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IMMUNOBIOLOGY
Direct inhibition of CD40L expression can contribute to the clinical efficacy
of daclizumab independently of its effects on cell division
and Th1/Th2 cytokine production
James T. Snyder,1 Jijia Shen,1 Hooman Azmi,1 Jeannie Hou,2 Daniel H. Fowler,2 and Jack A. Ragheb1
1Laboratory
of Immunology, National Eye Institute and 2Experimental Transplantation and Immunology Branch, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, MD
Humanized anti-CD25 antibodies (eg, daclizumab) have been successfully used to
treat several autoimmune diseases. Paradoxically, IL-2 blockade in mice can induce autoimmunity. An interspecies difference in the relative contribution of IL-2 to
CD25ⴙ T regulatory cell (CD25ⴙTreg) versus CD25ⴙ effector cell function might
explain this conundrum. Consistent with
this are reports that daclizumab inhibits
human CD25ⴙ effector cell cytokine production by blocking the expression of
CD40L. However, in mice, IL-4 and IL-12
regulate CD40L expression. As human
Th1/Th2 cytokine production is also dependent on IL-2, daclizumab’s inhibition
of CD40L expression could be due to an
indirect, rather than a direct, effect of IL-2.
Here, we clarify the mechanisms underlying CD40L expression. In contrast to the
mouse, human CD40L is regulated by
CD28 signaling and IL-2, not the principal
Th1/Th2-polarizing cytokines. We find that
CD40L is expressed on naive and memory
cells and inhibited by daclizumab independently of cell division. Collectively,
our results indicate that daclizumab could
inhibit CD25ⴙ effector T-cell function in
vivo by directly blocking CD40L expression. This difference between mice and
human may help explain the paradoxical
effects of IL-2R blockade in the 2 species.
(Blood. 2007;109:5399-5406)
© 2007 by The American Society of Hematology
Introduction
Monoclonal antibodies (mAb) directed against cytokines, cytokine
receptors, adhesion molecules, and costimulatory ligands are an
emerging therapy in both transplantation and autoimmune disease.1,2 Recent reports have described the successful use of a
humanized anti-CD25 mAb (daclizumab) in the treatment of
several autoimmune diseases.3-11 Daclizumab prevents binding of
IL-2 to its high-affinity receptor and subsequent signaling but does
not cause depletion of CD25⫹ cells. The clinical utility of such a
mAb was not apparent a priori as the generation and function of
CD25⫹CD4⫹ T regulatory cells (CD25⫹Tregs), which are thought
to be indispensable for immune tolerance, are reportedly IL-2
dependent.12-14 In fact, anti–IL-2/IL-2R antibodies can induce
autoimmunity in mice.14-16 This paradox raises important questions
about how daclizumab exerts its clinical efficacy and what the
requirements are for CD25⫹Treg function in humans. Understanding the mechanistic basis of daclizumab’s actions should provide
critical insights into the relative contribution of IL-2R signaling to
CD25⫹Treg and CD25 effector cell function and their roles in
maintaining human immune homeostasis.
CD40 ligand (CD40L; CD154) is a well-recognized costimulatory molecule that is expressed on CD4⫹ T cells shortly after
activation. This early expression of CD40L appears to require only
a TCR signal. More recently, it has been demonstrated that there is
a second, CD28-dependent phase of expression at 48 hours and that
this biphasic pattern of CD40L expression is essential to its
physiologic function.17,18 While early interaction of CD40L with its
receptor (CD40) on B cells initiates a program of B-cell activation,
Ig secretion, isotype switching, and B-cell memory formation,
sustained CD40L/CD40 signaling inhibits these same processes.17,19 Similarly, CD40L/CD40 interactions on antigenpresenting cells play an important role in promoting T-cell activation and Th1 differentiation, however, constitutive expression is
associated with the development of T-cell lymphoproliferative
abnormalities.20,21
Late CD40L expression in activated peripheral blood mononuclear cell (PBMC) cultures is almost completely inhibited by
daclizumab, leading to the supposition that the CD28 effect on
CD40L is mediated through IL-2R signaling. However, this
assumption is challenged by contradictory data in the mouse that
IL-2 has no effect on CD40L expression. Rather, IL-4 and IL-12 are
reported to counterregulate the late phase of CD40L expression
with IL-4 inhibiting and IL-12 promoting expression. Because IL-2
receptor blockade also profoundly inhibits both Th1 and Th2
cytokine production in PBMC cultures, the effect of daclizumab on
human CD40L expression could be direct or mediated indirectly
through IL-4 and IL-12 as reported in the mouse.18 It was also
unclear whether early- and late-phase CD40L might represent
differential expression on naive and memory CD4 T-cell subsets or
be a consequence of cell division.
Here, we address the Th1/Th2 cytokine dependence of human
CD40L expression and examine expression on naive and memory
cells and the relationship of that expression to CD28 costimulation,
cell division, and IL-2R signaling. We observe fundamental
differences in the regulation of CD40L between mice and humans,
a finding that has important implications for multiple models of
Submitted December 19, 2006; accepted February 12, 2007. Prepublished
online as Blood First Edition Paper, March 7, 2007; DOI 10.1182/blood-200612-062943.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2007 by The American Society of Hematology
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
SNYDER et al
immune function and disease. The presented data provide information that is important to understanding the contribution of IL-2 to
effector T-cell function. The direct inhibition of CD40L expression
by daclizumab that we describe here, and the resulting inhibition of
CD40L/CD40 effector functions we reported earlier, suggests that
this mechanism could play a vital role in the clinical activity of
IL-2R–blocking mAbs. The clinical efficacy of daclizumab in
multiple autoimmune diseases and the in vitro results reported here
suggest that in vivo inhibition of CD25⫹ effector T-cell function
may be more clinically relevant than any inhibition of CD25⫹
Tregs that occurs.
Patients, materials, and methods
Subjects
Buffy coat fractions were obtained from leukopheresed subjects belonging
to the National Institutes of Health healthy adult donor pool (IRB-approved
protocol no. 99-CC-0168). Umbilical cord blood was obtained through the
National Institutes of Health cord blood program (IRB-approved protocol
no. 01-DK-0122). All specimens were processed and plated into the
appropriate cultures within 18 hours of collection.
Cell culture reagents
Complete media (Biosource, Rockville, MD) consisting of RPMI 1640
supplemented with penicillin (100 U/mL), streptomycin (100 U/mL),
L-glutamine (2 mM), and 10% fetal bovine serum (FBS; Hyclone, Logan,
UT) was used for all stimulations. Lymphocyte separation media were
purchased from Mediatech (Herndon, VA). Ficoll-Paque was from Amersham Biosciences (Uppsala, Sweden).
Media used for Th1 versus Th2 cell polarization
All CD4⫹ cells were cultured in X-Vivo 20 media supplemented with 5%
human AB serum (Gemini Bioproducts, Woodland, CA). The Th1 culture
condition included the following: rhuIL-12 (2.5 ng/mL; Peprotech, Rocky
Hill, NJ); rhuIL-2 (1000 IU/mL; Chiron, Emeryville, CA); and anti–IL-4
antibody. Murine antihuman IL-4 antibody was generated from the B-cell
hybridoma, MP4.25D2.11 (ATCC, Manassas, VA); the hybridoma was
grown in serum-free X-Vivo 20 media, and the supernatant was collected
and used without further purification. The supernatant was tested for its
ability to compete for recombinant human IL-4 (rhuIL-4) in an enzymelinked immunosorbent assay (ELISA; BioSource, Camarillo, CA), and an
amount of supernatant sufficient to neutralize 6 ␮g/mL rhuIL-4 was added
to the Th1 cultures. The Th2 culture condition included rhuIL-4 (1000
IU/mL; Peprotech) and rhuIL-2 (20 IU/mL).
manufacturer’s directions. Antihuman CD45RO and CD45RA MicroBeads
(Miltenyi Biotec, Auburn, CA) were used for negative selection of
CD45RA⫹ and CD45RO cells, respectively, from purified CD4⫹ T cells on
an AutoMacs instrument (Miltenyi Biotec). Antihuman CD3 MicroBeads
(Miltenyi Biotec) were used to deplete PBMCs of T cells on an AutoMacs
instrument. Depletion of these cells was more than 95%. Purified CD4⫹
T cells were added back to CD3-depleted PBMCs in the same proportion as
they were originally present in the PBMCs.
Isolation of CD4ⴙ T cells for Th1/Th2 polarization
B cells and CD8⫹ T cells were depleted by 2 rounds of negative selection
using anti-CD20 and anti-CD8 murine antihuman antibodies (Nexell,
Irvine, CA) in combination with sheep antimouse magnetic beads using an
Isolex device (Nexell). The enriched CD4⫹ T cells, which were more than
95% pure by flow cytometry (⬍ 0.1% CD8⫹ T-cell contamination), were
then cryopreserved under liquid nitrogen conditions in X-Vivo 20 media
(BioWhittaker; Walkersville, MD) containing 5% DMSO, 5% pentastarch,
and 40% human serum albumin.
PBMC stimulations with plate-bound anti-CD3
PBMCs (1 ⫻ 106 cells/mL) in complete media were added to flat-bottom
6-well (4 to 8 mL) or 24-well (2 mL) tissue culture plates (Costar,
Cambridge, MA) and stimulated with immobilized OKT3 (plates previously coated at room temperature overnight at 2 ␮g/mL) ⫾ soluble
anti-CD28 antibody at 2 ␮g/mL in the presence of various cytokines and/or
antibodies. Additional antibodies were used at the following final concentrations based on the suppliers’ recommendations and our own titration data:
antihuman IL-12 (2 ␮g/mL), IL-12R␤1 (0.5 ␮g/mL), IFN-␥ (1 ␮g/mL),
IFN-␥R␣ (5 ␮g/mL), IL-4 (4 ␮g/mL), and CD25 (daclizumab, 10 ␮g/mL).
The final concentration of recombinant cytokines used is indicated in the
results. Trypan blue staining of the cultures indicated that under all
stimulation conditions there was no change in viability (⬎ 80%) over
the period of the experiments.
CD3, CD28 bead-based CD4ⴙ T-cell expansion
At culture initiation, frozen CD4⫹ T cells were thawed, and stimulated with
tosylated magnetic beads (Dynal, Oslo, Norway) conjugated with antihuman CD3 (OKT3) and antihuman CD28 (clone 9.3) antibodies at a cell to
bead ratio of 1:3. Antibody-coated beads were prepared as previously
detailed.22 Initial T-cell concentration was 0.3 ⫻ 106 cells/mL; cell concentration and volume were measured by Coulter Multisizer II (Beckman
Coulter, Fullerton, CA). Cell density was maintained below 1 ⫻ 106
cells/mL by addition of cytokine-replete Th1- or Th2-specific media;
however, IL-12 was added to Th1 cultures at culture initiation only. On day
12 of culture, CD4⫹ cells were restimulated with anti-CD3, anti-CD28–
coated beads in cytokine-replete media (cell to bead ratio, 1:3).
Recombinant proteins in PBMC cultures
Human rIL-4 and rIL-12 were purchased from R&D Systems (Minneapolis,
MN) and rIL-2, from Roche (Indianapolis, IN).
Antibodies in PBMC cultures
Humanized anti-Tac (HAT; anti-CD25; daclizumab) was a generous gift
from Hoffmann-La Roche (Nutley, NJ). Antibodies to CD28 (clone
CD28.2), CD80 (clone 307.4), CD86 (clone IT2.2), IL-12R␤1 (clone
2.4E6), IFN-␥ (clone B27), IFN-␥R␣ (clone GIR-208), and IL-4 (clone
MP4-25D2) were purchased from BD Pharmingen (San Diego, CA), as
were the isotype controls. Anti–IL-12 antibody was purchased from R&D
Systems. OKT3 was obtained from Ortho Biotech (Raritan, NJ).
Flow cytometric analysis
Flow cytometric analysis was performed according to standard protocols.
Fluorochrome-labeled anti-CD4 (APC), anti-CD40L (PE), and isotype
control antibodies were purchased from BD Pharmingen. Cells were stained
with the appropriate antibodies according to the manufacturer’s suggestions. Viaprobe (BD Biosciences) was added to gate out dead cells. All
incubations were performed at 4°C in staining buffer, and cells were washed
twice and fixed in 1% paraformaldehyde in PBS. Stained cell populations
were acquired using a FACScalibur flow cytometer (Becton Dickinson, San
Jose, CA). Data were analyzed using CellQuest (Becton Dickinson). Color
compensation settings were made with each round of staining using
appropriate fluorochrome-conjugated antibodies.
PBMC fractionation
PBMCs were isolated from leukopheresed buffy coat fractions by density
gradient centrifugation using lymphocyte separation media. Purified CD4⫹
T cells were isolated directly from buffy coats using the RosetteSep
antibody cocktail (StemCell Technologies, Vancouver, BC) according to the
Statistical analysis
The nonparametric rank-sum analysis of Wilcoxin was used to determine
the P value in those experiments (Figure 6A) where the number of donors
was sufficiently large, otherwise, a paired t test was performed.
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
DACLIZUMAB DIRECTLY INHIBITS CD40L EXPRESSION
5401
to CD80/86-blocked cultures restored CD40L expression to the
level seen on CD28-costimulated cells (Figure 1B). These results
demonstrate that CD28 signaling affects CD40L expression indirectly
and that the effect of CD28 costimulation on CD40L expression is
wholly mediated through its augmentation of IL-2 production.
Both naive and memory cells exhibit biphasic
CD40L expression
Figure 1. CD28-dependent CD40L expression is mediated via IL-2R signaling.
(A) CD28-costimulated PBMCs were cultured with anti-CD80/86 or anti-CD25 mAb.
(B) TCR-stimulated PBMCs were cultured with anti-CD80/86 mAb ⫾ rIL-2. rIL-2 was
added at 24 hours. All control cultures were incubated with the appropriate isotype
mAb. Gating was performed on CD4⫹ lymphocytes. The means ⫾ SE of 4 donors are
shown.
Adult PBMCs contain both naive (CD45RA⫹CD45RO⫺) and
memory (CD45RA⫺CD45RO⫹) CD4⫹ T cells. Memory cells are
generally considered to have a lower threshold for activation,
possibly diminishing or negating their need for a costimulatory
signal. We thus hypothesized that CD28-independent CD40L at 6
hours may reflect expression on activated memory cells, while
CD28-dependent late expression occurs on naive cells. We initially
addressed this question in unfractionated PBMCs using 4-color
flow cytometry. Results from a donor with approximately 70%
CD45RO⫹CD4⫹ cells are shown in Figure 2. In unstimulated
PBMCs (0 hours), gating on CD40L⫹CD4⫹ cells (1.6% of CD4⫹
lymphocytes) reveals that expression is almost entirely on memory
cells. However, at 6 hours after activation, CD40L is expressed
proportionately on naive and memory cells (34.2% of CD4⫹
lymphocytes), indicating that naive and memory cells have a nearly
equivalent capacity to express CD40L early.
To assess the memory phenotype of CD40L⫹ cells at later time
points, CD4⫹ T cells were separated into pure CD45RO and
CD45RA single-positive populations. To recapitulate the PBMC
culture milieu, we reconstituted CD3-depleted PBMCs with either
pure CD45RO⫹ or CD45RA⫹ CD4⫹ T cells prior to stimulation. As
shown in Figure 3A, both activated CD45RO⫹ and CD45RA⫹ cells
exhibit biphasic CD40L expression. While in this donor, CD45RO⫹
and CD45RA⫹ cells express CD40L equally, on average, expression was slightly greater on CD45RA⫹ cells at early and late time
points (Figure 3B). These findings indicate that naive and memory
cells have a nearly equivalent capacity to express CD40L and that
late-phase expression on both is CD28 dependent.
To confirm that naive cells exhibit biphasic CD40L expression,
we examined CD40L expression in umbilical cord blood, the only
bona fide source of naive human T cells. Unfractionated umbilical
cord blood mononuclear cells, consisting of more than 95%
CD45RA⫹CD45RO⫺ CD4⫹ lymphocytes, were stimulated under
Results
rIL-2 restores CD40L expression in CD80/86 blocked cultures
Earlier studies had demonstrated that IL-2R signaling is required
for late CD40L expression but had not ascertained whether CD28
signaling is itself necessary. To address this question, we asked if
rIL-2 could substitute for CD28 costimulation. As CD28 is
constitutively expressed on CD4⫹ T cells and PBMCs express
variable levels of the CD28 ligands CD80 and CD86, we performed these experiments in the presence of blocking anti-CD80
and anti-CD86 mAbs. As a control, we confirmed that the blocking
mAbs did not have inhibitory effects other than those due to
disrupting interactions with CD28. As shown in Figure 1A, the
blocking mAbs to CD80/86 had no effect on CD40L expression in
the presence of anti-CD28 mAb, while daclizumab severely
inhibited CD40L expression after 6 hours. By contrast, in PBMC
cultures stimulated with anti-CD3 alone, blocking CD80/86 inhibited CD40L expression after 6 hours (Figure 1B). Addition of rIL-2
Figure 2. Equivalent expression of early CD40L on naive and memory cells. The
memory phenotype of CD4⫹ cells (left panels) and CD40L⫹ CD4⫹ cells (right panels)
was differentiated in unfractionated resting (0 hour) and CD28-costimulated PBMCs
using 4-color flow cytometry. Results from a representative donor are shown.
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SNYDER et al
Figure 3. Naive and memory cells both express CD40L early and late. (A) CD40L
expression in stimulated, reconstituted PBMC cultures of pure CD45RA⫹ or CD45RO⫹
CD4⫹ T cells from a single donor. (B) Early- and late-phase CD40L expression in
reconstituted PBMCs from 13 donors. Due to overlap of the data points, not all
symbols are visible. Solid bars indicate the mean. (C) Umbilical cord blood
mononuclear cells were stimulated ⫾ anti-CD25 mAb. The means ⫾ SE of 4 donors
are shown.
BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
also had no impact on the number of CD40L-expressing cells
(Figure 4B) or the level (MFI) of CD40L expression (Figure 4C).
Having ruled out involvement of IL-4, we assessed the possible
contribution of the Th1-polarizing cytokine IL-12. On average,
CD28-costimulated PBMCs produce 25 pg/mL IL-12p70 at 48
hours. When IL-12–neutralizing and IL-12R–blocking mAbs sufficient to completely neutralize 100 pg/mL rIL-12 (or inhibit 750
pg/mL by 50%) were added to such cultures, there was no effect on
CD40L expression (Figure 4D). Parallel experiments to assess the
role of IFN-␥ demonstrated that, as in the mouse, IFN-␥ has no
impact on human CD40L expression (data not shown). The
addition of rIL-12 has been reported to enhance late CD40L
expression in mouse, thus we tested if high-dose IL-12 could alter
CD40L expression.17 As shown in Figure 4E, rIL-12 did significantly promote CD40L expression in TCR-stimulated cultures at
48 hours (P ⫽ .012) and at 72 hours (P ⫽ .004). This effect of
rIL-12 was also apparent in the presence of CD28 costimulation at
48 hours (P ⫽ .001) and 72 hours (P ⫽ .007). However, unlike
rIL-2, even 100 ng/mL rIL-12 could not raise the level of CD40L in
TCR-stimulated cultures to that observed with CD28 costimulation. Because endogenous IL-12 production in our PBMC cultures
is dependent on CD4⫹ T cells and IL-2, we asked whether the
ability of added rIL-12 to promote CD40L expression is itself IL-2
dependent. As shown in Figure 4F, when daclizumab was added to
rIL-12–treated PBMCs, CD40L expression was markedly inhibited
at 48 hours (P ⫽ .013) and 72 hours (P ⫽ .03), but not to the level
seen in daclizumab-treated cultures without rIL-12 (TCR/CD28/
the same culture conditions used for adult PBMCs. As shown in
Figure 3C, activated neonatal CD4⫹ cells express CD40L at early
and late time points, and such expression exhibits the same pattern
of CD28 and IL-2 dependence as that shown by adult PBMCs. This
temporal pattern of expression is consistent with the report that
naive mouse CD4⫹ T cells exhibit biphasic CD40L expression with
the same kinetics as PBMCs.17
Daclizumab blocks CD40L expression independently of its
inhibition of Th1/Th2 cytokine production
CD40L is important for both humoral and cellular immune
responses and thus it is plausible that Th1 and/or Th2 cytokines
may play a role in either positively or negatively regulating its
expression. In the mouse, IL-4 and IL-12 counterregulate the late
phase of CD40L expression with IL-4 inhibiting and IL-12
promoting expression, but, remarkably, IL-2 is reported to have no
effect.17 Daclizumab abolishes late CD40L expression and profoundly inhibits both Th1 and Th2 cytokine production in CD28costimulated PBMCs.18 Thus IL-2 could be acting directly to
promote late human CD40L expression or indirectly through the
action of IL-2–dependent cytokines such as IL-4, IFN-␥, and
IL-12, as reported in the mouse.17 To distinguish between these
possibilities, we first added to PBMC cultures anti–IL-4 mAb
sufficient to neutralize 10 ng/mL rIL-4. We had previously found
that activated PBMCs produce 0.02 to 0.10 ng/mL IL-4 at 48 hours.
Despite the use of a 100-fold excess of IL-4–neutralizing mAb,
there was no effect on CD40L expression (Figure 4A). This
demonstrates that IL-4 in situ does not regulate early or late CD40L
expression. In mouse T-cell cultures, addition of rIL-4 is reported to
inhibit late CD40L expression; so we tested if high-dose rIL-4
could alter human CD40L expression.17 Addition of 25 ng/mL
rIL-4, a concentration 250-fold greater than that observed in situ,
Figure 4. Biphasic CD40L expression is IL-4 independent. PBMCs were stimulated with OKT3 ⫾ anti-CD28 mAb. Gating was performed on CD4⫹ lymphocytes. (A)
Anti–IL-4 or isotype control mAb was added to parallel cultures. (B-C) Costimulated
PBMCs were cultured ⫾ rIL-4. The mean ⫾ SE of 4 donors are shown. (D) Anti–IL-12
and anti–IL-12R mAb or isotype controls were added to parallel cultures. The mean ⫾
SE of 3 donors are shown. (E) Stimulated PBMC cultures ⫾ rIL-12. The means ⫾ SE
of 5 donors are shown. (F) Stimulated PBMC cultures ⫾ rIL-12 ⫾ anti-CD25 mAb.
The mean ⫾ SE of 5 donors are shown.
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
DACLIZUMAB DIRECTLY INHIBITS CD40L EXPRESSION
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Daclizumab inhibits CD40L expression independently
of cell proliferation
Figure 5. Cytokine profile of Th1- and Th2-polarized CD4ⴙ T cells. Purified CD4⫹
T cells were stimulated on day 0 with bead-adsorbed anti-CD3 and anti-CD28 mAb
under Th1- or Th2-polarizing conditions. On day 12 cells were harvested and
restimulated for 24 hours with mAb-coated beads under neutral conditions, and
culture supernatants were assayed by cytokine ELISA. The mean ⫾ SE of 16 donors
are shown.
␣CD25 vs TCR/CD28/␣CD25 ⫹ rIL-12, P ⫽ .008 at 48 hours).
Thus, while endogenous IL-12 production in this PBMC system is
itself IL-2 dependent, high-dose rIL-12 can moderately enhance
CD40L expression in an IL-2–independent fashion.
Unlike immunized mice, human PBMCs do not display highly
skewed Th1 or Th2 phenotypes. Thus, we also examined CD40L
expression on CD4⫹ T cells expanded under Th1- or Th2polarizing conditions. After 12 days, cultures were again stimulated
in fresh media under neutral or polarizing conditions. Cells
restimulated under neutral culture conditions were assayed to
confirm they had acquired a polarized phenotype (Figure 5). Cells
restimulated under polarizing conditions were assessed for CD40L
expression 1 and 4 days later. On day 12, resting Th1-polarized
CD4⫹ T cells have significantly more (P ⫽ .03) CD40L than do
Th2 cells, which expressed baseline levels of CD40L (Figure 6A).
However, upon restimulation, both Th1- and Th2-polarized CD4⫹
T cells abundantly express CD40L (day 13). By day 16, while
CD40L remains elevated on Th1- and Th2-polarized CD4⫹ T cells,
there was again significantly more CD40L on Th1 cells (P ⫽ .001).
Of interest, day-12 polarized CD4⫹ T cells restimulated under
neutral culture conditions have a pattern of CD40L expression
similar to that found on cells restimulated under polarizing
conditions (Figure 6B).
Figure 6. CD40L expression is skewed on polarized Th1/Th2
cells. Purified CD4⫹ T cells were stimulated on day 0 with beadadsorbed anti-CD3 and anti-CD28 mAb under Th1- or Th2-polarizing
conditions. (A) On day 12, CD40L expression was measured and cells
were restimulated as on day 0 (n ⫽ 16). CD40L expression was
measured again on day 13 (n ⫽ 16) and day 16 (Th1 n ⫽ 5; Th2
n ⫽ 16). The mean ⫾ SE is shown. (B) CD40L expression was
measured at 0 hours (day 12) and following restimulation under the
same nonpolarizing conditions used for PBMC cultures. Data are
expressed relative to the 6-hours Th1 sample. Due to overlap of the
data points, not all symbols are visible. Solid bars indicate the mean
(n ⫽ 4). Standard error bars are shown.
To ascertain whether CD40L expression is coupled to cell division
in naive or memory cells, we again used reconstituted PBMCs
consisting of either pure CD45RA⫹ or CD45RO⫹ CD4⫹ T cells
that had been CFSE labeled. At 6 hours, no CD4⫹ T cells had
divided and thus early CD40L expression is independent of cell
division (data not shown). At 48 hours, about half of the CD4⫹ T
cells have divided at least once. The distribution of CD40L on cells
that had undergone 0, 1, or 2 cell divisions at 48 hours reflected the
donors’ proliferative capacity and increased with cell division. This
is illustrated by the donor shown in Figure 7, in whom 92%
(11%/12%) of CD45RA⫹ cells that divided twice were CD40L⫹,
whereas 55% (17%/31%) of the cells that had not divided
expressed CD40L. In daclizumab-treated cultures, CD40L expression was inhibited on cells that had not divided as well as those that
had. Replication beyond one cell division was completely blocked
by daclizumab, but the number of cells dividing once was
unaffected. By contrast, on cells that had divided once, CD40L
expression was inhibited 80%. On average, results for CD45RO⫹
cells were similar to those obtained with CD45RA⫹ cells (Tables 1
and 2). These results demonstrate that while late-phase CD40L
expression is proportionately greater on cells that have divided,
division is not necessary for CD40L expression and inhibition by
daclizumab is independent of blocking cell division, establishing
that IL-2 affects CD40L expression independently of cell
proliferation.
Discussion
CD40L is an essential molecule involved in promoting B- and
T-cell responses, and the consequences of human CD40L deficiency are evident in the X-linked hyper-IgM syndrome.23 It has
long been recognized that CD40L is rapidly expressed and
down-regulated on CD4⫹ T cells following TCR engagement.24-27
More recent data from our lab and the Randall lab have shown that
both human and mouse CD4⫹ T cells exhibit a biphasic pattern of
CD40L expression, indicating that its regulation and function are
more complex than originally envisioned (Lee et al17 and McDyer
et al18). In the mouse, it has been clearly established that the
biphasic character of CD40L expression is critical to its physiologic function.17,19 Human data also support the premise that
early- and late-phase CD40L are functionally distinct as CD40Ldependent IL-12 production is completely abolished by inhibiting
only the late-phase of CD40L expression.18 Human CD40L expression is biphasic both temporally and mechanistically. There is an
initial CD28-independent peak at 6 hours that is followed by a
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
SNYDER et al
Figure 7. Daclizumab inhibits CD40L expression independently
of cell division. Reconstituted PBMC cultures of pure, CFSE-labeled
CD45RO⫹ or CD45RA⫹ CD4⫹ T cells were CD28 costimulated ⫾
anti-CD25 Ab. On the left, a dot plot of such a CD28-costimulated
culture of CD45RA⫹ cells at 48 hours is shown. The number of cell
divisions (0, 1, 2) as measured by CFSE dilution is indicated. On the
right, the distribution of divided cells and CD40L on those cells, in the
presence (daclizumab) and absence (control) of anti-CD25 is shown.
nadir at 24 hours and a second, CD28-dependent peak of expression at 48 to 72 hours. Here, we have expanded upon those
preliminary observations by showing that biphasic expression is an
integral aspect of CD40L regulation and not a result of cell division
or differential expression on naive and memory CD4⫹ cells. On the
contrary, though CD40L expression on resting CD4⫹ T cells is
almost entirely restricted to memory cells, upon activation adult
naive and memory CD4⫹ cells have a nearly equivalent capacity to
express CD40L early and late and are equally dependent upon
CD28 signaling. While memory cells are widely regarded to be less
dependent on costimulation, late-phase CD40L and (by implication) IL-2 expression clearly remain dependent upon a CD28
signal. Although the use of surface markers to distinguish naive and
memory cells may never be unequivocal, CD45RA and CD45RO
are widely accepted as a means of broadly differentiating these
populations. To confirm the capacity of naive CD4⫹ cells to express
biphasic CD40L, rather than rely on additional surface markers (eg,
CD11a, CD27, CD62L, CCR7) that disputably distinguish between
memory and naive cells, we examined CD40L expression on
neonatal CD4⫹ cells, the only certain source of naive human T
cells. While there are many reports that neonatal CD4⫹ T cells are
not fully immunocompetent, we thought that examining CD40L
expression on these cells may nonetheless be informative.28-34
Indeed, we found that umbilical CD4⫹ T cells, like naive adult
CD4⫹ T cells, exhibit early CD28-independent and late CD28dependent CD40L expression upon T-cell activation. Our results
are consistent with the report that primary mouse CD4⫹ T cells
exhibit biphasic CD40L expression with kinetics identical to what
we have reported in PBMCs.17
The studies presented here also provide information that is
important to understanding the mechanistic basis of late CD40L
expression and the contribution of IL-2 to effector T-cell
function. The ability of rIL-2 to substitute for CD28 costimulation establishes that CD28 signaling does not directly regulate
CD40L expression on human CD4⫹ T cells and further demonstrates that the CD28-mediated regulation of late CD40L
expression is dependent upon IL-2. The level of IL-2 expression
in the culture impacts CD40L expression at all times after 6
hours to the extent that in some donors the nadir at 24 hours may
not be apparent without blocking IL-2R. While these results
show that IL-2 is necessary for late CD40L expression, and we
have demonstrated that such expression is not a result of
IL-2–driven proliferation, they do not establish that IL-2
directly regulates CD40L expression. Furthermore, though the
kinetics of biphasic CD40L expression in mice and humans are
identical, IL-2 is reported to have no effect on the expression of
mouse CD40L. Rather, in the mouse, IL-4 and IL-12 counterregulate the late phase of CD40L expression with IL-4–inhibiting
and IL-12– promoting expression.17 Because Th1 and Th2
cytokine production in PBMC cultures is profoundly dependent
upon IL-2, our earlier experiments did not distinguish whether
the effect of IL-2 on human CD40L expression was a direct one
or mediated indirectly through IL-4 and IL-12 as reported in the
mouse.18 While IL-4 has no effect on human CD40L expression,
we did find that, as previously reported for purified human T
cells, exogenous rIL-12 augments CD40L expression in primary
PBMC cultures. While the effect of rIL-12 is partially IL-2
independent, rIL-12 cannot substitute for CD28 or IL-2R
signaling. Additionally, CD40L is expressed at a higher frequency and for a sustained period in polarized Th1 CD4⫹ T-cell
cultures relative to their Th2 counterpart. These differences are
not dependent on the continued presence of IL-4 or IL-12
(Figure 6B), suggesting that such changes may reflect the
differentiation state of these long-term cultured cells. The
counterregulation of CD40L expression by IL-4 and IL-12 in the
mouse has been interpreted in the context of a model in which
late-phase (ie, IL-12 driven) CD40L expression, in contrast to
early-phase expression, inhibits B-cell activation and immunoglobulin production.17 These observations in the mouse are in
contradistinction to the reports that in humans, rIL-12 promotes
B-cell proliferation and immunoglobulin production in a CD40Ldependent fashion.35,36 While we have not attempted to exhaustively rule out a role for other cytokines in regulating CD40L
expression, we can conclude that the effect of IL-2 is not
Table 1. Daclizumab inhibits CD40L expression independently
of cell division in naive CD4ⴙ T cells
Table 2. Daclizumab inhibits CD40L expression independently
of cell division in memory CD4ⴙ T cells
CD45RA
% Divided
Cell divisions at 48 hrs
CD45RO
2
1
0
Total
14 (6)
47 (7)
39 (13)
100%
Daclizumab
0
35 (11)
65 (11)
100%
% CD40L⫹
2
1
0
Total
13 (5)
40 (8)
17 (3)
70%
Control
0
7 (3)
11 (3)
18%
Daclizumab
Control
Control
Daclizumab
Results shown are mean (⫾ SE) of 4 donors.
% Divided
Cell divisions at 48 hrs
2
1
0
Total
7 (4)
54 (4)
40 (6)
100%
Daclizumab
0
37 (6)
64 (6)
100%
% CD40L⫹
2
1
0
Total
5 (3)
38 (6)
20 (2)
63%
0
13 (5)
14 (3)
27%
Control
Results shown are mean (⫾ SE) of 4 donors.
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BLOOD, 15 JUNE 2007 䡠 VOLUME 109, NUMBER 12
DACLIZUMAB DIRECTLY INHIBITS CD40L EXPRESSION
mediated through the principal IL-2–dependent Th1/Th2 cytokines and that even pharmacological doses of IL-12 play a
subordinate role.
Although Lee et al17 found that IL-2 does not impact mouse
CD40L expression, several reports do indicate a role for CD28/IL-2
signaling.17 While early CD40L expression is unaltered in the
CD28⫺/⫺ mouse, B7 blockade in vitro and in vivo has been shown
to inhibit CD40L expression, indicating that CD28 signaling plays
a role at later time points.37-40 Additionally, CD28⫺/⫺ mice have
been shown to have severely impaired IL-12 production at 48
hours. Correspondingly, in human PBMCs, CD28-mediated upregulation of IL-12 is dependent upon IL-2R signaling.18,41 Finally,
IL-2 and STAT5 in naive mouse T cells have been shown to play an
essential role in IL-4 production.42-45 It is unclear how to reconcile
these earlier demonstrations of the IL-2 dependence of CD40L,
IL-12, and IL-4 expression in the mouse, collectively or individually, with the report that mouse CD40L expression is entirely
independent of IL-2 but regulated by IL-4 and IL-12. It is striking
that the regulation of such a fundamental molecule as CD40L may
be as divergent as has been described in mice and humans, yet there
is precedence for genetically encoded immunologic differences
between these species.46
Humanized monoclonal antibodies directed against the CD25
subunit of the high-affinity IL-2R have been shown to have
clinical utility in several distinct autoimmune entities.3-11 These
reports were quite unexpected as IL-2 blockade in mice can
induce autoimmune disease, apparently as a consequence of
inhibiting CD25⫹Treg function.14-16,47 The immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance
(IPEX) syndrome, an autoimmune disorder with phenotypic
similarities to CD25⫹Treg deficiency in the mouse, establishes
that CD25⫹Tregs have a significant role in establishing immune
tolerance during the major period of human lymphocyte development.48 However, evidence that CD25⫹Tregs are of functional
importance in human adults is lacking.49 Despite the results of
the IL-2–blocking experiments in mice, it remains controversial
whether extrathymic CD25⫹Tregs require IL-2 in vivo.50,51 The
therapeutic success of daclizumab in autoimmune disorders and
our earlier demonstration that it blocks CD28-dependent CD40L
expression and CD40L-dependent IL-12 production, as well as
drastically reduces IFN-␥ production, suggest that inhibition of
IL-2–dependent effector mechanisms might explain the apparently paradoxical clinical efficacy of the antibody. Collectively,
the data presented here indicate that the inhibitory activity of
daclizumab is in all likelihood due to a direct effect of IL-2 on
CD40L expression. These findings suggest that human
CD25⫹Tregs in the periphery either do not require IL-2 or that,
on balance, inhibition of IL-2R signaling in CD25⫹CD4⫹
effector cells is more clinically significant than inhibition of
CD25⫹Treg function.
In summary, understanding the mechanistic basis of daclizumab’s inhibitory activity has provided important insights into
the regulation of CD40L expression and the contribution of
5405
IL-2R signaling to CD25⫹ effector T-cell function. These
findings, and our previous reports that daclizumab inhibits the
effects of CD40L/CD40 signaling, in conjunction with the
demonstrated clinical efficacy of daclizumab, challenge the
notion that in humans the primary function of IL-2 is to promote
and sustain CD25⫹Tregs.52 Additional human data, such as the
well-known immunotherapeutic effect of administered IL-2 in
melanoma and renal carcinoma, also cast doubt on concepts of
CD25⫹Tregs that have been developed in the mouse.53 Inhibition of IL-2 effector functions, in addition to less well-defined
mechanisms such as the expansion of natural killer (NK)
regulatory cells, is likely to contribute to the therapeutic effect
of daclizumab.54,55 Our findings reveal a potentially fundamental difference between humans and mice in the regulation of
CD40L expression that has profound implications for mouse
models of B-cell maturation, transplant tolerance, allergy, and
autoimmune disease. Beyond their research implications, these
differences have direct clinical ramifications as well. Studies of
CD40L in the mouse would suggest that anti–IL-12 therapy,
which has demonstrated clinical efficacy in autoimmune disease, could act in part by down-regulating CD40L expression.56,57 Such an inference would suggest that anti–IL-12
therapy might represent an alternate means by which to inhibit
CD40L function, an immunomodulatory strategy that has been
shown to induce long-term graft tolerance in a primate allotransplant model.58 On the contrary, our data in primary human T
cells suggest that therapies directed against IL-2, but not IL-12,
are more likely to be effective in modulating CD40L expression
and might represent an alternative strategy to anti-CD40L
mAb-based therapy and its associated adverse effects.58
Acknowledgments
We thank George Reed for assistance with the statistical analysis,
Elizabeth Joyal for cord blood collections, and John McDyer, John
O’Shea, Robert Seder, and Tom Waldmann for critical reading of
the paper.
Authorship
Contribution: J.A.R. conceived and designed the experiments,
analyzed and interpreted the data, and wrote the paper; J.T.S., J.S.,
and H.A. designed and performed experiments, collected data,
and analyzed and interpreted results; J.H. and D.H.F. conceived,
executed, and analyzed the results of the Th1/Th2 experiments
reported in the paper; J.T.S., J.S., and D.H.F. contributed to writing
the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Jack A. Ragheb, Bldg 10, Rm 10N113, 10
Center Dr, MSC-1857, Bethesda, MD 20892-1857; e-mail:
[email protected].
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2007 109: 5399-5406
doi:10.1182/blood-2006-12-062943 originally published
online March 7, 2007
Direct inhibition of CD40L expression can contribute to the clinical
efficacy of daclizumab independently of its effects on cell division and
Th1/Th2 cytokine production
James T. Snyder, Jijia Shen, Hooman Azmi, Jeannie Hou, Daniel H. Fowler and Jack A. Ragheb
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