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The Journal of Immunology, 2006, 177: 2072-2079.
Copyright © 2006 by The American Association of Immunologists

Characterization of (4-Hydroxy-3-Nitrophenyl)Acetyl (NP)-Specific Germinal Center B Cells and Antigen-Binding B220– Cells after Primary NP Challenge in Mice1

Kristy L. Wolniak*,{dagger}, Randolph J. Noelle{ddagger} and Thomas J. Waldschmidt2,*,{dagger}

* Department of Pathology and {dagger} Interdisciplinary Graduate Program in Immunology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242; and {ddagger} Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03755


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previous studies examining the primary germinal center (GC) response to SRBC in mice demonstrated a steady ratio of IgM+ to isotype-switched GC B cells and a persistent population of GC B cells with a founder phenotype. These characteristics held true at the inductive, plateau, and dissociative phases of the GC response, suggesting a steady-state environment. To test whether these characteristics apply to the primary response of other T cell-dependent Ags, the present study examined the GC response after challenge with (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Multiparameter flow cytometric analysis was used to assess the phenotype of splenic NP-reactive cells at multiple time points after immunization. Results of these studies demonstrated the characteristics of the SRBC-induced GC reaction to be fully maintained in the NP response. In particular, there was a steady ratio of nonswitched to switched B cells, with the majority of NP-reactive GC B cells displaying IgM. In addition, a substantial frequency of B220– NP-binding cells was observed in the spleen at later time points after NP challenge. Although these cells were IgE+, they were found to express both {kappa} and {lambda} L chains and display the high-affinity IgE Fc (Fc{epsilon}RI) receptor, suggesting that this population is not of B cell origin. Adoptive transfer studies further demonstrated the B220– NP-binding subset to be derived from the myeloid lineage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Formation of a specific high-affinity Ab response is critical for effective T cell-driven humoral immunity. Efficient generation of such an Ab response depends on the germinal center (GC),3 an inducible structure found in secondary lymphoid tissue after Ag challenge. Upon immunization, Ag-specific B cells are activated in the T cell zones and migrate to B cell follicles (1, 2, 3, 4). Within the follicle, B cells rapidly proliferate and initiate the GC response, where they interact with Ag-specific CD4+ Th cells (5) and follicular dendritic cells (6, 7). GC B cells undergo clonal expansion, somatic hypermutation (SHM), positive selection of high-affinity variants, and negative selection of low-affinity and self-reactive cells (8, 9, 10, 11, 12). Commitment to memory B cells and long-lived plasma cells also depends upon the GC environment and provides long-term protection against the offending pathogen (8, 9, 10, 11, 12).

Although a significant number of studies have investigated the initiation, function, and molecular events within GCs, few have focused on detailing the stages of GC B cell differentiation. In studies of human GCs, multiparameter flow cytometry has successfully delineated the progressive stages of GC B cell differentiation (13, 14). In particular, characterization of surface marker gain and loss enabled definition of functionally distinct GC B cell subsets, and a comprehensive linear model of GC B cell maturation (14). Based on these reports, our laboratory applied the method of sequential multiparameter flow cytometric analysis to fully characterize the primary murine GC response after SRBC immunization. Using a range of Ig, activation and differentiation markers, GC B cells were examined in BALB/c mice at various time points postchallenge (4, 15). Defining GC B cells as B220+ and peanut agglutinin (PNA)high, these studies revealed the persistence of an IgD+CD23highCD38high GC B cell subset throughout the response. In addition, the ratio of IgM+ to IgG+ GC B cells was unchanged during the GC reaction. From the emergence of GCs at day 4 to their dissolution several weeks postimmunization, IgM+ and isotype-switched GC B cells were maintained at an approximate ratio of 3:1. Only the BLA-1 marker was found to be dynamic, with expression ranging from >90% on early GC B cells to <50% 3 wk postchallenge. Together, these findings indicated the primary murine GC response to be better characterized as a steady-state rather than a dynamic process (4, 15).

Although the GC reaction in the mouse has been examined after challenge with a number of T cell-dependent Ags, our classical understanding of murine GCs is derived from the work of Kelsoe and colleagues (2, 12, 16, 17, 18). These investigators used the clonally restricted response to (4-hydroxy-3-nitrophenyl)acetyl (NP) in Ighb mice, where immunization with NP-protein conjugates leads to recruitment of B cells primarily expressing VH186 and {lambda} L chains, and is therefore useful in analyzing Ag-specific GC B cells. Kelsoe and coworkers (2, 12, 16, 17, 18) detailed the development of the primary GC response to NP-chicken {gamma}-globulin in C57BL/6 mice using immunohistology and molecular analyses. In their resulting model, interaction of Ag-specific B and T cells initially gives rise to foci of Ab forming cells at the periphery of the periarteriolar lymphoid sheath (2). GCs emerge simultaneously with periarteriolar lymphoid sheath foci at day 4, peak on day 12, and diminish thereafter. SHM begins in GC B cells at the end of the first week following immunization, and together with BCR-driven selection, results in preferential survival of high-affinity clones as the response matures (17, 18). Curiously, immunohistologic analysis revealed almost all GC B cells to express IgG by the second week postchallenge (2). This finding suggested isotype switching to be dynamic in contrast to GCs induced after SRBC challenge. Kelsoe and colleagues (12) additionally demonstrated that high-affinity memory cells and long-lived plasma cells are products of the GC. Importantly, these studies provided the framework for subsequent investigation of Ag-specific GC B cells.

To determine whether the characteristics of the SRBC-driven GC response are applicable to the well-characterized clonally restricted NP GC reaction, we performed sequential multiparameter flow cytometric analyses on splenocytes from NP-keyhole limpet hemocyanin (KLH)-immunized C57BL/6 mice. Ag-specific B cells were identified flow cytometrically by their ability to bind 4-hydroxy-3-iodo-5-nitrophenylacetate (NIP) conjugated to PE. In addition to normal mice, the NP response of genetically altered mice bearing transgenic NP-specific H chains was assessed. These studies revealed the NP-induced GC reaction to exhibit the same attributes as those previously described for GCs formed after SRBC immunization. There was a steady ratio of IgM+ to isotype-switched GC B cells and a persistence of IgD+CD23highCD38high GC B cells throughout the response. In addition, the frequency of cells expressing the BLA-1 Ag decreased as the response matured.

An interesting element of the NP-specific response is the recently identified population of B220– Ag-binding cells that appear in the spleen and bone marrow after primary and secondary immunization. Studies by McHeyzer-Williams and coworkers (19, 20) identified two populations of B220– NP-binding cells that express either IgG or IgE. Single-cell molecular analyses revealed the IgG-expressing B220– NP-binding cells have undergone SHM, suggesting these cells to be GC-derived (19). Additionally, adoptive transfer studies provided evidence that these cells are capable of giving rise to Ab-secreting cells (19). Of interest, subsequent reports by two groups identified a similar population of B220– Ag-binding cells following immunization with allophycocyanin or PE (21, 22). These latter studies concluded that B220– Ag-binding cells were not B cell in origin, and captured Ag via FcR-bound Ab (21, 22). Flow cytometric analyses of the NP-specific GC B cell response in our experiments demonstrated a similar population of B220– Ag-binding PNA+ cells. This subset appeared following immunization and persisted throughout and beyond the GC response. Detailed analyses revealed these cells to express IgE in combination with both {kappa} and {lambda} L chains, and to be negative for all common B cell markers. Additional experiments suggested this population to be nonlymphoid in nature and to bind Ag-specific Ab though Fc{epsilon}RI.


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

Six- to 8-wk-old female C57BL/6 mice were purchased from the National Cancer Institute (Frederick, MD) and housed in the University of Iowa specific pathogen-free facility. Anti-NP H chain V(D)J transgenic (quasi-monoclonal (QM)) mice, provided by Dr. M. Wabl (University of California, San Francisco, CA) (23) were backbred onto the C57BL/6Jh–/–J{kappa}–/– strain for nine generations to produce QM.B6 mice (24). QM.B6 females were crossed with wild-type C57BL/6 males to produce F1 offspring used in these experiments. The (QM x C57BL/6)F1 mice are referred to as QM{kappa}.Fcer1g–/– mice lacking the {gamma}-chain subunit of Fc{epsilon}RI were purchased from Taconic Farms and LT{alpha}–/– mice were obtained from The Jackson Laboratory. RAG-1–/– and CD45.1 mice on the C57BL/6 background were provided by Dr. S. Perlman (University of Iowa, Iowa City, IA) and Dr. A. Schlueter (University of Iowa, Iowa City, IA), respectively. All protocols using mice were approved by the Institutional Animal Care and Use Committee.

Abs and reagents for flow cytometry

The following mAbs were prepared in our laboratory and used for flow cytometric studies: RA3-6B2, a rat IgG anti-mouse B220; b76, a rat IgG anti-mouse IgM; 11-26, a rat IgG anti-mouse IgD; B3B4, a rat IgG anti-mouse CD23; 53-10.1, a rat IgG anti-mouse BLA-1 (25); clone 90, a rat-IgG anti-mouse CD38 (provided by Dr. J. Kearney, University of Alabama, Birmingham, AL); ID3, a rat anti-mouse CD19 (kindly provided by Dr. D. Fearon, University of Cambridge, Cambridge, U.K.); M1/69, a rat IgG anti-mouse CD24 (heat-stable Ag); 187.1, a rat IgG1 anti-mouse {kappa} L chain; M5114, a rat IgG anti-mouse MHC class II; 7E9, a rat IgG anti-mouse CD21/35 (provided by Dr. T. Kinoshita, Osaka University, Osaka, Japan); EM95, a rat IgG anti-mouse IgE; PK136, a mouse IgG anti-mouse NK1.1; M1/70, a rat IgG anti-mouse CD11b; 9F3, a rat IgG anti-mouse CD44; FD441.8, a rat IgG anti-mouse CD11a; and CY 34.1.1, a mouse IgG anti-mouse CD22. Abs were semipurified from HB101 serum-free supernatants by 50% ammonium sulfate precipitation and conjugated to biotin (Sigma-Aldrich), R-PE (Molecular Probes), Texas Red (Molecular Probes), or Cy5 (Amersham Biosciences) using standard procedures. Biotin-conjugated R11-153, a rat IgG anti-mouse {lambda}1 L chain, biotin-conjugated Jo2, a hamster IgG anti-mouse CD95, biotin-conjugated 281-2, a rat IgG anti-mouse Syndecan-1, fluorescein-conjugated HM79b, a hamster IgG anti-mouse CD79b and R-PE-conjugated 53-7.313, a rat IgG anti-mouse CD5 were purchased from BD Pharmingen. LO-MG1-2, a rat IgG anti-mouse IgG1 was obtained from Zymed. Biotin-conjugated affinity-purified goat anti-mouse IgG2b-specific Ab was purchased from Southern Biotechnology Associates. Fluorescein-conjugated PNA was obtained from Vector Laboratories. Biotin-conjugated MAR-1, a hamster anti-mouse Fc{epsilon}R1 {alpha}-chain was purchased from eBioscience. R-PE-conjugated streptavidin (Southern Biotechnology Associates) and Texas Red-conjugated streptavidin (Jackson ImmunoResearch Laboratories) were used as secondary reagents.

NIP-PE conjugation

NIP-PE was generated by conjugating R-PE to NIP-OSu (Biosearch Technologies). R-PE was dialyzed against 0.1 M sodium borate (pH 9.2). NIP-OSu was diluted in dimethylformamide and added to R-PE at molar ratio of 20:1. The reaction mixture was incubated at 4°C for 4 h and subsequently dialyzed against PBS to remove unconjugated NIP.

Immunizations

C57BL/6, QM{kappa}, Fcer1g–/–, and reconstituted RAG-1–/– mice were injected i.p. with 100 µg of NP-KLH (Biosearch Technologies) precipitated in alum. C57BL/6 and Fcer1g–/– mice were injected i.p. with 400 µg of NP-KLH in 100 µl of Ribi adjuvant (Corixa).

Cell preparation and flow cytometric analysis

Splenocytes were harvested on days 4, 6, 8, 10, 12, 18, and 24 postchallenge. Mice were anesthetized with halothane and sacrificed with cervical dislocation. Spleens were obtained and ground between frosted slides to create a cell suspension in 1x balanced salt solution. Splenocytes were washed and resuspended in staining buffer (SB: balanced salt solution, 5% bovine calf serum, and 0.1% sodium azide), followed by density centrifugation over Fico/Lite-LM (Atlanta Biologicals) to obtain viable mononuclear cells. To stain splenocytes for multiparameter flow cytometric analysis, 1–2 x 106 cells suspended in SB were added to 10 µl of rat serum and 20 µg of 2.4G2 (anti-Fc{gamma}R) to prevent background staining mediated by FcR binding. The appropriate primary Abs, NIP-PE, and PNA-FITC were added to the cells and incubated for 20 min on ice. The cells were washed twice in SB, and secondary streptavidin reagent was added to detect biotinylated Abs. The cells were again incubated on ice for 20 min, washed twice in SB, and resuspended in fixative (1% formaldehyde in 1.25x PBS). Flow cytometric analysis was performed on a FACSVantage SE flow cytometer (BD Biosciences) equipped with the argon ion laser (488-nm line at 300 mW) and a dye laser using Rhodamine 6G pumped by a secondary argon ion laser (595-nm line at 300 mW). All samples were fluorescence compensated with positive controls to ensure the absence of spectral overlap. Dead cells and debris were excluded with forward and orthogonal scatter gating. A total of 100,000–500,000 cells was analyzed per sample. All data were acquired using DESK software provided by W. Moore (Stanford University, Stanford, CA) and analyzed using FlowJo software (Tree Star). Fluorescence data are displayed as four decade logarithmic plots.

Bone marrow transfers

RAG-1–/– (CD45.2) mice on the C57BL/6 background were irradiated with 250 rad. A total of 5 x 106 mononuclear bone marrow cells from wild-type C57BL/6 congenic mice (CD45.1) was transferred into the RAG-1–/– mice retro-orbitally. Reconstituted RAG-1–/– mice were immunized after 8 wk of rest.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The NP-specific GC B cell response in C57BL/6 mice

To determine whether the features of the SRBC GC reaction apply to the NP-induced GC response, splenocytes from NP-KLH-immunized C57BL/6 mice were analyzed by multiparameter flow cytometry. Mice were challenged with NP-KLH in Ribi adjuvant, and splenic mononuclear cells were examined 4–18 days postimmunization. In wild-type C57BL/6 mice, Ribi adjuvant was found to induce the strongest NP response, as previously observed (19). NP-specific B cells were identified with NIP conjugated to PE. Immunization of C57BL/6 mice with NP-KLH generated a population of NIP-binding splenocytes not observed in naive mice (Fig. 1A). Within the NIP-binding subset, a population of B220+PNAhigh GC B cells was easily identified (Fig. 1B) (15, 26). Their status as GC B cells is confirmed by expression of GL7 and CD95 (data not shown) (27, 28). Consistent with previous studies, a majority of the NIP-binding GC B cells was found to use {lambda} L chains (Fig. 1C). NP-induced GC B cells were further examined for expression of surface markers found to delineate subsets in the SRBC GC response. NIP-binding B220+PNAhigh cells were therefore tested for expression of IgM, IgD, IgG1, CD23, CD38, and BLA-1 (4, 15). Fig. 1C shows these markers to define NP-induced GC B cell subpopulations based on either plus/minus or high/low expression patterns. As listed in Table I, the NP GC response appeared at day 4, peaked at day 12, and persisted into the third week postimmunization, similar to previous studies (2). Accordingly, NP GC B cell subsets were enumerated on days 4, 8, 12, and 18 (Fig. 1D). As observed after SRBC challenge, the ratio of nonswitched (IgM+) to switched (IgG+) GC B cells remained largely unchanged during the course of the NP GC response (~4:1). In addition, a surprising frequency of NIP-binding B220+ PNAhigh B cells expressed IgD and high levels of CD23 and CD38, hallmarks of founder GC B cells. Importantly, the frequency of IgD+CD23highCD38high GC B cells remained relatively constant. Consistent with the SRBC response, the frequency of CD23highCD38high GC B cells slightly exceeded that of IgD+ GC B cells, suggesting loss of IgD before down-regulation of CD23 and CD38 (4, 15). Collectively, the results show the primary NP GC response to exhibit steady-state characteristics. As expected, the only marker to display a dynamic pattern of expression was BLA-1. Fig. 1D demonstrates BLA-1 to be expressed on nearly all NIP-binding GC B cells early in the response, with progressive loss as the reaction matures. This result is again consistent with BLA-1 loss on GC B cells after SRBC challenge (4, 15).


Figure 1
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  		FIGURE 1. Kinetics and maturation of the NP-specific GC response in C57BL/6 mice. Mice were immunized i.p. with NP-KLH in Ribi adjuvant. Splenocytes were harvested at the designated time points postchallenge and analyzed by multiparameter flow cytometry. A, Splenocytes from an unimmunized mouse were stained with anti-B220 and NIP-PE. The dot plot shows background levels of NIP-binding B cells. B, Representative dot plots of splenocytes analyzed 12 days postimmunization. The left panel represents cells stained with anti-B220 and PE and illustrates background staining directed against PE. The middle panel shows cells stained with anti-B220 and NIP-PE and demonstrates the marked increase in NIP-binding cells after NP-KLH challenge. The far right panel shows anti-B220 and PNA staining on the gated NIP-PE binding population (indicated in the middle panel). The B220+PNAhigh cells (within the boxed area on the right panel) represent NP-reactive GC B cells. C, Representative histograms of NP-induced GC B cells analyzed 12 days postimmunization. Cells were stained with anti-B220, NIP-PE, PNA, and the indicated markers. Histograms are derived from the B220+ NIP-binding PNAhigh gated population. Vertical lines illustrate where positive/negative or high/low determinations were made for the bar graphs shown in D. D, Frequencies of IgM+, IgG1+, IgD+, CD23high, CD38high, and BLA-1high cells within the NP-induced GC B cell population at days 4, 8, 12, and 18 postchallenge. Bar graphs represent means ± SD. Four mice were analyzed at each time point.

 

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  		Table I. Frequency: of NIP-binding GC B cells following NP-KLH immunization

 
The NP-specific GC response in transgenic mice bearing NP-specific H chains

In addition to examining the NP GC response in wild-type C57BL/6 mice, we further tested mice transgenic for NP-specific BCRs. Specifically, we examined the GC response in (QM x C57BL/6)F1 mice. The QM or quasi-monoclonal strain was developed by insertion of a rearranged NP-specific VHDJH, element (17.2.25) into the JH locus (23). These transgenic knock-in mice were bred onto a JH–/– and JK–/– background, and therefore use endogenous {lambda} L chains to generate NP-specific Ag receptors (23). When initially testing QM mice bred onto the C57BL/6 background (24), we observed an early robust GC response after NP-KLH challenge (29). However, the NP GC response abruptly dissipated at days 6–7 postimmunization, preventing full kinetic analysis (29). The abortive GC response is not observed in F1 offspring of QM mice bred with wild-type C57BL/6 mice. (QM x C57BL/6)F1 (referred to as QM{kappa}) mice have normal H chain and {kappa} chain loci contributed by the wild-type parent, with the result that ~30% of the B cells bind NIP instead of the entire B cell compartment (Fig. 2). QM{kappa} mice were challenged with NP-KLH precipitated with alum, and the splenic GC response was examined at various times postimmunization. Alum was used for the QM{kappa} experiments because this adjuvant proved to be more effective than Ribi in eliciting a NP GC response in this strain. As expected, NIP-binding GC B cells were observed after challenge, although their frequency was similar to that observed in wild-type mice (Table I). Importantly, as shown in Fig. 2A, NP-induced GC B cells were PNAhigh and used primarily {lambda} L chains. Using a multicolor protocol, subsets of NIP-reactive GC B cell subsets were enumerated based on IgM, IgG1, IgD, CD23, CD38, and BLA-1 expression (Fig. 2B). QM{kappa} mice were tested at days 4, 8, 12, and 18 after immunization (Fig. 2C). Similar to NP-KLH-challenged C57BL/6 mice, the NP GC response in QM{kappa} mice is characterized by a steady ratio of nonswitched (IgM+) to switched (IgG1+) B cells, a relatively constant proportion of IgD+CD23highCD38high cells, and a reduction of BLA-1 late in the response. Although the NP GC response in wild-type and QM{kappa} transgenic mice exhibited similar traits, subtle differences were noted. In particular, the frequency of nonswitched and founder GC B cells were slightly higher in QM{kappa} mice, and BLA-1 loss occurred with a delayed kinetics. Importantly, however, the NP-induced GC response in both C57BL/6 wild-type and QM{kappa} transgenic mice displayed characteristics nearly identical with GCs observed after SRBC immunization.


Figure 2
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  		FIGURE 2. Kinetics and maturation of the NP-specific GC response in QM{kappa} mice. Mice were immunized i.p. with NP-KLH precipitated in alum. Splenocytes were harvested at the designated time points postchallenge and analyzed by multiparameter flow cytometry. A, Representative plots of QM{kappa} splenocytes at day 12 postimmunization. The left panel shows cells stained with anti-B220 and PE, and illustrates background staining directed against PE. The second plot shows splenocytes stained with anti-B220 and NIP-PE and demonstrates the significant frequency of NIP binding B cells due to the expressed transgene. The third panel shows anti-B220 and PNA staining on the gated NIP-PE binding population (indicated in the second panel). The B220+PNAhigh cells (within the boxed area on the right panel) represent NP-reactive GC B cells. The far right panel shows {lambda} L chain expression on the gated B220+ NIP-binding PNAhigh population. B, Representative histograms of NP-induced GC B cells analyzed 12 days postimmunization. Cells were stained with anti-B220, NIP-PE, PNA, and the indicated markers. Histograms are derived from the B220+ NIP-binding PNAhigh gated population. Vertical lines illustrate where positive/negative or high/low determinations were made for the bar graphs shown in C. C, Frequencies of IgM+, IgG1+, IgD+, CD23high, CD38high, and BLA-1high cells within the NP-induced GC B cell population at days 4, 8, 12, and 18 postchallenge. Bar graphs represent means ± SD. Four mice were analyzed at each time point.

 
Characterization of B220– NIP-binding cells

In both C57BL/6 (Fig. 3) and QM{kappa} mice (data not shown) challenged with NP-KLH, a population of B220– NIP-binding spleen cells was consistently observed. As illustrated in Fig. 3, this subset of Ag-binding cells was typically present by day 8 postchallenge and persisted through the second and third weeks of the response. The generation of B220– NIP-binding cells occurred whether Ribi (Fig. 3A) or alum (B) was used as adjuvant, although alum typically resulted in a more prominent population. Examination of bone marrow from NP-KLH immunized mice also revealed B220– NIP-binding cells (data not shown). This subset is similar to previously reported B220– Ag-binding cells, the identity of which is controversial (19, 20, 21, 22). Accordingly, we characterized the surface phenotype of the B220– NIP-binding subset in C57BL/6 mice after immunization. Expression of selected markers is shown in Fig. 4 with a complete listing of results in Table II. This population was found to be negative for markers expressed on all or most mature B cells including CD19, CD22, CD23, CD24 (heat stable Ag), CD38, CD79b (Igbeta), and MHC class II. Although they expressed intermediate levels of PNA ligand, the B220– NIP-binding cells were BLA-1– and CD95–, two markers expressed strongly on GC B cells. This subset was also negative for CD138, a marker of plasma cells. In addition to the lack of any B cell markers, the B220– NIP-binding population did not express NK1.1 or CD5, markers displayed on NK and T cells. Of interest, the cells were CD11ahigh, CD11blow, CD44high, and pan CD45+, indicating hemopoietic derivation. Although the survey was not exhaustive, the composite phenotype suggests this subset not to be of the B cell, T cell, or NK cell lineages, but a member of the myeloid family.


Figure 3
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  		FIGURE 3. Appearance of a B220– NIP-binding population following immunization with NP-KLH. Adult C57BL/6 mice were immunized i.p. with either NP-KLH in Ribi adjuvant (A) or NP-KLH precipitated in alum (B). Splenocytes were harvested at days 4, 8, 12, and 18 postchallenge, stained with anti-B220 and NIP-PE, and analyzed by flow cytometry. As illustrated in both panels, a population of B220– NIP-binding cells appeared at day 8 postimmunization (indicated in each plot) and was observed at all subsequent time points analyzed. This population is not seen in unimmunized mice. Results are representative of at least four mice per time point.

 

Figure 4
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  		FIGURE 4. B220– NIP-binding cells do not express B cell markers. For all markers except IgG2b, C57BL/6 mice were immunized with NP-KLH precipitated in alum and splenocytes were harvested on day 12. For IgG2b staining, splenocytes were obtained from mice challenged 12 days prior with NP-KLH in Ribi adjuvant. Cells were stained with anti-B220, NIP-PE, and the indicated markers. Expression of the various markers (bold histograms) is derived from the B220– NIP-binding gated cells. Isotype controls are shown as light histograms. Results are representative of at least three mice.

 

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  		Table II. Phenotype of B220– NIP-binding cellsa

 
The B220– NIP-binding cell population bears surface IgE and both {kappa} and {lambda} L chains

Previous studies examining B220– NIP-binding cells induced after challenge had identified the Ag receptor as either IgG or IgE. We therefore tested the splenic B220– Ag-binding cells for a range of Ab isotypes after immunization with NP-KLH. None of the cells was positive for IgM, IgD, IgG1, or IgG2b (Fig. 4 and Table II). For most experiments, alum was used in combination with NP-KLH because this adjuvant induced a more pronounced population of B220– NIP-binding cells. Studies were also performed with the Ribi adjuvant, however, in an effort to induce B220– IgG-bearing cells (19). Regardless of the adjuvant used, B220– NIP-binding cells bearing IgG were not observed. As illustrated in Fig. 5A, the entire population stained brightly for IgE and expressed {lambda} L chains. Curiously, virtually all of the B220– NIP-binding cells displayed both {lambda} and {kappa} L chains (Fig. 5B). Given the marked expression of IgE on these cells, and the presence of both L chain classes, we further tested whether the NP-binding Abs could have been passively absorbed. Toward this end, Fig. 5C shows that exogenous IgE could be bound by these cells. Because the B220– NIP-binding population was negative for CD23 (or Fc{epsilon}RII; Table II), this absorption was likely the result of binding to the high-affinity Fc{epsilon}RI. Fig. 5D demonstrates these cells to indeed express the {alpha}-chain of the Fc{epsilon}RI. Taken together, it would appear that IgE anti-NP Abs present on this population result from binding to the high-affinity IgE FcR rather than synthesized internally and expressed as a transmembrane receptor.


Figure 5
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  		FIGURE 5. B220– NIP-binding cells bind IgE Ab through the high-affinity IgE FcR. C57BL/6 mice were immunized with NP-KLH precipitated in alum and splenocytes were harvested on day 12. Cells were stained with anti-B220, NIP-PE, and either anti-IgE and anti-{lambda} L chain (A), anti-{kappa} L chain and anti-{lambda} L chain (B), IgE (C), or an Ab directed against the {alpha}-chain of the high-affinity IgE FcR (D). The dot plots or histograms in A–D are derived from the B220– NIP-binding gated population. In C, cells were exposed to a biotin-conjugated IgE mAb (A3B1) during the primary incubation followed by washing and a secondary incubation with Texas Red-avidin. The bold histogram represents biotin-IgE plus avidin staining, whereas the light histogram is derived from cells incubated with the avidin conjugate alone. In D, the bold histogram represents {alpha}-chain staining, whereas the light histogram is the isotype control. Results are representative of two mice.

 
The B220– NIP-binding cell population is absent following immunization of Fcerg1–/– mice

In light of the above evidence suggesting the B220– NIP-binding cells to absorb Ag-specific IgE via Fc{epsilon}RI, Fcerg1–/– mice were immunized with NP-KLH in alum or Ribi and their splenocytes were examined 12 days postchallenge (peak of the response). Fcerg1–/– mice lack the {gamma}-chain subunit of the high-affinity IgE FcR, thereby disabling its expression. As shown in Fig. 6, splenocytes from wild-type C57BL/6 mice contained both B220+ and B220– NIP-binding cells. Whereas the B220+ NIP-binding cells were still present at the expected frequency in the spleens of Fcerg1–/– mice, the B220– population was no longer observed (Fig. 6).


Figure 6
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  		FIGURE 6. B220– NIP-binding cells are not observed in Fcegr1–/– mice. Wild-type C57BL/6 and Fcegr1–/– mice were immunized i.p. with either NP-KLH in Ribi adjuvant (A) or NP-KLH precipitated in alum (B). Splenocytes were harvested at day 12, stained with anti-B220 and NIP-PE, and analyzed by flow cytometry. Results are representative of two mice per adjuvant group.

 
B220– NIP-binding cells are generated in the absence of GCs, and can be derived from nonlymphoid populations

Phenotypic characterization of B220– NIP-binding cells, along with examination of Fcerg1–/– mice, suggested these cells to be a nonlymphoid population. Because B220– Ag-binding cells have been proposed to be an atypical memory cell (19, 20), we tested whether this population could be induced in mice unable to form GCs. Memory B cells are a product of the GC reaction (30) and thus should not be generated in mice where GC structures are unable to form after immunization. LT{alpha}–/– mice on the C57BL/6 background were therefore immunized with NP-KLH in alum and spleen cells examined 12 days after immunization. Although B cells in LT{alpha}–/– mice can undergo normal isotype switching, they are unable to generate GC responses (31, 32, 33). Despite this functional lesion, LT{alpha}–/– mice generated a normal frequency of B220– NIP-binding cells (data not shown), indicating this population does not require differentiation within the GC environment.

As a direct means to test whether B220– Ag-binding cells arise independently of B cells, RAG-1–/– mice on the C57BL/6 (CD45.2) background were lightly irradiated and given bone marrow from wild-type CD45.1 C57BL/6 mice. After 8 wk of rest, chimeric mice were immunized with NP-KLH and splenocytes assessed for both CD45.1+ and CD45.2+ NIP-binding cells. In CD45.2+ RAG-1–/– recipients of CD45.1+ wild-type bone marrow, the myeloid compartment will be CD45.1/CD45.2 mixed, whereas mature B cells and T cells will be exclusively donor (CD45.1+) derived. Thus, if B220– NIP-binding cells are a product of B cell activation, they should only express the CD45.1 allele. If, in contrast, the B220– Ag-binding population is of myeloid origin, their CD45 expression pattern should be mixed, and reflect the state of myeloid chimerism in these mice. Fig. 7 demonstrates a normal distribution of B220+ and B220– NIP-binding cells in the spleens of immunized mice 12 days postchallenge. As expected, all the B220+ NIP-binding cells were CD45.1+ and hence donor-derived (Fig. 7). Of interest, the B220– NIP-binding population was both host (CD45.2+) and donor (CD45.1+) derived. Fig. 7 further shows the prominent population of B220– NIP-binding cells typically observed in bone marrow after NP-KLH challenge was also split between host- and donor-derived cells. Importantly, the CD45.1 to CD45.2 ratio of splenic and bone marrow B220– NIP-binding cells in reconstituted mice was nearly identical with that observed for total CD11b+ cells. These data are consistent with the B220– NIP-binding population being of myeloid rather than B cell origin. Although it is formally possible that a portion of the CD45.1+ B220– NIP-binding cells could be B cell-derived, the CD45.2+ B220– NIP-binding subset must arise from nonlymphoid cells.


Figure 7
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  		FIGURE 7. B220– NIP-binding cells can be generated in Rag1–/– mice. A total of 5 x 106 CD45.1+ bone marrow cells was transferred into lightly irradiated (250 rad) CD45.2+ Rag1–/– mice. Recipient mice were immunized with NP-KLH precipitated in alum 8 wk posttransfer. Twelve days after challenge, splenocytes and bone marrow cells were harvested, stained with anti-B220, NIP-PE, anti-CD45.1 and anti-CD45.2, and analyzed by flow cytometry. A, B220+ NIP-binding spleen cells were exclusively derived from CD45.1+ wild-type donor cells. B220– NIP-binding spleen cells were composed of both CD45.1+ donor and CD45.2+ recipient cells. B, B220– NIP-binding bone marrow cells were composed of both CD45.1+ donor and CD45.2+ recipient cells. In this recipient, the peripheral blood CD11b+ population was composed of 57% CD45.1+ and 43% CD45.2+ cells. The splenic B220– NIP-binding population is composed of 59% CD45.1+ and 41% CD45.2+ cells. Results are representative of four adoptively transferred mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although a number of previous investigators have used flow cytometry to analyze the NP-specific GC response in mice (34, 35, 36, 37, 38, 39), the present report is the first to systematically assess the maturation of NP-reactive B cell subsets through the entire primary GC reaction. Using a panel of markers previously found to delineate GC populations after SRBC challenge, both C57BL/6 and transgenic QM{kappa} mice were examined at multiple time points after NP-KLH challenge. The results demonstrated that NP GC responses exhibit the same attributes as GC B cells induced with SRBC. The experiments further revealed that a prominent B220–, NIP-binding population commonly found after NP challenge is of nonlymphoid origin, and is able to capture Ag through passively absorbed IgE Abs.

In response to SRBC challenge, the observed GC reaction had a number of characteristics that reflect a steady-state environment (4, 15). The ratio of nonswitched IgM+ to switched (primarily IgG1+) GC B cells remained steady (at ~3:1) throughout the GC response. In addition, a subset of GC B cells with a founder phenotype (IgD+CD23highCD38high) was detectable at a steady frequency throughout the reaction. The only GC marker found to display dynamic expression after SRBC immunization was BLA-1, with the majority of GC B cells expressing high levels early in the response followed by progressive loss. Given the central role of the NP response in our current understanding of murine GCs, we tested whether the same characteristics found in SRBC-induced GCs held true after NP challenge. Using NIP-conjugated PE to detect NP-reactive B cells, the GC response was examined at days 4, 8, 12, and 18. Although the total number of NIP-binding GC B cells was low in frequency (Table I), they were easily detectable by flow cytometry enabling detailed study. Of interest, the ratio of nonswitched IgM+ to switched GC B cells was constant throughout the response, with IgM-bearing cells dominating the reaction (Figs. 1 and 2). Although the frequency of IgM+ GC B cells is higher than that observed in the SRBC reaction, the pattern is the same. In addition, a population of putative GC founder cells (IgD+CD23highCD38high GC B cells) persisted at a frequency of 40–60% at all time points (Figs. 1 and 2). Finally, BLA-1 was shown to decay on the NIP-binding GC population as the response matured, a feature also seen with SRBC-induced GC B cells. Taken together, the results indicate that the attributes of the primary murine GC response detailed in the SRBC system also apply to the clonally restricted NP reaction.

Compared with previous studies examining NP-specific GC B cells, the central characteristics of the GC reaction found in the present study were nearly identical (2, 12, 16, 17, 18, 34, 35, 36, 37, 38, 39). In particular, NIP-binding GC B cells first appeared at days 4–6, peaked in frequency at day 12, and began to dissipate by day 18 (2). The majority of the NIP-binding GC B cells was also found to display {lambda} L chains (2). One major difference noted in our findings was the steady presence of nonswitched IgM+ GC B cells throughout the reaction (Figs. 1 and 2). Previous work had suggested that switching in the primary GC response to NP is dynamic, with virtually all GC B cells expressing IgG as the response matured (2). Although it is not evident why the proportion of switched GC B cells differed between the two studies, the varying results might be explained by the technical readouts. Whereas Jacob et al. (2) used immunohistochemistry to analyze GC responses, we applied the technique of multiparameter flow cytometry. Although the former technique has the advantage of assessing individual GCs in situ, the latter allows for exclusive analysis of B cells thereby eliminating the contribution of cytophilic Abs bound to follicular dendritic cells. Using flow cytometry, we documented IgM+ cells to compose the majority of GC B cells throughout both the SRBC (4, 15) and NP response (Figs. 1 and 2). This also held true when challenging mice with a range of Ags, including KLH, PE, and viral particles (Ref. 4 and our manuscript in preparation). When examining GCs with immunohistology, results have been mixed. Although some investigators found IgG+ B cells to dominate the GC response (2, 40, 41), others have found a significant proportion of IgM+ B cells within the GC at the height of the reaction (42, 43). The reason for these contrasting histologic observations is not clear, although studies showing IgG-bearing GC B cells as the prominent population examined Ag-specific clonally restricted or transgenic responses (2, 36, 37), whereas reports demonstrating IgM+ GC B cells to constitute a large fraction of the GC used complex Ags leading to a diverse array of responding clones (38, 39).

An interesting and unexpected finding in the flow cytometric characterization of the NP response was the presence of B220– cells among the NIP-binding, PNA+ population. These cells appeared by day 8 following challenge with NP-KLH in either alum or Ribi adjuvant, and persisted throughout and beyond the primary GC response (Fig. 3). B220– NIP-binding cells were also easily identifiable in the bone marrow (Fig. 7). Phenotypic analysis of this population revealed an absence of all major B cell surface markers including CD79b, a component of the BCR complex (Fig. 4). Although the expression of pan CD45, CD44, and CD11a confirmed their hemopoietic origin, further testing revealed this population to belong to the myeloid rather than lymphoid lineage. Of interest, the Ab displayed on the B220– NIP-binding cells was almost entirely IgE, with no evidence for IgM, IgD, or IgG. Additional analysis showed the cells to express both {kappa} and {lambda} L chains, strongly indicating the Ag-binding IgE Ab to be captured rather than synthesized internally (Fig. 5). This was confirmed by the ability to load additional IgE onto the cells, the expression of the high-affinity IgE FcR, and by the absence of this population in NP-immunized Fcerg1–/– mice (Figs. 5 and 6). Additional experiments revealed the normal presence of B220– NIP-binding cells after NP-KLH challenge of LT{alpha}–/– mice (data not shown). Because these mice are unable to generate GCs (31, 32, 33), the B220– Ag-binding cells observed in the present study are unlikely to represent a post-GC memory population. Finally, after NP-KLH challenge, B220– NIP-binding cells of host origin were identified in the spleen and bone marrow of RAG-1–/– mice reconstituted with wild-type bone marrow, clearly demonstrating that this population can arise independent of B cells (Fig. 7).

In previous studies by McHeyzer-Williams and colleagues (19, 20), two populations of atypical B220– Ag-binding cells were observed after NP-KLH immunization. One subset was CD11blowIgE+CD79b– with the other exhibiting a CD11bhighIgG+CD79b+ phenotype. The former population appears identical with the B220– NIP-binding cells identified in the present study. Although McHeyzer-Williams and coworkers (19, 20) concluded that the B220– IgE+ NP-binding cells compose a unique B cell memory subset, our results identify these cells as an Fc{epsilon}RI-expressing myeloid population. Our findings are consistent with the recent report by Mack et al. (21) in which B220– Ag-binding cells were found in the blood, bone marrow, and spleen after challenge with allophycocyanin. They documented these cells to bind Ag through Fc{epsilon}RI-captured IgE Ab and exhibit a phenotype consistent with murine basophilic granulocytes (21). Although we were able to easily identify the CD11blowIgE+CD79b– cells reported by McHeyzer-Williams et al. (19) and Driver et al. (20) after NP-KLH challenge, we were unable to document the CD11bhighIgG+CD79b+ population observed in their studies. Regardless of whether alum or Ribi was used as adjuvant, neither IgG+B220– nor IgE–B220– Ag-binding cells were observed at any time point postchallenge. Because IgG1+ (Figs. 1 and 2) and IgG2b+ (data not shown) NP-binding B220+ B cells were easily documented, the failure to detect IgG+B220– cells is unlikely due to technical issues. This observation is similar to the findings of Mack et al. (21) who likewise reported an inability to detect an IgE– B220– Ag-binding population after allophycocyanin immunization. Of interest, Bell and Gray (22) did observe IgG+B220– Ag-capturing cells after challenge with PE. However, these investigators concluded the population to consist of myeloid cells and passively bind Ag-specific IgG through high-affinity IgG FcRs.

Finally, it is evident that IgE Abs are readily generated during primary B cell responses resulting in armed Fc{epsilon}RI+ myeloid cells. Their presence in both bone marrow and spleen leads to the question of physiologic significance. As originally documented by Paul and coworkers (44, 45, 46), and recently confirmed by Mack et al. (21), these Fc{epsilon}RI-bearing cells are capable of rapidly producing Th2 cytokines subsequent to cross-linking of surface IgE. In the presence of Ag, therefore, this population may play a key role in determining the local availability of Th2 cytokines, and thus influence the fate of activated B cells. In addition, Th2 cytokines elaborated by Fc{epsilon}RI+ myeloid cells may aid in maintaining an overall Th1-Th2 balance either locally or systemically. In a recent report by Kang et al. (47), IgE production was found to be essential in blunting exaggerated Th1 cytokine production and local airway inflammation. Myeloid cells armed with Ag-specific IgE may also be intended for immune protection mediated by type I hypersensitivity reactions. A proportion of splenic Fc{epsilon}RI+ myeloid cells have been shown to contain preformed histamine and to release significant levels of this mediator upon culture with IL-3 (46). Of interest, Han et al. (18) found that administration of soluble NP-protein conjugates 9 days after primary NP challenge resulted in marked systemic anaphylactic reactions. This observation underscores the potential for IgE Ab production during a primary Th cell-driven B cell response, and the multiple roles this isotype can play in immune regulation and protection.


   Acknowledgments
 
We thank Dr. Matias Wabl for providing QM mice, Dr. Stanley Perlman for the RAG-1–/– mice, and Dr. Annette Schlueter for the CD45.1 mice. We also acknowledge the expert technical assistance of Teresa Duling and Lorraine Tygrett.


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


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

1 This work was supported by National Institutes of Health Grants RO1 AA014400 (to T.J.W.) and R37 AI26296 (to R.J.N.). Back

2 Address correspondence and reprint requests to Dr. Thomas J. Waldschmidt, Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA 52242. E-mail address: thomas-waldschmidt{at}uiowa.edu Back

3 Abbreviations used in this paper: GC, germinal center; SHM, somatic hypermutation; PNA, peanut agglutinin; NP, (4-hydroxy-3-nitrophenyl)acetyl; KLH, keyhole limpet hemocyanin; NIP, 4-hydroxy-3-iodo-5-nitrophenylacetate; QM, quasi-monoclonal. Back

Received for publication October 5, 2005. Accepted for publication May 18, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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