Maintenance of the CD40-related immunodeficient response
in hyper-IgM B cells immortalized with a LMP1-regulated
Kristina T. Lu,* Rebecca L. Dryer,* Charles Song,†and Lori R. Covey*,1
*Department of Cell Biology and Neuroscience, Rutgers, The State University of New Jersey, Piscataway;
and†Harbor General Hospital, University of California, Los Angeles, Torrance
(pt1) with non-X-linked hyper-immunoglobulin M
syndrome revealed a CD40-mediated defect in B
cell activation that resulted in low CD23 expres-
sion and absence of germ-line transcription and
were complemented in vitro by a high threshold of
sustained signaling through CD40. To further an-
alyze 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 ex-
pressed it under the control of a tet-inducible pro-
moter (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/CD38hiand re-
verted to a CD23hi/CD38lophenotype upon ex-
tended growth in culture, suggesting that the CD40
defect was reversed by selection and/or constitu-
tive expression of LMP1. In contrast, pt1-LCLtet
cells retained the CD23lo/CD38hiphenotype after
extended periods of culture and failed to up-regu-
late CD23 in response to CD40 signals. Analysis of
pt1-LCLtetcells 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. To-
gether, 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. J. Leukoc.
Biol. 78: 620–629; 2005.
Our previous investigation of a patient
Key Words: B cell proliferation ? B cell activation ? lymphoblas-
toid ? signal transduction ? viral transformation
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 [3–8]. 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 trans-
mission (reviewed in ref. ). Examples include HIGM2,
which results from mutations in the “activation-induced cyti-
dine deaminase” (AID) protein required for CSR and somatic
mutation [12–14]; HIGM3, which is caused by mutations in
CD40 ; uracil-N glycosylase (UNG)-dependent HIGM; and
a second X-linked form associated with anhidrotic ectodermal
dysplasia, associated with mutations in the NF-?B essential
modulator (NEMO) or inhibitor of ?B kinase-? gene (IKK?)
[16–18]. The engagement of either the CD40 signal transduc-
tion 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-
We have previously characterized lymphocyte expression
and function in a female patient (pt1) with a diagnosis of
non-X-linked HIGM syndrome . 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? transcription, and CSR but normal CD80 expres-
sion 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 ,
strongly suggested that the pt1 defect was related directly to an
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: email@example.com
Received March 19, 2005; revised April 7, 2005; accepted May 15, 2005;
620Journal of Leukocyte Biology
Volume 78, September 2005
0741-5400/05/0078-620 © Society for Leukocyte Biology
inability of B cells to integrate a normal threshold of CD40
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 lymphoblas-
toid 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 anti-
gen-1 (LFA-1) LFA-3, and an array of cytokines involved in B
cell differentiation from lymphoblastoid to plasmacytoid cells
[20–28]. In vitro infection of resting B cells by EBV efficiently
drives cells out of quiescence and into a continuously dividing
CD23hi/CD38lopopulation that undergoes limited differentia-
tion 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) [30–35]. These adaptor
proteins are required for transmitting signals from CD40 (and
other TNFR family members) for selective proliferation, acti-
vation, and apoptosis (reviewed in ref. ). 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
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 re-
ceptor FcERII, which is expressed rapidly on B cells in re-
sponse to IL-4 and T cell contact (reviewed in ref. ). In
EBV infection, CD23 mRNA and protein expression are rap-
idly 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. ).
In this work, we demonstrate that the activation defect
relating to decreased CD23 expression in pt1 B cells is re-
versed 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
LCLtetcells 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
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 . HH514 is
a single-cell clone of the Burkitt’s lymphoma cell line P3HR1 . 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
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 (1?107), which were added to 5 ml supernatant harvested from the
EBV-transformed marmoset cell line B95-8 . Following a 1-h incubation at
37°C, 5 ml RPMI-Complete and 5 ?g 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  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-Krup-
pel-associated box (tetR-KRAB), in a tetracycline-dependent manner. In ad-
dition, 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 promot-
ers and therefore not regulated by tetR-KRAB.
Briefly, HH514 cells (1?107) were cotransfected with 20 ?g p1852 plasmid
and 10 ?g pCMV-BZLF1. Electroporation was performed at 960 ?F 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 ?
106cells/well in a 96-well plate in RPMI-Complete medium supplemented
with 1 ?g/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 ?g/ml). The absence of P3HR1
helper virus in immortalized B cell clones was verified by polymerase chain
reaction (PCR) analysis .
Surface expression analysis
LCLs or LCLtetcells (5?105), 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% NaN3/1? phosphate-buffered saline [PBS; fluorescein-activated
cell sorter (FACS) wash],followed by incubation with 5 ?g heat-aggregated IgG
to inhibit nonspecific binding. Cells were incubated for 45 min at 4°C with
saturating amounts of fluorescein isothiocyanate (FITC)-conjugated monoclo-
nal antibodies (mAb) against CD23, CD38, CD40, and CD11a (Ancell, Bay-
port, 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 1? PBS, and
analyzed by FACS using an Epics Profile II flow cytometer (Coulter Electron-
ics, Hialeah, FL) or a FACScan (Becton Dickinson, Mountain View, CA).
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 sep-
arated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, transferred onto polyvinylidene difluoride membrane (Schlei-
cher and Schuell, Keene, NH), and detected with antibodies against LMP1 and
EBNA2 (Dako, Carpinteria, CA). Horseradish peroxidase-conjugated second-
Lu et al.
Altered CD40-mediated proliferation in HIGM B cells 621
ary antibodies were used for detection by enhanced chemiluminescence (Am-
ersham, Piscataway, NJ).
293 and 293/CD40L membrane isolation
Cell membranes were isolated according to previously published techniques
. Briefly, 293 or 293/CD40L cells were incubated in DMEM Complete
medium containing 100 ?g/mL ?-methyl-D-mannoside (Sigma Chemical Co.,
St. Louis, MO) at 37°C for 1 h. Cells were washed twice in ice-cold 1? PBS
containing 100 ?g/mL ?-methyl-D-mannoside, resuspended at 5 ? 106
cells/mL in ice-cold homogenization buffer [100 ?g/mL ?-methyl-D-manno-
side, 20 mM Tris-Cl, 10 mM NaCl, 0.1 mM MgCl2,0.1 mM phenylmethylsul-
fonyl fluoride (Sigma Chemical Co.), 0.5 ?g/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 ? 107cell equivalents/100 ?l Complete medium.
Parallel cultures of LCLs were established at 5 ? 104cells/well/100 ?l 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 incorpo-
ration. Cells were pulsed with 0.5 ?Ci [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
For comparison of two samples, a two-tailed Student’s t-test was used. Signif-
icance was set at P ? 0.05. Data in figures and tables are shown as mean ?
SD unless otherwise indicated.
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 . Since CD23 expression is gener-
ally 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 activa-
tion-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 in-
consistent with the phenotype of the fully activated LCL and
more closely resembles the pt1 primary B cells upon CD40L
stimulation (ref.  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
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 pos-
sibility, 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.
Establishment of LCLtetcells
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 condition-
TABLE 1. Analysis of Surface Activation Markers in LCLsa
MHC class II
aLCLs were analyzed for B cell lineage (CD20 and CD40), activation
(CD23, CD80, and CD86), and cell adhesion (CD54 and CD11a) proteins, as
well as CD38 (a cell-surface molecule found down-regulated on the majority of
LCLs) and MHC class II by FACS approximately 5 weeks post-transformation.
Numbers represent percent-positive/MFI and are representative of three inde-
pendent pt1-LCL populations.
622Journal of Leukocyte Biology
Volume 78, September 2005
ally 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 re-
quired for in vitro immortalization of B cells . 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 affect-
ing 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).
To confirm that a LMP1-mediated function was regulated
directly by tet in the LCLtetpopulation, 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 LCLtetpopulations (middle and bot-
tom panels) as well as in the pt1-LCLtetpopulation (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 re-
sults indicate that the pt1-LCLtetcells are dividing in re-
sponse to signals through LMP1 and that if there is an
intrinsic defect in the CD40-mediated proliferation, it is not
recapitulated with LMP1 signaling.
Fig. 1. pt1 LCL expresses a unique phenotype
with respect to activation markers. pt1 (open pro-
file) and control C3121-LCLs (darkly shaded pro-
file) were stained with mAb against human CD23,
CD38, CD40, CD54, CD80, and CD86 and ana-
lyzed for surface expression by flow cytometry. The
y-axis represents cell number, and the x-axis rep-
resents relative fluorescence intensity. Lightly
shaded profile represents isotype control.
Fig. 2. Pt1-LCL CD23/CD38 expression with ex-
tended 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, respec-
tively. The stippled line represents the isotype con-
trol for each antibody.
Lu et al.
Altered CD40-mediated proliferation in HIGM B cells623
Maintenance of defective CD23 expression
To determine the pattern of CD23 and CD38 expression,
pt1-LCLtetand control LCLtetpopulations 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 expres-
sion, 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-LCLtetcells 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-LCLtetcells and only a modest decrease
in CD23 expression in D11-LCLtetand C2-LCLtetcells (com-
pare 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 LCLtetlines is relatively indepen-
dent of changes in LMP1 expression and that signaling through
CD40 can induce a small shift in expression in the LCLtetlines.
In the presence of LMP1, there was a high level of surface
CD38 expression in pt-1-LCLtetcells. 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 concom-
itant 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-LCLtetcells. The loss of this LMP1/
CD40-mediated function suggests that an additional factor
Fig. 3. Characterization of conditional LMP1 expression in LCLtetcells. (A) Investi-
gation of LMP1 and EBNA2 expression in conditionally transformed LCLtetcells.
pt1-LCLtet(top panels) and control D11- and C2-LCLtetcells (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
LCLtetlines with respect to LMP1 expression. Pt1 and control LCLtetcells 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 tetracy-
cline. 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
624Journal of Leukocyte Biology
Volume 78, September 2005
required for CD38 down-regulation is absent in pt1-LCLtet
cells. It is important that these findings support the use of
LCLtetlines 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-
To further study the CD40 defect in pt1 B cells, we measured
and compared the CD40-specific proliferation response of pt1-
LCLtetcells to control LCLtetcells. Our initial work demon-
strated a minimal difference in the growth of pt1-LCLtetand
control LCLtetlines under different conditions of LMP1 expres-
sion (Fig. 3B). However, results from Figure 4 suggested that
low CD23 expression was not complemented in pt-LCLtetcells
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. Thus, these lines provide an ideal
system to study the effect of CD40 activation on proliferation in
the presence or absence of LMP1 signaling. LCLtetcells 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 (un-
stimulated) or 293/CD40 cells expressing CD40L (CD40-stim-
ulated). As shown in Figure 5A, costimulation of pt1 and
control LCLtetpopulations with LMP1 and 293/CD40L mem-
branes 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-LCLtetcells. 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-LCLtetcells
had significant increases in proliferation in response to CD40L,
pt1-LCLtetcells 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 af-
fects 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.
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. ). Ac-
cordingly, constitutive LMP1-dependent signaling through the
TRAFs may mask defective responses that are otherwise evi-
dent 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/CD38hiphenotype observ-
Fig. 4. Maintenance of the primary CD23/CD38
expression profile in pt1-LCLtetcells with LMP1 or
CD40L stimulation. Pt1-LCLtetand 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. LCLtetcells were then
stained with FITC-labeled CD23 mAb (left panels)
or FITC-labeled CD38 mAb (right panels) and an-
alyzed 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.
Lu et al.
Altered CD40-mediated proliferation in HIGM B cells625
able in pt1 primary B cells and therefore have limited use with
respect to studying the defect in impaired CD40 signaling. In
contrast, pt1-LCLtetcells cultured in the presence or absence
of LMP1 or CD40L maintain the specific CD23lo/CD38hiphe-
notype 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 LCLtetcells and that these signals fail to appreciably
modulate the expression of CD38 or CD23 in pt1-LCLtetcells.
Finally, we show that CD40-induced proliferation is also se-
verely reduced in the pt1-LCLtetcells and that normal levels of
cell cycling are achieved only by signaling through LMP1 and
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 path-
ways to drive B cell proliferation and differentiation (reviewed
ref. ). Specifically, mice lacking CD40 and expressing a
transgenic LMP1 restore many, but not all, of the CD40-
specific functions . 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 [53–55]. The mechanistic basis of these
stronger signals is likely related to the fact that signaling
through CD40, but not LMP1, results in ubiquitin- and pro-
teasome-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 LCLtetcells.
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 LCLtetcells. 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
[59–63]. Accordingly, EBNA2 has been shown to be necessary
for the efficient expression of LMP1 in B cells [64–66], 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 immortal-
ization, there would be a strong, selective advantage for growth
of LMP1?/CD23?cells. In contrast, in the LCLtetsystem,
LMP1 is expressed from a heterologous promoter, and there-
fore, 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 popu-
lation would remain predominantly CD23–.
Recent data support the premise that LMP1 mediates pro-
liferation through the induction of c-myc and Jun AP-1 family
members. In particular, c-myc expression is up-regulated
Fig. 5. Identification of a CD40-mediated proliferation defect in pt1-LCLtetcells. (A) Control and pt1-LCLtetcells 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) LCLtetlines 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,
626Journal of Leukocyte Biology
Volume 78, September 2005
within 30 min of LMP1 signals . 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 comple-
mentation of the proliferation defect only with LMP1 reinforces
the idea that a higher threshold of signal is required to over-
come the downstream defect. It is interesting that we see a
decrease in proliferation in control and pt1-LCLtetcells when
they receive signals through LMP1 and CD40 compared with
LMP1 alone. This suggests that when pt1-LCLtetcells 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. Dis-
section of the LMP1 and CD40 signaling pathways leading to
c-myc expression in control LCLtetand pt-LCLtetlines is cur-
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
LCLtetcells 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 nor-
mal and immunodeficient B cells.
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 Com-
mission 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 Dis-
eases, 2 T32 AI07403, awarded to Dr. Sidney Pestka, Univer-
sity 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 Fug-
man (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
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