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A Costimulatory Function for T Cell CD40
1
Melissa E. Munroe* and Gail A. Bishop
2
*
†‡
CD40 plays a significant role in the pathogenesis of inflammation and autoimmunity. B cell CD40 directly activates cells, which
can result in autoantibody production. T cells can also express CD40, with an increased frequency and amount of expression seen
in CD4
ⴙ
T lymphocytes of autoimmune mice, including T cells from mice with collagen-induced arthritis. However, the mech
-
anisms of T cell CD40 function have not been clearly defined. To test the hypothesis that CD40 can serve as a costimulatory
molecule on T lymphocytes, CD40
ⴙ
T cells from collagen-induced arthritis mice were examined in parallel with mouse and human
T cell lines transfected with CD40. CD40 served as effectively as CD28 in costimulating TCR-mediated activation, including
induction of kinase and transcription factor activities and production of cytokines. An additional enhancement was seen when both
CD40 and CD28 signals were combined with AgR stimulation. These findings reveal potent biologic functions for T cell CD40 and
suggest an additional means for amplification of autoimmune responses. The Journal of Immunology, 2007, 178: 671– 682.
T
he 50-kDa membrane receptor of the TNFR superfamily,
CD40, is expressed by APC, including dendritic cells,
macrophages, and B lymphocytes (1, 2). The ligand for
CD40, CD154, is expressed on activated T cells and allows for
interactions with APC during the cognitive phase of the immune
response, as well as directing effector T cell-dependent B cell ac-
tivation (3). Such interactions have been directly implicated in
autoimmunity. Blocking CD40-CD154 interactions has been
shown to either prevent or alleviate such diseases as insulin de-
pendent diabetes mellitus (4), arthritis (5), and systemic lupus er-
ythematosus (6), the latter two benefiting from abrogated T cell-
dependent autoantibody production (7, 8).
The role of CD40 as a direct signal receptor has now been ex-
panded to T cells. Shortly after CD154 was cloned, it was dem-
onstrated that CD154 can augment mitogen and TCR-mediated
proliferation of CD4
⫹
and CD8
⫹
T cells (9), although the lack of
CD154-specific Abs at that time precluded further investigation.
Although a specific biologic role for CD40 on CD8
⫹
T cells re
-
mains undefined (10 –13), it has been shown that autoimmune-
prone strains of mice have increased numbers of CD40
⫹
CD4
⫹
T
cells compared with normal strains (14). The most extensively
studied of these is the NOD mouse (15–17).
To date, the physiologic role(s) of CD40 on T cells has not been
characterized, nor have the mechanisms by which CD40 affects T
cell function been defined. We have previously studied CD40 as an
important signaling molecule on B lymphocytes, delivering signals
alone and synergistically with the BCR (18), leading to NF-
B and
JNK pathway activation and subsequent proliferation, secretion of
cytokines, Ig production and isotype switching (reviewed in Refs.
19 and 20). Like B cells, T cells have been shown to use TNFR
family members as costimulatory molecules for Ag receptor stim-
ulation (reviewed in Refs. 21 and 22). We hypothesized that co-
stimulation is a plausible role for CD40 on T cells. In the studies
presented here, we determined that T cell CD40 augmented CD3
and CD3 plus CD28-mediated cytokine production in T cells from
mice which have developed collagen-induced arthritis (CIA),
3
as
well as in T cell lines stably transfected with CD40. Although
CD40 signals alone did not activate NFAT or IL-2 secretion, CD40
ligation markedly augmented CD3 and CD3 plus CD28 responses.
As in B cells, T cell CD40 was able to efficiently bind the adaptor
proteins TNFR-associated factors (TRAF), activate NF-
B and
AP-1 pathways, and stimulate TNF-
␣
secretion. Taken together,
these findings reveal that CD40 can act as a powerful signaling
receptor on T as well as B lymphocytes, a function that may have
important implications for T-B interactions in autoimmune
diseases.
Materials and Methods
Cells
The mouse T cell line 2B4.11 (23) and human T cell line Jurkat (24) have
been described previously. Cell lines and their stable transfectants express-
ing hCD40 were maintained in RPMI 1640 containing 10% FCS (Hy-
Clone), 10
M 2-ME, and antibiotics. These subclones are referred to as
2B4.hCD40 and J.hCD40. Hi5 insect cells expressing hCD154 have been
described and characterized previously (25, 26). These cells grow at 26°C
and rapidly die to form membrane fragments at 37°C and therefore do not
overgrow cell cultures.
Stable transfections
Cell lines were stably transfected with a previously reported hCD40 ex-
pression plasmid (27) as described previously (28). G418-resistant clones
were analyzed for expression of hCD40 using a FACScan flow cytometer
(BD Biosciences) and mean channel fluorescence (MCF) determined using
FlowJo software.
Reagents
Recombinant mouse TNF-
␣
, IL-2, and IFN-
␥
were purchased from Pep-
roTech. Streptavidin-HRP was purchased from Jackson ImmunoResearch
Laboratories. ELISA TMB peroxidase substrate was purchased from KPL.
Tosylactivated Dynabeads for Ab conjugation (per manufacturer’s instruc-
tions) were purchased from Dynal Biotech. PMA and ionomycin were
purchased from Sigma-Aldrich.
*Department of Microbiology and
†
Department of Internal Medicine, University of
Iowa, Iowa City, IA 52242; and
‡
Veterans Affairs Medical Center, Iowa City, IA
52242
Received for publication June 21, 2006. Accepted for publication October 17, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health and the
Veterans’ Administration (to G.A.B.) and postdoctoral fellowship support provided
by the American Heart Association and the American Cancer Society (to M.E.M.).
2
Address correspondence and reprint requests to Dr. Gail A. Bishop, 2193B MERF,
Department of Microbiology, University of Iowa, Iowa City, IA 52242. E-mail ad-
dress: gail-bishop@uiowa.edu
3
Abbreviations used in this paper: CIA, collagen-induced arthritis; CII, type II
chicken collagen; MCF, mean channel fluorescence; TRAF, TNFR-associated factor.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
Antibodies
The 1C10 (anti-mCD40, rat IgG2a), 72-2 (rat IgG2a isotype control), and
G28-5 (anti-hCD40, mouse IgG1) hybridomas were purchased from the
American Type Culture Collection. MOPC-31c (mouse IgG1 isotype con-
trol) was from Sigma-Aldrich. Polyclonal rabbit anti-TRAF2 Ab was from
MBL. Polyclonal mouse anti-yy1 and polyclonal rabbit anti-TRAF3, anti-
TRAF1, anti-TRAF6, and anti-hCD40 Abs were from Santa Cruz Biotech-
nology. Polyclonal rabbit anti-I
B
␣
, anti-phosphorylated I
B
␣
, anti-
NF
B2 p100/p52, anti-JNK, and anti-phosphorylated JNK Abs were from
Cell Signaling Technology. Mouse anti-actin Ab (C4) was from Chemicon
International. Peroxidase-labeled goat anti-rabbit and goat anti-mouse IgG
Abs were from Jackson ImmunoResearch Laboratories. Anti-mouse CD3
(145-2C11; Armenian hamster IgG1), anti-human CD3 (OKT3; mouse
IgG2a), anti-mouse CD28 (37.51; hamster IgG), anti-human CD28
(CD28.2; mouse IgG1), and relevant isotype control Abs were purchased
from eBioscience. PE-labeled anti-mouse CD3 (145-2C11; Armenian ham-
ster IgG1), FITC labeled anti-mouse CD40 (HM40-3; Armenian hamster
IgM), anti-human CD40 (5C3; mouse IgG3), anti-human CD154 (TRAP1,
mouse IgG1), anti-mouse CD80 (16-10A1; Armenian hamster IgG2), anti-
mouse CD86 (GL1; rat IgG2a), anti-mouse CD95 (Jo2; Armenian hamster
IgG2), and relevant isotype control Abs were purchased from BD Pharm-
ingen. FITC labeled anti-mouse CD25 (PC61.5; rat IgG1), anti-mouse
CD54 (YN1/1.7.4; rat IgG2b), and anti-mouse CD11
␣
(M17/4; rat IgG2a)
Abs were purchased from eBioscience. Biotin-labeled anti-mouse CD154
(MR1; Armenian hamster IgG) and relevant isotype control Abs were pur-
chased from eBioscience. Alexa Fluor 488-labeled streptavidin was pur-
chased from Molecular Probes/Invitrogen Life Technologies. Anti-mouse
IL-2 and IFN-
␥
(coating and biotinylated) ELISA Abs were purchased
from Caltag Laboratories. Anti-human IL-2 and anti-mouse TNF-
␣
(coat-
ing and biotinylated) ELISA Abs were purchased from eBioscience.
Mice/CIA induction
Female C57BL/6 mice were purchased at 5– 8 wk of age from the National
Cancer Institute. Mice were housed in a specific pathogen-free barrier fa-
cility with restricted access, and all procedures were performed as ap-
proved by the University of Iowa Animal Care and Use Committee. CIA
was induced based on the methods of Campbell et al. (29). Briefly, mice
were either left naive, immunized in the tail s.c. with 100
g of type II
chicken collagen (CII; Sigma-Aldrich) dissolved in 10 mM acetic acid and
emulsified in IFA (Sigma-Aldrich) containing 5 mg/ml H37 RA heat-killed
mycobacteria (CFA; Difco Laboratories), or immunized with 10 mM acetic
acid emulsified in CFA. Mice were monitored for limb erythema and swell-
ing and paws measured (each paw recorded individually; four measure-
ments per mouse) with calipers two to three times per week (4026F; Mi-
tutoyo) (29, 30). All mouse studies were reviewed and approved by the
University of Iowa Animal Care and Use Committee.
T cell isolation
T cells were isolated from mouse spleens 70 days postimmunization.
Briefly, spleens from euthanized mice were teased apart with forceps,
erythrocytes lysed in ACK buffer, and remaining cells placed over a T cell
enrichment column per manufacturer’s protocol (R&D Biosystems). T
cells were enriched to ⬃90% purity as determined by flow cytometry (see
Table I). Primary mouse T cells were cultured in Click’s medium contain-
ing 1% nutridoma-SP (Roche), 10
M 2-ME, and antibiotics, or stained for
CD3 (PE) and CD40 (FITC) and analyzed by flow cytometry.
NF-
B/NFAT/AP-1 dual luciferase reporter assays
2B4.hCD40 or J.hCD40 cells (1.5 ⫻ 10
7
) were transiently transfected with
20
gof4⫻ NF-
B, 40
gof4⫻ NFAT, or 40
gof7⫻ AP-1 luciferase
reporter plasmid (31), and 1
gofRenilla luciferase vector (pRL-null;
Promega) by electroporation. Cells were rested on ice for 15 min, then
stimulated (2 ⫻ 10
6
cells/ml) for 6 h (NF-
B) or 24 h (NFAT/AP-1) with
10
g/ml anti-hCD40 or isotype controls, and/or 5 ⫻ 10
5
beads/ml anti-
CD3 or anti-CD3
⫹
CD28-coated Dynabeads. After stimulation, cells were
pelleted, lysed, and assayed for relative luciferase activity (NF-
B, NFAT,
or AP-1:Renilla) per manufacturer’s protocol (Promega) using a Turner
Designs 20/20 luminometer, with settings of a 2-s delay followed by a
10-s read.
I
B
␣
/JNK assays
2B4.hCD40 or J.hCD40 cells (2 ⫻ 10
6
) were stimulated for indicated times
with culture medium, 10
g of anti-hCD40 Ab (or respective isotype con-
trols) and/or 5 ⫻ 10
5
beads/ml anti-CD3 or anti-CD3 plus CD28-coated
Dynabeads) to induce phosphorylation and degradation of the proteins
blotted. The cells were pelleted by centrifugation, lysed and analyzed by
SDS PAGE and Western blotting. Peroxidase-labeled Abs were visualized
on Western blots using a chemiluminescent detection reagent (Pierce).
NF-
B2 activation
2B4.hCD40 or J.hCD40 cells (2 ⫻ 10
6
) were stimulated for indicated times
with culture medium, 10
g of anti-hCD40 Ab (or respective isotype con-
trols) and/or 5 ⫻ 10
5
beads/ml anti-CD3 or anti-CD3 plus CD28-coated
Dynabeads) to induce RelB activation, and processing of p100 to p52. The
cells were pelleted by centrifugation and cytoplasmic and nuclear fractions
isolated as described previously (32). Samples were analyzed by SDS-
PAGE and Western blotting. Peroxidase-labeled Abs were visualized on
Western blots using a chemiluminescent detection reagent.
FIGURE 1. CD40 expressed on T cells from mice
with CIA. A, C57BL/6 mice were immunized with CFA
only, CII plus CFA, or remained naive, as described in
Materials and Methods. Paws were measured two to
three times per week from days 21–70 postimmuniza-
tion. Mice receiving CII plus CFA had significant (p ⬍
0.001) paw swelling compared with CFA or naive con-
trols. Data represent two experiments (4 mice/group/
experiment; 8 mice/32 paws/data point total). B,
Spleens from mice in A were pooled (2 spleens/pool, 4
pools/group), and T cells were isolated as described in
Materials and Methods. Cells were stained with anti-
mouse CD40 (FITC) and anti-mouse CD3 (PE) or iso-
type control Abs and analyzed by flow cytometry.
Quadrants were drawn based on staining with isotype
control Abs.
672 SIGNALING BY CD40 IN T CELLS
Cytokine ELISA
Primary mouse T cells or 2B4.hCD40 or J.hCD40 cells (4 ⫻ 10
5
) were
stimulated at optimal, empirically derived time points with culture me-
dium, 1
g/ml anti-hCD40 Ab and/or anti-CD28, or plate-bound anti-CD3
(or respective isotype controls). Cytokine concentrations in culture super-
natants were determined by ELISA, using cytokine-specific coating Abs
and biotinylated detection Abs. Streptavidin-HRP binding to biotinylated
detection Abs was visualized with TMB substrate and the reaction was
stopped with 0.18 M H
2
SO
4
. Plates were read at 450 nm by a Spectra
-
Max
250
Reader (Molecular Devices). Data were analyzed with SoftMax
Pro software (Molecular Devices); unknowns were compared with a stan-
dard curve containing at least five to seven dilution points of the relevant
recombinant cytokine on each assay plate. In all cases, the coefficient of
determination for the standard curve (r
2
) was ⬎0.98. ELISA unknowns
were diluted to fall within the standard values.
TRAF recruitment to receptors in detergent-insoluble
microdomains (Rafts) and immunoprecipitation.
2B4.hCD40 or J.hCD40 (1 ⫻ 10
7
) cells were stimulated with 10
g of anti-hCD40 Ab (or isotype control Abs) or Hi5 cells ex-
pressing hCD154 (or Hi5 cells expressing WT baculovirus; 1:4
Hi5 cells:lymphocytes) for 15 min at 37°C to induce recruitment of
TRAFs to membrane rafts and allow formation of CD40 signaling
complexes, as described previously (33). Detergent (1% Brij 58)-
soluble and insoluble fractions were separated as described previ-
ously (34). Samples of soluble and insoluble lysates were reserved
for SDS-PAGE separation and analysis by Western blotting. The
remainder of the lysates were immunoprecipitated with protein
G-Sepharose beads (Amersham Biosciences) prewarmed with anti-
hCD40 Ab for3hat4°C. The immunoprecipitation complexes
were washed four times with lysis buffer before separation by
SDS-PAGE and analysis by Western blot.
Up-regulation of cell surface proteins
In experiments evaluating activation-induced up-regulation of surface pro-
teins, 2B4.11 or 2B4.hCD40 cells were incubated with the indicated stimuli
in 96-well plates (1–2 ⫻ 10
5
cells/well). After 48–72 h, the cells were
washed, then incubated for 20 min on ice in PBS-0.5% FCS-0.02% sodium
azide containing 2.5 mM EDTA. EDTA treatment helped to dissociate cell
aggregates formed upon CD40 stimulation. Following the EDTA incuba-
tions, cells were washed and stained (in the absence of EDTA) with Abs for
analysis by flow cytometry.
Statistical analyses
Analyses were performed with GraphPad Instat software. A two-tailed
paired Student’s t test was used to determine significance between groups
in CIA experiments, for cytokine ELISA, surface molecule up-regulation
experiments, and luciferase reporter assays.
Results
CD40 expression on T cells from mice with CIA
CIA is a frequently used mouse model of inflammatory rheumatoid
arthritis that is both Ab and T cell dependent (35, 36). It has been
previously demonstrated that mouse strains prone to autoimmune
diabetes have an increased number of T cells expressing CD40 that
correlates with development of pathology (14). We examined
FIGURE 2. CD40 enhances CD3 and CD3 plus CD28-mediated cyto-
kine production in T cells from mice with CIA. Splenic T cells from
C57BL/6 mice immunized with CII plus CFA, CFA only, or remaining
naive were isolated as described in Materials and Methods. Cells were
stimulated with plate-bound anti-CD3 ⫾ anti-CD28 and/or anti-mouse
CD40 Abs (compared with medium (Med)/isotype control (IC)). Culture
supernatants were collected and assayed for IL-2 (A, 48 h), IFN-
␥
(B,72h),
or TNF-
␣
(C, 24 h) by ELISA. Data represent the mean ⫾ SEM of du-
plicate samples of three independent experiments. There was no difference
between medium and isotype control samples.
Table I. Increased number of CD40
⫹
T cells in CIA mice
a
CII/CFA CFA Only Naive
Splenocytes (pre-T cell isolation)
Percentage of CD3
⫹
T cells
33.19 ⫾ 0.63 33.52 ⫾ 0.28 34.44 ⫾ 0.58
No. of CD3
⫹
T cells (⫻10
6
)
5.67 ⫾ 0.17 5.11 ⫾ 0.17 4.81 ⫾ 0.35
Percentage of CD3/CD40
⫹
T cells
9.31 ⫾ 0.52
b
4.30 ⫾ 0.37 4.02 ⫾ 0.46
No. of CD3/CD40
⫹
T cells (⫻10
5
)
15.90 ⫾ 1.54
c
6.55 ⫾ 0.60 6.06 ⫾ 0.85
Negatively selected T cells
Percentage of CD3
⫹
T cells
88.67 ⫾ 1.56 87.41 ⫾ 3.28 89.01 ⫾ 1.67
No. of CD3
⫹
T cells (⫻10
6
)
3.85 ⫾ 1.75 3.24 ⫾ 0.30 3.68 ⫾ 0.34
Percentage of CD3/CD40
⫹
T cells
7.53 ⫾ 0.70
d
2.96 ⫾ 0.26 2.80 ⫾ 0.56
No. of CD3/CD40
⫹
T cells (⫻10
5
)
3.31 ⫾ 0.48
e
1.11 ⫾ 0.17 1.40 ⫾ 0.11
a
T cells were isolated as described in Materials and Methods from spleens of C57BL/6 mice receiving CII/CFA, CFA only,
or remaining naive (four mice per group for two experiments; two spleens per pool, four pools total) 70 days postimmunization.
Cells were stained with anti-mouse CD40 (FITC) and anti-mouse CD3
⑀
(PE) or isotype control mAbs and analyzed by flow
cytometry. Spleens from CII/CFA mice had significantly more CD3
⫹
CD40
⫹
T cells than either CFA or naïve controls.
b
p ⬍ 0.01 (CII/CFA compared with CFA only or naive).
c
p ⬍ 0.001 (CII/CFA compared with CFA only or naive).
d
p ⫽ 0.01 (CII/CFA compared with CFA only or naive).
e
p ⬍ 0.05 (CII/CFA compared with CFA only or naive).
673The Journal of Immunology
whether this was also true during the inflammatory process of CIA
development. C57BL/6 mice injected with CII/CFA developed
significant paw swelling ( p ⬍ 0.001) compared with mice given
CFA only or naive controls (Fig. 1A). Splenic T cells from these
mice were isolated and evaluated for dual expression of CD3 and
CD40. A representative FACS plot of isolated T cells is presented
in Fig. 1B; quantitation is presented in Table I. Small numbers of
cells in the FACS samples that were CD3
⫺
were also CD40
⫺
and
therefore unlikely to be APC (Fig. 1B and Table I, line 5). Data in
Table I demonstrate that there are more than twice as many CD40
⫹
T cells in the spleens of mice that received CII/CFA compared
with mice that received CFA only or naive controls ( p ⱕ 0.01 for
percentage, line 3, or absolute number, line 4). This is true whether
evaluating the percentage or absolute numbers of CD3
⫹
CD40
⫹
cells as a part of the whole spleen (Table I, lines 3 and 4) or after
enrichment of T lymphocytes (Table I, lines 7 and 8). This phe-
nomenon was not due to an alteration in the splenic T cell popu-
lation as a whole, or post-T cell isolation after CII/CFA immuni-
zation. There was no significant difference between immunization
groups in the percentage (first line) or absolute numbers (second
line) of CD3
⫹
T cells, whether evaluating mixed splenocyte, or
isolated T cell populations (Table I).
T cell CD40 as a costimulatory molecule
The findings discussed above raise the possibility that CD40 ex-
pressed by T cells may play a role in T cell activation. Experiments
presented in Fig. 2 explored whether CD40 engagement could aug-
ment CD3 or CD3 plus CD28-mediated cytokine production by
splenic T cells isolated from mice immunized with CII/CFA. Be-
cause minimal cytokine production was detected from cells stim-
ulated with medium alone, isotype control Abs, or anti-CD3 Ab, it
is unlikely that the small amounts of residual non-T cells found
after T cell enrichment (Fig. 1 and Table I) are APC. As expected,
anti-CD3 Ab induced a modest amount of IL-2 (Fig. 2A), and
anti-CD28 Ab significantly enhanced CD3-mediated IL-2 produc-
tion in all three experimental groups (CD3 vs CD3 plus CD28:
naive, p ⫽ 0.02; CFA only, p ⫽ 0.01; CII/CFA, p ⬍ 0.0001).
CD40 stimulation alone did not induce any appreciable IL-2 pro-
duction. However, in T cells from mice immunized with CII/CFA,
CD40 significantly enhanced the level of CD3 ( p ⫽ 0.002)- and
CD3 plus CD28 ( p ⫽ 0.009)-mediated IL-2 production. This en-
hancement did not occur in the largely CD40-negative T cells from
naive or CFA-treated mice because there was no significant dif-
ference in IL-2 produced between T cells treated with agonists for
CD3 vs CD3 plus CD40 or CD3 plus CD28 vs CD3 plus CD28
plus CD40.
In addition to IL-2 production, we also evaluated the ability of
CD40 to contribute to the production of the proinflammatory cy-
tokines IFN-
␥
(Fig. 2B) and TNF-
␣
(Fig. 2C) by T cells from CIA
mice compared with controls. As with IL-2 secretion, CD28 en-
hanced CD3-mediated IFN-
␥
(Fig. 2B, p ⱕ 0.001 for all groups)
and TNF-
␣
(Fig. 2C, p ⱕ 0.001 for all groups) production in T
cells from all three mouse groups. Unlike IL-2, anti-CD40 alone
induced a significant amount of both IFN-
␥
(Fig. 2B, p ⫽ 0.001)
and TNF-
␣
(Fig. 2C, p ⬍ 0.0001), but only in T cell cultures from
mice immunized with CII/CFA and not in cultures from control
mice. CD40 significantly enhanced CD3 and CD3 plus CD28-me-
diated IFN-
␥
(Fig. 2B, p ⱕ 0.02 for both stimuli) and TNF-
␣
( p ⱕ
0.01 for both stimuli) production, but only in mice immunized with
CII/CFA. These data indicate that CD40 can act as a TCR co-
stimulator and that it can cooperate in a nonredundant manner with
CD28 to further enhance T cell cytokine production.
The above ex vivo experiments contained a mixed population of
CD40-expressing and nonexpressing T cells (Fig. 2), with a rela-
tively small percentage of T cells expressing CD40 (Fig. 1 and
Table I). This small percentage limited detailed molecular charac-
terization of CD40 function on CD40-expressing T cells, although
the data presented in Fig. 2 indicate that this population has sig-
nificant biologic activity distinct from that of CD4
⫹
T cells that do
not express CD40. We thus wanted to complement these experi-
ments with stimulation of homogeneous populations of CD40
⫹
T
cells, as well as explore CD40 signaling pathways. Because of
limiting numbers of CD40
⫹
T cells in CIA mice that would require
potentially function-altering positive selection for isolation,
CD40
⫹
T cell lines were a desirable alternative. We thus stably
transfected mouse 2B4.11 (2B4.hCD40) and human Jurkat
(J.hCD40) T cell lines with hCD40 (Fig. 3, A and B; MCF for
2B4.hCD40 ⫽ 704.66 vs 147.48 for 2B4.11; MCF for J.hCD40 ⫽
245.72 vs 199.11 for Jurkat) and evaluated the ability of CD40 to
activate IL-2 production (Fig. 4, A and B). Interestingly, even with
PMA/ionomycin stimulation, there was no detectable CD154 ex-
pression by either 2B4.hCD40 or J.hCD40 cells (Fig. 3, C and D),
although we see CD154 expression by Hi5 insect cells infected
with a baculovirus encoded to express CD154 (data not shown).
Because these clones do not express CD154, autocrine CD40 stim-
ulation does not contribute to subsequent findings.
We first evaluated the ability of the transfected 2B4.hCD40 and
J.hCD40 to secrete cytokines in response to CD3 ⫾ CD28 and/or
CD40 stimulation (Fig. 4). To closely mimic the design of our
mouse ex vivo experiments, in which biologic responses to CD40
FIGURE 3. Expression of hCD40 and CD154 on 2B4.11 and Jurkat cell
lines. A and B, 2B4.11 (A) or Jurkat (B) cells were transfected with DNA
encoding for hCD40. Cells were stained with anti-human CD40 and ana-
lyzed by flow cytometry. Gray profiles represent anti-hCD40 mAb staining
of untransfected cells; black profiles represent staining of transfected cells.
There was no significant staining of cells with an isotype control Ab. C and
D, 2B4.hCD40 (C) or J.hCD40 (D) cells remained untreated or were
treated for 6 h with 1
g/ml PMA plus 200
M ionomycin, then stained
with anti-CD154 and analyzed by flow cytometry. Dashed profiles repre-
sent staining of untreated cells with isotype control mAb; gray profiles
represent anti-CD154 staining of untreated cells; black profiles represent
anti-CD154 staining of PMA-ionomycin-treated cells. There was no sig-
nificant staining of PMA-ionomycin-treated cells with an isotype control
Ab. Similar results were seen at 24 and 48 h (data not shown).
674 SIGNALING BY CD40 IN T CELLS
were made by small percentages of CD40
⫹
T cells, CD40 trans
-
fected T cells were cocultured with their untransfected parent cell
lines at various ratios ranging from 6 to 100% transfected cells.
CD40 stimulation alone of cells stably expressing the transfected
receptor did not induce IL-2 production (Fig. 4, A and B). While
CD28 ligation appropriately enhanced CD3-mediated IL-2 produc-
tion in all T cell clones, CD40 stimulation enhanced IL-2 produc-
tion via CD3 or CD3 plus CD28 only if cells stably expressing
CD40 were present in the culture well, similar to responses of
primary T cell cultures from CIA mice (Fig. 2B). CD40 was able
to act in costimulatory fashion with CD3 with only 6% of CD40
⫹
cells in the coculture ( p ⫽ 0.01 when 6% 2B4.hCD40 present, p ⬍
0.0001 at 100% 2B4.hCD40; p ⬍ 0.001 when 6% J.hCD40
present, p ⬍ 0.0001 at 100% J.hCD40), just as was seen in primary
T cells from CIA mice (Fig. 2B). Enhancement of CD3 plus CD28-
mediated IL-2 production by CD40 increased with greater num-
bers of transfected cells in the well ( p ⫽ 0.02 at 100%
2B4.hCD40; p ⫽ 0.005 when 6% J.hCD40 present, p ⬍ 0.0001 at
100% J.hCD40). Of particular interest is that CD40 could ulti-
mately enhance CD3-mediated IL-2 production to a degree similar
to that of CD28 in 2B4.hCD40 cells (Fig. 4A) and significantly
better in J.hCD40 cells (Fig. 4B, p ⬍ 0.0001 at 100% J.hCD40).
Thus, the response of both 2B4.hCD40 and J.hCD40, even when
only 6% of the T cells in each culture were CD40
⫹
cells, closely
paralleled the responses seen in freshly isolated T cells, validating
these cell lines as useful experimental models for the study of
CD40 function in T cells.
In addition to IL-2 production, we evaluated the ability of CD40
to contribute to the production of proinflammatory cytokines
TNF-
␣
(Fig. 4C) and IFN-
␥
(Fig. 4D). These cytokines were
readily detected in culture supernatants of stimulated 2B4.11 or
2B4.hCD40 cells, but not Jurkat cells, which have been propagated
as an IL-2-producing human leukemia T cell line (24) and which
require overexpression of other proteins to induce TNF-
␣
(37) and
IFN-
␥
(38) secretion. Unlike IL-2, CD40 stimulation alone was
able to stimulate both production of TNF-
␣
( p ⫽ 0.02 when 6%
2B4.hCD40 cells were present, p ⫽ 0.001 at 100%) and IFN-
␥
( p ⫽ 0.02 when 6% 2B4.hCD40 cells were present, p ⬍ 0.0001 at
100%) in those cells stably expressing CD40. Similar to IL-2,
CD40 stimulation of T cells expressing CD40 was able to augment
both CD3 and CD3 plus CD28-mediated cytokine production.
The role of T cell CD40 in up-regulation of cell surface
molecules
CD40 signaling is known to up-regulate a number of cell surface
molecules (27), including CD11
␣
(LFA-1), CD54 (ICAM-1), and
CD95 (Fas), all of which play significant roles in T cell activation
(39 – 41). We also evaluated the ability of CD40 to contribute to
CD80 (B7-1) and CD86 (B7-2) up-regulation, an important com-
ponent of activation by other CD40-expressing cells (42), as well
as CD25, a marker of T cell activation (43). We compared baseline
expression of these cell surface molecules on 2B4.11 and
2B4.hCD40 cells, as well as receptor-specific up-regulation of
these molecules postactivation (Fig. 5). Both 2B4.11 and
2B4.hCD40 expressed basal CD80 (Fig. 5C), CD86 (Fig. 5D), and
CD11
␣
(LFA-1; Fig. 5E), with higher expression in 2B4.hCD40
cells. CD3 plus CD28 stimulation induced up-regulation of all sur-
face molecule tested in both 2B4.11 and 2B4.hCD40 cells (right
graph in each panel, p ⱕ 0.01 for all groups). CD40 signals alone,
and in conjunction with CD3 or CD3 plus CD28 stimulation, in-
duced up-regulation of all surface molecules tested in 2B4.hCD40
cells, but not the nontransfected 2B4.11 cell line. CD40 signals
augmented CD3-mediated up-regulation of cell surface molecules
similar to CD28 stimulation in 2B4.hCD40 cells ( p ⬍ 0.01 CD3 vs
CD3 plus CD28 or CD3 plus CD40 for all groups, no significant
difference in response between CD3 plus CD28 and CD3 plus
CD40 stimulation), with a maximal response achieved when triple
CD3 plus CD28 plus CD40 stimulation was given. Interestingly,
CD40 signaling induced a maximal enhancement of the costimu-
latory response in 2B4.hCD40 cells in conjunction with CD3 plus
CD28 to up-regulate CD25 expression (Fig. 5A).
The role of T cell CD40 in activation of cytokine signaling
pathways
Experiments presented above demonstrate that CD40 can act as a
costimulatory molecule to enhance CD3 and CD3 plus CD28-me-
diated T cell activation. NFAT, AP-1, and NF-
B are known to be
involved in the activation of several T cell proinflammatory cyto-
kine genes, including those encoding IL-2 (44– 46), IFN-
␥
(47,
48), and TNF-
␣
(45, 49). We and others have demonstrated that
CD40-mediated activation of B lymphocytes involves AP-1 (50,
51) and NF-
B (31, 52) signaling pathways, and there is evidence
FIGURE 4. CD40 enhances CD3 and CD3 plus
CD28-mediated cytokine production in 2B4.hCD40
and J.hCD40 lines. 2B4.11 ⫾ 2B4.hCD40 (A, C, and D)
or Jurkat ⫾ J.hCD40 (B) cells were stimulated with
plate-bound anti-CD3 ⫾ anti-CD28 and/or anti-CD40
Abs (compared with medium (Med)/isotype control
(IC)). Culture supernatants were collected after 24
(TNF-
␣
), 48 (IL-2), or 72 (IFN-
␥
) h and assayed for
IL-2 (A and B), TNF-
␣
(C), or IFN-
␥
(D) by ELISA.
Data represent the mean ⫾ SEM of triplicate samples
from two independent experiments. There was no dif-
ference between medium and isotype control samples.
675The Journal of Immunology
of CD40-mediated NFAT activation (51, 53). We asked if this is
also the case for T cell CD40.
CD3 or CD40 signals alone induced minimal NFAT reporter
gene activation compared with control stimuli in 2B4.hCD40 cells
(Fig. 6A), while CD28 in conjunction with CD3 signals induced an
increased response ( p ⫽ 0.001) compared with medium/isotype
controls or CD3 single stimulation in 2B4.hCD40 cells (Fig. 6A;
p ⬍ 0.0001 compared with medium/isotype controls or CD3 in
J.hCD40 cells, Fig. 6B). While CD40 signaling was able to en-
hance CD3-mediated NFAT activation ( p ⬍ 0.001 in both
2B4.hCD40 and J.hCD40 cells), it was not to the same degree as
CD28-mediated enhancement. The greatest NFAT response was
achieved via engagement of all three receptors: CD3 plus CD28 plus
CD40 ( p ⫽ 0.007 compared with CD3 plus CD28, p ⬍ 0.001 com-
pared with CD3 plus CD40 in 2B4.hCD40 cells, p ⫽ 0.017 compared
with CD3 plus CD28, p ⫽ 0.003 compared with CD3 plus CD40 in
J.hCD40 cells). Although CD3 signals alone were able to trigger in-
creased NFAT activity in J.hCD40 cells ( p ⫽ 0.001; Fig. 6B), CD28
and CD40 signals augmented this response in a manner similar to
their effects in 2B4.hCD40 cells.
We next investigated the ability of CD40 to signal via AP-1 in
T cells (Fig. 6). Unlike NFAT (Fig. 6), CD40 signals alone acti-
vated AP-1 ⬃2-fold over control stimuli in 2B4.hCD40 cells (Fig.
7A; p ⫽ 0.005). While CD40 signals enhanced CD3-mediated
AP-1 activation in both cell lines ( p ⬍ 0.001 in 2B4.hCD40 cells;
p ⫽ 0.009 in J.hCD40 cells), CD40 was a less efficient costimu-
lator in this response than CD28 (CD3 vs CD3 plus CD28: p ⫽
0.007 in 2B4.hCD40 cells; p ⫽ 0.005 in J.hCD40), and the
FIGURE 5. CD40 enhances CD3
and CD3 plus CD28-mediated cell
surface molecule up-regulation.
2B4.11 or 2B4.hCD40 cells were
stimulated with plate-bound anti-
CD3 ⫾ anti-CD28 and/or anti-CD40
Abs (compared with isotype control)
for 48 –72 h, then analyzed by flow
cytometry to determine expression
levels of surface proteins. Histograms
(gray ⫽ isotype control; black ⫽ sur-
face molecule) represent baseline ex-
pression (stimulated with isotype con-
trol Ab), with corresponding median
channel fluorescence (⌬MCF ⫽ MCF
of surface molecule ⫺ MCF of iso-
type control staining) represented in
the left graph of each panel. Changes
in MCF for each surface molecule
tested (poststimulation minus base-
line) is represented in the right graph
of each panel. A–F, Data represent the
mean ⫾ SEM of MCF from two in-
dependent experiments.
676 SIGNALING BY CD40 IN T CELLS
maximal response was again achieved with simultaneous engage-
ment of CD3 plus CD28 plus CD40 ( p ⫽ 0.02 compared with CD3
plus CD28, p ⫽ 0.03 compared with CD3 plus CD40 in
2B4.hCD40 cells, p ⫽ 0.01 compared with CD3 plus CD28, p ⫽
0.002 compared with CD3 plus CD40 in J.hCD40 cells). CD40
signals in B cells strongly activate phosphorylation of the AP-1
family member c-jun, via activation of JNK (54 –56). We evalu-
ated the ability of T cell CD40 to contribute to JNK activation
alone or in combination with CD3 or CD3 plus CD28. Strikingly,
in both 2B4.hCD40 and J.hCD40 cells (Fig. 8, A and B), only when
CD40 was engaged was strong phosphorylation of JNK observed,
with no phosphorylation seen via CD3 signaling and minimal
phosphorylation via CD3 plus CD28 signaling.
It is well established that CD40 signals lead to NF-
B activation in
B cells (52, 56–58) and we asked if this was also true for T cell CD40.
CD40 alone initiated a strong NF-
B response in 2B4.hCD40 (Fig.
9A, p ⬍ 0.001) and J.hCD40 (Fig. 9B, p ⬍ 0.001) cells, while CD3
evoked a minimal response compared with controls. CD3 plus CD40
activated NF-
B more than CD40 alone (CD40 vs CD3 plus CD40:
p ⫽ 0.03 in 2B4.hCD40 cells, p ⫽ 0.02 in J.hCD40 cells) and 2.5- to
5-fold greater than CD3 plus CD28 (CD3 plus CD28 vs CD3 plus
CD40: p ⫽ 0.005 in 2B4.hCD40 cells; p ⫽ 0.004 in J.hCD40 cells),
while the combination of CD3 plus CD28 plus CD40 signals provided
an additional 20% increase in NF-
B activation ( p ⬍ 0.001 compared
with CD3 plus CD28, p ⫽ 0.03 compared with CD3 plus CD40 in
2B4.hCD40 cells; p ⫽ 0.01 compared with CD3 plus CD28, p ⫽ 0.04
compared with CD3 plus CD40 in J.hCD40 cells).
Early events in NF-
B activation have been shown to include
phosphorylation and degradation of I
B
␣
(59), and recent studies
suggest that an alternate pathway for activating NF-
B (NF-
B2),
in which p100 is processed to p52 and shuttled to the nucleus by
RelB, is also used by some TNFR family members, including
CD40 (56, 58). To test which NF-
B activation pathways are used
by CD40 in T cells, we assayed for I
B
␣
phosphorylation and
degradation (NF-
B1; Fig. 10) or processing of p100 to p52 with
nuclear translocation of p52 and RelB (NF-
B2; Fig. 11). Using
densitometry to normalize I
B
␣
values to the loading control actin
(Fig. 10B), CD40 engagement alone stimulated phosphorylation
and degradation of I
B
␣
with up to 100% greater efficiency than
CD3 or CD3 plus CD28, with an even greater increase when CD40
signals were combined with CD3 or CD3 plus CD28. Similar re-
sults were seen in J.hCD40 cells (data not shown). With respect to
the NF-
B2 pathway, in both 2B4.hCD40 (Fig. 11) and J.hCD40
(data not shown), CD40 stimulation alone, but not CD3, resulted in
processing of p100 to p52 (Fig. 11, A and B) and translocation of
p52 and RelB to the nucleus (Fig. 11, C and D). Using densitom-
etry to normalize p100, p52, and RelB values to the loading control
actin (Fig. 11B, cytoplasmic fraction) or yy1 (Fig. 11D, nuclear
FIGURE 7. CD40 activates and enhances CD3 and CD3 plus CD28-
mediated AP-1 activation. 2B4.hCD40 (A) or J.hCD40 (B) cells were tran-
siently transfected with 7⫻ AP-1-luciferase and Renilla-luciferase reporter
plasmids, rested on ice for 30 min, then stimulated for 24 h. Cells were
stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal beads
armed with anti-CD3 or anti-CD3/anti-CD28 Abs. Relative luciferase ac-
tivity (AP-1:Renilla) of stimulus vs control cell groups was calculated as
the mean ⫾ SEM of duplicate samples from two independent experiments.
There was no difference between medium and isotype control samples.
FIGURE 6. CD40 enhances CD3 and CD3 plus
CD28-mediated NFAT activation. 2B4.hCD40 (A)or
J.hCD40 (B) cells were transiently transfected with 4⫻
NFAT-luciferase and Renilla-luciferase reporter plas-
mids, rested on ice for 30 min, then stimulated for 24 h.
Cells were stimulated with anti-hCD40 or isotype con-
trol (IC) Abs or Dynal beads armed with anti-CD3 or
anti-CD3 plus anti-CD28 Abs. Relative luciferase ac-
tivity (NFAT:Renilla) of stimulus vs control was cal-
culated as the mean ⫾ SEM of duplicate samples from
three independent experiments. There was no difference
between medium and isotype control samples.
677The Journal of Immunology
fraction), we observed that CD40 and CD28 augmented CD3-me-
diated activation of NF-
B2 to a similar degree, whereas the com-
bination of CD3 plus CD28 plus CD40 gave maximal stimulation.
TRAF association of T cell CD40
CD40, like other members of the TNFR superfamily, relies on the
association of adaptor molecules, TRAFs, for downstream signal-
ing events, including activation of kinases and transcription fac-
tors, production of cytokines, up-regulation of surface molecules,
and various aspects of the humoral response (20). However, the
characteristics of TRAF association with CD40 have been shown
to differ between B cells, macrophages, dendritic cells, and epi-
thelial cells (19). It was thus important to determine CD40-TRAF
associations in T cells. In mouse B cells, we have previously dem-
onstrated that TRAFs 1, 2, 3, and 6 associate with either endoge-
nous mouse CD40 or transfected hCD40, following receptor liga-
tion (19, 60 – 62). We therefore compared the ability of TRAFs to
move into membrane lipid rafts (Fig. 12, A and B, left panel) and
associate with hCD40 (Fig. 12, A and B, right panel) in Brij sol-
uble (cytoplasmic) and insoluble (lipid raft) fractions in T cells.
TRAF2 and TRAF3 moved efficiently into the lipid raft fraction (left
panel) and associated with CD40 (right panel) in both 2B4.hCD40
(Fig. 12A) and J.hCD40 (Fig. 12B) cells, similar to their recruitment
in B cells (61). This was true if hCD40 was engaged by agonistic Abs
or by CD154, although more efficient movement (left panel) and re-
ceptor association (right panel) of TRAF1 and TRAF6 occurred when
hCD154 was the stimulus, as previously reported (63, 64).
Discussion
All B cells express CD40, and its functions on B cells have been
the subject of much study. Much less well appreciated is that ac-
tivated T cells can also express CD40, and the roles and mecha-
nisms of action of T cell-expressed CD40 still represent a signif-
icant knowledge gap. Various TNFR family members have been
shown to act as coregulatory molecules on T cells, some possibly
contributing to inflammatory disease (65). This role has been pre-
viously established for CD40 on B cells (66), and we demonstrate
here that this is also true for T cell CD40. We see a reproducible
increase in CD40 expression on T cells from mice with the chronic
inflammatory disorder CIA (Fig. 1 and Table I). Although the in-
crease in CD40
⫹
cells in CIA vs control mice is modest compared
with genetically predisposed autoimmune-prone strains of mice
(14, 17), it is consistent with the FACS profile of T cells from
normal mice (9, 17, 67). Strains of mice that are prone to sponta-
neous autoimmune disease have robust expression of CD40 by T
cells, whereas nondisease prone strains do not (14), so it is not
surprising that C57BL/6 mice do not have as robust proportions of
CD40
⫹
T cells as strains that develop autoimmune diseases rela
-
tively early in life. A normal mouse strain can induce functional
CD40 on T cells when encountering an Ag/danger signal (67), but
this level still does not reach that of mouse strains prone to spon-
taneous autoimmune disease, which have likely been receiving
chronic stimulation since early in life. However, findings presented
here show that T cell CD40 can contribute to enhanced T cell
activation even in individuals who do not have strong genetic pre-
disposition to autoimmunity.
Importantly, this CD40 is capable of T cell stimulation, indicat-
ing that it may have significant functional consequences (9, 14, 15,
67). Initially, this was a surprising finding. However, the Ag-spe-
cific precursor frequency for naive T cells is ⬃1/1 ⫻ 10
5
(100
cells/spleen) (68, 69), a small population capable of significantly
expanding and producing a sufficient protective adaptive immune
response upon antigenic stimulation. This response ultimately
leads to the survival of ⬃5% of the activated T cells (⬃1 ⫻ 10
5
cells) to serve as a memory pool after the contraction phase (69).
Interestingly, in the CIA model, at 70 days postimmunization, we
can isolate a population of 3– 4 ⫻ 10
5
CD40
⫹
T cells per spleen
(Table I) that are capable of CD40-mediated activation of cytokine
FIGURE 8. CD40 activates and
enhances CD3 and CD3 plus CD28-
mediated JNK phosphorylation.
2B4.hCD40 (A) or J.hCD40 (B)
cells were rested at 37°C for 30 min,
then stimulated for 5–60 min with
anti-hCD40 or isotype control (IC)
Abs or Dynal beads armed with anti-
CD3 or anti-CD3 plus anti-CD28
Abs. Cells were pelleted, lysed, and
lysates analyzed by SDS-PAGE and
Western blot for phosphorylated (P.)
and total (T.) JNK. Similar results
were obtained in two independent
experiments.
FIGURE 9. CD40 activates and enhances CD3 and CD3 plus CD28-
mediated NF-
B activation. 2B4.hCD40 (A) or J.hCD40 (B) cells were
transiently transfected with 4⫻ NF-
B-luciferase and Renilla-luciferase
reporter plasmids, rested for 15 min on ice, then stimulated for 6 h. Cells
were stimulated with anti-hCD40 or isotype control (IC) Abs or Dynal
beads armed with anti-CD3 or anti-CD3 plus anti-CD28 Abs. Relative
luciferase activity (NF-
B:Renilla) of stimulus vs control was calculated as
the mean ⫾ SEM of duplicate samples from two independent experiments.
There was no difference between medium and isotype control samples.
678 SIGNALING BY CD40 IN T CELLS
production (Fig. 2), despite only representing ⬃7% of the isolated
T cell population (Table I). This finding was recapitulated in ex-
periments whereby CD40
⫹
T cells were mixed with CD40
⫺
T
cells in a controlled fashion and a significant CD40-mediated re-
sponse was seen with as few as 6% CD40
⫹
T cells in culture
(Fig. 4).
Like other costimulatory TNFR family members expressed on T
cells (70), while CD40 itself cannot induce IL-2 production, it
augments the CD3 response and gives maximal stimulation to-
gether with CD3 plus CD28 signals (Figs. 2 and 4). This is true in
both T cells from CIA mice (Fig. 2) and in T cell lines expressing
CD40 (Fig. 4, A and B), even when transfected CD40
⫹
cells are
mixed with CD40
⫺
(untransfected) T cells to give a similar small
percentage of CD40
⫹
cells as that observed in CIA mice. Impor
-
tantly, anti-CD40 stimulation does not yield a positive cytokine
response in T cells lacking CD40, although their response to CD3
plus CD28 stimulation is similar to that of CD40
⫹
T cells (Figs. 2
and 4). Activation of the transcription factor NFAT is critical to
IL-2 production (44). Also consistent with a role as a costimulator,
CD40 itself cannot induce NFAT activation (Fig. 6), but can aug-
ment these responses to CD3 and CD3 plus CD28 ligation. It is
likely that the TCR complex provides calcium-mediated signaling
that is necessary, but not sufficient, for T cell activation and IL-2
production (71), while CD40 provides costimulatory signaling via
NF-
B and AP-1, necessary to activate NFAT and subsequent IL-2
production (44, 72, 73).
FIGURE 10. CD40 activates and enhances I
B
␣
phosphorylation/degradation. A, 2B4.hCD40 cells were
stimulated with anti-hCD40 or isotype control (IC) Abs
or Dynal beads armed with anti-CD3 or anti-CD3 plus
anti-CD28 Abs. Cells were pelleted, lysed, and lysates
analyzed by SDS-PAGE and Western blot for phos-
phorylated and total I
B
␣
. B, Density of bands as a
proportion of cells treated with medium (med) alone
and normalized to the value of the actin bands shown in
B. Similar results were obtained in two independent
experiments.
FIGURE 11. CD40 activates and enhances CD3 and
CD3 plus CD28-mediated nuclear translocation of p52
and RelB in the NF-
B2 pathway. 2B4.hCD40 (A–D)
cells were stimulated with anti-hCD40 or isotype con-
trol (IC) Abs or Dynal beads armed with anti-CD3 or
anti-CD3/anti-CD28 Abs. Cells were pelleted, cyto-
plasmic and nuclear fractions isolated, and lysates an-
alyzed by SDS-PAGE and Western blot for p100/p52
and RelB. Blots were stripped and reblotted for actin
(cytoplasmic fractions; A and B) or yy1 (nuclear frac-
tions; C and D) as loading controls. B, Density of bands
as a proportion of cells treated with medium (med)
alone and normalized to the value of the actin bands
shown in A. D, Density of bands as a proportion of cells
treated with medium (med) alone, normalized to the
value of the yy1 bands shown in C. Similar results were
obtained in two independent experiments.
679The Journal of Immunology
Both CD40 (74) and CD28 (75) contribute to cell activation via
association with lipid rafts. CD40 signals use TRAF adaptor mol-
ecules in B cells (61, 64), and we demonstrate in Fig. 12 that the
same is true for T cell CD40. TRAFs 1, 2, 3, and 6 bind CD40
within the Brij insoluble (raft) fraction upon engagement with ag-
onistic anti-CD40 Ab or membrane-bound CD154. This suggests
that TRAFs are a critical component of T cell CD40 signaling,
providing a key difference from CD28-mediated signaling. As in B
cells (50), T cell CD40 efficiently activates up-regulation of cell
surface molecules (Fig. 5), both canonical and noncanonical
NF-
B pathways (Figs. 10 and 11), AP-1 (Fig. 7), and the AP-1
activator JNK (Fig. 8). CD40 as a costimulatory molecule is as
effective as CD28 at signaling via AP-1 and severalfold more ef-
ficient at signaling via NF-
B, with maximal increase in both re-
sponses when stimulating T cells with agonists for CD3 plus CD28
plus CD40. This suggests that CD40 and CD28 may have different
molecular mechanisms leading to activation of AP-1 and NF-
B,
as has been proposed when comparing CD28 and other TNFR
family members on T cells (76). Importantly, it shows that CD40
can provide more powerful enhancement of NF-
B, a transcription
factor that induces cytokine genes with particular potency, than
other T cell costimulators. This is also seen in the activation of
JNK, which in the two T cell lines tested, was seen only when a
CD40 costimulus was included.
The above findings suggest that CD40 can increase the potency
and number of signaling pathways available to T cells that express
it. This is important when considering threshold requirements for
developing autoimmune disease and associated chronic inflamma-
tion. The presence of CD40-expressing T cells or autoantibodies is
not enough to develop autoimmunity. NOR mice, like their NOD
counterparts, have CD40-expressing T cells, yet do not develop
disease (17). Similarly, the presence of autoantibodies does not
necessarily indicate pathogenesis (77–79). Cooperation between
cell types and signals from the environment to the immune re-
sponse determine development of disease (80, 81).
Like many autoimmune diseases, including diabetes (82), ar-
thritis (83), and lupus (7), CIA requires cooperation between T and
B cells in its pathogenesis (84), and CD40 maybe a potent co-
stimulator in this process. CD40 expressed by B and T cells may
use similar molecular mechanisms, described above, to contribute
to the pathogenesis of inflammation in autoimmune disease. CD40
on both B cells (18, 85– 89) and T cells (this study) synergizes with
AgRs to enhance lymphocyte activation, cytokine production
(Figs. 2 and 4), and up-regulation of cell surface molecules (Fig.
5). CD40 signaling leads to isotype switching and autoantibody
production in B cells (90). In T cells, it has been demonstrated that
CD40 engagement leads to TCR revision within germinal centers
(67), skewing the T cell population further toward autoimmunity
(14 –16).
A proinflammatory cytokine environment is critical for reaching
the threshold of autoimmune disease development (65, 91). CD40
engagement in either T or B cells leads to TNF-
␣
secretion, as
shown in Figs. 2C and 4C and (56, 92). We have previously re-
ported that CD40 signaling to B cells is partially mediated by
TNF-
␣
binding to TNFR2 (56, 92) and hypothesize this also to be
true of T cell CD40, as it has been demonstrated that TNF-
␣
acts
via TNFR2 to lower the threshold of T cell activation and IL-2
production (93–95). Taken together, the presence of increased
numbers of CD40
⫹
T cells in autoimmune mice and the demon
-
stration that CD40 can act as an effective costimulatory receptor on
T cells suggest that blockade of CD40-CD154 interactions can
abrogate the pathogenesis of autoimmune disease on several lev-
els. This has been demonstrated in the experimental autoimmune
encephalomyelitis model of multiple sclerosis, whereby disease
development is dependent on CD40 signaling, particularly in the
absence of CD28 (96). In the HgCl
2
-induced autoimmunity model,
CD28 is unable to overcome the lack of CD40 signaling to induce
disease (97).
CD40 may not just be a TCR costimulatory molecule on T cells
but may make these cells effective APC by up-regulating cell sur-
face molecules (Fig. 5). Increased levels of not only CD40, but
other costimulatory molecules such as CD80, CD86, ICAM-1, and
LFA-1, may also be pivotal in autoimmune disease development
(97). Although beyond the scope of this study, it would be inter-
esting to investigate the costimulatory capacity of activated CD40-
expressing T cells for CD40-negative T cells, as well as for other
cell types, including APC. It is quite possible that activation of
CD40 on T cells lowers the threshold of disease development but
is not sufficient for it to occur, requiring CD40 activation on APC
for cytokine production and on B cells for autoantibody secretion.
In this way, CD40 would prove central to autoimmune disease
development and pathogenesis, influencing not only the cognitive
activation of APC but also T cells. As seen in Fig. 2, results in
enhanced effector proinflammatory cytokine production that along
with CD40 activation on B cells would lead to autoantibody pro-
duction and, ultimately, chronic inflammation.
Disclosures
The authors have no financial conflict of interest.
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FIGURE 12. CD40-mediated TRAF recruitment. 2B4.hCD40 (A)or
J.hCD40 (B) cells were stimulated for 15 min with anti-hCD40 Ab (vs
isotype control Ab; IC) or Hi5 insect cells expressing hCD154 (vs WT
baculovirus; WT). Detergent soluble (S) and insoluble (I) lysates were
prepared as described in Materials and Methods. Seventy percent of the
lysates were immunoprecipitated with protein G-Sepharose armed with
anti-hCD40. Lysates and immunoprecipitates were separated by SDS-
PAGE and analyzed by Western blot. Similar results were obtained in two
identical experiments.
680 SIGNALING BY CD40 IN T CELLS
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682 SIGNALING BY CD40 IN T CELLS
