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and CD40 Ligand Expression in Human T Cells: Disruption of Both IL-12-Dependent and -Independent Pathways of IFN- Production
* Laboratory of Immunology, National Eye Institute,
Critical Care Medicine Department, Clinical Center, and
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and
Guy’s, King’s, and St. Thomas’ Hospital, London, United Kingdom.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (Tac/CD25) of the IL-2R are an
emerging therapy in both transplantation and autoimmune disease.
However, the mechanisms underlying their therapeutic efficacy have not
been fully elucidated. Therefore, we examined the affect of IL-2R
blockade on Th1 and Th2 cytokine production from human PBMC. Addition
of a humanized anti-Tac Ab (HAT) to activated PBMC cultures
inhibited IFN-
production from CD4 and CD8 T cells by 80–90%. HAT
partially inhibited production of TNF- and completely inhibited
production of IL-4, IL-5, and IL-10. Furthermore, IL-12, a central
regulatory cytokine that induces IFN-, was undetectable in treated
cultures. As T cell-dependent induction of IL-12 is regulated via
CD40/CD40 ligand (CD40L) interactions, we examined the affect of
HAT on CD40L expression. We found CD40L expression to be biphasic with
an early (6 h) peak that is CD28/IL-2-independent, but a later peak (48
h) being CD28/IL-2-dependent and inhibited by HAT. Similarly, IFN-
production at 6 h was CD28/IL-2-independent but
CD28/IL-2-dependent and inhibited by HAT at 48 h. Nonetheless,
addition of rCD40L or exogenous IL-12 to HAT-treated cultures could not
restore IFN- production. The IFN- deficit in such cultures
appears to be due to a direct inhibition by HAT of IL-12-independent
IFN- production from T cells rather than altered expression of
either the IL-12R
1 or IL-12R2 chains. These data demonstrate that
IL-2 plays a critical role in the regulation of Th1 and Th2 responses
and impacts both IL-12-dependent and -independent IFN- production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (CD25) of the high affinity IL-2R, is generally expressed at
low levels on a small percentage of circulating PBMC from normal human
donors (1). However, it is rapidly up-regulated upon T
cell activation (2). The observation that elevated levels
of the IL-2R are found on the PBMC of patients in the midst of
allograft rejection, as well as patients with a multitude of neoplastic
or autoimmune diseases, suggested that the high-affinity IL-2R might be
a useful target for therapeutic intervention (3). Although
the original anti-Tac Ab that was used clinically could block the
binding of IL-2 to its receptor, the medical utility of this
murine Ab was severely hampered by its short half-life in human serum
and the rapid induction of neutralizing anti-mouse Abs (4, 5). The development of daclizumab, a humanized anti-Tac Ab
(HAT),2 has largely
alleviated these problems (6). HAT has been used mostly in the renal transplant setting, where clinical studies have shown it to be a safe and effective therapy in reducing acute rejection episodes in allograft recipients. HAT has also been used successfully as part of a glucocorticoid-free immunosuppressive regimen in an islet cell transplant clinical study (7). Most of the clinical experience with HAT has been limited to the administration of five doses at the time of transplantation. Recently, the use of HAT in patients with autoimmune disease has been reported (8, 9). In one study, patients with moderate to severe uveitis requiring multiple immunosuppressive drugs to control their disease were started on HAT therapy. Nine of these 10 patients were successfully tapered off all their immunosuppressive medications and maintained on monthly infusions of HAT as a sole agent (9). Six of the nine responders have now been disease-free on HAT monotherapy for nearly 4 years (J. A. Ragheb, unpublished observations).
The mechanisms by which HAT produces its therapeutic effect are
incompletely understood. Furthermore, evidence from certain mouse
models would suggest that not only might IL-2R blockade not be
therapeutic, but it could also actually be detrimental (10, 11). Thus, it was of interest to explore how HAT may inhibit the
potent immune responses seen in allograft rejection and aggressive
forms of autoimmune disease. Early studies with human PBMC showed that
anti-Tac Ab profoundly inhibited T cell proliferation without
affecting IL-2 production and markedly diminished Ab production in cell
cultures stimulated with mitogen (4). Biochemically, this
is a consequence of blocking IL-2R occupancy and thus preventing a
series of downstream signal transduction events (12). To
examine whether other immunologic mechanisms might contribute to the
clinical efficacy of HAT therapy, we tested the affect of the Ab on Th1
and Th2 cytokine production from activated T cells in vitro. In
order to assess any impact HAT may have on APC-T cell interactions,
PBMC cultures were used in most of the studies reported in this work.
The results demonstrate that IL-2R blockade leads to an inhibition of
both Th1 and Th2 cytokine production. Since IFN-, the key Th1
effector molecule, has been implicated in the pathogenesis of both
allograft rejection and certain autoimmune processes, we
focused on the mechanisms by which HAT inhibits the production of this
cytokine (13, 14, 15, 16). Our studies demonstrate that HAT
inhibits IFN- production in vitro from activated PBMC through both
IL-12-dependent and -independent mechanisms. Inhibition of
IL-12-dependent IFN- appears to be the result of diminished IL-12 in
HAT-treated primary PBMC cultures. In addition, these studies reveal
that expression of both CD40 ligand (CD40L) and IFN- is biphasic,
consisting of IL-2-dependent and -independent components. Finally, we
show that IL-12-independent IFN- production from purified
lymphocytes is inhibited by HAT, demonstrating a direct role for IL-2
in the regulation of IFN- production from human T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Buffy coat fractions were obtained from leukopheresed subjects belonging to the National Institutes of Health (Bethesda, MD) normal donor pool (protocol no. 99-C-0168). All specimens were processed and plated into the appropriate cultures within 18 h of collection.
Reagents
Complete media consisting of RPMI 1640 supplemented with 10% heat-inactivated human AB sera (Sigma-Aldrich, St. Louis, MO), penicillin (100 U/ml), streptomycin (100 U/ml), and L-glutamine (2 mM) was used for all stimulations. Lymphocyte separation medium was purchased from Organon Teknika (Durham, NC). OKT3 was purchased from Orthobiotech (Raritan, NJ). Staphylococcus aureas Cowan strain (SAC) was purchased from Calbiochem (La Jolla, CA).
Recombinant cytokines
Human rIL-12 was purchased from R&D Systems (Minneapolis, MN).
Trimeric soluble CD40L molecule (CD40LT) was a generous gift
from Immunex (Seattle, WA). Human rIFN- and human rIL-2 were
purchased from BD PharMingen (San Diego, CA).
Antibodies
Humanized anti-Tac (zenapax) was a generous gift from Hoffmann-La Roche (Nutley, NJ). Mouse anti-human CD40L was a generous gift from Immunex. Anti-CD28 Ab (mAb 9.3) was purchased from BD PharMingen.
PBMC fractionation and stimulation
PBMC were isolated from leukopheresed buffy coat fractions by density gradient centrifugation using lymphocyte separation media. PBMC (1 x 106 cells/ml) were then added to 96-well (200 µl) flat-bottom or 24-well (2 ml) tissue culture plates (Costar, Cambridge, MA) and stimulated with immobilized OKT3 (plates previously coated at room temperature overnight) ± soluble anti-CD28 Ab in the presence of various cytokines and/or Abs. Trypan blue staining of the cultures indicated that under all stimulation conditions there was no change in cell number or viability (80%) over the 48-h course of the experiments. Abs were used at the following final concentrations (micrograms per milliliter) unless specified otherwise in the text: OKT3 = 2, CD28 = 2, HAT = 10. It should be noted that the 10% human AB serum in the culture medium contains 400–1300 µg/ml of IgG1 and thus also serves as a normal serum control in our experiments. rIL-2 was used at a 100 U/ml final concentration. Isolation of purified CD4 and CD8 T cells was performed using MACS-positive selection columns for CD4 and CD8 T cells (Miltenyi Biotec, Auburn, CA). Prior to positive selection of CD4 T cells, PBMC were either treated with L-leucine methyl ester (Sigma-Aldrich) or fractionated on a MACS CD4-negative selection column in order to deplete non-T cells. Purity for CD4 and CD8 T cells was >99%. Purified T cells were then stimulated as above.
Preparation of whole cell lysates for immunoprecipitations and Western blots
Following stimulation, cells were washed once with ice-cold PBS and then lysed on ice for 20 minutes in a lysis buffer containing 1% Nonidet P40, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 2 µg/ml aprotinin (ICN Pharmaceuticals, Costa Mesa, CA), 100 µg/ml AEBSF (ICN Pharmaceuticals). For immunoprecipitations, an Ab to Jak3 (SantaCruz Biotechnology, Santa Cruz, CA) was added to lysates and incubated overnight at 4°C. Ab-protein complexes were then captured from the lysates by adding protein-A Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) and incubating with rotation at 4°C for 4 h. The beads were then washed twice with ice-cold PBS and boiled in reducing sample buffer and the immunoprecipitated proteins were resolved on a 7.5% polyacrylamide gel. For the detection of phosphorylated Stat5 proteins, whole cell lysates were prepared by lysing the cells directly in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromophenol blue), boiled for 5 min and proteins were resolved on a 7.5% polyacrylamide gel.
For Western blots, proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) by electroblotting (Invitrogen, San Diego, CA). Membranes were first blocked in PBS containing 5% BSA and 0.1% Tween 20 (Calbiochem) and then were incubated with either an anti-phosphotyrosine Ab, PY99 (SantaCruz Biotechnology) or an anti-phospho-Stat5 Ab, Tyr694 (New England Biolabs, Beverly, MA). Following detection of the phosphorylated proteins, the membranes were stripped in a solution containing 2% sodium dodecyl sulphate, 100 mM 2-ME, 62.5 mM Tris-HCl, pH 6.8, for 10 min at 50°C. Membranes were then washed and blocked in PBS containing 5% nonfat dried skimmed milk and were incubated with either Janus kinase (Jak)3 or Stat5 (BD Transduction Laboratories, Lexington, KY) antibodies. Western blots were developed using SuperSignal chemiluminescent substrate (Pierce, Rockford, IL).
Measurement of cytokine production
Tissue culture supernatants were collected at 48 h for
IFN-, TNF-, IL-2, IL-4, IL-5, IL-10, and IL-12p70 and stored at
-70°C until used. An IL-12-specific ELISA that detects the p70
heterodimer was purchased from R&D Systems with a lower limit of
detection, 7.8 pg/ml. IFN- content was determined by a two-step
ELISA assay consisting of biotinylated detecting Ab. Both the
anti-human IFN- capture and detection Abs were purchased from
Pierce, with a lower limit of detection of 41 pg/ml.
Multicytokine assessment
The BD PharMingen cytometric bead array (CBA) was used to
simultaneously assay for the cytokines IFN-, TNF-, IL-2, IL-4,
IL-5, and IL-10 from tissue culture supernatants. This assay uses flow
cytometry to measure soluble analytes in a particle-based immunoassay
and was carried out as previously described (17). The
lower limit of detection for all cytokines in this assay was 20
pg/ml.
Surface flow cytometric analysis
PBMC were cultured (1 x 106 cells/ml) in
24-well plates and stimulated with OKT3 ± anti-CD28 Ab. Flow
cytometric analysis was performed according to standard protocols.
Fluorochrome-labeled anti-CD3, anti-CD4, anti-CD8,
anti-CD14, anti-CD40L, anti-CD69, anti-IFN-, and
isotype control Abs were purchased from BD PharMingen.
Fluorochrome-labeled anti-CD25 Ab (clone 7G7) that does not share
the same binding epitope as HAT, was purchased from Ancell (Bayport,
MN). Staining for IL-12R1 and IL-12R2 was performed using rat
anti-IL-12R1 and IL-12R2 Abs along with a rat IgG2a isotype
control Ab, all purchased from BD PharMingen. Cells were stained with
the appropriate Abs according to the manufacturer’s suggestions. All
incubations were performed at 4°C in staining buffer, and cells were
washed twice between incubations. Stained cell populations were
acquired using a FACSCalibur flow cytometer (BD Biosciences, Mountain
View, CA). Data were analyzed using either CellQuest (BD Biosciences)
or FlowJo software (TreeStar, Cupertino, CA). Color compensation
settings were made with each round of staining using appropriate
fluorochrome-conjugated Abs.
Intracellular cytokine detection
Detection of intracellular cytokine by flow cytometry was performed according to protocol (18). Briefly, stimulated PBMC (4 x 106 cells) were treated with brefeldin-A (Sigma-Aldrich) at a final concentration of 10 µg/ml to inhibit cytokine secretion. At 4 h of incubation, the cells were washed twice and fixed in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized. Fixed and permeabilized cells were blocked in a solution of PBS with 0.2% saponin (25% sapogenin in content; Sigma-Aldrich), 1 mM CaCl2, 1 mM MgSO4, 0.05% (w/v) NaN3, 1% BSA, pH 7.4, with 5% nonfat dry milk overnight at 4°C. Cells were then aliquoted at 1 x 106 per tube and washed once in a solution of PBS/saponin. The pellet was resuspended in PBS/saponin/milk containing Abs for staining and incubated for 30 min at 4°C in the dark. Samples were then washed twice in PBS/saponin, resuspended in 300 µl PBS/BSA 0.1%. Gating was performed on the desired cell populations and 10,000–30,000 events were collected.
Statistical analysis
Normally distributed continuous variable comparisons are done using the Student t test using Microsoft Excel (Redmond, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2, the key mitogen produced by T cells themselves, is often
categorized as a Th1 cytokine. However, previous studies have
demonstrated that T cells possess the ability to produce IL-2 prior to
terminal differentiation. Therefore, using HAT, we assessed the impact
of IL-2R blockade and thus IL-2 signaling on the regulation of multiple
Th1/Th2 cytokines in stimulated human PBMC. To quantitate the effect of
HAT on the production of multiple cytokines simultaneously, a CBA assay
was performed to measure IFN-, TNF-, IL-10, IL-5, IL-4, and IL-2
at 48 h. Results from two of five donors who exhibited either a
more pronounced Th1 response (donor 1) or a more pronounced Th2
response (donor 2) are shown in Table 1
.
Fresh PBMC were stimulated with OKT3 ± anti-CD28 Ab in the
presence or absence of HAT for 48 h. Supernatants were then
collected and assayed for the principal Th1/Th2 cytokines. Low to
undetectable levels of all the cytokines were found in unstimulated
PBMC at 48 h. Stimulation with OKT3 resulted in detection of all
six cytokines in most of the donors. The addition of anti-CD28 Ab
enhanced IL-2 production, as well as the Th1 cytokines IFN- and
TNF-, and the Th2 cytokines IL-4, IL-5, and IL-10. In stimulated
PBMC cultures treated with HAT, there was, as expected, no inhibition
of IL-2 (data not shown) (4). In contrast, the addition of
HAT diminished IFN- production by 80–90% in all donors under all
stimulatory conditions. TNF- was partially inhibited by HAT in most
cases but this varied from 2- to 3-fold among the studied donors. In
addition, the presence of HAT under all culture conditions completely
abrogated production of the Th2 cytokines IL-4, IL-5, and IL-10,
underscoring the critical role of IL-2 in regulating these
cytokines.
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To demonstrate that HAT was blocking IL-2R signaling in our PBMC
cultures, we examined the activation state of two signal transduction
molecules associated with signaling through the high-affinity IL-2R.
Fig. 1, A and B,
demonstrates that HAT blocks tyrosine phosphorylation of STAT5 without
affecting the total amount of cellular STAT5. Similarly, Fig. 1, C and D, demonstrates that HAT inhibits Jak3
activation without altering the level of Jak3 protein.
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The findings demonstrate that most T cells are activated at 48 h in HAT-treated cultures, indicating that inhibition is not likely the result of an early block in T cell activation. Collectively, these results, along with HAT’s inability to inhibit IL-2 production, support the conclusion that in our system, HAT is acting specifically through its inhibition of IL-2R signaling.
HAT inhibits IFN- production from both CD4 and CD8 T cells
Numerous studies have implicated exuberant Th1 responses and
IFN- in the immunopathogenesis of certain autoimmune disorders and
cell-mediated allograft rejection. Therefore, we focused in this study
on the role of IL-2 in regulating human IFN- production under
conditions of polyclonal T cell activation. PBMC were stimulated with
OKT3 ± anti-CD28 Ab or exogenous IL-2 in the presence or
absence of HAT. IFN- was measured by ELISA at the peak of production
(48 h). As shown in Fig. 2A,
PBMC stimulated with OKT3 produced 11,560 ± 2,125 pg/ml of
IFN- whereas unstimulated PBMC did not produce any detectable
IFN- protein. The addition of anti-CD28 Ab or exogenous IL-2 to
stimulated PBMC cultures resulted in a 2- to 3-fold enhancement
of IFN- production compared to cultures stimulated with OKT3
alone. The addition of HAT to PBMC cultures severely inhibited IFN-
at 48 h under all stimulation conditions. The inability of
exogenous IL-2 to enhance IFN- production in the presence of HAT
further demonstrates that inhibition is a consequence of IL-2R
blockade. Moreover, the results indicate that the majority of IFN-
production is IL-2-dependent and that the CD28 enhancement of IFN-
is mediated wholly through IL-2.
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-producing T cells are inhibited by HAT in these PBMC
cultures. PBMC were stimulated with OKT3 ± anti-CD28 for
48 h in the presence or absence of HAT and stained for
intracellular IFN-. As shown in Fig. 2
B, IFN-
production from both CD4 and CD8 T cells was inhibited by HAT. In
addition, inhibition of IFN- production was observed in CD45RA and
CD45RO CD4 T cell populations (data not shown). HAT inhibits IL-12p70 production from CD28-costimulated PBMC
IL-12 is a potent regulator of Th1 responses and IFN-
production from both T cells and NK cells. IL-12p70, composed of the
p40 and p35 chains, is the biologically active heterodimer necessary
for optimal induction of Th1 responses. Previous work has demonstrated
that interactions between APC and T cells via CD40/CD40L are essential
for APC production of IL-12 via the T cell-dependent IL-12 biosynthesis
pathway. We thus sought to determine whether HAT-mediated inhibition of
IFN- might be due to a reduction in IL-12 production. Fig. 3A illustrates the average
IL-12 production from the PBMC of eight normal donors. PBMC were
stimulated with high-dose OKT3 (10 µg/ml) and anti-CD28 (10
µg/ml) Ab in the presence or absence of HAT or anti-CD40L Ab. In
these experiments, the addition of anti-CD40L Ab serves as a
positive control for IL-12 inhibition. After 48 h, supernatants
were collected and assayed for IL-12p70 protein by ELISA. There was no
detectable IL-12 in supernatants from unstimulated PBMC (data not
shown). IL-12 production from stimulated PBMC (medium) produced a mean
of 24 ± 3 pg/ml. The addition of either HAT or anti-CD40L Ab
to stimulated PBMC cultures blocked IL-12 production. These data
indicate that IL-12 production from PBMC in response to T cell-specific
stimuli is highly IL-2-dependent.
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. Alternatively, monocytes were
stimulated with SAC and IFN- to model IL-12 production via the
microbial pathway. IL-12p70 was assayed at 48 h by ELISA. As shown
in Fig. 3B, the addition of HAT to pure monocyte cultures
had no effect on IL-12 production via either pathway. This further
demonstrates that HAT-mediated inhibition of IL-12 from PBMC cultures
is directly related to its limiting effect on T cell activation.
Therefore we examined whether CD40L expression on activated CD3 T cells
might be IL-2-dependent, thus potentially explaining, at least in part,
the inhibition of IL-12 production by HAT.
CD28 costimulation enhances late, but not early, CD40L surface
expression and IFN- production in an IL-2-dependent manner
To examine the role of CD28 and IL-2 on CD40L surface expression,
PBMC were stimulated with OKT3 ± anti-CD28 Ab in the presence
or absence of HAT. CD40L surface expression was generally assessed at
6, 12, 24, 36, and 48 h using flow cytometric analysis. As
reported previously by others, a low level of CD40L expression was
detected on freshly isolated CD3 T cells (data not shown). T cells
stimulated with OKT3 alone exhibited maximal expression of CD40L at
6 h, declining until reaching a nadir at 24 h (Fig. 4A). By contrast, the addition
of anti-CD28 Ab to OKT3-stimulated cultures enhanced CD40L
expression 4-fold at 48 h (Fig. 4B). The inclusion of
HAT abrogated the CD28-mediated enhancement of CD40L expression at
48 h and inhibited expression in both OKT3-stimulated and
CD28-costimulated cultures at all time points except 6 h (Fig. 4).
A similar pattern was observed in all the donors tested. These findings
demonstrate that CD40L expression is up-regulated by CD28
costimulation, at late but not early times, in an IL-2-dependent
fashion.
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expression using
intracellular cytokine staining at early and late time points in
CD28-costimulated PBMC (Fig. 5). As was
observed for CD40L expression, IFN- production in CD4 and CD8 cells
was not inhibited by HAT at 6 h. In marked contrast, at later time
points IFN- production was nearly abolished in HAT-treated cultures.
These results demonstrate that early expression (6 h) of CD40L and
IFN- is not inhibited by HAT while at later times CD28-mediated
enhancement of CD40L and IFN- expression occurs through an
IL-2-dependent mechanism.
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production in activated
PBMC cultures
To determine whether the reduced levels of IL-12 observed in
HAT-treated cultures accounted for all of the inhibition of IFN-
production, we tested the ability of exogenous IL-12 to restore IFN-
production in such cultures. PBMC were activated with OKT3 and
anti-CD28 Ab with varying doses of exogenous IL-12 in the presence
or absence of HAT. Supernatants were collected at 48 h and assayed
for IFN- production by ELISA. Representative results from one of
eight donors tested are shown in Fig. 6.
At all doses, IL-12 failed to restore IFN- in HAT-treated cultures
to the level seen in cultures without HAT. These data suggest that
HAT-mediated inhibition of IFN- production is not due solely to
diminished IL-12 production.
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in activated PBMC cultures treated with HAT
CD40/CD40L interactions, in addition to inducing IL-12,
up-regulate APC expression of several molecules associated with T cell
activation and/or costimulation. Thus the inability of exogenous IL-12
to fully restore IFN- production might be due to insufficient
costimulation from monocytes in HAT-treated PBMC cultures. To test this
possibility, we assessed the ability of CD40LT to restore IFN-
production from activated PBMC cultures treated with HAT. PBMC were
stimulated with OKT3 and anti-CD28 Ab for 48 h in the presence
or absence of HAT, along with CD40LT. IL-12 and IFN- content were
determined by ELISA. As shown in Fig. 7A, over a dose range of
CD40LT, CD28-costimulated cultures produced between 500 and 1100 ng/ml
of IFN-. The addition of HAT to the cultures strikingly reduced
IFN- to a range of 34–120 ng/ml. In contrast, when IL-12 production
was assessed in the same cultures shown in Fig. 7A, the
level of IL-12p70 was unaffected by the presence of HAT (Fig. 7B). Thus, exogenous CD40/CD40L stimulation could fully
restore IL-12 production in the presence of HAT, but failed to restore
IFN- production. These data suggest that HAT-mediated inhibition of
IFN- production is not due solely to diminished CD40/CD40L
interactions or the resulting loss of IL-12 production.
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1 and IL-12R2 surface expression on
CD28-costimulated human T cells is IL-2-independent
The failure of either exogenous IL-12 or CD40LT to restore IFN-
in CD28-costimulated PBMC cultures suggested that T cells either could
not respond optimally to IL-12 and/or that HAT directly inhibited
IFN- production from T cells. To ascertain whether the inability of
exogenous IL-12 to fully restore IFN- production in HAT-treated
cultures might be due to diminished levels of IL-12R, we examined the
surface expression of both chains of the receptor by flow cytometry.
PBMC were stimulated with OKT3 (0.2, 2, or 10 µg/ml) and
anti-CD28 Ab in the presence or absence of HAT. Cells were then
harvested at 48 h and stained immediately for CD3 and IL-12R1
or IL-12R2. Independent of the dose of OKT3, the addition of HAT did
not lead to a significant change in the surface expression of either
the IL-12R1 or the IL-12R2 chain in six donors. The same was true
at 72 h (data not shown). Results from a representative donor are
shown in Fig. 8A. While the
addition of HAT had no effect on IL-12R expression in these donors, it
did inhibit IFN- production in the same culture (Fig. 8B).
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production from purified CD4 and CD8
T cells
To determine whether IFN- might be produced from T cells
independently of IL-12, and if so, whether such production is inhibited
by HAT, we purified CD4 and CD8 T cells from PBMC so as to remove the
IL-12-producing APC. Purified CD4 and CD8 T cells, along with whole
PBMC from the same donor, were stimulated with OKT3 and anti-CD28
Ab in the presence or absence of HAT. As shown in Fig. 9, only the unfractionated PBMC
population showed demonstrable IL-12-dependent IFN- as evidenced by
a 60% reduction in IFN- production when anti-IL-12 Ab was added
to culture 531–11(28,531–11,596 pg/ml). However, the addition of
anti-IL-12 Ab to the purified CD4 and CD8 T cells cultures had no
significant effect on IFN-, indicating that IFN- production in
these cultures occurred in an IL-12-independent manner. In contrast,
the addition of HAT in this experiment resulted in a >90% inhibition
of IFN- production from PBMC 531–1(28,531–1,232 pg/ml). In the same
donor, the addition of HAT to CD4 T cell cultures resulted in a 6-fold
decrease in IFN- 970–1(8,970–1,433 pg/ml) and a 2-fold inhibition of
IFN- from CD8 T cell cultures 814–1(2,814–1,390 pg/ml). Thus, HAT
inhibits IL-12-independent IFN- production from purified human T
cells demonstrating a direct role for IL-2R signaling in the regulation
of IFN- expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. Earlier studies had noted that anti-Tac inhibits
IFN- production from T cell clones or PHA-stimulated PBMC but did
not explore the basis of this inhibition (21, 22). In our
studies, intracellular cytokine staining at the peak of IFN-
production (48 h) revealed that HAT inhibits IFN- expression in both
CD4 and CD8 T cells, independent of CD28 costimulation. In contrast,
early production of IFN- (6 h) was largely independent of IL-2 (or
CD28 costimulation; data not shown). The lack of CD28-mediated
enhancement at 6 h is consistent with the suggestion that there
exists a population of Th1 memory and/or effector cells that do not
require B7/CD28 interactions for IFN- production. Such cells may
either be independent of costimulatory signals or dependent on
costimulatory signals that are as yet incompletely characterized
(23).
IL-12, a critical regulatory cytokine for IFN- induction, can be
induced via a microbial pathway or through a CD40/CD40L-dependent T
cell pathway (14, 24). Studies in both the human and mouse
have reported that CD28 costimulation or IL-2 have a role in the
regulation of IL-12p40 production from APC (25, 26, 27). In
this study, we report that HAT treatment completely abrogates
detectable IL-12p70 production from OKT3/CD28-stimulated PBMC while
having no effect on pure monocyte cultures. These results demonstrate
that CD28 costimulation of IL-12 production is mediated through the
IL-2R and highlight the critical role of endogenous IL-2 in the
regulation of IL-12. Collectively, these functional data raise the
question of whether CD28 costimulation and/or IL-2 regulates CD40L
expression, if not initially, then possibly at later time points.
While studies using CD40L promoter driven reporters in both the mouse and human have found expression to be upregulated by CD28 costimulation, results from the majority of cellular studies in the mouse have been divided (28, 29). Data from the CD28 knockout mouse clearly demonstrate that CD28 is not required for the induction or early (6–10 h) expression of CD40L (26, 30). Despite the phenotype of the CD28-/- mouse, B7 blockade in vitro and in vivo has been shown to inhibit CD40L expression. This finding raises the possibility that as yet unidentified B7 counterreceptor(s) may costimulate CD40L expression or that CD28 costimulation in the mouse, as we observe for human PBMC, is needed for sustained surface expression of CD40L (30, 31, 32, 33).
Conflicting reports on the temporal expression of human CD40L can probably be attributed to more limited data sets. The earliest studies reported that at 24 h, surface expression was weak, with subsequent studies showing that CD40L mRNA peaks much earlier (34, 35, 36). In CD28-costimulated cells CD40L expression peaked later and then decreased (37). While costimulation greatly augmented the accumulation of CD40L mRNA, its affect on CD40L surface expression was much more modest, advancing the peak of expression without altering the absolute level (34). In none of the above studies was it determined whether the increase in CD40L expression was a direct consequence of CD28 signaling or mediated via IL-2.
In long term (14 day) cultures of human CD4 cells stimulated with anti-CD3/anti-CD28-coupled beads it has been shown that IL-2 alone can reinduce the expression of CD40L (38). In comparison to plate bound anti-CD3 and anti-CD28 costimulation at 6 h, Ab-coupled beads induce CD40L expression to a higher level in a greater number of naive CD4 cells and that expression is sustained beyond 96 h. It has also been reported that at 24 and 48 h, CD40L on purified CD4 T cells stimulated with plate bound anti-CD3 is up-regulated by IL-2 (39).
In our studies we examined the regulation of human T cell CD40L
expression by both CD28 and IL-2. By examining early, intermediate, and
late time points in the presence and absence of CD28 costimulation our
studies reveal that CD40L exhibits a biphasic pattern of surface
expression. This pattern of CD40L expression bears a striking
resemblance to that of IFN-. Despite this correlation, the
relationship between late CD40L and IFN- expression is unclear, as
it would be expected that CD40L expression should precede that of
IFN-. Our results unify and clarify the disparate results reported
in earlier human studies on CD40L regulation and demonstrate that the
CD28-mediated enhancement of CD40L expression is wholly dependent upon
IL-2R signaling. Whether our findings indicate that CD28 regulation of
CD40L expression in the human differs from that in the mouse awaits an
analysis of CD40L expression in the mouse at later time points under
similar stimulatory conditions.
The biological activities of IL-12 are mediated via a specific
high-affinity receptor composed of the IL-12R1 and IL-12R2
subunits (40, 41, 42). The IL-12R2 subunit on T cells
appears to be the critical determinant involved in maintaining IL-12
responsiveness and controlling Th1 commitment (43, 44).
Investigators have previously reported that B7/CD28 costimulation
and/or IL-2 play a role in the regulation of both IL-12R subunits in
mouse and man.
Studies in mice have shown enhanced mRNA expression for both IL-12R
subunits in activated T cells in the presence of B7 accessory cells
(45, 46). Furthermore, it has been demonstrated in mice
that CD28 costimulation, acting through IL-2, is necessary for both the
normal expression of IL-12R2 and subsequent differentiation of
autoreactive effector cells (26). This requirement for
CD28 signaling may impact not only the level of IL-12R surface
expression, but also the receptors ability to signal as well
(47).
In human PBMC, IL-12R is not present on the surface of unstimulated
cells, but can be induced by exogenous IL-2 alone. By contrast,
induction of the IL-12R on PHA blasts appears to be largely
IL-2-independent (48, 49). In OKT3-stimulated PBMC,
anti-CD28 Ab has been reported to augment IL-12R1 surface
expression and IL-12 binding; however, these authors did not determine
if this enhancement was IL-2-dependent (49).
Our studies demonstrate that IL-12R levels are undiminished on
HAT-treated PBMC and thus cannot account for the deficit in IFN-
production from these cultures. The results indicate that the surface
expression of both IL-12R subunits is independent of endogenous
IL-2/IL-12R signaling in CD28-costimulated PBMC. These findings are
consistent with the early observations of Desai et al.
(48). Our findings may also suggest that there is
differential regulation of the IL-12R2 chain between mouse and
human, as has recently been elucidated for regulation of IFN-
receptor signaling in these two species, however, ascertainment of this
will require further study (50).
Despite the unaltered surface expression of the IL-12R subunits on
activated T cells, exogenous IL-12 failed to restore IFN- in
HAT-treated cultures. In addition, the IL-12 dose response curves
obtained in the control and HAT-treated cultures are parallel,
suggesting that the inability to restore IFN- is not due to a defect
in IL-12R signaling. Furthermore, IFN- production could not be
restored by adding CD40L trimer, thus demonstrating that the deficit
was not due to a lack of CD40/CD40L-dependent costimulation from APC in
the IL-12/HAT-treated cultures (51). The ability of
exogenous CD40/CD40L stimulation to fully restore IL-12 in HAT-treated
cultures demonstrates that inhibition of CD40L expression limits IL-12
production. In contrast, the inability of CD40LT to restore IFN-
suggests that a component of HAT-mediated inhibition of IFN- is
IL-12-independent.
Earlier studies with human PHA blasts or unstimulated PBL cultures
examined the role of CD28 stimulation or IL-12 and/or IL-2 in the
induction of IFN- (25, 52, 53, 54). Those studies found
that upregulation of IFN- production by exogenous IL-12 was enhanced
by an anti-CD28 Ab and inhibited by CTLA4-Ig. Interestingly,
costimulation enhanced IFN- production synergistically when an IgM
anti-CD28 Ab was used but not when the IgG2a anti-CD28 Ab
employed in our studies was used (54). However, these
investigators concluded that CD28 costimulation in combination with
IL-12 drives IFN- production by a mechanism that is largely
independent of IL-2 production. Similar findings have been reported in
murine Th1 clones (55). In contrast, we conclude that in
CD28-costimulated PBMC cultures, a significant component of IFN-
production is dependent upon IL-2R signaling, even in the presence of
maximal amounts of IL-12 or CD40L signaling. This conclusion led us to
examine whether IL-12-independent, IL-2-dependent IFN- might account
for this observation. Using highly purified CD4 and CD8 T cell
populations stimulated with anti-CD3 and anti-CD28, we show
that in part, IL-12-independent IFN- production is indeed
IL-2-dependent.
Thus, we conclude that IL-2R signaling impacts both the IL-12-dependent
and -independent T cell pathways of IFN- production. In addition, we
find that the production of both Th1 and Th2 cytokines in
CD28-costimulated PBMC cultures is severely inhibited by HAT. Previous
reports in both the mouse and human have demonstrated that IL-2 plays
an important role in regulating cytokine responses (10, 11, 21, 56, 57, 58, 59, 60). In naive mouse cells, Th1/Th2 cytokine expression is
cell cycle-dependent, suggesting that the role of IL-2 is to drive such
cells to cycle (61). By contrast, previously activated
cells that have not undergone cell division can express CD25 and
produce IFN- upon restimulation to the same extent as cells that had
divided earlier (62). We find in a bulk population of
human PBMC that inhibiting IL-2R signaling blocks cytokine production
prior to cell division and, at least for IFN-, this occurs both in
presumably naive (CD45RA) and memory (CD45RO) cells. We are presently
exploring the relationship between cell division, cytokine production,
and IL-2R signaling at the single cell level using CFSE staining.
While the ability of HAT to inhibit cytokine production may have been predicted from studies of the IL-2 and CD25 knockout mice, the therapeutic use of HAT would never have been predicated on the phenotype of those mice, which actually develop autoimmune disease. The long-term safe and efficacious use of HAT in our patient population clearly demonstrates that results from the mouse must be extrapolated to the human with great caution. This apparent contradiction might be explained by the recent observation that mice whose T cells are IL-2R-deficient in the periphery, but not in the thymus, do not develop autoimmune disease (63). The demonstration that HAT can simultaneously inhibit the production of both Th1 and Th2 cytokines suggests that it may have a clinical role in the treatment of severe allergic type diseases in addition to its current usage in the transplant and autoimmune disease settings (64).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HAT, humanized anti-Tac Ab; CD40L, CD40 ligand; SAC, S. aureus Cowan strain; CD40LT, trimeric soluble CD40L molecule; Jak, Janus kinase; CBA, Cytometric Bead Array.
Received for publication March 12, 2002. Accepted for publication June 26, 2002.
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