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(Journal of Leukocyte Biology. 2003;73:764-770.)
© 2003 by Society for Leukocyte Biology

IgD-receptor (IgD-R) cross-linking partially protects murine T cells from dexamethasone-induced apoptosis

Seetha M. Lakshmi Tamma*,{dagger} and Richard F. Coico*

* Department of Microbiology and Immunology, CUNY Medical School, New York, New York; and
{dagger} Department of Biomedical Sciences, C. W. Post, Long Island University, Brookville, New York

Correspondence: Richard F. Coico, Ph.D., Chairman, Department of Microbiology and Immunology, CUNY Medical School, 138 Street and Convent Avenue, New York, NY 10031. E-mail: coico{at}med.cuny.edu


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ABSTRACT
 
Based on our previous findings that immunoglobulin D (IgD) receptor (IgD-R) cross-linking with oligomeric IgD (IgD-R-xL) led to T cell activation, we examined the effect of IgD-R-xL on the expression of Fas antigen and apoptosis induction. In splenic T cells, IgD-R-xL followed by dexamethasone (dex) treatment resulted in a decreased percentage of Fas-positive cells as well as a decreased mean fluorescence intensity (P<0.05) when compared with cells treated with dex alone. There are significant differences in annexin–fluorescein isothiocyanate (FITC) and phosphatidylinositol (PI) staining between samples treated with dex alone and IgD-R-xL followed by dex-treated samples (P<0.05), suggesting a protective role for IgD-R-xL. No significant differences are seen in Fas antigen expression, annexin–FITC staining, and/or PI staining in murine T hybridoma (7C5) cells cultured under similar conditions (P<0.07). We hypothesize that ligation of IgD-R may predispose antigen-specific T lymphocytes for survival during primary immune responses when IgD-positive B cells serve as antigen-presenting cells.

Key Words: IgD • T cell activation • Fas


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INTRODUCTION
 
Immunoglobulin D (IgD) is a major antigen receptor isotype coexpressed with IgM on the surface of mature B lymphocytes in mice, humans, and other species. It has been demonstrated that IgD also serves as a ligand for IgD-specific receptors (IgD-R) expressed on CD4+ T cells and that deglycosylated IgD fails to bind to these receptors [1 2 3 4 5 ]. Moreover, N-acetylgalactosamine blocks the binding of IgD-R to IgD-coated target cells, suggesting that IgD-R have lectin-like properties [6 7 8 ]. IgD-R+ T cells display several enhanced, functional properties as compared with control CD4+ T cells. The most striking function of IgD-R+ T cells concerns their enhanced ability to help B cells respond to antigens [1 , 5 , 9 10 11 ]. Taken together, these studies suggest that IgD-R may mediate bidirectional signaling during cognate T–B interactions.

Most human and murine FcRs are members of the Ig superfamily, and others belong to the lectin families (e.g., IgD-R) [12 13 14 15 16 17 ]. Many FcRs trigger cellular responses using the same transduction pathways as antigen receptors [18 ]. We have reported that IgD-R transmit intracellular signals when cross-linked with oligomeric IgD (IgD-R-xL), resulting in T cell activation [19 ]. Our results demonstrate that IgD-R-xL leads to tyrosine phosphorylation of p56 Lck and phospholipase C (PLC)-{gamma}1 in T cell hybridoma, 7C5, as well as in normal T cells from BALB/c mice treated with oligomeric IgD in vivo and then exposed to oligomeric IgD in vitro [19 ].

Glucocorticoids have been extensively used in the treatment of malignant lymphoproliferative disorders, as they inhibit proliferation and induce cell death [20 ]. The effects of glucocorticoids on apoptosis have been determined in T cells [21 , 22 ], monocytes [23 ], basophils [24 ], eosinophils [25 ], thymocytes [26 ], dendritic cells (DC) [27 ], multiple myeloma cells [28 ], and chronic lymphocytic leukemic cells [29 ]. Activated T cells are susceptible to induction of apoptosis or programmed cell death in response to ligation of several cell-surface receptors such as CD2, CD3, CD4, and Fas (CD95). Nishimoto et al. [30 ] have reported that the apoptosis of murine T cells is regulated by a mechanism different from that of thymocytes and found that dexamethasone induced apoptosis in murine splenic T cells and thymocytes. This is in contrast to other agents such as phorbol esters, cyclic adenosine monophosphate agonists, and calcium ionophores, which induced apoptosis in T cells to a lesser extent than in thymocytes [30 ]. Fas antigen, CD95, is a 36-KD transmembrane glycoprotein, which belongs to the nerve growth factor/tumor necrosis factor receptor family of surface molecules [31 ]. Several studies have suggested that Fas antigen may mediate apoptosis [32 33 34 ]. Through apoptotic cell death [33 ], an anti-Fas monoclonal antibody has been shown to effectively kill murine cell lines transfected with the human Fas antigen cDNA. In response to T cell receptor (TCR) engagement, CD95 has been shown to be up-regulated, followed by CD95L up-regulation [35 36 37 38 39 40 ]. Binding of CD95 to CD95L triggers clonal deletion (by apoptosis) of the cycling T lymphocytes [35 36 37 38 39 40 ].

Given the functional consequences of up-regulation of IgD-R on CD4+ T cells following cross-linking of basal receptors with oligomeric IgD and the ability of IgD-R to transmit intracellular signals associated with T cell activation, the effects of IgD-R-xL on induction of apoptosis in CD4+ T cells were analyzed. As activated CD4+ T cells have been suggested to be eliminated with rapid kinetics via Fas/Fas ligand (CD95/CD95L)-mediated apoptosis, Fas antigen expression following IgD-R-xL and levels of apoptosis induction in CD4+ IgD-R+ murine T cell hybridoma 7C5 as well as in splenic CD4+T cells from BALB/c mice was also studied. The data indicated that prior ligation of IgD-R appears to protect splenic T cells at least partially from undergoing accelerated apoptosis.


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MATERIALS AND METHODS
 
Antibodies and reagents
Phorbol 12-myristate 13-acetate, ionomycin, dexamethasone, and avidin–fluorescein isothiocyaante (FITC) were purchased from Sigma Chemical Co. (St. Louis, MO); anti-CD3, phycoerythrin-conjugated anti-CD4 antibody, murine Fas (Jo2 clone) and Fas-ligand (MFL3 clone) antibodies, and annexin V-FITC were purchased from PharMingen (San Diego, CA); and propidium iodide (PI) was purchased from Molecular Probes (Eugene, OR).

Purification and biotinylation of IgD
TEPC-1017 myeloma cells secreting oligomeric IgD [41 ] were maintained by intraperitoneal transfer in pristane-primed BALB/c mice. Ascites were collected and were subjected to ammonium sulfate precipitation and affinity chromatography by using a Griffonia simplicifolia-1 Sepharose column (EY Laboratories, San Mateo, CA), and IgD was eluted with 0.1 M D-galactose [42 ]. IgD was biotinylated according to the method of Lisanti and Sargiacomo [43 ].

Mice
Four- to 8-week-old female, BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA).

Cell culture
7C5 is an SJL lymphoma-specific hybridoma (CD4+ T) cell line established by Tsiagbe et al. [44 ] in Dr. G. J. Thorbecke’s laboratory and was a generous gift. Cells were cultured in Eagle’s minimal essential medium (EMEM) and tumor cocktail. [EMEM (520 ml), 7.5 g D-glucose, 75 ml 50x modified Eagle’s medium (MEM) essential amino acids, 37.6 ml 100x MEM nonessential amino acids, 100 ml 10x L-glutamine, and 8.5 g Na2HCO3were mixed; pH was adjusted to 7.0; and 100 ml 10x penicillin/streptomycin, 500 mg gentamycin, and 8.5 µl 2-mercaptoethanol (2-ME) were added and adjusted to 1 L with EMEM.] Cells were cultured in medium containing 10% fetal calf serum (FCS). Splenic T cells were purified by panning or by immunomagnetic separation as described before [10 ]. Cells were adjusted to 1 x 106/ml in RPMI medium containing 10% FCS, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were treated as follows: medium; cross-linked with anti-CD3; cross-linked with oligomeric IgD as described [19 ]; preligated with oligomeric IgD for 2 h and cultured in the presence of dexamethasone (50 ng/106 cells); and cultured in the presence of dexamethasone alone (50 ng/106 cells). Cultures were set up in duplicates: One set of cells cultured under different conditions was harvested after 48 h to analyze Fas antigen; the other set of cells was harvested after 72 h to analyze apoptosis.

Flow cytometry
For direct staining, 1 x 106 cells were washed twice with phosphate-buffered saline (PBS) and incubated with 0.5–1 µg biotinylated murine IgD for 30 min on ice. Cells were washed 3x with PBS and were incubated with avidin–FITC (1:100) for 30 min on ice, washed 2x, and resuspended in fresh PBS + 0.01% NaN3. A total of 10,000 cells were analyzed in a manually set lymphocyte gate on a Coulter Elite cell sorter (Coulter Electronics, Hialeah, FL). FcR blocker was used to eliminate background staining.

Cells were labeled with anti-Fas antibody following culture for 48 h under different culture conditions according to instructions from the manufacturer. Cells were then washed and analyzed by flow cytometry. After converting the mean log fluorescence intensity to linear fluorescence intensity, we compared each positive sample back to its isotype-matched control. We analyzed the number of cells positive for Fas antigen and also determined the amount of antigens per cell and the mean fluorescence intensity (MFI) by using the Fas antibody divided by the MFI of the control antibody for each treatment.

Apoptosis
Annexin–FITC staining
In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) translocates from the inner to the outer surface of the plasma membrane, thereby exposing PS to the external cellular environment [45 ]. Annexin V is a 35–36 KD Ca 2+-dependent, phospholipid-binding protein that has a high affinity for PS and binds to cells with exposed PS. Thus, fluorochrome-conjugated annexin (e.g., annexin–FITC) serves as sensitive probe for flow cytometric analysis of cells that are in the early stages of apoptosis. The detection of apoptotic cells by annexin is technically simple and can be performed on unfixed cells. One major concern with this assay is the lack of a control molecule to assess the amount of nonspecific binding, and it has been recommended in the kit to use unconjugated annexin to block the binding of labeled annexin–FITC. Cells were washed twice with cold PBS and resuspended in 1x binding buffer (10x binding buffer: 0.1 M HEPES/NaOH, pH 7.4, 1.4 mM NaCl, 25 mM CaCl2, dilited to 1x before use) at a concentration of 1 x 106 cells/ml and stained according to the manufacturer’s instructions. The solution (100 µl) was transferred to a 5-ml culture tube; annexin V–FITC was added gently to cells and was mixed; and cells were incubated for 15 min at room temperature in the dark. Then, 400 µl 1x binding buffer was added to each tube. Samples were analyzed by flow cytometry (Epics Elite, Coulter Electronics). Unstained cells and cells treated first with unconjugated annexin V followed by annexin V–FITC staining were used as controls.

Staining with PI
PI staining was used to assess late stages of apoptosis [46 , 47 ]. Apoptotic measurement involved staining for nuclear content with PI, which identified a subdiploid peak (Ao) as representative of apoptotic cells [48 49 50 51 ]. Briefly, the cells were washed and resuspended in Hanks’ balanced saline solution (HBSS). To this cell suspension, 0.5 ml RNase solution (1 mg/ml) was added, followed by 1 ml PI (2 µg/ml in HBSS). Cells were incubated overnight at 4°C in the dark. Cell debris and cell clumps were excluded by gating for single cells under forward and side-light scatter, and PI fluorescence of individual cells was determined. The machine is triggered on PI fluorescence equal to 25% of the fluorescence of the G0 peak (G0 peak set at channel 400; discriminator set on channel 100). Apoptosis was determined by quantitating the percentage of cells in the subdiploid peak (Ao). Stained cells were also examined under UV microscope.

Trypan blue dye exclusion
Cells were harvested after 48 h and 72 h cultures as described earlier. A sample of cells was incubated for 5 min with 0.1% trypan blue (Life Technologies, Gaithersburg, MD), examined by light microscopy with a minimum of 100 total cells counted per slide, and scored as able to exclude the dye (live) or unable to exclude the dye (apoptotic).

Statistical analysis
Statistical analysis was performed using the Student’s t-test.


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RESULTS
 
IgD-R are constitutively expressed on 7C5 T hybridoma cells
Cells were stained with biotinylated IgD, followed by avidin–FITC. T hybridoma cells constitutively express much higher levels of IgD-R than resting splenic T cells [52 ]. Preincubation of cells with unconjugated IgD resulted in a significant inhibition of IgD-R and biotinylated IgD interaction, confirming the specificity of IgD to IgD-R (data not shown). Staining data confirm expression of IgD-R on 7C5 T hybridoma cells and its specificity to IgD. About 35–40% of splenic T cells are positive for IgD-R (data not shown), in agreement with previous findings [10 ].

Preligation with oligomeric IgD followed by dexamethasone treatment results in down-modulation of Fas antigen expression in splenic T cells
Fas–Fas-L interactions have been shown to send death signals and induce apoptosis [37 38 39 ]. Cross-linking of other cell-surface receptors had been shown to induce Fas antigen up-regulation and apoptosis induction [48 , 50 , 53 ]. 7C5 cells were cultured for 48 h under various conditions, and Fas antigen expression was measured as described in Materials and Methods.

As shown (Table 1 ), there is increased Fas antigen expression in 7C5 cells cultured under various conditions. Fas antigen is up-regulated even when cells were cultured in the absence of any treatment. Fas antigen expression is significantly higher in dexamethasone-treated samples (P<0.05) when compared with medium controls. However, no significant differences are seen between samples treated with dexamethasone alone and samples pretreated with oligomeric IgD followed by dexamethasone treatment (IgD-R-xL-dex; P<0.07) or between IgD-R-xL samples and IgD-R-xL-dex treatment (P<0.06).


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  		Table 1. Fas Antigen Expression Following IgD-R Cross-Linking

Isolation, culture, and staining of splenic T cells were as described in Materials and Methods. Representative staining data are shown in Figure 1 . In general, the levels of Fas-antigen expression were significantly lower under all conditions in splenic T cells from BALB/c when compared with 7C5 cells (Table 1) . The data indicate that IgD-R-xL appears to down-modulate the levels of Fas antigen expression in dexamethasone-treated samples, where IgD-R are preligated with oligomeric IgD in splenic T cells. However, no such down-modulation is seen in CD4+ murine T hybridoma 7C5 cells.



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  		Figure 1. Preligation of IgD-R followed by dexamethasone results in down-modulation of Fas antigen expression in splenic T cells. Representative data showing Fas antigen expression. Splenic T cells from BALB/c mice were cultured in medium alone, IgD-R-xL, treated with dexamethasone, or IgD-R-xL-dex treatment. Cells were cultured for 48 h, stained for Fas antigen expression, and analyzed by flow cytometry as described in Materials and Methods. Shaded histograms indicate various treatments: (A) IgD-R-xL, (B) dexamethasone-treated, (C) IgD-R-xL-dex treatment; unshaded histograms represent medium control.

Preligation with oligomeric IgD followed by dexamethasone treatment results in decreased annexin–FITC staining in splenic T cells
Murine T hybridoma 7C5 cells were cultured for 72 h, and cells were washed and stained with annexin–FITC as described in Materials and Methods. Representative staining data are shown in Figure 2 . As shown in Table 2 , there is increased annexin–FITC staining in 7C5 cells and splenic T cells cultured under various conditions. No significant differences are seen between samples treated with dexamethasone alone and IgD-R-xL-dex treatment (P<0.08) in 7C5 cells.



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  		Figure 2. Preligation with oligomeric IgD followed by treatment with dexamethasone results in decreased annexin–FITC staining in splenic T cells. Figure shows representative data showing annexin–FITC staining. 7C5 cells or splenic T cells from BALB/c mice were cultured in medium alone, IgD-R-xL, treated with dexamethasone, or IgD-R-xL-dex treatment as indicated. Cells were cultured for 72 h to analyze early stages of apoptosis by annexin–FITC staining as described in Materials and Methods. As indicated, the left panel represents 7C5 cells, and the right panel represents splenic T cells.


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  		Table 2. Annexin–FITC and PI Staining Following IgD-R Cross-Linking

Splenic T cells were cultured for 72 h and stained with annexin–FITC to assess early apoptosis (Table 2) . There are significant differences among dexamethasone-treated samples, IgD-R-xL, and IgD-R-xL-dex (P<0.05). In splenic T cells, it is more apparent that preligation with oligomeric IgD down-modulated staining with annexin–FITC in subsequently dexamethasone-treated samples and protected cells from showing early signs of apoptosis.

Preligation with oligomeric IgD and subsequent treatment with dexamethasone resulted in decreased levels of apoptosis in splenic T cells
7C5 cells and splenic T cells were cultured for 72 h and stained with PI as described in Materials and Methods [48 49 50 51 ]. Treatment with dexamethasone induced a characteristic hypodiploid apoptotic (Ao) DNA peak, whereas sodium azide treatment, which induces necrosis, failed to induce this hypodiploid (Ao) DNA peak. Cells in the Ao fraction (Figs. 3 and 4 ) represent cells in early (containing fragmented nuclei but with intact membrane integrity) and later stages of apoptosis. As shown in Figure 3 , by 72 h, there is increase in apoptosis (Ao peak) in dexamethasone-treated samples when compared with all the other treatments as well as at various time points. No significant differences are seen between samples treated with dexamethasone alone and IgD-R-xL-dex treatment (P<0.06) in 7C5 cells (Table 2) . Therefore, in 7C5 cells, IgD-R-xL does not appear to protect cells from dexamethasone-induced apoptosis (Figs. 3 and 4) .



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  		Figure 3. Kinetics of PI staining in 7C5 cells, which were harvested at various time points as indicated, stained with PI, and analyzed by flow cytometry as described in Materials and Methods. Figure shows representative data of PI staining of 7C5 cells.



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  		Figure 4. Preligation of IgD-R followed by dexamethasone treatment protects splenic T cells from undergoing apoptosis. Representative data showing PI staining of splenic T cells, which were cultured in medium alone, IgD-R-xL, treated with dexamethasone, or IgD-R-xL-dex treatment as indicated. Cells were cultured for 72 h to analyze late stages of apoptosis by PI staining as described in Materials and Methods.

Splenic T cells were isolated, cultured for 72 h, and stained with PI as indicated (Table 2) . Representative staining data are shown in Figure 4 . As shown in Figure 4 and Table 2 , it is more apparent that preligation with oligomeric IgD protected cells from undergoing apoptosis. Therefore, it appears that IgD-R-xL provides some levels of protection from apoptosis, perhaps in T cells activated for the first time or cells undergoing primary activation.

Morphologic examination of identical samples showed the presence of cells with characteristics typical of cells that underwent apoptotic cell death.


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DISCUSSION
 
IgD-R and IgD interactions have been shown to play important regulatory roles in T cell and B cell responses. Previously, it has been shown that increased expression of IgD-R on T cells resulted in increased antibody responses to T-dependent antigens, and interaction of IgD-R with oligomeric IgD or membrane IgD resulted in antigen-specific T cell clonal expansion [10 ]. Certain T hybridoma cells, such as 7C5, have been shown to constitutively express higher levels of IgD-R than resting splenic T cells [36 ]. Other coreceptor cross-linking has been shown to transmit partial intracellular signals independently or to interfere in TCR/CD3-mediated signals, resulting in anergy or apoptosis induction [50 , 54 ]. However, unlike CD4 preligation [53 , 55 ], preligation of IgD-R with subsequent stimulation through CD3 resulted in tyrosine phosphorylation of intracellular proteins and suggests that IgD-R does not interfere in TCR/CD3-mediated signals [19 ].

We examined whether IgD-R-xL would result in expression of the Fas antigen, which has been shown to be associated with apoptosis induction [37 , 39 ]. The levels of Fas antigen expressions were higher in dexamethasone-treated samples as compared with samples treated with oligomeric IgD alone or IgD-R-xL-dex treatment in splenic T cells. Similarly, IgD-R-xL-dex treatment resulted in significantly reduced levels of apoptosis (P<0.05). These studies suggest that IgD-R-xL may result in expression of death signal(s) only in cells that have already been primed to undergo apoptosis or are in the early stages of apoptosis. It appears to be the case, especially in 7C5 cells, where IgD-R-xL would only appear to accelerate the process.

Glucocorticoids are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells [20 21 22 23 24 25 26 27 28 29 ]. From the literature, it is clear that dexamethasone induces apoptosis, and several mechanisms have been reported for the induction. Schmidt et al. [23 ] reported that apoptosis required the activation of caspases and that CD95 and CD95L were up-regulated in a dose-dependent manner in human peripheral blood monocytes. Zhang et al. [25 ] reported that dexamethasone could activate c-jun NH2-terminal kinase and p38 mitogen-activated protein kinase in a time-dependent manner but not extracellular-regulated kinase in eosinophils and concluded that dexamethasone-induced apoptosis is regulated by caspases but not through the common apoptosis-related caspases-3 and -8 as in other cell types. Buratta et al. [56 ] demonstrated that treatment of mouse thymocytes with dexamethasone enhanced the incorporation of [3H] serine into phosphatidyl serine, and treatment with dexamethasone also enhanced the activity of the serine base-exchange enzyme and apoptosis. Others [26 ] concluded that dexamethasone-induced apoptosis involves sequential activation of phosphoinositide-specific PLC, acidic sphingomyelinase, and caspases. Pruschy et al. [57 ] reported that the proto-oncogene c-fos mediates apoptosis in murine T lymphocytes induced by dexamethasone and suggested that c-fos enhanced p53-dependent radiation and p53-independent dexamethasone-induced apoptosis in murine T lymphocytes. Therefore, all or some of these mechanisms may be responsible for dexamethasone-induced apoptosis in splenic T cells and 7C5 T hybridoma cells. The possible reason for the failure of IgD-R and IgD interactions to protect 7C5 cells from dexamethasone-induced apoptosis appears to be that as it is a cell line, many cells might have been at a terminal-differentiation stage.

Several other agents or molecules have been shown to down-regulate dexamethasone-induced apoptosis. For example, Kim et al. [27 ] reported that agonistic CD40 antibody completely inhibited dexamethasone-induced apoptosis in DC, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Chauhan et al. [58 ] reported that interleukin-6 inhibits dexamethasone-induced apoptosis but not ionizing radiation-induced apoptosis in multiple myeloma cells. Similarly, Weng et al. [59 ] reported that staphylococcal enterotoxin B (SEB)-induced proliferation and apoptosis are resistant to the treatment of dexamethasone in murine lymphocytes and concluded that this is a result of high levels of cytokines produced in the SEB-stimulated cultures, suggesting that cytokines play a role in down-regulating the effects of dexamethasone. Similarly IgD-R and IgD interactions appear to play a role in down-regulating dexamethasone-induced apoptosis in splenic T cells.

In light of our studies using splenic T cells with up-regulated IgD-R, which display enhanced, proliferative responses when IgD+ B cells present antigen [10 ], it is of interest to speculate that up-regulation of IgD-R may allow B cells to present antigens without delivering a death signal. Evidence to support these IgD-R-mediated, regulatory mechanisms would suggest that ligation of IgD-R (expressed by normal IgD-R+T cells) by IgD (expressed by antigen-presenting B cells) may ensure survival of antigen-primed T cells during early activation events in vivo. If such a mechanism is at work, the functions of IgD and IgD-R take on new significance. Future studies are aimed at dissecting these possible regulatory properties of IgD and IgD-R.


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ACKNOWLEDGEMENTS
 
This study was supported in part by Grant 5G12RR03060 from the National Institutes of Health (Bethesda, MD). We greatly appreciate the excellent technical help of Igor Toporosky and Gregory Richard. The authors dedicate this paper to Dr. G. J. Thorbecke, who dedicated her life to training and helping immunologists.

Received October 15, 2002; revised February 19, 2003; accepted February 24, 2003.


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