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Originally published online as doi:10.1189/jlb.0305159 on June 16, 2005

Published online before print June 16, 2005
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(Journal of Leukocyte Biology. 2005;78:620-629.)
© 2005 by Society for Leukocyte Biology

Maintenance of the CD40-related immunodeficient response in hyper-IgM B cells immortalized with a LMP1-regulated mini-EBV

Kristina T. Lu*, Rebecca L. Dryer*, Charles Song{dagger} and Lori R. Covey*,1

* Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway; and
{dagger} Harbor General Hospital, University of California, Los Angeles, Torrance

1Correspondence: Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, 604 Allison Road, Piscataway, NJ 08854. E-mail: covey{at}biology.rutgers.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous investigation of a patient (pt1) with non-X-linked hyper-immunoglobulin M syndrome revealed a CD40-mediated defect in B cell activation that resulted in low CD23 expression and absence of germ-line transcription and class-switch recombination. These deficiencies were complemented in vitro by a high threshold of sustained signaling through CD40. To further analyze the signaling defect in pt1 B cells, two types of Epstein-Barr virus lymphoblastoid cell lines (LCLs) were generated that either constitutively expressed the viral transforming protein latent membrane protein-1 (LMP1; pt1-LCL) or expressed it under the control of a tet-inducible promoter (pt1-LCLtet). Because LMP1 signals through the CD40 pathway, the pt1-LCL and pt1-LCLtet lines allow comparison of downstream functions in response to either constitutive LMP1 signals or regulated LMP1 and CD40 signals. Immortalized pt1-LCLs were initially CD23lo/CD38hi and reverted to a CD23hi/CD38lo phenotype upon extended growth in culture, suggesting that the CD40 defect was reversed by selection and/or constitutive expression of LMP1. In contrast, pt1-LCLtet cells retained the CD23lo/CD38hi phenotype after extended periods of culture and failed to up-regulate CD23 in response to CD40 signals. Analysis of pt1-LCLtet cells in response to the CD40 signals in the presence or absence of LMP1 revealed that mitogenic activation resulted only from LMP1 and not CD40, indicating a difference in the response of pt1 B cells to these two distinct signals. Together, these data demonstrate that the pt1-LCLtet cells maintain the CD40-related defect and provide a unique approach to study the independent effects of LMP1- and CD40-directed signals.

Key Words: B cell proliferation • B cell activation • lymphoblastoid • signal transduction • viral transformation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Primary hyper-immunoglobulin M (IgM; HIGM) syndrome is a heterogeneous group of rare immunodeficiencies defined by normal-to-elevated serum IgM levels with corresponding low-to-absent levels of circulating IgG, IgA, and IgE antibodies. The defect in the production of most isotypes renders affected individuals susceptible to recurrent and severe bacterial and viral infections (reviewed in refs. [1 , 2 ]). The most prevalent form of HIGM syndrome is X-linked and associated with an impaired CD40 ligand (CD40L) gene function [3 4 5 6 7 8 ]. The lack of "switched" antibodies in HIGM1 patients is a consequence of the absence of CD40L:CD40 contact, which is required for antigen-selected B cells to undergo class-switch recombination (CSR; reviewed in refs. [9 , 10 ]). Less-common forms of HIGM occur sporadically or as an effect of autosomal recessive transmission (reviewed in ref. [11 ]). Examples include HIGM2, which results from mutations in the "activation-induced cytidine deaminase" (AID) protein required for CSR and somatic mutation [12 13 14 ]; HIGM3, which is caused by mutations in CD40 [15 ]; uracil-N glycosylase (UNG)-dependent HIGM; and a second X-linked form associated with anhidrotic ectodermal dysplasia, associated with mutations in the NF-{kappa}B essential modulator (NEMO) or inhibitor of {kappa}B kinase-{gamma} gene (IKK{gamma}) [16 17 18 ]. The engagement of either the CD40 signal transduction pathway or CD40-mediated functions, such as CSR, in different forms of HIGM underscores the critical role CD40 plays in bringing about the development of humoral and cell-mediated immunity.

We have previously characterized lymphocyte expression and function in a female patient (pt1) with a diagnosis of non-X-linked HIGM syndrome [19 ]. Our analysis revealed a major defect associated with B cell activation that involved a subset of CD40-induced responses. Specifically, we observed selective impairment of CD40-mediated CD23 expression, germ-line I{gamma} transcription, and CSR but normal CD80 expression in primary B cells activated with CD4+ T cells. These observations, along with results showing normal CD40 and AID in pt1 B cells (data not shown) and complementation of the defect in vitro by extended signaling through CD40 [19 ], strongly suggested that the pt1 defect was related directly to an inability of B cells to integrate a normal threshold of CD40 activation signals.

To extend our understanding of the pt1 B cell phenotype, we sought to use a long-term culture system that recapitulated the CD40 signaling defect. To this end, experiments were initiated to establish Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) that retained many of the properties of activated B cells including the expression of B cell activation molecules CD23, CD30, and CD44, intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1) LFA-3, and an array of cytokines involved in B cell differentiation from lymphoblastoid to plasmacytoid cells [20 21 22 23 24 25 26 27 28 ]. In vitro infection of resting B cells by EBV efficiently drives cells out of quiescence and into a continuously dividing CD23hi/CD38lo population that undergoes limited differentiation upon autonomous growth in culture [21 , 29 ]. One of the six viral proteins essential for immortalization is latent membrane protein-1 (LMP1), which acts as a constitutively active receptor by signaling through the tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) [30 31 32 33 34 35 ]. These adaptor proteins are required for transmitting signals from CD40 (and other TNFR family members) for selective proliferation, activation, and apoptosis (reviewed in ref. [36 ]). Expression of LMP1 in B cells induces many of the same cellular functions as CD40 and therefore, to a large extent, engages common signaling pathways to target a specific set of activation-induced genes.

In our current study, we have used two different sets of LCLs to extend our understanding of the pt1 defect relative to CD23 expression and proliferation. CD23 is the low-affinity IgE receptor FcERII, which is expressed rapidly on B cells in response to IL-4 and T cell contact (reviewed in ref. [37 ]). In EBV infection, CD23 mRNA and protein expression are rapidly induced and remain at a high level in the vast majority of LCLs [22 , 23 ]. CD23 has been implicated in playing a central role in EBV immortalization based on the observations that transformed cells only arise from infected cells that express CD23, and a nontransforming mutant virus fails to induce CD23 in infected cells [23 , 38 ]. In addition, resting B cells are induced to proliferate in response to CD40 ligation and LMP1 signaling during T-dependent responses (reviewed in ref. [39 ]).

In this work, we demonstrate that the activation defect relating to decreased CD23 expression in pt1 B cells is reversed in standard pt1 LCLs after an extended time in culture. In contrast, establishment of pt1 EBV-transformed B cell lines with regulated LMP1 expression resulted in a cell population that retains low CD23 expression and fails to respond to proliferative signals through CD40. These findings validate the LCLtet cells as a model system to study both normal and affected CD40-mediated signaling pathways within the context of EBV immortalization of human B cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines and culturing conditions
The 293 cell line is derived from human embryonic kidney cells [American Type Culture Collection (ATCC), Manassas, VA]. The CD40L expressing a 293 cell line (293/CD40L) was constructed as described previously [40 ]. HH514 is a single-cell clone of the Burkitt’s lymphoma cell line P3HR1 [41 ]. WI38, a human fibroblast cell line, was purchased from ATCC. B cell lines were grown in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% L-glutamine, and 1% penicillin/streptomycin (RPMI-Complete). The 293, 293/CD40L, and WI38 lines were grown in Dulbecco’s modified Eagle’s medium-DMEM Complete media in the same conditions as described above.

Generation of LCLs
Normal donor and pt1 samples of peripheral blood were separated by Ficoll-Hypaque gradient centrifugation to recover peripheral blood mononuclear cells PBMCs (1x107), which were added to 5 ml supernatant harvested from the EBV-transformed marmoset cell line B95-8 [42 ]. Following a 1-h incubation at 37°C, 5 ml RPMI-Complete and 5 µg cyclosporine A were added. The cultures were incubated for 14–21 days until aggregates were visible. Control LCLs (C3121, C3688, and C1125) were randomly selected from cultures that were transformed on approximately the same date as pt1 B cells. Control- and pt1-LCLs were continually expanded in RPMI-Complete for 1 month prior to analysis. For surface expression studies, cells were analyzed at 5 weeks post-transformation. For time-course studies, cells were grown for an additional 4 weeks in culture.

Mini-EBV transformation (LCLtet) of primary B cells with recombinant plasmid p1852 was performed as described previously [43 ] with modifications. The p1852 mini-EBV plasmid consists of the coding sequence of the wild-type LMP1 gene under the control of an artificial promoter whose activity can be regulated by a chimeric transcriptional repressor, tetracycline repressor-Kruppel-associated box (tetR-KRAB), in a tetracycline-dependent manner. In addition, 10 other viral genes, generally expressed in the latent phase of the EBV life-cycle, are included in the plasmid and expressed under their own promoters and therefore not regulated by tetR-KRAB.

Briefly, HH514 cells (1x107) were cotransfected with 20 µg p1852 plasmid and 10 µg pCMV-BZLF1. Electroporation was performed at 960 µF and 250 V. Virus released into the supernatant was collected 5 days later. PBMCs isolated from peripheral blood samples by centrifugation on a Ficoll-Paque gradient were infected with virus from HH514 and plated at a dilution of 5 x 106 cells/well in a 96-well plate in RPMI-Complete medium supplemented with 1 µg/ml tetracycline and 1 mM sodium pyruvate on a lethally irradiated (50 Gy) WI38 feeder cell layer. For the first 4 weeks of culture, medium was also supplemented with cyclosporin A (0.5 µg/ml). The absence of P3HR1 helper virus in immortalized B cell clones was verified by polymerase chain reaction (PCR) analysis [44 ].

Surface expression analysis
LCLs or LCLtet cells (5x105), cultured in the presence or absence of soluble human CD40L (500 ng/ml, Peprotech Inc., Rocky Hill, NJ), were washed in 3% FCS/0.1% NaN3/1x phosphate-buffered saline [PBS; fluorescein-activated cell sorter (FACS) wash], followed by incubation with 5 µg heat-aggregated IgG to inhibit nonspecific binding. Cells were incubated for 45 min at 4°C with saturating amounts of fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAb) against CD23, CD38, CD40, and CD11a (Ancell, Bayport, MN) and biotin-conjugated mAb against CD20, CD54, CD80, CD86, major histocompatibility complex (MHC) II (Ancell), or the matched isotype controls. Cells were washed in FACS wash, and biotin-conjugated samples were further incubated with phycoerythrin-conjugated streptavidin for 30 min at 4°C. Cells were washed, fixed with 1% paraformaldehyde in 1x PBS, and analyzed by FACS using an Epics Profile II flow cytometer (Coulter Electronics, Hialeah, FL) or a FACScan (Becton Dickinson, Mountain View, CA).

Protein immunoblots
Cell extracts were prepared by lysis in 50 mM Tris-HCl (pH 8)/1 mM EDTA/1% Nonidet P-40/150 mM NaCl. Equal amounts of protein were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membrane (Schleicher and Schuell, Keene, NH), and detected with antibodies against LMP1 and EBNA2 (Dako, Carpinteria, CA). Horseradish peroxidase-conjugated secondary antibodies were used for detection by enhanced chemiluminescence (Amersham, Piscataway, NJ).

293 and 293/CD40L membrane isolation
Cell membranes were isolated according to previously published techniques [45 ]. Briefly, 293 or 293/CD40L cells were incubated in DMEM Complete medium containing 100 µg/mL {alpha}-methyl-D-mannoside (Sigma Chemical Co., St. Louis, MO) at 37°C for 1 h. Cells were washed twice in ice-cold 1x PBS containing 100 µg/mL {alpha}-methyl-D-mannoside, resuspended at 5 x 106 cells/mL in ice-cold homogenization buffer [100 µg/mL {alpha}-methyl-D-mannoside, 20 mM Tris-Cl, 10 mM NaCl, 0.1 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co.), 0.5 µg/mL DNase-I (Sigma Chemical Co.)], and homogenized to detach the membranes. Cell debris was separated from the membranes over a 41% surcrose cushion by ultracentrifugation for 1 h at 26000 rpm. The interfacial membrane band was isolated, washed in RPMI, and pelleted by centrifugation at 35000 rpm for 45 min. Membranes were resuspended at 3 x 107 cell equivalents/100 µl Complete medium.

Proliferation assay
Parallel cultures of LCLs were established at 5 x 104 cells/well/100 µl in flat-bottom 96-well plates with 293 or 293/CD40L membranes (at a final dilution of 1:10) for 24 h to assay cellular growth by [3H]thymidine incorporation. Cells were pulsed with 0.5 µCi [3H]thymidine (Perkin Elmer, Boston, MA) during the last 6 h of culture before lysis by one round of freezing and thawing. Cells were harvested onto a glass fiber filtermat with a semiautomatic cell harvester (Skatron Instruments, Sterling, VA) and counted on a Beckman LS analyzer.

Statistical analysis
For comparison of two samples, a two-tailed Student’s t-test was used. Significance was set at P< 0.05. Data in figures and tables are shown as mean ± SD unless otherwise indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Pt1-LCLs show a partially activated phenotype with respect to a subset of cell-surface proteins
To generate standard LCLs, pt1 and control PBMCs were infected with EBV and cultured continuously for a period of 3–5 weeks prior to analysis. Determination of viral expression by Western analysis for EBV-transforming proteins EBNA1, EBNA2, EBNA2A, EBNA3A, EBNA3C, and LMP1 revealed no difference in pt1-LCLs relative to control lines (data not shown). As noted above, a clear defect in pt1 primary B cells was their failure to express CD23 at a normal level after activation with CD40L [19 ]. Since CD23 expression is generally highly elevated in LCLs, we sought to characterize the phenotype of pt1-LCLs relative to control LCLs, focusing on the expression of CD23 as well as other lineage- and activation-specific markers. For control lines, we chose LCLs with a range of phenotypes with respect to surface Ig expression and growth in culture (C3121, IgG+; C3688, IgM+; and C1125, IgM+). In particular, we analyzed two different IgM+ lines to establish a range of marker expression in transformed B cells from control individuals. As presented in Table 1 and shown representatively in Figure 1 , the expression of CD23 [both percent-positive cells and mean fluorescent intensity (MFI)] was down-regulated markedly in the pt1 relative to control LCLs. Other surface markers such as CD11a, CD80, and MHC II, which are normally induced in response to EBV infection, fell within the normal range of control LCLs. However, the MFIs of CD54 (ICAM-1) and CD40 were reduced, and the MFI of CD86 was increased relative to controls. Also of note was that CD38, which is generally down-regulated on LCLs, was highly expressed on pt1 and C3688. A profile of reduced CD23, CD40, ICAM-1, and elevated CD38 expression is inconsistent with the phenotype of the fully activated LCL and more closely resembles the pt1 primary B cells upon CD40L stimulation (ref. [19 ] and data not shown). Additionally, the finding that CD80 expression is indistinguishable from control LCL levels extends our previous finding that CD40-induced CD80 expression in primary B cells is not affected by the pt1 mutation [19 ].


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  		Table 1. Analysis of Surface Activation Markers in LCLsa

 


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  		Figure 1. pt1 LCL expresses a unique phenotype with respect to activation markers. pt1 (open profile) and control C3121-LCLs (darkly shaded profile) were stained with mAb against human CD23, CD38, CD40, CD54, CD80, and CD86 and analyzed for surface expression by flow cytometry. The y-axis represents cell number, and the x-axis represents relative fluorescence intensity. Lightly shaded profile represents isotype control.

 
Extended signaling through LMP1 changes the phenotypic profile of the pt1-LCLs
The fact that LMP1 constitutively signals through the CD40 pathway suggested that a subset of pt1 defective responses that revert with sustained signaling, may also be reversed by LMP1 expression over an extended period of time. To test this possibility, pt1-LCL (CD23lo/CD38hi) and control C3121-LCL (CD23hi/CD38lo) populations were analyzed over a 4-week period for changes in CD23 and CD38 expression. As shown in Figure 2 (upper panels), there was a notable change in CD23 expression in both percent-positive and MFI in the pt1-LCL population (from 13.8%/27 to 79.1%/68.6) and little change in the percent-positive of the control population over the period studied (98%/354.7–97.9%/143.1). Within the same time course, we also observed a decrease in the expression of CD38 in pt1-LCLs (from 94.4%/113.8 to 88%/42.3) and control LCLs (from 4.2%/22.8 to 2%/8.1; lower panels). The increase in CD23 and decrease in CD38 expression are consistent with the pt1-LCL population acquiring a more plasmacytoid phenotype resulting from constitutive signaling through LMP1 or the selective outgrowth of a small LMP1+/CD23+ population.



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  		Figure 2. Pt1-LCL CD23/CD38 expression with extended growth in culture. pt1-LCLs (lightly shaded) and C3121-LCLs (darkly shaded) were analyzed for surface expression of CD23 (upper panels) and CD38 (lower panels) at weekly intervals over a 4-week time period by FACS. Numbers above and below the indicator bar represent the percentage of positively stained pt1 and control cells, respectively. The stippled line represents the isotype control for each antibody.

 
Establishment of LCLtet cells
To circumvent the loss of the CD40 defect, as measured by low CD23 expression, we used an alternative EBV-based system that uses a recombinant EBV genome (p1852) that conditionally immortalizes B cells by expressing LMP1 under the control of a tet-inducible promoter. In this system, all other viral genes are expressed under their own promoters and at a level required for in vitro immortalization of B cells [43 ]. Primary pt1 and control B cells were infected with virus stocks of virion-packaged p1852, and single-cell clones were expanded in medium containing tet to allow for LMP1 expression. The presence of only the p1852 plasmid in the B cell clones was confirmed by PCR analysis (data not shown). IgM+/IgD– pt1 (pt1-LCLtet) and control mini-EBV clones (IgM+ D11-LCLtet and IgM– C2-LCLtet) were transferred from tet-containing (tet+) media and cultured in the absence of tetracycline (tet–) for 3 days before culturing the cells for an additional 4 days in tet+ media. Total extracts were collected from cultures at each time-point, and LMP1 expression was examined by Western blot analysis (Fig. 3A ). The p1852 plasmid contains only the LMP1 gene under the control of the tetR-KRAB repressor. To verify that only LMP1 expression is regulated by tet, the same blot was analyzed for EBNA2 expression. In the absence of tet, LMP1 expression was dramatically decreased by Day 1 and remained low-to-absent during the time-period examined (left panels). During the same time-period, the levels of EBNA2 remained unchanged in each clone. Addition of tet led to the reinduction and sustained expression of LMP1 without affecting EBNA2 expression (right panels). We also examined other viral genes by Western blot analysis or quantitative PCR and found the expression levels were not affected by the addition or removal of tetracycline (data not shown).



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  		Figure 3. Characterization of conditional LMP1 expression in LCLtet cells. (A) Investigation of LMP1 and EBNA2 expression in conditionally transformed LCLtet cells. pt1-LCLtet (top panels) and control D11- and C2-LCLtet cells (middle and bottom panels, respectively) were cultured for 3 days without tet followed by readdition of tet for another 4 days. Total cell extract was isolated each day and analyzed by Western blot using mAb to LMP1 and EBNA2. (B) Proliferation analysis of pt1 and control LCLtet lines with respect to LMP1 expression. Pt1 and control LCLtet cells were cultured in parallel for 6 days in the continuous presence (+tet, LMP1 on), continuous absence (–tet cont, LMP1 off), or the absence and then readdition (–/+tet) of tetracycline. Cells were pulsed with [3H]thymidine 6 h prior to the end of each time-point and then lysed and analyzed for proliferation. Results are expressed as the mean counts per minute (CPM) and SD of triplicate cultures. Results are representative of three similar experiments.

 
To confirm that a LMP1-mediated function was regulated directly by tet in the LCLtet population, cell proliferation was measured in cultures grown in the continuous presence (+tet cont, solid black line), continuous absence (–tet cont, solid gray line), or the absence and then readdition (–/+tet, stippled line) of tet during a 6-day period (Fig. 3B) . In the +tet cont cultures, there was a similar level of sustained proliferation in control LCLtet populations (middle and bottom panels) as well as in the pt1-LCLtet population (top panel). In contrast, there was an initial, gradual growth arrest, which corresponded directly to the cessation of LMP1 expression in the –/+tet cultures. Upon readdition of tet at day 3, proliferation increased to the +tet cont levels, paralleling the reinduction of LMP1 expression. These results indicate that the pt1-LCLtet cells are dividing in response to signals through LMP1 and that if there is an intrinsic defect in the CD40-mediated proliferation, it is not recapitulated with LMP1 signaling.

Maintenance of defective CD23 expression in pt1-LCLtet cells
To determine the pattern of CD23 and CD38 expression, pt1-LCLtet and control LCLtet populations were surface stained and FACS analyzed after a continuous 4-week culture in tet+ media followed by an additional 3-day incubation in tet+ or tet– media. Also, to assess the extent of CD23 and CD38 expression after stimulation with CD40L in the absence of LMP1 expression, cells incubated in tet– media for 3 days were transferred into media containing soluble CD40L for an additional day. As shown in Figure 4 , pt1-LCLtet cells grown in the presence of tet (+LMP1, top left panel, black line) express a lower level of CD23 on their surfaces compared with both control cell lines (middle and bottom left panels, black lines). When cells were shifted to tet– media, there was no change in the expression pattern of CD23 in pt1-LCLtet cells and only a modest decrease in CD23 expression in D11-LCLtet and C2-LCLtet cells (compare top left to middle and bottom left panels, dashed lines). Upon stimulation with CD40L in the absence of LMP1, CD23 levels increased slightly in pt1 and control C2 cells only (left panels, gray line). Together, these results reveal that the level of CD23 expression in the LCLtet lines is relatively independent of changes in LMP1 expression and that signaling through CD40 can induce a small shift in expression in the LCLtet lines.



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  		Figure 4. Maintenance of the primary CD23/CD38 expression profile in pt1-LCLtet cells with LMP1 or CD40L stimulation. Pt1-LCLtet and control LCLtet cells were cultured in tet+ continuously or tet– media for 3 days. For CD40L stimulation, LCLtet cells in tet– media were cultured for an additional 16 h with soluble CD40L. LCLtet cells were then stained with FITC-labeled CD23 mAb (left panels) or FITC-labeled CD38 mAb (right panels) and analyzed by FACS. The peaks represent positively stained cells cultured in the presence of LMP1 (+LMP1, black line), absence of LMP1 (–LMP1, stippled line), or absence of LMP1 plus CD40L (–LMP1+CD40L, gray line), and the shaded peaks represents the corresponding isotype control.

 
In the presence of LMP1, there was a high level of surface CD38 expression in pt-1-LCLtet cells. However, similar to our CD23 observation, there was no measurable change in CD38 expression in the absence of LMP1 or upon restimulation with CD40L (top right panel). In contrast, both control lines showed low CD38 expression in the presence of LMP1 and a concomitant and rapid increase in the absence of LMP1 (middle and bottom right panels). Addition of CD40L resulted in the down-regulation of CD38 in the control cells, returning to levels at or below that seen with LMP1 (middle and bottom right panels, gray line). These experiments revealed that signaling through LMP1 or CD40 can down-regulate CD38 expression in the control but not in the pt1-LCLtet cells. The loss of this LMP1/CD40-mediated function suggests that an additional factor required for CD38 down-regulation is absent in pt1-LCLtet cells. It is important that these findings support the use of LCLtet lines to study the defect in pt1 B cells and to analyze similarities and differences in LMP1 and CD40 signaling.

CD40-mediated proliferation is defective in pt1-LCLtet cells
To further study the CD40 defect in pt1 B cells, we measured and compared the CD40-specific proliferation response of pt1-LCLtet cells to control LCLtet cells. Our initial work demonstrated a minimal difference in the growth of pt1-LCLtet and control LCLtet lines under different conditions of LMP1 expression (Fig. 3B) . However, results from Figure 4 suggested that low CD23 expression was not complemented in pt-LCLtet cells in the presence of LMP1 or CD40 signaling. A hallmark feature of CD40 activation is that it stimulates proliferation of non-EBV-immortalized B cells and inhibits growth of EBV lines by arresting cells in G0/G1 [46 ]. Thus, these lines provide an ideal system to study the effect of CD40 activation on proliferation in the presence or absence of LMP1 signaling. LCLtet cells were cultured with either tet (Fig. 5A , "LMP1 on") or without tet for 3 days (Fig. 5B , "LMP1 off") and then further cocultured for an additional 24 h with isolated membranes from 293 cells (unstimulated) or 293/CD40 cells expressing CD40L (CD40-stimulated). As shown in Figure 5A , costimulation of pt1 and control LCLtet populations with LMP1 and 293/CD40L membranes resulted in decreased proliferation compared with cells costimulated with 293 membranes alone. This suggests that negative proliferative signals transduced through CD40, in the context of sustained LMP1 signaling, are integrated normally in pt1-LCLtet cells. In contrast, when cells were cultured in tet– media for 3 days and then stimulated with CD40L-expressing membranes, a different response was observed between pt1 and control cells. Although control D11- and C2-LCLtet cells had significant increases in proliferation in response to CD40L, pt1-LCLtet cells showed no significant change under these conditions (Fig. 5B) . The limited growth response of pt1-LCLtet cells indicates that the CD40-related defect also directly affects proliferation. This was further confirmed by assaying the proliferative response of primary pt1 B cells to CD40L and observing a distinct defect in CD40-induced cell growth (data not shown). These results provide further evidence for impaired CD40 signaling in pt1 B cells and support the hypothesis that the defect is manifested in aberrant proliferation, as well as low-to-absent CD23 expression and CSR. Also, we demonstrate that unlike CD23 expression, defective proliferation can be complemented by signals through LMP1 but not through CD40.



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  		Figure 5. Identification of a CD40-mediated proliferation defect in pt1-LCLtetcells. (A) Control and pt1-LCLtet cells were cultured in the continuous presence of tet and further subcultured for an additional 24 h with 293 membranes (unstimulated, solid bars) or 293/CD40L membranes (CD40-stimulated, shaded bars). Proliferation was measured by [3H]thymidine incorporation of triplicate samples, as outlined in Materials and Methods. Results are expressed as the mean CPM ± SD of two independent experiements. (B) LCLtet lines grown without tet for 3 days were cultured in the absence (solid bars) or presence of CD40L (shaded bars) for 24 h. Results are expressed as the mean CPM ± SD of four independent experiments. Significance is shown as * and **, reflecting P values of <0.05 and <0.01, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Identifying novel defects that involve CD40 signaling can be challenging, given that EBV, which is the primary method for immortalizing human B cells, appropriates the CD40 pathway in a ligand-independent manner (reviewed in ref. [47 ]). Accordingly, constitutive LMP1-dependent signaling through the TRAFs may mask defective responses that are otherwise evident under regulated levels of signal. In this report, we used conventional and tet-regulated, EBV-mediated transformation to immortalize B cells from a non-X-linked HIGM patient. A comparison of phenotypic and functional characteristics of at least three different clones of pt1 and control LCL populations revealed a number of novel findings. First, we found that conventional LCLs lose the CD23lo/CD38hi phenotype observable in pt1 primary B cells and therefore have limited use with respect to studying the defect in impaired CD40 signaling. In contrast, pt1-LCLtet cells cultured in the presence or absence of LMP1 or CD40L maintain the specific CD23lo/CD38hi phenotype over an extended period of time. Second, we observed that signals through LMP1 and CD40 separately regulate the expression of CD38 and to a much lesser degree, CD23, in control LCLtet cells and that these signals fail to appreciably modulate the expression of CD38 or CD23 in pt1-LCLtet cells. Finally, we show that CD40-induced proliferation is also severely reduced in the pt1-LCLtet cells and that normal levels of cell cycling are achieved only by signaling through LMP1 and not CD40.

Overall, these results are consistent with the pt1 defect residing in the CD40-mediated signal-transduction pathways leading to CD23 expression and mitogenic activation. Also, our results support a model of sustained signaling through tet-regulated LMP1 expression, complementing some aspects of the pt1 defect (i.e., proliferation) but not others (i.e., CD23 expression). Observed differences in the response to CD40 and/or LMP1 signals extend previous findings that these two molecules use separate as well as overlapping signaling pathways to drive B cell proliferation and differentiation (reviewed ref. [48 ]). Specifically, mice lacking CD40 and expressing a transgenic LMP1 restore many, but not all, of the CD40-specific functions [35 ]. Also, CD40L activation of CD40 in mouse B cells induces a subset of RNAs that are not up-regulated in LMP1-activated B cells [49 , 50 ]. These different responses may be explained by the fact that distinct subsets of adaptor molecules bind with different affinities to LMP1 and CD40 [51 , 52 ], and LMP1 produces amplified and/or sustained activation responses relative to CD40 in both in vitro and in vivo experiments [53 54 55 ]. The mechanistic basis of these stronger signals is likely related to the fact that signaling through CD40, but not LMP1, results in ubiquitin- and proteasome-dependent TRAF2 and TRAF3 degradation [53 , 56 ] and that LMP1 forms a higher order complex than the trimeric signaling complex induced by CD40L-CD40 ligation [57 , 58 ].

Given the potency of LMP1 signals, we were surprised to find that restoration of high levels of CD23 expression was observed only in conventional LCLs and not in the LCLtet cells. One explanation for this finding may be that the expression is not being complemented by LMP1 directly, but rather, there is a distinct growth advantage in the traditional LCLs for CD23+ cells. This growth advantage could be related to overlapping pathways of regulation of LMP1 and CD23, only in LCLs and not in mini-EBV-transformed LCLtet cells. For example, after EBV infection of B cells and immediate expression of EBNA2 and EBNA5, there is a distinct lag in LMP1 expression, which appears to precede or coincide with initiation of DNA synthesis [59 60 61 62 63 ]. Accordingly, EBNA2 has been shown to be necessary for the efficient expression of LMP1 in B cells [64 65 66 ], and LMP1 is critical for EBV-mediated B cell proliferation and transformation [43 , 67 ]. If a common signaling or transcription factor required for LMP1 and CD23 expression is affected in pt1 B cells, then under conditions of standard EBV immortalization, there would be a strong, selective advantage for growth of LMP1+/CD23+ cells. In contrast, in the LCLtet system, LMP1 is expressed from a heterologous promoter, and therefore, the expression of CD23 and LMP1 is formally "un-linked." Thus, the small number of CD23+ cells would not necessarily be at a selective growth advantage, and the population would remain predominantly CD23–.

Recent data support the premise that LMP1 mediates proliferation through the induction of c-myc and Jun AP-1 family members. In particular, c-myc expression is up-regulated within 30 min of LMP1 signals [50 ]. CD40 activation turns on many of the same genes involved in cell-cycle regulation in LMP1- and CD40-activated B cells, induction of c-myc appears to be independent of new protein synthesis [49 , 50 ]. In light of these findings and the fact that the pt1 defect appears to be B cell-specific, we would hypothesize that c-myc is induced by LMP1 but not by CD40 signals. The fact that we see complementation of the proliferation defect only with LMP1 reinforces the idea that a higher threshold of signal is required to overcome the downstream defect. It is interesting that we see a decrease in proliferation in control and pt1-LCLtet cells when they receive signals through LMP1 and CD40 compared with LMP1 alone. This suggests that when pt1-LCLtet cells are being induced to proliferate through LMP1, they are able to respond to CD40 signaling, further supporting the idea that the signal provided by LMP1 is overcoming the proliferation defect. Dissection of the LMP1 and CD40 signaling pathways leading to c-myc expression in control LCLtet and pt-LCLtet lines is currently ongoing.

In summary, our findings support the use of the mini-EBV system as a viable means to maintain the immunodeficient phenotype in long-term culture. Defects in CD23 expression and CD40-mediated cell proliferation demonstrate impaired CD40 activation and signaling in the pt1 B cells. Thus, the LCLtet cells provide a functional model system to further localize the defect and study the independent effects of LMP1- and CD40-mediated signals on downstream responses in normal and immunodeficient B cells.


   ACKNOWLEDGEMENTS
 
This work was supported by a National Institutes of Health (NIH) grant (AI37081) and a Busch Biomedical Research grant from Rutgers University to L. R. C. and K. T. L. was supported by a predoctoral fellowship from the New Jersey State Commission on Cancer Research. R. L. D. was supported in part by a training grant, Virus-Host Interactions in Eukaryotic Cells, from NIH-National Institute of Allergy and Infectious Diseases, 2 T32 AI07403, awarded to Dr. Sidney Pestka, University of Medicine and Dentistry of New Jersey. We are grateful to pt1 and her family for their ongoing participation in these studies. We acknowledge the generosity of Dr. Douglas Fugman (Rutgers University Cell Repository) for help in generating the EBV-LCLs and Dr. Susan Rittling for assistance with statistical analysis (Rutgers University). Also, we thank Drs. Wolfgang Hammerschmidt (GSF-National Research Center for Environmental Health), Bill Sugden (University of Wisconsin), and George Miller (Yale University) for their generosity in providing the p1852 and pCMV-BZLF1 plasmids and HH514 cells, respectively. We also thank Drs. Ameesha Batheja (Johnson and Johnson Research Institute), Jeffery T. Sample (St. Jude’s Childrens Research Hospital), and Ingrid K. Ruf (St. Jude’s Childrens Hospital) for their contributions in the early stages of this project. Finally, we acknowledge past and present members of the Covey laboratory for insightful criticism and continuous support.

Received March 19, 2005; revised April 7, 2005; accepted May 15, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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