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The Journal of Immunology, 1999, 163: 4150-4159.
Copyright © 1999 by The American Association of Immunologists

Expression, Regulation, and Function of B Cell-Expressed CD154 in Germinal Centers1

Amrie C. Grammer*, Richard D. McFarland{dagger}, Jonathan Heaney*, Bonnie F. Darnell{dagger} and Peter E. Lipsky2,*

* Harold C. Simmons Arthritis Research Center and Departments of Internal Medicine and {dagger} Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75235


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38positiveIgD+ pre-germinal center (GC)/GC founder, CD38positive GC and CD38-IgD- memory populations, and were also found in the CD38-IgD+ naive and CD38brightIgD+ plasmablast subsets, but not in the CD38brightIgD- plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-{kappa}B, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38positiveIgD- GC B cells to CD38-IgD- memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bcells express CD154/CD40 ligand following activation in vitro by ionomycin and phorbol ester (1, 2, 3) as well as by the polyclonal B cell activators Staphyloccus aureus Cowan I (1, 2) or LPS (4, 5). The finding that CD154 expressed by activated B cells mediates homotypic CD154-CD40 interactions resulting in aggregation, clonal expansion, and differentiation into Ig-secreting cells (1, 2, 4, 5) suggests that up-regulation of this molecule might play an essential role in propagating humoral responses in vivo. Further support for this possibility is the finding that circulating B cells from patients with autoimmune diseases, such as systemic lupus erythematosus, characterized by polyclonal B cell activation express CD154 (5, 6, 7). Moreover, certain B cell lymphomas express CD154 in a functional manner (8, 9, 10, 11). These considerations prompted an examination of whether CD154 is expressed physiologically in vivo at sites of B cell activation in secondary lymphoid tissue as well as a delineation of the regulation of B cell CD154 expression and its functional activity.

Signaling through CD40 is important in the formation and perpetuation of the germinal center (GC)3 reaction since mice deficient in CD40 (12, 13, 14) or its ligand (CD154) (14, 15, 16) do not form functional GCs following immunization with T-dependent (TD) Ag. Additionally, humans with X-linked hyper-IgM syndrome (HIgMXL) syndrome, who cannot express a functional CD154 (reviewed in Refs. 17, 18) do not form functional GCs in response to TD Ag (19, 20).

The GC is one of the structures in which maturation of the humoral response to Ag occurs, fostering somatic hypermutation, selection, and isotype switching of activated B cells (reviewed in Refs. 21, 22, 23). Although CD154-CD40 interactions are essential for initiation and propagation of the GC reaction, there are stages of the GC reaction that appear to proceed in the absence of CD154+ T cells. CD154+ T cells are absent from the dark zone (DZ), where rapid B cell proliferation and somatic hypermutation occur, and are found infrequently if at all in the basal light zone (LZ) of the GC (24, 25, 26, 27, 28), where high avidity Ag-binding B cells are rescued from apoptosis (29, 30). Despite the paucity of CD154-expressing T cells, an established GC rapidly disassembles when CD154-CD40 interactions are blocked (31). One possible explanation for this finding is that cells other than T cells express CD154 in the DZ and LZ of GCs in secondary lymphoid tissues. Activated CD154-expressing B cells are prime candidates to provide the essential CD40-mediated signals in these regions.

To test this hypothesis requires an examination of CD154 expression by B cells in secondary lymphoid organs. In humans, tonsils have been employed extensively to understand GC behavior, despite their chronic inflammatory and often infected character (reviewed in Ref. 32). Therefore, the current experiments were undertaken to determine whether CD154 is expressed in situ by tonsillar B cells, to examine the nature of signals that regulate B cell CD154 expression, and to investigate the functional activity of CD154 expressed by human B cells. The data clearly indicate that CD154 is expressed by tonsillar B cells, is up-regulated by engagement of surface Ig (sIg) or CD40 itself, and is likely to be of great importance in propagating GC reactions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Tonsils were minced and digested in RPMI medium (Life Technologies, Grand Island, NY) containing 210 U/ml collagenase type I (Worthington Biochemical, Lakewood, NJ) and 90 KU/ml DNase (Sigma, St. Louis, MO) for 30 min at 37°C. Following filtration through a wire mesh, the cells were washed twice in 20% NHS-RPMI and once with 10% NHS-RPMI. Mononuclear cells (MNC) were obtained by centrifugation of heparinized venous blood or digested tonsil tissue over diatrizoate/Ficoll gradients (Sigma). Blood was obtained from healthy adult volunteers or an HIgMXL patient previously demonstrated to lack functional CD154 expression. The mutations in CD154 of this HIgMXL patient have been reported (Refs. 1, 33 ; EMBL accession number X96710). In some cases, MNCs were depleted of NK cells and monocytes and separated into T cell-enriched and B cell-enriched populations as described (1). The B cell population was further purified by positive selection on a Ceprate streptavidin column (CellPro, Bothell, WA) following staining of cells with biotinylated anti-CD19 mAb (Coulter, Hialeah, FL). Alternatively, the B cell population was purified by negative selection on a magnetic column (StemCell Technologies, Vancouver, Canada), following staining of cells with a mixture of dextran cross-linked mAb specific for glycophorin A, CD2, CD3, CD14, CD16, and CD56, followed by exposure to a magnetic colloid covalently linked to anti-dextran mAb. The resultant population of B cells was analyzed by flow cytometry and found to be >97% sIg+ (FITC-conjugated anti-polyvalent Ig Ab, Caltag, South San Francisco, CA; FITC-conjugated anti-{kappa} mAb, Becton Dickinson, San Jose, CA; and PE-conjugated anti-{lambda} mAb, Coulter) and <3% CD3+ or CD4+ (FITC; PerCP, Becton Dickinson). To eliminate remaining T cells from the purified B cell population, where indicated, cells were stained with FITC-conjugated anti-CD3 mAb (Sigma) and sorted for the CD3-negative population using the FACStarPlus (Becton Dickinson).

Culture conditions

B cells were cultured (1 x 105/well) in U-bottom 96-well microtiter plates (Costar, Cambridge, MA) in RPMI medium supplemented with penicillin G (200 U/ml), gentamicin (10 µg/ml), and 10% FCS. In some cases, cells were activated with 10 ng/ml PMA (Sigma) and 1.34 µM ionomycin (Calbiochem, La Jolla, CA). sIg was cross-linked with anti-IgM, anti-IgD (Biosource International, Camarillo, CA), or glycine-conjugated Sepharose beads. Alternatively, sIg was engaged with 10 µg/ml of a F(ab')2 of anti-human IgM or polyvalent Ig Ab (Jackson ImmunoResearch, West Grove, PA) or whole mouse anti-human IgM mAb (DA4.4, American Type Culture Collection (ATCC), Manassas, VA).

CD40 was engaged with membranes from Sf9 cells infected with recombinant baculovirus encoding murine CD154 and prepared as previously described (34, 35). Specificity of the effects of this reagent was demonstrated by incubation with or without 5–10 µg/ml of either MR1, a hamster anti-mouse CD154 mAb (kind gift of Dr. Randolph Noelle, Dartmouth Medical School, Lebanon, NH) or 2C11, a control hamster anti-mouse CD3 mAb (ATCC) that has no reactivity with murine CD154 or human lymphocytes.

To analyze the effects of CD154, cells were incubated in the presence of 10 µg/ml of a mouse anti-hCD154 mAb (5c8; Ref. 25 ; kind gift of Biogen, Boston, MA), or P1.17 (ATCC), an isotype-matched control mouse mAb. In some cases, cells were incubated with intact humanized mouse anti-hCD154 mAb (h24-31; kind gift of the IDEC, San Diego, CA), F(ab) fragments of h24-31 generated by pepsin digestion, or h24-31 conjugated to Sepharose beads.

Pooled human Ig (hIg, Sandoglobulin, Novartis, East Hanover, NJ), F(ab) fragments of hIg, glycine conjugated to Sepharose beads, or membranes from Sf9 cells infected with wild-type baculovirus were used as controls.

Inhibitors

Calcineurin-dependent NF-AT activation was inhibited with cyclosporine (Cy, 100 ng/ml; Novartis), MKK1 activation was inhibited with PD98059 (200 µM; Calbiochem), and NF-{kappa}B activity was blocked with an inhibitor of proteosome-mediated I{kappa}B degradation, lactacystein (100 µM; Calbiochem). New protein synthesis was inhibited with cycloheximide (1 µg/ml; Sigma).

Flow cytometric analysis

Cells were stained with mAb as previously described (1) and analyzed with a FACScan, a FACScalibur, a FACStarPlus, or a FACS Vantage flow cytometer (Becton Dickinson). CD154 expression was analyzed with biotinylated CD40.Ig (kind gift of Dr. Marilyn Kehry, Boehringer Ingelheim, Ridgefield, CT) or biotinylated h2431 followed by either streptavidin conjugated to PE (Becton Dickinson), 613, or 670 (Life Technologies). Alternatively, CD154 expression was analyzed with 8976-PE (Becton Dickinson) or with 2443 (kind gift of Dr. Randolph J. Noelle) or unconjugated 8976, followed by biotinylated rat anti-mouse Ig and streptavidin-PE. In some cases, B cells were acid washed as described (1) to remove bound CD40 before staining with anti-CD154 mAb. Subsets of tonsillar B cells were delineated with Ab specific for CD19 (PerCP, APC, Becton Dickinson; biotinylated, Coulter), IgD (FITC, Caltag; PE, Southern Biotechnology, Birmingham, AL), CD38 (HB7, APC or PE, Becton Dickinson; HIT2, FITC, Caltag), CD23 (biotinylated, The Binding Site, San Diego, CA; FITC, Becton Dickinson), TdT (FITC, Supertech, Rockville, MD), Ki67 (Biogenics, Ramon, CA) followed by anti-mouse IgG1 (FITC, The Binding Site), CD44 (A3D8, biotinylated, ATCC), and CD77 (Biodesign, Kennebunk, ME) followed by goat F(ab')2 anti-rat IgM FITC (Jackson ImmunoResearch). Isotype-matched mAb were used as controls. Analysis was performed using CellQuest and Paint-a-Gate Software (Becton Dickinson).

RNA extraction, cDNA synthesis, and PCR analysis

RNA and cDNA were prepared as previously described (1). PCR analysis was made semiquantitative by varying the number of amplification cycles and performing dilutional analysis so that there was a linear relationship between the amount of cDNA used for each reaction and the intensity of the band obtained by Southern hybridization of the PCR product. PCR reactions and Southern blotting were performed as described (1). MgCl2 was used at a concentration of 2 mM for G6PD and TCR{alpha} and 2.5 mM for CD154. The primers used for the PCR reactions were: CD154: L5 and III3 (1); G6PD: 5'-ACC TAC AAG TGG GTG AAC CC-3' and 5'-CTT GGC AGC TGA GGA ATG TAG C-3'; and TCR{alpha}: 5'-GAA CCC TGA CCC TGC CGT GTA CC-3' and 5'-ATC ATA AAT TCG GGT AGG ATC C-3'. In some cases, PCR products were purified using the QIAquick-spin PCR purification kit (Qiagen, Chatsworth, CA) following the manufacturer’s instructions and subjected to nested PCR using primers II5' and II3' (1). The following probes were used for Southern blotting: CD154, Ia3' or III5' (1); G6PD, 5'-ATT GAC CTC AGC TGC ACA TTC C-3'; and TCR{alpha}, 5'-GTC ACT GGA TTT AGA GTC TCT C-3'.

Analysis of B cell function

Proliferation was analyzed by [3H]thymidine incorporation as previously described (1). The percentage of live and apoptotic cells in a given population was analyzed by hypotonic propidium iodide staining as previously described (36).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Freshly isolated tonsillar B cells express a higher density of CD154 than in vitro-activated peripheral B cells

Initial experiments demonstrated that tonsillar B and T cells in situ and freshly isolated ex vivo express CD154 at equivalent densities without in vitro stimulation (Figs. 1Go and 2). The density of CD154 expression by tonsillar T and B cells is lower than that expressed by peripheral blood T cells activated in vitro and higher than that expressed by in vitro-activated peripheral blood B cells as assessed by binding of a CD40.Ig construct (1) or the anti-CD154 mAb 8976 (Fig. 1GoA), 2431, and 2443 (data not shown). The specificity of anti-CD154 mAb was demonstrated by the finding that preincubation with excess CD40.Ig decreased both the mean fluorescence intensity (MFI) (Fig. 1GoA) and the percentage of cells stained with these mAb (data not shown). Additionally, both FACS analysis and immunohistochemistry demonstrated that the anti-CD154 mAb employed in these studies did not stain T or B cells isolated from a donor with HIgMXL syndrome (data not shown) lacking functional CD154 expression (1, 33). It is important to note that tonsillar B cells not only expressed the CD154 epitopes recognized by the various mAb, but had functionally active CD154 in that it bound a CD40.Ig construct (Fig. 1GoA). Finally, in agreement with the staining data, RT-PCR and Southern blotting determined that activated peripheral blood T cells expressed the greatest CD154 mRNA, tonsillar T and B cells expressed moderate CD154 mRNA, and activated peripheral blood B cells expressed the least CD154 mRNA (Fig. 1GoB). Importantly, T cell contamination of the B cells was ruled out since mRNA for the {alpha}-chain of the TCR (TCR{alpha}) could not be amplified from these B cell samples (data not shown).



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  		FIGURE 1. Freshly isolated tonsillar B cells express a higher density of CD154 than in vitro-activated peripheral blood B cells. A, CD154 expression on CD3+ T cells and CD19+ B cells was assessed by FACS analysis following incubation with or without CD40. Ig and staining with FITC-conjugated mAb to CD19 or CD3 and PE-conjugated anti-CD154 mAb (89-76). Tonsillar MNCs were stained immediately following isolation. Highly purified peripheral blood T and B cells were stained following culture for 18 h with ionomycin and PMA. The results of one of two experiments with similar findings are shown as density of CD154 expression by the respective populations (MFI). B, Analysis of CD154 mRNA was performed by RT-PCR using varying amounts of cDNA from freshly isolated tonsillar T and B cells as well as from highly purified peripheral T and B cells following stimulation for 18 h with ionomycin and PMA. Following Southern blotting analysis, the intensity of the CD154 PCR product band was quantitated by AMBIS analysis and plotted as the relative density of PCR product as a function of the amount of initial cDNA.

 
All tonsils studied contained both T and B cells, although the percentages of these subsets varied with the donor (Fig. 2GoA). Routinely, tonsil MNCs contained a significantly higher percentage of B cells compared with T cells (Fig. 2GoA), and a greater percentage of tonsillar B cells expressed CD154 compared with tonsillar T cells (Fig. 2GoB), although the density of CD154 expressed was equivalent (Fig. 1GoC). Furthermore, all tonsils contained CD154-expressing B cells, but somewhat surprisingly only some tonsils contained CD154-expressing T cells (data not shown). Finally, immunohistochemical analysis of serial sections of tonsil tissue confirmed that T cells expressing CD154 were localized to the interfollicular regions (25) and that B cells expressing CD154 were found in the IgD-positive mantle zone and the GC itself. In addition, CD154 was expressed by IgD-positive plasmablasts in the interfollicular and lymphoepithelial regions (data not shown).



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  		FIGURE 2. Comparative density of CD154 expressed by tonsillar B and T cells. Freshly isolated MNCs from eight individual tonsils were assessed for the percentage of T and B cells (A), the percentage of these lymphocyte populations that express CD154 (B), and the relative density of CD154 expression (% maximum MFI of either cell type determined as a fraction) by tonsillar CD19- T cells and CD19+ B cells (C). FACS analysis was performed after staining with PE-conjugated anti-CD154 mAb (8976) and FITC-conjugated anti-CD19 mAb. Similar results were found when CD154 expression by the CD3+ and CD3- populations were analyzed (data not shown). Data are expressed as the mean ± SEM of results from eight tonsils. Significance was determined by paired two-sample Student’s t test.

 
Expression of CD154 by tonsillar B cell subsets

Initial analysis of tonsillar B cell staining by FACS confirmed that they could be contained in the previously described (22, 23, 37) CD38-IgD+ naive, CD38-IgD- memory, CD38+IgD+ mantle zone/pre-GC/GC founder, CD38+IgD- GC, and CD38bright plasma cell subsets (Fig. 3Go). Furthermore, immunohistochemical analysis of frozen tonsil sections confirmed that the mantle zone and GC contained CD38+ cells that were IgD+ and IgD- respectively, whereas CD38bright cells were found in the interfollicular and lymphoepithelial regions (data not shown).



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  		FIGURE 3. Subsets of B cells from multiple tonsils characterized by expression of CD38 and IgD. Freshly isolated MNCs or negatively selected B cells from ten individual tonsils were assessed for CD19+ subpopulations by FACS analysis following staining with FITC-conjugated anti-IgD, PerCP-conjugated anti-CD19, and APC-conjugated anti-CD38. Naive, memory, and GC subsets are indicated as previously described (22 23 37 ). The mean ± SEM percentages of CD19+ cells in each subset defined by CD38 and IgD expression from ten tonsils are depicted in B. A plot of a tonsil containing all eight subpopulations is shown in A.

 
Further analysis of the FACS data revealed eight populations of B cells defined by expression of IgD and CD38. It should be noted that there was considerable heterogeneity in the distribution of B cell subsets in individual tonsils. Thus, some tonsils contained all eight B cell populations (n = 2), whereas other tonsils contained only some (n = 8). A plot of a tonsil containing all eight subsets is shown in Fig. 3GoA, along with the distribution of CD19+ tonsillar B cells into those subsets in ten different tonsils (Fig. 3GoB). Novel subpopulations of CD38lowIgD+ and CD38lowIgD- tonsillar B cells were observed following staining with the HB7 anti-CD38 (Fig. 3GoA) but not the HIT2 anti-CD38 mAb (data not shown). Analysis of tonsillar B cell subpopulations for expression of CD154 revealed that all tonsillar B cell subsets displayed CD154 to varying degrees, with the exception of CD38bright/+++IgD- cells (Fig. 4GoA). Further analysis revealed there was no significant difference in the percentage of CD154-expressing cells observed in the IgD+ and IgD- subsets of tonsillar B cells, 35.4 ± 28.4% (range 3.8–93.6%) and 64.7 ± 28.4% (range 6.4–96.2%), respectively (p = 0.08, n = 10). However, a significantly higher percentage of B cells expressing CD154 was found in the CD38+IgD- subpopulation (27.3 ± 17.2%; range 0.7–48.7%) when compared with all other tonsillar B cell subsets (p = 0.02, n = 10). Moreover, a significantly lower percentage of CD154+ B cells was found in the CD38-IgD- memory (17.4 ± 11.0%; range 0.1–39.3%), CD38+IgD+ (14.1 ± 8.0%; range 1.9–26.3%), CD38++IgD+(12.4 ± 8.6%; range 0–53.6%), and CD38++IgD- subpopulations (14.8 ± 8.7%; range 0.6–28.2%) when compared with the CD38+IgD- subpopulation (p < 0.05, n = 10). The lowest percentage of CD154+ B cells was observed in the CD38-IgD+ naive (5.9 ± 3.8%; range 0–11.6%) and CD38+++IgD+ (6.2 ± 10.0%; range 0.1–25.9%) subpopulations (p < 0.05, n = 10). Finally, CD154-expressing B cells were never found in the CD38+++IgD- subpopulation (0.5 ± 0.5%; range 0–1.7%; n = 10). The greatest CD154 density was observed in activated naive B cells, mantle zone/pre-GC/GC founder (CD38+/++IgD+), as well as in the CD38+IgD- and CD38+++IgD+ populations. Moreover, the average density of CD154 was significantly greater on CD38-IgD+ naive B cells and CD38+IgD- B cells when compared with CD38-IgD- memory B cells and CD38++IgD- GC B cells (data not shown).



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  		FIGURE 4. Expression of CD154 by tonsillar B cell subsets. Freshly isolated MNCs or negatively selected B cells from individual tonsils were assessed for CD154 expression by FACS analysis following staining with PE-conjugated anti-CD154 (8976), PerCP-conjugated anti-CD19, APC-conjugated anti-CD38, and FITC-conjugated anti-IgD (A) or FITC-conjugated anti-CD23 (B). The mean ± SEM percentages of CD154-expressing cells in each CD19+ B cell subset defined by CD38 and IgD expression from ten tonsils (A) or CD38 and CD23 expression from five tonsils (B) are depicted.

 
CD23 expression by tonsillar B cell subpopulations defined by IgD and CD38 was also examined, since previous studies had defined this Ag as a marker of mantle zone/pre-GC/GC founder cells (reviewed in Ref. 22). Although CD23-expressing B cells were found in all tonsillar subpopulations defined by IgD and CD38 (data not shown), a significantly higher percentage (p = 0.02, n = 5) of the CD38+ and CD38++ subsets expressed CD23 (11.5 ± 7.5%, range 1.3–20.5%, and 6.4 ± 6.2%, range 0.6–18.1, respectively) when compared with the CD38- and CD38+++ subpopulations (2.5 ± 1.6%, range 0.4–4.4%, and 1.4 ± 2.6%, range 0–6.5%, respectively). This trend was observed regardless of IgD expression. In addition, there was no significant difference in the overall percentage of cells expressing CD23 when the IgD+ and IgD- B cells were compared (12.6 ± 12.6%, range 0–30.3% vs 13.9 ± 11.8%, range 0.4–29.1%; p = 0.26, n = 5).

Analysis of CD154 expression by tonsillar B cell subpopulations defined by CD38, IgD, and CD23 (Fig. 4GoB) revealed that a significantly greater percentage (p = 0.05, n = 5) of CD154-expressing cells was observed in CD23+CD38+ tonsillar B cells (32.0 ± 13.6%; range 10.8–52.2%) when compared with CD23-CD38+ tonsillar B cells (11.6 ± 6.6%; range 0.3–18.9%). Moreover, within CD23-expressing cells, a significantly (p < 0.03, n = 5) higher percentage of CD154-expressing cells was found in the CD38+ and CD38++ subsets (32.0 ± 13.6%, range 10.8–52.2%, and 19.0 ± 13.3%, range 5.5–42.4%, respectively) when compared with the CD38- and CD38+++ subpopulations (6.6 ± 4.0%, range 1.6–10%, and 5.2 ± 8.2%, range 0–21.1%, respectively). By contrast, examination of CD154 expression in CD23- tonsillar B cells revealed that a significantly higher percentage (p < 0.02, n = 5) were CD38-, CD38+, and CD38++, (11.1 ± 6.8%, range 0–19.1%; 11.6 ± 6.6%, range 0.3–18.9%; 14.6 ± 8.5%, range 2.6–25.4%), than were CD38+++ (0.3 ± 0.4%, range 0–0.5%).

Engagement of CD40 or sIg on human B cells up-regulates CD154 expression

To examine whether engagement of CD40 or sIg induces CD154 expression, B cells were initially analyzed following ligation of these receptors. It is important to emphasize that engagement of CD40 was accomplished with Sf9 membranes expressing recombinant murine CD154 that is not recognized by the anti-human CD154 mAb used for detection. Furthermore, experiments were conducted using an amount of mCD154 previously shown to induce a variety of functional outcomes (34, 35, 36). Analysis by FACS (Fig. 5Go) and fluorescence microscopy (data not shown) demonstrated that engagement of CD40 or sIg on B cells induced expression of CD154. Moreover, there was a relationship between the amount of mCD154 used for stimulation and the level of subsequent CD154 expression (Fig. 5GoB).



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  		FIGURE 5. Engagement of CD40 or sIg on B cells induces surface expression of CD154. Highly purified peripheral blood B cells (1 x 105) were cultured for 18 h with medium alone, 10 µg/ml anti-Ig, or 2 µl membranes from Sf9 cells expressing baculovirus-encoded recombinant mCD154 (left). Right, Highly purified peripheral blood B cells were cultured for 18 h with medium alone or with 2 or 6 µl of membranes from Sf9 cells expressing baculovirus-encoded recombinant mCD154. Human CD154 expression was assessed by FACS after staining with PE-conjugated anti-CD154 (8976) and FITC-conjugated anti-CD19.

 
Induction of CD154 requires new protein synthesis and is inhibited by cyclosporine, PD98059, or lactacystein

To investigate potential signaling mechanisms involved in regulating CD154 expression in human B cells, highly purified peripheral blood B cells were activated with Sf9 membranes expressing recombinant mCD154 or with anti-Ig in the presence or absence of various inhibitors (Fig. 6Go). Engagement of CD40 or sIg induced CD154 protein expression and mRNA (Fig. 6Go). Importantly, T cell contamination of the peripheral B cells was ruled out since TCR{alpha} mRNA could not be amplified from these samples (Fig. 6GoB). Moreover, specificity for the effect of mCD154-expressing Sf9 membranes was documented, since all effects were blocked by an anti-mCD154 mAb (Fig. 6GoB, data not shown). Cycloheximide inhibited induction of surface CD154 (p < 0.02) following engagement of sIg or CD40, demonstrating that expression requires new protein synthesis (Fig. 6GoA). Additionally, CD154 induced by engaging sIg or CD40 was blocked with lactacystein (p < 0.01; Fig. 6Go, A and B), an inhibitor of proteosome-mediated degradation of I{kappa}B. By contrast, inhibiting calcineurin with cyclosporine or MKK1 activity with PD98059 interfered with induction of CD154 on peripheral B cells following engagement of sIg (p = 0.05) but not CD40 (p = 0.29) (Fig. 6Go, A and B). Of note, similar results were observed when sIg was engaged with either soluble anti-IgM, anti-IgD, or polyvalent Ig alone or conjugated to Sepharose beads (data not shown).



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  		FIGURE 6. Role of AP-1/NF-AT and NF-{kappa}B in regulating CD154 expression by activated peripheral B cells. Highly purified peripheral blood B cells (1 x 105) were cultured with medium alone, 10 µg/ml anti-Ig, or membranes from Sf9 cells expressing recombinant mCD154 in the presence or absence of the 100 ng/ml Cy, 200 µM PD98059, 30 µM lactacystein, or 1 µg/ml cycloheximide. Human CD154 expression was assessed by (A) FACS analysis after staining cells with FITC-conjugated anti-CD19 and PE-conjugated anti-hCD154 (8976) or by (B) RT-PCR and Southern blotting of isolated mRNA. Data in A are expressed as the mean of two to seven experiments ± SEM. Significance was determined by paired two-sample Student’s t test. The blot in B is representative of three experiments with similar results.

 
Homotypic CD154-CD40 interactions between GC tonsillar B cells play a role in differentiation to the memory phenotype

To examine the contribution of homotypic CD154-CD40 interactions between tonsillar B cells during differentiation to the memory B cell phenotype, highly purified CD38++IgD- and CD38+IgD- GC B cells were cultured in vitro in the presence of a saturating concentration of anti-CD154 mAb or an isotype-matched control mAb. GC B cells spontaneously differentiated into memory B cells during a 3-day incubation, with 48% of CD38+IgD- and 8% of CD38++IgD- cells becoming CD38-IgD- during this time period. Of note, the emergence of CD38-IgD- memory B cells was partially inhibited by blocking homotypic CD154-CD40 interactions with an anti-CD154 mAb (p < 0.05; Fig. 7Go). As an additional control, the impact of the anti-CD154 mAb on the spontaneous in vitro differentiation of CD38-IgD+ naive tonsillar B cells into CD38+IgD+ pre-GC/GC founder B cells was examined. No inhibition was noted (data not shown). To ensure that the impact of anti-CD154 mAb on memory B cell differentiation was not secondary to an effect on B cell viability or apoptosis, CD38-IgD- memory B cells were analyzed only in the nonapoptotic population detected by staining with propidium iodide, and a similar effect was noted.



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  		FIGURE 7. Homotypic CD154-CD40-mediated interactions between GC B cells play a role in survival and differentiation to the memory subset. Following staining with APC-conjugated anti-CD19 or biotinylated anti-CD19 and strepavidin-670, PE-conjugated anti-CD38, and FITC-conjugated anti-IgD, CD38++IgD- and CD38+IgD- GC B cells were sorted using a FACStarPlus or FACS Vantage flow cytometer. Representative pre- and post-sort analysis of tonsillar B cells is shown in the left panel. Tonsillar CD38++IgD- or CD38+IgD- B cells (1 x 105; purity of 98–99%), analyzed immediately (initiation) for the presence of CD38-IgD- memory B cells, were cultured in the presence of 10 µg/ml of the anti-CD154 mAb, 5c8, or an isotype-matched control mAb, P1.17 (control). Following a 3-day incubation, the presence of CD38-IgD- B cells in the nonapoptotic cells, determined by propidium iodide staining, was analyzed for the presence of CD38-IgD- memory B cells. Data are expressed as the mean ± SEM. Significance was determined by paired two-sample Student’s t test.

 
CD40- and sIg-induced CD154 plays a functional role in B cell responses

The final experiments examined whether CD154 expressed by B cells was involved in functional responses in vitro. Whereas whole anti-CD154 mAb inhibited anti-Ig- or CD154-induced DNA synthesis of highly purified peripheral B cells obtained from some normal donors (Fig. 8GoA), anti-CD154 mAb had no effect on anti-Ig- or CD154-induced DNA synthesis of peripheral B cells from a donor with HIgMXL syndrome (Fig. 8GoB) previously documented not to express functional CD154 (1, 33). It should be noted that similar results were observed when sIg was engaged with either soluble anti-IgM or anti-IgD alone or either anti-Ig conjugated to Sepharose beads (data not shown). Specificity for the effect of mCD154-expressing Sf9 membranes was documented, since all effects were blocked by an anti-mCD154 mAb (data not shown). Although anti-CD154 inhibited anti-Ig (n = 4 of 16) and CD154 (n = 4 of 4) induced proliferation of B cells from some donors, in other donors (n = 9 of 16), intact anti-CD154 costimulated anti-Ig-induced DNA synthesis of highly purified normal peripheral B cells and in others (n = 3 of 16) had little positive or negative impact (data not shown). To analyze this apparent functional heterogeneity further, additional experiments were conducted with F(ab) fragments of anti-CD154 as well as anti-CD154 conjugated to Sepharose beads. Whereas F(ab) fragments of anti-CD154 consistently blocked DNA synthesis of normal peripheral B cells following engagement of sIg or CD40 (Fig. 8Go, C and D), engaging CD154 with anti-CD154 conjugated to Sepharose beads significantly costimulated anti-Ig-induced, but not CD154-induced, DNA synthesis of normal peripheral B cells (Fig. 8GoC).



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  		FIGURE 8. Functional activity of B cell-expressed CD154. Highly purified peripheral blood B cells (1 x 105) from a normal donor (A, C, and D) or a donor with HIgMXL syndrome (B) were cultured for 3 days with medium alone, 10 µg/ml anti-Ig (A, B, and C), or membranes from Sf9 cells expressing recombinant mCD154 (A, B, and D) in the presence or absence of intact anti-human CD154 mAb (A and B), a F(ab) fragment of an anti-CD154 mAb (C and D), or Sepharose beads conjugated with anti-CD154 mAb (C and D). Proliferation was assessed by [3H]thymidine incorporation. Data are expressed as the mean ± SEM. Representative experiments are shown. The results of one of four experiments are shown for A, two experiments for B, and three experiments for C and D. Experiments with intact 24-31 anti-CD154 mAb were performed in the presence of anti-Ig 16 times and in the presence of membranes from Sf9 cells expressing recombinant mCD154 4 times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tonsillar B cells expressed CD154 at a similar density to tonsillar T cells both in situ and ex vivo. B cell CD154 expression could be induced following engagement of either CD40 or sIg in a manner that required new protein synthesis and signaling pathways that predominantly involved AP-1/NF-AT and NF-{kappa}B. Furthermore, CD154-mediated B cell homotypic interactions played a role in promoting differentiation of GC cells to memory B cells. Finally, B cell-expressed CD154 costimulated functional responses by acting as a ligand for CD40 as well as a direct signaling molecule.

Freshly isolated tonsillar B cells express a level of CD154 mRNA and protein equivalent to that expressed by tonsillar T cells and a much higher level of CD154 mRNA and protein than peripheral B cells after in vitro stimulation (Figs. 1Go and 2Go). The difference in CD154 expression by B cells isolated from the tonsil compared with that by peripheral B cells stimulated in vitro suggests that there may be signaling molecules present in inflamed secondary lymphoid tissue that costimulate up-regulation of CD154 in tonsillar B cells (38, 39, 40, 41, 42, 43). It is also possible that tonsillar B cells have differentiated sufficiently so that signals provided by ligation of sIg or CD40 directly induce exaggerated expression of CD154. Whether enhanced expression of CD154 by tonsillar B cells reflects intrinsic features of the B cells themselves and/or a facilitating influence of the tonsillar milieu remains to be determined.

Previous studies had defined tonsil B cell subsets by their expression of IgD and CD38 (22, 23, 37, 44). This study extends these findings by demonstrating that CD38+ tonsillar B cells can be divided into novel populations of CD38low and CD38intermediate cells by flow cytometry (Fig. 3GoA) with the HB7 anti-CD38 mAb. Follicular mantle zone/pre-GC/GC founder cells are known to be CD38+IgD+ cells (22, 45). In the current study, these cells were found within CD38+IgD+ and CD38++IgD+ tonsillar B cell populations (Figs. 3Go and 4Go) since the highest percentage of cells expressing the previously defined marker of mantle zone pre-GC/GC founder cells, CD23 (22, 45, 46), was found in both subsets (Fig. 4GoB). With regard to expression of CD154 by tonsillar B cell subsets, it should be noted that the majority of CD154-expressing cells observed in the CD38+IgD+ and CD38++IgD+ populations were CD23+, with the greatest expression noted in the former (Fig. 5Go), consistent with their status as activated pre-GC/GC founder cells. Moreover, the highest percentage of B cells expressing CD154 was found in the CD38+IgD- population when compared with all other tonsillar B cell subsets (Fig. 4GoA). This CD38+IgD- subset was also Ki67-, TdT-, CD77low, and CD44high (data not shown) consistent with the designation of this population as centrocytes (22, 23, 47). Finally, a higher percentage of centrocytes expressed CD154 than was noted for the CD38++IgD- population (Fig. 4GoA) that was Ki67+, TdT+, CD77high, and CD44low (data not shown), consistent with the designation of this population as centroblasts (22, 23, 45, 47).

Engagement of CD40 or sIg on B cells induced CD154, and the expression of this molecule clearly contributed to ongoing responses. Whereas intact anti-CD154 mAb consistently had no effect on anti-Ig- or CD154-induced proliferative responses from an HIgMXL donor (Fig. 8GoB), the results with normal donors suggested that anti-CD154 may have exerted multiple effects on activated B cells, blocking CD154-CD40-mediated costimulation in some experiments and functioning as a direct costimulator in others. The results using F(ab) fragments of anti-CD154 or CD154 coupled to Sepharose beads provide a potential explanation for these findings. Whereas the anti-CD154 24-31 F(ab) fragment consistently blocked anti-Ig-induced proliferation, the 24-31 anti-CD154 mAb conjugated to Sepharose beads consistently costimulated. It should be noted that somewhat different from the results noted with anti-Ig stimulation, proliferation following CD40 engagement was blocked with both the whole anti-CD154 mAb and the F(ab) of the anti-CD154 mAb; costimulation was not observed, even with anti-CD154-conjugated Sepharose beads. Thus, CD40 ligation-induced proliferation appears to be dependent on the endogenous expression of CD154 and ongoing CD154-CD40 interactions. Costimulation via CD154 engagement was not observed either because the initial signaling was insufficient to induce this pathway of costimulation or because the CD40 and CD154 signaling pathways are redundant. By contrast, engagement of sIg-induced CD154 expression and also CD154-mediated bidirectional costimulation via CD40 or CD154 or both. The effect of the intact anti-CD154 mAb was likely to reflect the dominance of the CD40 or CD154 signaling pathway and could depend on the intensity of the signal generated from ligation of sIg or the density of CD154 expressed or both. Although preliminary work has documented the capacity of CD154 engagement to induce a variety of proximal signaling events in other cell types (48, 49, 50, 51, 52, 53), this is the first example of the ability of CD154 to mediate B cell signaling directly. The potential role of CD154 as a signaling receptor on B cells is consistent with the previous finding that injection of a CD40.Ig construct into a mouse genetically deficient in CD40 induced small, but quantifiable, GCs after immunization and also enhanced in vivo production of IgM following immunization of normal mice (54, 55).

After engagement of sIg or CD40, B cell CD154 expression was induced by specific pathways of transcriptional regulation. Of importance, engagement of both sIg and CD40 induced de novo synthesis of CD154 and not reexpression of preformed protein from intracellular stores, as has been reported for tonsillar (27) and synovial (56) T cells and anti-Ig-stimulated murine splenic B cells (4). Utilization of specific inhibitors demonstrated that engagement of sIg on resting, peripheral B cells induced CD154 expression by means of signaling pathways involving calcineurin and therefore likely leading to nuclear translocation of NF-ATc. This finding parallels the effects noted in T cells (38, 57, 58, 59, 60). Moreover, NF-AT motifs in the 5' promoter region of the CD154 gene in both the mouse (61) and human (62) have been shown to bind NF-ATp/c-NF-ATn/AP-1 complexes in nuclear extracts derived from activated T cells and to control transcription of the gene. Although NF-AT has been thought to be a T cell-specific transcription factor, there is a growing body of evidence that B cells can also be induced to activate NF-AT in a Cy-sensitive manner by a variety of stimuli, including engagement of sIg (63, 64, 65, 66, 67, 68). The current data indicate that this pathway plays an essential role in induction of B cell CD154 expression following engagement of sIg, and perhaps in permitting anti-Ig-activated B cells to employ CD154 as a costimulatory molecule.

In addition to the apparent role of NF-AT in regulating B cell CD154 expression following engagement of sIg, NF-{kappa}B activation played an important role in up-regulating the expression of CD154 following ligation of either CD40 or sIg. In this regard, examination of the published sequence of the human CD154 promoter (GenBank/EMBL accession number L47983; Ref. 62) reveals the presence of five potential NF-{kappa}B binding sites (69), including at least one within the proximal CD154 promoter necessary for PMA and Con A-driven transcription of the CD154 gene in Jurkat T cells (62). Importantly, ligation of both sIg and CD40 is known to activate NF-{kappa}B in B cells (reviewed in Ref. 69). Moreover, CD40-mediated induction of CD154 mRNA in Daudi B cells was blocked by the src kinase inhibitor, herbimycin A (3), previously shown to interfere with CD40-induced activation of NF-{kappa}B (70).

The current data suggest that CD154 expression observed by tonsillar B cells may be the result of in vivo signaling through CD40 or the sIg complex during an immune response initially induced by T-dependent Ags. Ligation of CD40 on naive B cells in the interfollicular zone of tonsils by T cells expressing CD154 may induce CD154 expression on the tonsillar B cells themselves. Engagement of CD40 on naive B cells by CD154-expressing T cells has been shown to lead to expression of CD23 (71) and CD38 (35). Of interest, in the current study, the highest percentage of CD23-expressing cells was observed in the CD38+ and CD38++ subsets (data not shown). Moreover, a significantly greater percentage of CD154-expressing cells was observed in the CD23+ portion of CD38+ B cells when compared with those that were CD23- (Fig. 4GoB). These observations suggest the possibility that induction of these molecules might be induced coordinately following CD40 ligation and the initiation of GC reactions.

Previous studies have demonstrated that maintenance of GC reactions requires ongoing signaling through the CD154-CD40 coreceptors. Specifically, the entire GC, including the DZ, rapidly disassembles following administration of an anti-CD154 mAb to an immunized mouse (31), even though T cells are largely absent from the DZ of GCs in the mouse or human (24, 25, 26, 27, 28). One explanation for this finding is that CD154 expressed by GC B cells may sustain clonal expansion in the absence of T cells. This hypothesis is strengthened by our previous finding that CD154 expression on B cells leads to homotypic CD154-CD40 interactions and DNA synthesis (1, 2) and the current finding that CD40-induced proliferation of B cells is partially mediated by subsequent interactions involving the CD154-CD40 coreceptors (Fig. 8Go). In addition, since ligation of CD40 on B cells induces CD154 expression (Figs. 5Go, 6Go), homotypic CD154-CD40 interactions between B cells may sustain CD154 expression on B cells in an autocrine or paracrine manner independent of T cells after initial activation. Further evidence that CD154-CD40 interactions between tonsillar B cells propagate GC reactions is provided by the finding that in vitro differentiation of highly purified GC B cells to those with a memory phenotype was partially blocked by an anti-CD154 mAb (Fig. 7Go). It should be noted that the impact of anti-CD154 was documented only by phenotypic analysis. However, there is currently no other means to dissect centrocytes from memory cells, and, therefore, it is likely, but not definitely proven, that this phenotypic change implies an impact of anti-CD154 on memory cell differentiation. In addition, it should be emphasized that the effect of homotypic B cell interactions mediated by CD154-CD40 was only partial, implying other receptor-ligand pairs are likely to be involved in this process. Despite these caveats, the data are consistent with previous evidence that CD40 engagement on IgD- cells in the absence of purposeful CD38 ligation has been shown previously to down-regulate CD38 expression (35). These results, therefore, imply that homotypic B cell interactions mediated by CD154-CD40 interactions play a central role in the maturation of CD38++IgD- and CD38+IgD- GC cells to CD38-IgD- memory cells beyond any direct involvement of CD38 engagement. This conclusion is consistent with the previous observation that blocking CD154-CD40 interactions in vivo abolished Ag-stimulated clonal expansion of B cells in GCs (72, 73) and decreased the development of memory B cells (55). Furthermore, these findings extend previous reports demonstrating that CD40 ligation preferentially induced differentiation of CD38++IgD- and CD38+IgD- GC B cells to memory cells defined by phenotype as well as function (74), by suggesting that the source of CD154 in this phenomena may be the tonsillar B cell itself.

A final issue relates to the role of engagement of sIg during B cell maturation events in secondary lymphoid tissues that include initial activation of naive or recirculating memory B cells in the interfollicular or lymphoepithelial regions or following challenge with a TD or T-independent Ag, selection of B cells in the basal LZ of GCs, and maintenance of the memory B cell subset during a TD response or CD38brightIgD+ plasmablasts during a T-independent response (reviewed in Refs. 21, 22, 23, 24, 75). The current data provide a mechanism by which engagement of sIg without T cell stimulation may influence B cell function in a manner that simulates some aspects of TD responses (Figs. 5Go, 6Go, and 8Go). Thus, CD154 expression induced following sIg engagement in tonsillar B cell subsets may provide T cell equivalent stimulation by engaging CD40 on bystander B cells. In addition, CD154 expression by B cells may provide a bidirectional signaling mechanism that substitutes for the influence of activated T cells.


   Acknowledgments
 
We thank Rehana Hussain and Christine Pavlovitch for excellent technical assistance, Angelia Jones, Mela Wierzchowski and Ricardo Olivarez for assistance with tonsil tissue acquisition, Ellis Lightfoot and Dr. Saba Jamal for histological analysis of tonsil specimens, Dr. Kate Luby-Phelps for advice and assistance with the fluorescence microscopy experiments, Dr. Louis Picker for advice regarding flow cytometry, and Dr. Richard Wasserman for providing peripheral blood from an HIgMXL patient.


   Footnotes
 
1 This research was supported by National Institutes of Health (NIH) Grant AI-31229. A.C.G. was supported in part by NIH Postdoctoral Training Grant AR-18550. Back

2 Address correspondence and reprint requests to Dr. Peter E. Lipsky, Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8884. E-mail address: Back

3 Abbreviations used in this paper: GC, germinal center; MFI, mean fluorescence intensity; TD, T-dependent; DZ, dark zone; LZ, light zone; sIg, surface Ig; hIg, human Ig; Cy, cyclosporine; MNC, mononuclear cell; MKK1, mitogen-activated protein kinase kinase 1; LAC, lactacystein; HIgMXL syndrome, X-linked hyper-IgM syndrome. Back

Received for publication February 24, 1999. Accepted for publication July 29, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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