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. 2002 Aug;106(4):503–510. doi: 10.1046/j.1365-2567.2002.01435.x

Effect of cell density on in vitro mouse immunoglobulin E production

Dania Rabah 1,*, Daniel H Conrad 1
PMCID: PMC1782743  PMID: 12153513

Abstract

In vitro immunoglobulin E (IgE) production was found to be sensitive to increasing cell concentration in culture wells. While class switching to IgE is intact as suggested by surface IgE staining, ELISPOT analysis provided evidence that the differentiation of IgE committed B cells to the plasma cell stage was arrested at high cell doses. In fact, splitting the cells at higher concentrations after culture initiation increased IgE production. Cells plated at higher doses were found to be more prone to apoptosis as assessed by Annexin staining. Interestingly, inhibiting apoptosis by the use of the caspase inhibitor DEVD significantly increased IgE levels implicating apoptosis in the preferential deletion of IgE expressing cells. These data not only highlight the caveat against using a single B-cell dose for IgE production in vitro but also suggest for the first time a possible IgE regulatory mechanism mediated by cell density.

Introduction

Immunoglobulin E (IgE) is one of the five classes of antibodies and occurs only in mammals. It is scarce in the serum due to its relatively high turnover rate, but is one of the major isotypes produced locally during an immune response.1 The elevated levels of IgE during a parasitic infection in both humans and other mammals, together with the inability of mice lacking the IgE gene to clear the worm burden effectively, evoke the importance of IgE in parasitic infection.2 However, in developed countries, IgE is involved mostly in type I hypersensitivity.3 Given the direct involvement of IgE production in allergic conditions, several attempts are being made to modulate the synthesis of IgE.

Class switching to IgE can be separated into distinct steps starting with germline transcription, followed by a switch recombination event producing the mature IgE transcript. In the murine system the majority of IgE-expressing cells are generated by sequential switching from IgM to IgE via IgG1. Subsequently, IgE-committed B cells differentiate into plasma cells, which are effector cells that secrete high amounts of immunoglobulin and represent the terminal stage of B-cell development (reviewed in ref. 4). Several surface molecules, e.g. CD405,6 and CD27,7 and transcription factors, e.g. BSAP,8 Blimp-1,9 BCL-610 and XBP-1,11 have been implicated in terminal differentiation of B cells. This study reports a new variable that controls terminal differentiation in vitro, cell density. IgE production was found to be inversely proportional to the number of cells in the culture wells. In fact, high cell density prevented IgE-switched B-cell terminal differentiation by promoting their preferential deletion by apoptosis.

Materials and methods

Antibodies and reagents

M15, a mouse IgG1 anti-leucine zipper monoclonal antibody (mAb), was obtained from Immunex (Seattle, WA).12 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) was prepared in dimethylsulphoxide at a concentration of 5 mm as a stock solution and kept at −20° until use. Non-essential amino acids (NEAA), β-mercaptoethanol (2-ME), sodium pyruvate, HEPES and phorbol 12-myristate 13-acetate were all purchased from Sigma Chemicals (St Louis, MO). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories (Logan, UT). Recombinant murine interleukin-4 (IL-4) generated in the baculovirus system was a generous gift from Dr William Paul (National Institutes of Health, Bethesda, MD); recombinant murine IL-5 was purchased from R & D systems (Minneapolis, MN). CD40LT (recombinant CD40 ligand trimer) was obtained from Immunex.12 The cell permeable caspase 3 inhibitor, an aldehyde based 20aa polypeptide, DEVD-CHO, was purchased from Biomol (Plymouth Meeting, PA).

Animals and B-cell isolation and cell culture

BALB/c mice were purchased from the National Cancer Institute (Frederick, MD). All mice used in experiments were between 6 and 14 weeks of age. In a single-cell suspension, B lymphocytes isolated from disrupted spleens were negatively selected as described previously.13,14 Briefly, anti-CD5, anti-CD8 (both from Dr William Paul), anti-Thy-1.1 (TiB99), and guinea-pig complement (Life Technologies Inc., Gaithersburg, MD) were used to kill T cells. Subsequently, the cells were layered on a discontinuous Percoll gradient and resting B cells were collected from the 66–70% interface. B cells were then plated at different cell concentrations in 96-well plates (Costar, Cambridge, MA) in a volume of 200 µl of B-cell medium (RPMI-1640 containing 2 mm l-glutamine, 1 mm sodium pyruvate, 0·1 mm NEAA, 10 mm HEPES buffer, 5×10−5 m 2-ME, 100 µg/ml streptomycin, 100 U/ml penicillin and 10% FBS) and stimulated with 50 000 U/ml IL-4, 5 ng/ml IL-5, 0·1 µg/ml CD40LT and 0·1 µg/ml M15 at 37° in a 5% CO2 incubator. These activation conditions have been previously shown to be optimal for IgE production.15

Enzyme-linked immunosorbent assay (ELISA)

IgM, IgG1 and IgE levels were determined by ELISA as previously described.15 Briefly, IgE levels were assayed using rat anti-mouse IgE mAb B1E3 and R1E4 as the capture and biotinylated secondary antibodies, respectively. IgG1 and IgM levels were determined using an unlabelled primary goat anti-mouse antibody at 5 µg/ml and detected using a goat anti-mouse class-specific antibody coupled to alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). All ELISA assays were performed in Costar high-binding ELISA plates.

ELISPOT

The protocol used to quantify IgE-producing cells has been described previously.16 Briefly, ELISPOT Immulon 4 (Dynex, Chantilly, VA) plates were coated with B1E3 overnight then blocked with a solution of phosphate-buffered saline (PBS) with 5% FBS. Cells isolated on day 5 post-culture were added to the plates and incubated for 5 hr at 37° in a 5% CO2 incubator and then washed. Spots were detected using biotinylated R1E4, followed by Streptavidin-AP and finally 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate solution. The number of IgE-secreting cells was quantified by counting the number of blue spots per well and multiplying by the dilution factor, and was expressed as the number of antibody-forming cells (AFC) per million B cells.

Surface immunoglobulin determination and CFSE staining

Surface IgE and IgG1 expression was analysed by fluorescent labelling. To block non-specific binding, cells were incubated with 5 µg/ml anti-FcγRII mAb (2.4G2).17 Stimulated unstained cells were used to correct for autofluorescence. Direct labelling was then performed for surface IgE expression by incubating cells with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) – conjugated anti-IgE (Southern Biotechnology) for 45 min at 4°. Cells stimulated with CD40LT alone, which are unable to switch to IgE, were used as a control for non-specific IgE binding. IgG1 expression was detected using an indirect labelling technique; a primary biotinylated rat anti-mouse IgG1 was followed by FITC- or PE-labelled avidin. Non-specific controls were stained with FITC/PE-avidin only. Immediately before analysis, propidium iodide (PI) was added and the viable cell population was gated on forward and side scatter profile as well as PI− population.

CFSE and Annexin V staining

Resting B cells were labelled with CFSE as described previously.18,19 Briefly, B cells were washed and resuspended at 107 cells/ml in PBS containing 0·1% bovine serum albumin (BSA), CFSE was added to a final concentration of 10 µm, and cells were incubated for 10 min at 37° in the dark. The cells were subsequently washed twice with PBS/BSA and resuspended in complete B cell medium. Labelled cells were then plated in 96-well plates at 2·5×103 and 5×104 cells/well then stimulated with IL-4, CD40LT and IL-5 and cultured at 37° in a humid atmosphere containing 5% CO2. Activated B cells were then harvested at various times post-culture, washed, then analysed on FACScan (Becton Dickinson, Mansfield MA) using CellQuest.

Phosphatidyl serine exposure on apoptotic cells was measured as previously described.20 The ApoAlert Annexin V kit (Clonetech Laboratories Inc., Palo Alto CA) was used and the protocol recommended by the manufacturer was followed. Briefly, B cells, either freshly harvested or stained for surface immunoglobulin, were washed and then labelled with FITC-Annexin V for 15 min. Just before FACS analysis, PI (provided by the kit) was added to the samples to discriminate necrotic cells (Annexin V− PI+) from apoptotic cells (Annexin V+ PI− and Annexin V+ PI+).

Proliferation assay

On day 2 post-culture, or on the days indicated, B cells were pulsed using 1 µCi [3H]thymidine/well (ICN Biomedicals Inc., Costa Mesa, CA) for 8 hr. Cells were then harvested onto a Unifilter 96 plate (Packard Instrument Co. Meridian, CT) using a Filtermate 196 plate harvester (Packard Instrument Co.) and the [3H]thymidine incorporation into DNA was measured by reading the plate in a TopCount model B9902 (Packard Instrument Co.).

Results

IgE production is inhibited at high cell density in vitro

IgE production in vitro was consistently found to be sensitive to increasing B-cell concentration per well. This aspect was of particular interest because it was unique to the IgE isotype. As illustrated in Fig. 1(a), IgM production tended to increase with increasing B-cell concentrations. While IgG1 levels did not increase as a function of B-cell dose, they remained relatively constant (Fig. 1a) and only IgE levels dramatically dropped at cell concentrations above 2500 cells/well (Fig. 1b).

Figure 1.

Figure 1

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IgE production is inversely proportional to increasing B-cell concentration per well. Murine B cells from BALB/c mice were stimulated with CD40LT, IL-4 and IL-5 and plated in triplicate at increasing B-cell concentrations per well ranging from 250 to 105 cells/well. Supernatants were harvested on day 14 and IgM, IgG1 (a) and IgE (b) were assayed by ELISA. Error bars represent ± 1 SE. This figure depicts a representative experiment of 14 independent experiments performed.

Differentiation but not class switching to IgE is affected by cell density

We wished to locate the step where IgE is inhibited as a result of increasing cell density. Flow cytometry analysis of surface IgE showed that IgE expression was stimulated by CD40LT and IL-4 to the same extent in both high and low cell concentration conditions (Fig. 2a). These results indicate that the switching process is not affected by cell density, and therefore, point to a possible defect in differentiation to IgE-secreting plasma cells.

Figure 2.

Figure 2

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IgE AFCs are inhibited at higher cell concentrations. (a) Flow cytometry analysis of B cells plated at 2·5×103 (grey line) and 50×103 cells/well (black line) stimulated with CD40LT, IL-4 and IL-5 and harvested on day 4 post-culture. Negative control (cells stimulated with CD40LT alone) is depicted by the dotted line. This figure shows a representative experiment of four experiments performed. (b) B cells were stimulated and plated at increasing B-cell concentrations per well ranging from 250 to 104 cells/well. Cells at the different culture conditions were harvested and several dilutions were used in an ELISPOT assay as described in the Materials and methods section. Error bars represent ± 1 SE. This figure shows one of three independent experiments.

To address this possibility, after 5 days incubation equal numbers of live cells plated at high and low cell concentrations were collected and the number of IgE-forming cells was assessed by ELISPOT assay. The number of IgE AFCs dropped dramatically with increasing B-cell concentration (Fig. 2b) paralleling the drop in secreted IgE levels (Fig. 1b). Collectively, these results show that while switching to the IgE isotype is intact, the differentiation of IgE-committed B cells to the plasma cell stage is arrested at higher cell concentrations.

Splitting the cells on different days enhances IgE production

Since IgE production was inversely proportional to the number of cells in the culture wells we tried to reverse this inhibition by decreasing the cell number on various days after culture initiation. To this end, cell cultures were split on different days and IgE production at the various cell concentrations was compared before and after splitting. As shown in Fig. 3, there was a significant increase in IgE production at higher cell concentrations when the cells were split on days 2, 3 and 4. This experiment confirmed that the cell density is the major variable controlling differentiation to IgE-secreting plasma cells.

Figure 3.

Figure 3

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IgE levels can be enhanced by decreasing cell concentrations on different days after culture initiation. B cells were stimulated and plated in triplicate at increasing B-cell concentrations per well. Cells were divided on days 2, 3 and 4 or left unchanged. Supernatants were harvested on day 14 and IgE was assayed by ELISA. Error bars represent ± 1 SE. The P-values were determined by Student's t-test and statistical significance for P<0·01 are shown by an asterisk (*). This figure depicts a representative experiment of three independent experiments performed.

Apoptosis is responsible for the dramatic IgE inhibition at high cell doses

B cells were less viable at higher cell densities, as evidenced by Trypan blue exclusion assays (data not shown). The decreased viability was also reflected by a reduced proliferative response on a per cell basis, as shown by [3H]thymidine incorporation assays (Fig. 4a). Proliferation of cells plated at higher dose reached a plateau on day 3 then decreased on later days, whereas at the lower concentration conditions cells continued to proliferate 5 days after culture initiation (Fig. 4b). The decrease in proliferation was also contributed to by a slower cell division at high cell concentrations. CFSE staining showed that cells at high density entered earlier into the first division but subsequently divided at a slower rate than their low-cell-density counterparts (Fig. 4c).

Figure 4.

Figure 4

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Proliferation of B cells is inversely proportional to the number of B cells per well. B cells were stimulated with CD40LT, IL-4 and IL-5, and plated at increasing B-cell concentrations per well ranging from 250 to 105 cells/well. Cells were pulsed with [3H]thymidine for the last 8 hr of a 48-h incubation (a) or pulsed on days 2, 3, 4 and 5 (b). Counts per minute (c.p.m./cell=c.p.m./cell number per well on day 0. Error bars represent ± 1 SE. (c) B cells were stained with CFSE, or left unstained, then stimulated with CD40LT, IL-4 and IL-5 and plated at 103 cells/well and 105 cells/well. A negative control was stimulated with IL-4. Cells were harvested on the days indicated and cell division was analysed by flow cytometry. This figure shows a representative experiment out of three conducted.

Based on the above observations, together with the selective inhibition of IgE AFC, the possible differential programmed cell death of IgE-switched B cells at high cell concentrations was investigated. To address this hypothesis, B cells were double stained with PE-coupled anti-immunoglobulin and FITC-Annexin V, which detect immunoglobulin expression and apoptosis, respectively. As predicted, cells plated at higher cell density exhibited a higher percentage of apoptosis, more evident on day 5 post-culture, which translated into a fourfold increase in % apoptotic cells (Fig. 5a). Surface immunoglobulin analysis showed that surface IgE and IgG1 expression were comparable at both cell concentrations (Fig. 5b) corroborating earlier data that switching was not affected by cell density. These results also support sequential switching to the IgE isotype. In fact, there was no detectable IgE expression until day 5 post-culture (Fig. 5b), whereas switching to IgM and IgG1 occurred as early as day 3 after culture initiation (data not shown). When apoptotic cells were excluded by gating on PI− Annexin V−, leaving only switched B cells that will potentially reach the plasma-cell stage, the pattern of surface immunoglobulin expression approximated to some extent that of the secreted immunoglobulin profile. IgM is higher at high cell concentration, IgG1 is not affected and IgE is reduced (Fig. 1; Fig. 5c). These results indicate that apoptosis could provide an explanation for the preferential loss of IgE-switched cells under confluent culture conditions.

Figure 5.

Figure 5

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Apoptosis and immunoglobulin expression as a function of time. B cells were stimulated with CD40LT, IL-4 and IL-5 and plated at 2·5×103 cells/well (grey line) and 5×104 cells/well (black line). B cells harvested on days 4 and 5 and were stained with Annexin V-FITC (a) or anti-IgE-PE and biotinylated anti-IgM or anti-IgG1 followed by strepavidin-PE (b) or double-stained (c). The negative control is depicted by the dotted line. % non apoptotic (PI− Annexin−) immunoglobulin-expressing cells (c). This figure shows a representative experiment out of three experiments conducted.

Promoting cell survival enhanced IgE production

To establish a functional correlation between apoptosis and IgE expression, we attempted to reverse the inhibition of IgE at higher cell doses using apoptosis inhibitors. B cells were stimulated and treated with or without DEVD (caspase 3 inhibitor) or z-VAD-fmk (general caspase inhibitor), alone or in combination with media replenishment on days 3 to 7 post-culture. Media replenishment failed to restore IgE production at the high cell concentration conditions (data not shown). The z-VAD studies were inconclusive because this agent exhibited an antiproliferative effect when used for a long period of time, which masked its anti-apoptotic properties (data not shown). Treatment with DEVD on the other hand, significantly increased IgE production at higher cell doses. This is shown in Fig. 6 both as the amount of IgE produced (Fig. 6a) and as the percentage of maximum IgE seen (Fig. 6b). Although B cells treated with DEVD showed the same monophasic B-cell dose response, the curve was flatter, with only 60% inhibition of IgE at the highest concentration (105 cells/well) in contrast to 85% in control cells.

Figure 6.

Figure 6

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Treatment with DEVD reversed IgE inhibition at higher cell concentrations. B cells were stimulated with CD40LT, IL-4 and IL-5 and plated in triplicate at increasing B-cell concentrations per well ranging from 250 to 105 cells/well. The 500 nm DEVD was added every 12 hr on days 3–7 in a 10 µl volume. Supernatants were harvested on day 8 and IgE was assayed by ELISA. Error bars represent ± 1 SE. This figure depicts a representative experiment of three independent experiments performed. (b) Data from (a) are represented as percentage of maximal IgE value (Max) for each cell concentration under the different culture conditions. The P-values were determined by Student's t-test. Statistical significance for P-values <0·0005 and P<0·05 are shown by (**) and (*), respectively.

We also used the longer-lived B cells overexpressing the anti-apoptotic bcl-2 gene to address the same hypothesis. B cells from BCL-2 Tg mice were only slightly more resistant to apoptosis (23%) as assessed by Annexin V staining on day 5 post-culture, and this protective effect of the bcl-2 transgene proved insufficient to reverse IgE inhibition at higher cell doses (data not shown).

Collectively, these results suggest that the lack of differentiation to IgE-secreting plasma cells at the higher cell concentration conditions is due to a preferential apoptosis of IgE-switched B cells.

Discussion

In earlier studies, we noted that IgE production in vitro drops dramatically with increasing B-cell concentration per well (Fig. 1b).21,22 Switching to IgE is not affected by increasing the B-cell dose. Both germline transcription (data not shown) and expression of the mature IgE transcript on the cell surface (Fig. 2a) are induced with comparable efficiencies at both high and low cell densities. The defect lies in the terminal differentiation of IgE-committed B cells, which is arrested at higher cell concentration (Fig. 2b).

Decreasing cell concentrations by splitting the cells on different days post-culture was the only treatment capable of restoring IgE production at higher cell doses (Fig. 3). This observation implicated the increased cell–cell contact aspect of high cell concentration conditions in IgE inhibition.

B cells plated at high doses exhibited a decreased proliferative response (Fig. 4a,b), and are less viable than their low-density counterpart as assessed by trypan blue exclusion (data not shown). Indeed, Annexin V staining revealed a fourfold increase in apoptosis at higher cell concentrations (Fig. 5a). In the murine system, switching to IgG1 is not an end stage of differentiation. In fact, the majority of IgE-expressing cells are generated by sequential switching from IgM to IgE via IgG1.23,24 The sequential switching imparts a temporal separation in IgM, IgG1 and IgE production. IgE switching occurs after IgG1 with at least 48-h delay23 (Fig. 5b). IgE expression is detectable only on day 5 post-culture (Fig. 5b) when the majority of the cells are apoptotic at higher cell concentrations. Therefore one interpretation of the results could be based on the delay in switching to IgE, making IgE-expressing B cells more sensitive to apoptosis. However, we cannot rule out a specific contact-mediated apoptosis selective to IgE-expressing cells.

A variety of signalling pathways for apoptosis induction are operative, death-receptor-induced apoptosis is one of the best-studied examples. Activation of Fas, a type-1 membrane protein belonging to the tumour necrosis factor (TNF) receptor family, by Fas ligand triggers apoptosis in susceptible cells. The ‘death domain’ of Fas interacts with other proteins (FADD, caspase-8) forming a multimeric complex called DISC (death-inducing signalling complex), which in turn activates a class of highly specific cellular proteases termed caspases. Caspases play an important role in apoptotic execution (reviewed in ref. 25). Two different pathways of Fas-mediated apoptosis exist, one that bypasses the mitochondria directly, leading to activation of caspases, the other activating caspases downstream of mitochondrial apoptogenic activity.26 However, evidence of caspase-independent programmed cell death is also accumulating.27,28 Apoptosis is tightly regulated by transcriptional regulation of various genes. The most notable are the Fas-associated death-domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP),29 and the Bcl family members especially Bcl2 and Bcl-xL.30,31 The latter can inhibit all apoptogenic activity of mitochondria.32 In this system, treatment with the apoptosis inhibitor DEVD (Fig. 6) increased the level of IgE production at the high cell density conditions, thereby establishing a functional correlation between IgE expression and apoptosis.

Our results support death-receptor-mediated induction of apoptosis at higher cell doses that is independent from mitochondrial apoptogenic activity. First, apoptosis is enhanced at higher cell concentrations (Fig. 5a), which was not found to be a consequence of trophic factor depletion; neither increasing concentrations of CD40LT nor replenishing the media had an effect on IgE levels (data not shown). Second, the Bcl-2 transgene provided only a modest protection from apoptosis in murine B cells and failed to reverse IgE inhibition. Finally, splitting B cells after culture initiation enhanced IgE production (Fig. 3), implicating cell surface molecular signalling. Although Fas- and FasL-deficient mice showed the same extent of IgE inhibition at high cell concentration (data not shown), the involvement of other TNF-receptor-containing death domains cannot be excluded. Therefore, we hypothesize that at higher cell concentrations the latter molecules are activated through increased cell–cell contact, mediating a cascade of events culminating in the induction of apoptosis. Preferential loss of IgE-expressing cells could be due either to the delayed appearance of these cells or to a higher expression of the death-domain-containing receptor(s) on their cell surface or both.

In conclusion, this study highlights the serious caveat of using a single B-cell dose for IgE production in vitro and identifies cell density as an important variable regulating differentiation to IgE-producing plasma cells.

Acknowledgments

This work was supported in part by a graduate assistantship from the department of Microbiology and Immunology and grants AI 18697, AI 44163 from NIH. The authors acknowledge the excellent technical assistance of Ms Yee ChanLi in preparation of the reagents used for the IgE ELISAs. We also thank Drs Suzanne Barbour and Bing-Hung Chen for critical review of the manuscript and for helpful suggestions.

References

  • 1.Conrad DH, Tinnell SB, Kelly AE, Immunoglobulin E In. Kaliner MA. Current Review of Allergic Disease. Philadelphia: Blackwell Science; 1998. pp. 39–50. [Google Scholar]
  • 2.King CL, Jia XL, Malhotra I, Liu SF, Mahmoud AAF, Oettgen HC. Mice with a targeted deletion of the IgE gene have increased worm burdens and reduced granulomatous inflammation following primary infection with Schistosoma mansoni. J Immunol. 1997;158:294–300. [PubMed] [Google Scholar]
  • 3.Sutton BJ, Gould HJ. The human IgE network. Nature. 1993;366:421–8. doi: 10.1038/366421a0. [DOI] [PubMed] [Google Scholar]
  • 4.Bacharier LB, Jabara H, Geha RS. Molecular mechanisms of immunoglobulin E regulation. Int Arch Allergy Immunol. 1998;115:257–69. doi: 10.1159/000069456. [DOI] [PubMed] [Google Scholar]
  • 5.Randall TD, Heath AW, Santos-Argumedo L, Howard MC, Weissman IL, Lund FE. Arrest of B lymphocyte terminal differentiation by CD40 signaling: mechanism for lack of antibody-secreting cells in germinal centers. Immunity. 1998;8:733–42. doi: 10.1016/s1074-7613(00)80578-6. [DOI] [PubMed] [Google Scholar]
  • 6.Stuber E, Neurath M, Calderhead D, Fell HP, Strober W. Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells. Immunity. 1995;2:507–21. doi: 10.1016/1074-7613(95)90031-4. [DOI] [PubMed] [Google Scholar]
  • 7.Raman VS, Bal V, Rath S, George A. Ligation of CD27 on murine B cells responding to T-dependent and T-independent stimuli inhibits the generation of plasma cells. J Immunol. 2000;165:6809–15. doi: 10.4049/jimmunol.165.12.6809. [DOI] [PubMed] [Google Scholar]
  • 8.Neurath MF, Stuber ER, Strober WBSAP. A key regulator of B-cell development and differentiation. Immunol Today. 1995;16:564–9. doi: 10.1016/0167-5699(95)80078-6. [DOI] [PubMed] [Google Scholar]
  • 9.Turner CA, Mack DH, Davis MM. Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell. 1994;77:297–306. doi: 10.1016/0092-8674(94)90321-2. [DOI] [PubMed] [Google Scholar]
  • 10.Shaffer AL, Yu X, He Y, Boldrick J, Chan EP, Staudt LM. BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control. Immunity. 2000;13:199–212. doi: 10.1016/s1074-7613(00)00020-0. [DOI] [PubMed] [Google Scholar]
  • 11.Reimold AM, Iwakoshi NN, Manis J, et al. Plasma cell differentiation requires the transcription factor XBP-1. Nature. 2001;412:300–7. doi: 10.1038/35085509. 10.1038/35085509. [DOI] [PubMed] [Google Scholar]
  • 12.McGrew JT, Leiske D, Dell B, et al. Expression of trimeric CD40 ligand in Pichia pastoris: use of a rapid method to detect high-level expressing transformants. Gene. 1997;187:193–200. doi: 10.1016/s0378-1119(96)00747-0. [DOI] [PubMed] [Google Scholar]
  • 13.Gold MR, Law DA, DeFranco AL. Stimulation of protein tyrosine phosphorylation by the B-lymphocyte antigen receptor. Nature. 1990;345:810–3. doi: 10.1038/345810a0. [DOI] [PubMed] [Google Scholar]
  • 14.Campbell KA, Studer EJ, Kilmon MA, Lees A, Finkelman FD, Conrad DH. Induction of B cell apoptosis by co-crosslinking CD23 and sIg involves aberrant regulation of c-myc and is inhibited by bcl-2. Int Immunol. 1997;9:1131–40. doi: 10.1093/intimm/9.8.1131. [DOI] [PubMed] [Google Scholar]
  • 15.Cho S-W, Conrad DH. A new multivalent B cell activation model – Anti-IgD bound to FcτRI. Properties and comparison with CD40L-mediated activation. Int Immunol. 1997;9:239–48. doi: 10.1093/intimm/9.2.239. [DOI] [PubMed] [Google Scholar]
  • 16.Payet ME, Woodward EC, Conrad DH. Humoral response suppression observed with CD23 transgenics. J Immunol. 1999;163:217–23. [PubMed] [Google Scholar]
  • 17.Unkeless JC. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med. 1979;150:580–96. doi: 10.1084/jem.150.3.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Lyons AB, Parish CR. Determination of lymphocyte division by flow cytometry. J Immunol Methods. 1994;171:131–7. doi: 10.1016/0022-1759(94)90236-4. [DOI] [PubMed] [Google Scholar]
  • 19.Hasbold J, Lyons AB, Kehry MR, Hodgkin PD. Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4. Eur J Immunol. 1998;28:1040–51. doi: 10.1002/(SICI)1521-4141(199803)28:03<1040::AID-IMMU1040>3.0.CO;2-9. 10.1002/(sici)1521-4141(199803)28:03<1040::aid-immu1040>3.0.co;2-9. [DOI] [PubMed] [Google Scholar]
  • 20.Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994;84:1415–20. [PubMed] [Google Scholar]
  • 21.Rabah D, Grant S, Ma C, Conrad DH. Bryostatin-1 specifically inhibits in vitro IgE synthesis. J Immunol. 2001;167:4910–18. doi: 10.4049/jimmunol.167.9.4910. [DOI] [PubMed] [Google Scholar]
  • 22.Cho SW, Kilmon MA, Studer EJ, van der Putton H, Conrad DH. B cell activation and Ig, especially IgE, production is inhibited by high CD23 levels in vivo and in vitro. Cell Immunol. 1997;180:36–46. doi: 10.1006/cimm.1997.1174. [DOI] [PubMed] [Google Scholar]
  • 23.Hodgkin PD, Castle BE, Kehry MR. B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation. Eur J Immunol. 1994;24:239–46. doi: 10.1002/eji.1830240138. [DOI] [PubMed] [Google Scholar]
  • 24.Mandler R, Finkelman FD, Levine AD, Snapper CM. IL-4 induction of IgE class switching by lipopolysaccharide-activated murine B cells occurs predominantly through sequential switching. J Immunol. 1993;150:407–18. [PubMed] [Google Scholar]
  • 25.Dragovich T, Rudin CM, Thompson CB. Signal transduction pathways that regulate cell survival and cell death. Oncogene. 1998;17:3207–13. doi: 10.1038/sj.onc.1202587. [DOI] [PubMed] [Google Scholar]
  • 26.Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998;17:1675–87. doi: 10.1093/emboj/17.6.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kawahara A, Ohsawa Y, Matsumura H, Uchiyama Y, Nagata S. Caspase-independent cell killing by Fas-associated protein with death domain. J Cell Biol. 1998;143:1353–60. doi: 10.1083/jcb.143.5.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM, Henkart PA. Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity. 1997;6:209–15. doi: 10.1016/s1074-7613(00)80427-6. [DOI] [PubMed] [Google Scholar]
  • 29.Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by cellular FLIP [see comments] Nature. 1997;388:190–5. doi: 10.1038/40657. [DOI] [PubMed] [Google Scholar]
  • 30.Boise LH, González-García M, Postema CE, et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74:597–608. doi: 10.1016/0092-8674(93)90508-n. [DOI] [PubMed] [Google Scholar]
  • 31.Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322–6. doi: 10.1126/science.281.5381.1322. 10.1126/science.281.5381.1322. [DOI] [PubMed] [Google Scholar]
  • 32.Marzo I, Brenner C, Zamzami N, et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J Exp Med. 1998;187:1261–71. doi: 10.1084/jem.187.8.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]

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