381:751.[Medline]
15. Healy, J. I., C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645.[Medline]
16. Qin, D., J. Wu, K. A. Vora, J. V. Ravetch, A. K. Szakal, T. Manser, J. G. Tew. 2000. Fc receptor IIB on follicular dendritic cells regulates the B cell recall response. J. Immunol. 164:6268.[Abstract/Free Full Text]
17. Jiang, Y., S. Hirose, R. Sanokawa-Akakura, M. Abe, X. Mi, N. Li, Y. Miura, J. Shirai, D. Zhang, Y. Hamano, T. Shirai. 1999. Genetically determined aberrant down-regulation of FcRIIB1 in germinal center B cells associated with hyper-IgG and IgG autoantibodies in murine systemic lupus erythematosus. Int. Immunol. 11:1685.[Abstract/Free Full Text]
18. Jiang, Y., S. Hirose, M. Abe, R. Sanokawa-Akakura, M. Ohtsuji, X. Mi, N. Li, Y. Xiu, D. Zhang, J. Shirai, Y. Hamano, H. Fujii, T. Shirai. 2000. Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility. Immunogenetics 51:429.[Medline]
19. Brauweiler, A., I. Tamir, S. Marschner, C. D. Helgason, J. C. Cambier. 2001. Partially distinct molecular mechanisms mediate inhibitory FcRIIB signaling in resting and activated B cells. J. Immunol. 167:204.[Abstract/Free Full Text]
20. Takai, T., M. Ono, M. Hikida, H. Ohmori, J. V. Ravetch. 1996. Augmented humoral and anaphylactic responses in FcRII-deficient mice. Nature 379:346.[Medline]
21. Takai, T., M. Li, D. Sylvestre, R. Clynes, J. V. Ravetch. 1994. FcR chain deletion results in pleiotrophic effector cell defects. Cell 76:519.[Medline]
22. Vora, K. A., J. V. Ravetch, T. Manser. 1997. Amplified follicular immune complex deposition in mice lacking the Fc receptor -chain does not alter maturation of the B cell response. J. Immunol. 159:2116.[Abstract/Free Full Text]
23. Lentz, V. M., T. Manser. 2000. Self-limiting systemic autoimmune disease during reconstitution of T cell-deficient mice with syngeneic T cells: support for a multifaceted role of T cells in the maintenance of peripheral B cell tolerance. Int. Immunol. 12:1483.[Abstract/Free Full Text]
24. Kraal, G., I. L. Weissman, E. C. Butcher. 1982. Germinal centre B cells: antigen specificity and changes in heavy chain class expression. Nature 298:377.[Medline]
25. Kroese, F. G., W. Timens, P. Nieuwenhuis. 1990. Germinal center reaction and B lymphocytes: morphology and function. Curr. Top. Pathol. 84:103.
26. Vora, K. A., K. M. Tumas-Brundage, V. M. Lentz, A. Cranston, R. Fishel, T. Manser. 1999. Severe attenuation of the B cell immune response in Msh2-deficient mice. J. Exp. Med. 189:471.[Abstract/Free Full Text]
27. Shinall, S. M., M. Gonzalez-Fernandez, R. J. Noelle, T. J. Waldschmidt. 2000. Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens. J. Immunol. 164:5729.[Abstract/Free Full Text]
28. Cervenak, L., A. Magyar, R. Boja, G. Laszlo. 2001. Differential expression of GL7 activation antigen on bone marrow B cell subpopulations and peripheral B cells. Immunol. Lett. 78:89.[Medline]
29. Kimura, S., N. Tada, E. Nakayama, Y. Liu, U. Hammerling. 1981. A new mouse cell-surface antigen (Ly-m20) controlled by a gene linked to Mls locus and defined by monoclonal antibodies. Immunogenetics 14:3.[Medline]
30. Holmes, K. L., R. G. Palfree, U. Hammerling, III H. C. Morse. 1985. Alleles of the Ly-17 alloantigen define polymorphisms of the murine IgG Fc receptor. Proc. Natl. Acad. Sci. USA 82:7706.[Abstract/Free Full Text]
31. Schiller, C., I. Janssen-Graalfs, U. Baumann, K. Schwerter-Strumpf, S. Izui, T. Takai, R. E. Schmidt, J. E. Gessner. 2000. Mouse FcRII is a negative regulator of FcRIII in IgG immune complex-triggered inflammation but not in autoantibody-induced hemolysis. Eur. J. Immunol. 30:481.[Medline]
32. Tew, J. G., J. Wu, D. Qin, S. Helm, G. F. Burton, A. K. Szakal. 1997. Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol. Rev. 156:39.[Medline]
33. Kraal, G., M. Rep, M. Janse. 1987. Macrophages in T and B cell compartments and other tissue macrophages recognized by monoclonal antibody MOMA-2: an immunohistochemical study. Scand J. Immunol. 26:653.[Medline]
34. Oliver, A. M., F. Martin, J. F. Kearney. 1997. Mouse CD38 is down-regulated on germinal center B cells and mature plasma cells. J. Immunol. 158:1108.[Abstract]
35. Liu, Y. J., G. D. Johnson, J. Gordon, I. C. MacLennan. 1992. Germinal centres in T-cell-dependent antibody responses. Immunol. Today 13:17.[Medline]
36. Ravetch, J. V., L. L. Lanier. 2000. Immune inhibitory receptors. Science 290:84.[Abstract/Free Full Text]
37. Tarlinton, D. M., K. G. Smith. 2000. Dissecting affinity maturation: a model explaining selection of antibody-forming cells and memory B cells in the germinal centre. Immunol. Today 21:436.[Medline]
38. Tew, J. G., J. Wu, M. Fakher, A. K. Szakal, D. Qin. 2001. Follicular dendritic cells: beyond the necessity of T-cell help. Trends Immunol. 22:361.[Medline]
39. Bolland, S., J. V. Ravetch. 2000. Spontaneous autoimmune disease in Fc()RIIB-deficient mice results from strain-specific epistasis. Immunity 13:277.[Medline]
40. Szakal, A. K., K. L. Holmes, J. G. Tew. 1983. Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. J. Immunol. 131:1714.[Abstract]
41. Hardie, D. L., G. D. Johnson, M. Khan, I. C. MacLennan. 1993. Quantitative analysis of molecules which distinguish functional compartments within germinal centers. Eur. J. Immunol. 23:997.[Medline]
42. Kosco-Vilbois, M. H., H. Zentgraf, J. Gerdes, J. Y. Bonnefoy. 1997. To "B" or not to "B" a germinal center?. Immunol. Today 18:225.[Medline]

This article has been cited by other articles:

				F. Coffey, X. Liu, and T. Manser Primary Development and Participation in a Foreign Antigen-Driven Immune Response of a Chromatin-Reactive B Cell Clonotype Are Not Influenced by TLR9 or Other MyD88-Dependent TLRs J. Immunol., November 15, 2007; 179(10): 6663 - 6672. [Abstract] [Full Text] [PDF]

				M. Al-Alwan, Q. Du, S. Hou, B. Nashed, Y. Fan, X. Yang, and A. J. Marshall Follicular Dendritic Cell Secreted Protein (FDC-SP) Regulates Germinal Center and Antibody Responses J. Immunol., June 15, 2007; 178(12): 7859 - 7867. [Abstract] [Full Text] [PDF]

				Z. S. M. Rahman and T. Manser Failed Up-Regulation of the Inhibitory IgG Fc Receptor Fc{gamma}RIIB on Germinal Center B Cells in Autoimmune-Prone Mice Is Not Associated with Deletion Polymorphisms in the Promoter Region of the Fc{gamma}RIIB Gene J. Immunol., August 1, 2005; 175(3): 1440 - 1449. [Abstract] [Full Text] [PDF]

				Y. Aydar, S. Sukumar, A. K. Szakal, and J. G. Tew The Influence of Immune Complex-Bearing Follicular Dendritic Cells on the IgM Response, Ig Class Switching, and Production of High Affinity IgG J. Immunol., May 1, 2005; 174(9): 5358 - 5366. [Abstract] [Full Text] [PDF]

				K. Su, J. Wu, J. C. Edberg, X. Li, P. Ferguson, G. S. Cooper, C. D. Langefeld, and R. P. Kimberly A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Fc{gamma}RIIb Alters Receptor Expression and Associates with Autoimmunity. I. Regulatory FCGR2B Polymorphisms and Their Association with Systemic Lupus Erythematosus J. Immunol., June 1, 2004; 172(11): 7186 - 7191. [Abstract] [Full Text] [PDF]

				K. Su, X. Li, J. C. Edberg, J. Wu, P. Ferguson, and R. P. Kimberly A Promoter Haplotype of the Immunoreceptor Tyrosine-Based Inhibitory Motif-Bearing Fc{gamma}RIIb Alters Receptor Expression and Associates with Autoimmunity. II. Differential Binding of GATA4 and Yin-Yang1 Transcription Factors and Correlated Receptor Expression and Function J. Immunol., June 1, 2004; 172(11): 7192 - 7199. [Abstract] [Full Text] [PDF]

				T. Manser Textbook Germinal Centers? J. Immunol., March 15, 2004; 172(6): 3369 - 3375. [Abstract] [Full Text] [PDF]

				Y. Aydar, P. Balogh, J. G. Tew, and A. K. Szakal Altered Regulation of Fc{gamma}RII on Aged Follicular Dendritic Cells Correlates with Immunoreceptor Tyrosine-Based Inhibition Motif Signaling in B Cells and Reduced Germinal Center Formation J. Immunol., December 1, 2003; 171(11): 5975 - 5987. [Abstract] [Full Text] [PDF]

				Z. SM. Rahman, S. P. Rao, S. L. Kalled, and T. Manser Normal Induction but Attenuated Progression of Germinal Center Responses in BAFF and BAFF-R Signaling-Deficient Mice J. Exp. Med., October 20, 2003; 198(8): 1157 - 1169. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rao, S. P. Articles by Manser, T. Search for Related Content PubMed PubMed Citation Articles by Rao, S. P. Articles by Manser, T.