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Department of Environmental and Molecular Toxicology and Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF was significantly suppressed from
CD154-deficient mice, indicating a lack of T cell priming. However,
splenic cells from CD154 knockout mice induced comparable levels of
CD86 expression and IL-12 production when compared with their wild-type
littermates. The treatment of CD154-/- mice with the
agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed
to restore either the CTL or alloantibody responses to P815. Likewise,
immunization with B7-transfected P815 tumor cells failed to generate
expansion of the CTL effector population in CD154-/-
mice. These results suggest that the generation of allograft immunity
is dependent on the interaction of CD154 with CD40 but not primarily
for the activation of APCs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD154 is a member of the TNF family (7, 8), which includes
TNF-
, CD95 ligand, LT , LT ß, and the ligands for CD27, CD30,
OX-40, and 4-1BB (9). Expression of CD154 is found
primarily on activated CD4+ T cells
(10), but has also been reported on
CD8+ T cells, mast cells, and basophils
(10, 11, 12, 13). CD40, the receptor for CD154, is expressed on
APCs such as B cells (14), macrophages (15),
and dendritic cells (16). It is a member of the TNF
receptor family (14), which includes TNF-RI (p55), TNF-RII
(p75), CD95, CD27, CD30, OX-40, and 4-1BB (9, 14).The
interaction of CD154 with CD40 has been identified as a major
pathway for the activation of APC and is essential for the
generation of many cell-mediated immune responses
(17, 18, 19). Disruption of this pathway leads to increased
susceptibility to opportunistic pathogens, illustrated by the
prevalence of Pneumocystis and Cryptosporidium
infections in hyper-IgM syndrome patients who fail to express
functional CD154 (20, 21). Experimental animal models to
investigate this immunodeficiency have been established by
administering CD40-Ig fusion protein or antagonistic CD154 Abs to block
CD154:CD40 interactions (17). Recently, the creation of
CD154 and CD40 knockout mice have allowed further investigation of the
important role of this pathway in the development of effective immunity
(22, 23).
It has been shown that CD40 ligation induces accessory molecules such as CD86, CD80, CD54, and MHC II on APC populations (10, 24, 25). It has also been determined that stimulation via CD40 induces secretion of IL-12 from both dendritic cells and macrophages (26, 27, 28). Recently, several reports suggested that a primary role of activated Th cells in the generation of CD4-dependent cell-mediated immunity was to provide CD40 ligation on APC, presumably to increase B7 expression and IL-12 secretion (29, 30, 31). This activation step then empowered the APC to successfully activate CD8+ cells and drive their differentiation into CTL effectors. Therefore, we hypothesized that a lack of CD154:CD40 interaction would prevent generation of allograft immunity by failing to increase B7 expression and induce IL-12 secretion from APC. To test this hypothesis, we used the P815 tumor allograft model and followed the activation of B cells, macrophages, and dendritic cells from CD154-/- and wild-type mice over the course of the allogeneic response. However, despite a significantly reduced capacity of CD154 knockout mice to generate alloimmunity, we found no differences in the induction of costimulatory molecule expression or IL-12 secretion between CD154+/+ and CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to six-week-old male C57BL/6, CD154-/- (B6, 129-Cd40l), CD154+/+ wild-type (B6, 129 F2), and female DBA/2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in front of a laminar flow unit.
P815 mastocytoma cells
Wild-type P815 tumor cells, derived from a methylcholantherene-induced mastocytoma, were maintained by weekly passage in DBA/2 mice. CD86-transfected P815 (clone HTR.C/B7-2) and its vector-transfected control (clone HTR.C/C) were generously provided by Dr. Thomas Gajewski (University of Chicago Medical Center, Chicago, IL). Transfected cells were maintained in vitro in RPMI 1640 medium supplemented with 10% FBS (HyClone, Logan, UT) and 1 mM gentamicin at 37°C in 5% CO2.
Reagents
HPLC-purified, antagonistic anti-CD154 mAb (MR1) used for in
vivo blocking experiments, anti-CD4 (GK1.5) used for depletion
studies, and agonistic anti-CD40 mAb (FGK45) used for exogenous
CD40 ligation experiments were all kindly provided by Dr. Randolph J.
Noelle (Dartmouth Medical School, Lebanon, NH). Hamster IgG and rat IgG
were purchased from Cappel (Organon Teknika, West Chester, PA) and used
as Ig controls for the in vivo blocking and CD40 ligation experiments,
respectively. Spleen cell phenotypes were determined by flow cytometric
analysis using the following Abs: FITC-conjugated anti-CD54 (3E2),
FITC-conjugated anti-CD11c (HL3), FITC-conjugated anti-CD62L
(MEL-14), PE-conjugated anti CD86 (GL1), PE-conjugated anti-CD44
(IM7), PE-conjugated anti-CD54, Biotin-conjugated
anti-IAb (AF6-120.1), CyChrome-conjugated
anti-B220 (RA3-6B2), and CyChrome-conjugated anti-CD8
(53-6.7) from PharMingen (San Diego, CA); Tri-color-conjugated
anti-Mac-1 (M1/70.15) from Caltag Labs (Burlingame, CA), and the
second step reagent streptavidin-Red613 from Life Technologies
(Gaithersburg, MD). For cytokine ELISA, capture and biotinylated
detection Ab pairs and their respective standards were purchased from
PharMingen (San Diego, CA), except for IL-12, which was purchased from
R&D Systems (Minneapolis, MN), and IL-4, which was obtained from
Genzyme (Cambridge, MA).
Animal treatments
Studies to evaluate the role of CD154 in allograft rejection were conducted in CD154-/- mice and their wild-type littermates. Alternatively, C57BL/6 mice were treated i.p. with 250 µg of MR1 on day 0 relative to P815 immunization to block CD154:CD40 interactions. In vivo depletion of CD4+ T cells was performed by injecting mice with 0.25 mg GK1.5 on day -2 relative to CD86-P815 injection. Preliminary studies determined that >99% of naive (CD44highCD45RBlow) CD4+ T cells were depleted for at least 10 days using this treatment protocol. Mice were injected i.p. with either 1 x 107 tumor ascites P815 or CD86-transfected P815 cells on day 0. The protocol for providing exogenous ligation of CD40 involved i.p. injection of both CD154-/- and CD154+/+ mice with 5 µg of FGK45 on days 0, 2, 4, 6, and 8 relative to P815 injection. All mice were sacrificed by CO2 asphyxiation on days 5–8, or 10 relative to injection. Spleens were removed aseptically, and blood was collected by heart puncture into heparinized syringes. Plasma was separated by centrifugation and stored at -70°C.
Preparation of spleen cells
Single-cell suspensions were prepared by pressing the spleen between the frosted ends of two microscope slides. Erythrocytes were removed by hypotonic lysis. Cells were washed once and resuspended in cold HBSS/5% FBS with 20 mM HEPES, 50 µg/ml gentamicin, and 1.5 mM sodium pyruvate.
Flow cytometric analysis of spleen cells
Expression of the cell-surface proteins CD86, CD54, and I-Ab was determined from viable spleen cells and then further measured on gated populations of B220+, Mac-1+, and CD11c+ cells. A distinct population of spleen cells expressing high levels of CD11c (CD11chigh) was determined to represent dendritic cells by profiles of costimulatory molecule expression. These CD11chigh cells constitutively expressed high levels of CD86, CD54, and I-Ab similar to levels found on dendritic cells enriched over BSA-"dense" gradients as previously described (32). In a typical splenic preparation, 1.5–3.3% of the cells were CD11chigh, of which >88.4% were CD86+, >99.5% were CD54+, and >95.5% were I-A+. CTL effectors (CTLE)3 were identified by first gating on the CD8+ T cells and then identifying the CD44high/CD62Llow population, as previously described (33). Nonspecific binding was blocked with 10 µg of rat IgG, and then the cells were stained with optimal concentrations of fluorochrome-conjugated mAb. Appropriately labeled, isotype-matched Igs were used as controls for nonspecific fluorescence. Between 20,000 and 100,000 events were collected by listmode acquisition from freshly stained cells using a Coulter XL flow cytometer (Coulter Electronics, Hialeah, FL) and analyzed using WinList (Verity Software House, Topsham, ME).
CTL assay
The cytolytic activity of spleen cells against P815 tumor
(ascites) cells was measured in a standard 4-h
51Cr release assay as previously described
(34). The percentage of cytotoxicity at each E:T ratio was
calculated using the following equation:
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Cytotoxic Ab assay
Cytotoxic alloantibody titers were determined using a
complement-dependent 51Cr release assay as
previously described (34). Briefly, serial 2-fold
dilutions (1/10 to 1/2560) of heat-inactivated plasma were incubated
with 51Cr-labeled P815 cells for 20 min at 37°C
in 5% CO2. Low-Tox-M rabbit complement (1/12,
Cedarlane Laboratories, Hornby, Ontario, Canada) was added for 45 min
at 37°C. The amount of 51Cr released into the
supernatant was measured by counting and specific activity was
calculated. All samples were tested in duplicate on separate plates.
The Ab titer was defined as the highest dilution of plasma at which a
minimum of 20% specific cytotoxicity was measured.
Cytokine analysis
Spleen cells (1 x 107) were
incubated in RPMI/10% FBS supplemented with 1.5 mM sodium pyruvate, 20
mM HEPES, and 50 µg/ml gentamicin for 6 h with 1 x
106 P815 tumor cells at 37°C in 5%
CO2 in 1.5 ml polypropylene Eppendorf tubes that
were treated with silicone to prevent nonspecific production of IL-12
by adherent spleen cells. Supernatant cultures from P815 tumor cells
alone and naive spleen cells with P815 were also collected as controls
for each experiment. Levels of IL-2, IL-4, IL-12, and IFN- were
determined for each supernatant by using specific Ab sandwich ELISAs.
Secondary biotinylated detection reagents were complexed with
avidin-peroxidase and visualized with ABTS substrate. Absorbance was
measured at 405 nm using a plate reader (Bio-Tek Instruments, Wincoski,
VT), and cytokine values were then determined using Immunosoft software
(Dynatech Labs, Alexandria, VA). TNF levels were measured by standard
bioassay (34) using L929 fibroblasts.
Statistical analysis
Results are presented as the mean ± SE of six mice per
group unless indicated otherwise. Most experiments were repeated at
least once. ANOVA modeling was performed using Statview statistical
software (Abacus Concepts, Berkeley, CA). Comparisons between means
were made using the least significant difference multiple comparison
t test or Dunnet’s t test for pairwise
comparisons. Values of p 
0.05 were considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have established the importance of CD154 in the
generation of cell-mediated immunity, including models of allograft
rejection (36, 37). We investigated the role of CD154 in
the generation of immune responsiveness to allogeneic P815 tumor cells.
Following immunization of C57BL/6 mice with P815 tumor cells, the
concomitant development of CTL and alloantibody responses effectively
rejects these allogeneic cells. The allograft response peaks within 10
days of immunization and correlates with the generation of CTL activity
specific for H2-Dd-bearing cells
(34) and the emergence of alloantibodies in the plasma
(Fig. 1
, A and B).
CD154 knockout mice failed to generate allo-CTL activity following
immunization with P815 tumor cells (Fig. 1A). Similar
results were also observed in P815-immunized
CD154+/+ mice, which had been treated with the
anti-CD154 blocking Ab, MR1. Furthermore, the production of
cytotoxic alloantibody was suppressed by >90% in both the
CD154-/- and MR1-treated mice when compared
with their appropriate controls (Fig. 1B). Although it has
already been established that IgM is the isotype primarily responsible
for cytotoxicity in this alloantibody assay, we further evaluated the
effect of blocking CD154:CD40 interactions on the generation of other
alloantibody isotypes. Consistent with previously published reports
that illustrate the critical role of CD154 in the generation of humoral
immunity (22, 23, 38), blocking CD154 ligation of CD40
resulted in significant suppression of not only IgM, but also IgG2a and
IgG1 anti-P815 Abs, as measured by flow cytometry (data not
shown).
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The inability to mount an effective CTL response to allogeneic
P815 cells could be due to either lack of priming of T cells or
defective effector function. Therefore, we analyzed the splenic
CD8+ cells from P815-immunized
CD154-/- and CD154+/+
mice for the generation of cells bearing the CTLE
phenotype. Expression of the
CD8/CD44high/CD62Llow
phenotype correlates with cytolytic activity, as shown previously by
Mobley and Dailey (33). Ten days after P815 immunization,
CD154+/+ mice had generated 
3 x
107 CTLE while the number
of effector CTLs detected from the spleens of
CD154-/- mice did not differ significantly from
that found in nonimmunized mice (Fig. 2A). These results indicate
that the lack of CTL activity in CD154-/- mice
was due to an absence of CTLE and not an
inability of CTL cells to perform their effector function.
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, and TNF on
days 5–10 following immunization (34). As shown in Fig. 2
, B–D, increased levels of IFN- and TNF were observed
on days 6–8 and increased IL-2 production on days 5–8 from wild-type
but not CD154-deficient mice. The reduced cytokine production from
knockout mice continued through day 10 (data not shown) when both the
CTL and alloantibody assays were performed. Additionally, the
production of IL-4 was barely detectable on any day tested (data not
shown) and did not differ between wild-type and knockout mice. These
results indicate that a lack of CD154:CD40 interaction prevents proper
T cell priming although not via immune deviation (the switch from a
beneficial Th1 type of response to an inappropriate Th2 type) as
previously reported by Hancock et al. in the cardiac allograft model
(37). APC activation is unaffected in CD154-deficient mice following immunization with P815 tumor cells
To examine if disruption of the CD154:CD40 pathway would render
mice incapable of priming alloantigen-specific T cells by failing to
activate APC, we analyzed the production of IL-12 and induction of
costimulatory molecules from the spleen cells of immunized mice.
Ligation of CD40 has been shown to induce IL-12 production, primarily
from APC, thereby regulating the differentiation of activated T cells
into Th1 effector cells and promoting cell-mediated immunity
(39). Due to the considerable influence CD40-mediated
IL-12 production has on priming Th1 effector T cells and the absence of
Th1 priming previously described in CD154-deficient mice, we examined
the ability of spleen cells from P815-immunized mice to produce IL-12.
As shown in Fig. 3A, spleen
cells from both CD154 knockout and wild-type mice produced comparable
levels of IL-12 on days 5–8 postimmunization. Although levels were
higher in cultures from wild-type mice on day 8, these levels were not
significantly different from those produced by CD154 knockout mice, a
relationship that continued through day 10 (data not shown).
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B, comparable CD86 expression was exhibited on days 5–8
from CD154+/+ and
CD154-/- mice. Similarly, CD154 deficiency did
not alter normal Ag-induced increases in CD54 or
I-Ab expression on spleen cells on days 5–8
(data not shown). As it is unknown exactly which splenic APC population
is responsible for the activation of allo-specific T cells in the P815
model, expression of accessory molecules on
B220+, Mac-1+, and
CD11c+ cells was determined. Shown in
representative histograms in Fig. 3, C–E, CD154 deficiency
did not alter CD86 expression on any of these APC populations following
P815 immunization. Histograms are shown for day 6 data, but are
representative of all days tested (days 5–8). Ag-induced increases in
splenic CD86 expression were observed to be slight on the B cells (mean
channel fluorescence (MCF = 9), significant for the dendritic
cells (MCF = 44), while decreased expression was exhibited on the
macrophage population (MCF = 5.5). This apparent reduction in CD86
expression on Mac-1+ cells following P815
injection may be attributable to an influx of
Mac-1+ cells into the spleen (2% in naive mice
vs 10% in mice injected with P815. However, this increased
Mac-1+ population was seen in both the wild-type
as well as the knockout mice, yielding no explanation for the absence
of T cell priming in mice defective for CD154. Similar patterns of CD54
and I-Ab expression were also detected on days
5–8 for each of the splenic APC populations examined without
significant effects of CD154 deficiency (data not shown). Although
these markers of APC activation are by no means exhaustive, the data
taken together fail to establish any measurable differences in
CD40-mediated APC activation between P815-immunized CD154-deficient or
wild-type mice. The apparent lack of effect of the CD154 mutation on
induction of the accessory molecules CD86, CD54, and
I-Ab, combined with an unaltered capacity to
produce IL-12 in the knockout mice, suggests that CD40 interaction with
its ligand is not an essential requirement for activation of APC in
this model of allograft immunity. Exogenous ligation of CD40 fails to restore allograft immunity in CD154-deficient mice
Because CD154-/- mice are genetically
incapable of expressing functional CD154, we hypothesized that
exogenous ligation of CD40 would circumvent the need for its ligand and
restore allograft effector functions in these mice. To test this
hypothesis, both wild-type and CD154-deficient mice were immunized with
P815 tumor cells and then administered FGK45, an agonistic
anti-murine CD40 mAb. Treatment of mice with the 5-µg dose of
FGK45 produced highly elevated expression of CD86 on splenic APC and
significantly increased plasma levels of IL-12 within 48 h (Fig. 4, A and B).
Surprisingly, exogenous ligation of CD40 with FGK45 did not restore CTL
activity in CD154-/- mice to levels seen from
wild-type mice (Fig. 4C). Although a small increase in CTL
activity was observed in CD154-/- mice that had
received anti-CD40 treatment, this increase was not statistically
significant at any of the E:T ratios examined. It is important to note
that splenic T cells from CD154-/- mice are not
intrinsically incapable of generating CTL effector cells as shown in a
recent report by Buhlmann et al. In this report, spleen cells from
CD154-/- and wild-type mice generated
comparable CTL activity after being cultured in vitro for 6 days with
allogeneic stimulator cells (41). In addition to a lack of
restoration of the CTL response by anti-CD40, no enhancement of
cytotoxic alloantibody plasma titers was observed in
anti-CD40-treated CD154-/- mice when
compared with the appropriate controls (Fig. 4D).
Interestingly, treatment of CD154+/+ mice with a
5-µg dose of FGK45 partially suppressed the generation of cytotoxic
alloantibodies on day 10 after P815 immunization, yet had no adverse
effect on CTL activity. The dose of FGK45 administered to mice in these
experiments proved to be of critical importance as higher amounts of
anti-CD40 mAb (25 and 100 µg) produced moderate to severe
organ-specific toxicity including thymic atrophy, hepatic coagulative
necrosis and neutrophilia, and splenic lymphoproliferation within the
lymphoid follicles (D.M.S. and O. R. Hedstrom, unpublished
observations). Ultimately, any beneficial effects of increasing
costimulatory molecule expression and systemic IL-12 production by APCs
following anti-CD40 treatment failed to enhance the generation of
allograft effector function.
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We have previously reported that C57BL/6 mice depleted of
CD4+ T cells failed to generate CTL activity
following P815 immunization (34). However, when P815 cells
transfected to express high levels of murine CD86 (Fig. 5A) were used to immunize
CD4-depleted mice, increased numbers of CTL effector cells were
generated when compared with CD4-depleted mice injected with
vector-transfected P815 cells (Fig. 5B). The dual expression
of both H2-Dd (signal one) and B7 (signal two)
provides necessary stimulation of alloreactive
CD8+ cells to drive their differentiation from
precursor CTL (CTLp) into CTLE possessing full
cytolytic activity. To examine a possible role for CD154 in
CD8+ T cell activation, we immunized
CD154-/- mice with B7-transfected P815 tumor
cells. By providing the requisite two signals (Ag and costimulation)
for T cell activation, CD154 knockout mice (similar to CD4-depleted
mice) should be capable of generating CTLE unless
there is a requirement for CD154 in this process. On day 10
postimmunization, the numbers of CTLE were
determined for both CD154-/- and
CD154+/+ mice. In contrast to the results
observed in CD4-depleted mice, CD154-/- mice
injected with P815-CD86 failed to generate comparable numbers of
CTLE when compared with wild-type mice that had
received either P815-vector or P815-CD86 (Fig. 5C). Also,
following immunization with either P815-vector or P815-CD86,
CD154-/- mice failed to generate CTL activity
or splenic IFN- production comparable to that measured from
CD154+/+ mice (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, the mechanism by which disruption of the CD154:CD40 pathway suppresses cell-mediated immunity varies depending on the experimental model examined. Our results show that immunization of CD154-deficient mice with allogeneic P815 cells failed to properly prime allo-specific T cells. Inadequate priming of T cells due to the absence of CD154 has been shown in several other models of T-dependent immunity. Studies by Grewal et al. demonstrated suppression of Ag-specific expansion and effector cytokine production by CD4+ T cells from mice lacking CD154 (50). Likewise, Gray and coworkers showed that T cells activated in the absence of CD40 were unable to help normal B cells undergo Ig class switching or germinal center formation (51). While data exist implicating CD154 involvement in Th cell priming, studies examining its role in priming of CD8+ cells are less clear. A limited number of studies suggest that activation of CD8+ CTLs following viral infection is unaffected in CD154-/- mice (52, 53, 54). However, a role for CD154 was indicated for the maintenance of CTL memory cells, as the anti-viral memory CTL response was defective in mice deficient for CD154 (52). Currently, while the importance of CD154 in the generation of the allograft response is accepted, the precise role is unclear. In a study by Larsen et al., the predominant effect of CD154 blockade was found to be defective effector T cell function and not the priming of these cells (36). However, in a study by Hancock et al., T cells were found to have been inappropriately primed causing them to deviate from the normal Th1 phenotype into suppressive Th2 cells that produced increased levels of IL-4 and IL-10 (37). In our model, failure to generate alloimmunity was due to defective T cell priming and not due to immune deviation (as IL-4 levels were undetectable) or compromised T cell effector function (no CTLE were generated).
Currently, a potential explanation for the suppressed cell-mediated immunity observed in CD154-deficient mice revolves around inadequate APC activation attributable to deficient CD40 ligation. CD40 ligation has been shown to induce increased APC activity by up-regulating costimulatory molecule expression and increasing the production of IL-12 as well as other proinflammatory cytokines (24, 26, 27, 55). In CD154-deficient mice infected with an adenovirus, a lack of CD40 signaling resulted in a failure to generate both CTL and Ab responses (56). Reduced expression of CD86 was noted on spleen cells from these adenovirus-infected CD154 knockout mice. Moreover, treatment of these animals with an agonistic anti-CD40 mAb restored their immune responsiveness, an outcome the authors attributed to increased CD86 expression. Likewise, in a murine model of experimental allergic encephalomyelitis (EAE), Grewal et al. showed that CD154-deficient mice that carried a transgenic TCR for myelin basic protein failed to generate the disease after antigenic immunization (57). Similar to the adenoviral model, injection of these mice with APC-expressing transgenic B7 allowed subsequent development of acute EAE, indicating that CD154 was required for induction of costimulatory activity on the APC. These reports contrast with our results that suggest that APC activation is a CD40-independent event following immunization with allogeneic P815 tumor cells. Although some evidence exists identifying the dendritic cell as the key APC in our model (58), we evaluated costimulatory profiles for all putative splenic APC populations to best identify any effect of CD154 deficiency. However, no differences in CD86, CD54, or I-Ab induction were observed on the B220+, Mac-1+, or CD11c+ cells from CD154-/- and CD154+/+ mice following P815 immunization. Additionally, no effect was measured in the ability of spleen cells from both types of mice to produce IL-12 following activation. Although in contrast to the adenoviral and EAE models, these results are consistent with three reports examining the role of CD154 in a cardiac allograft model in which CD154-deficiency did not alter CD86 induction/expression (36, 37, 49). The recent study by Niimi et al. clearly demonstrated the importance of the CD40 interaction in the immune response to alloantigen and suggested that the effect of this pathway may be independent of its effect on the B7/CD28 pathway. These data taken together suggest that dependency of CD40 ligation for APC activation may be related to characteristics inherent to specific Ags. Uptake of Ags such as viruses and soluble proteins by APCs may require CD40 ligation to induce competent costimulatory activity, whereas ingestion of particulate Ags such as allogeneic cells may sufficiently activate APC and therefore have less dependence on CD40. This would provide one potential explanation for the normal expression of costimulatory molecules on APCs from CD154-/- mice in models of allograft rejection but not in other experimental systems.
The failure to restore allograft immunity in
CD154-/- mice immunized with P815 tumor cells
and treated with an agonistic anti-CD40 mAb is an interesting
result. If the only contribution of CD154 toward the generation of this
alloimmune response was to provide ligation of CD40, then it should
follow that providing that stimulus would restore allograft effector
functions. This result did not occur in our studies, possibly because
ligation of CD40 with agonisitic Abs did not reproduce the same
physiological effects as CD154. Also, it is possible that signaling
could be occurring directly into the T cells via CD154 following
ligation of CD40. Interestingly, many potentially therapeutic effects
were observed in our mice receiving anti-CD40 treatment. These
included induction of high levels of costimulatory molecule expression
on splenic APC combined with an increased production of IL-12.
Recently, it has been shown by Bennett et al. that similar
anti-CD40 treatment of CD4-depleted mice provided necessary signals
to induce competent cross-priming of Ag-exposed APC (30).
It is conceivable then that exogenous ligation of CD40 in our system
also enhanced cross-priming in our APC populations. However, no
restoration of CTL activity was seen in the P815-immunized
CD154-deficient mice that received anti-CD40 treatment, suggesting
that the defect may not reside in the APC but in the T cell. Our
results differ from those reported by Yang and Wilson (56)
and several recent reports in which anti-CD40 mAb treatment of
CD154-/- or CD4-depleted mice, restored CTL
activity (29, 30, 31, 56). However, all of these studies
evaluated CTL activity from anti-CD40-treated mice only after
driving potential CTLp in vitro for 5–7 days with Ag, APC, and even
added growth factors, making it difficult to properly compare their
results to those generated in our study. In addition to the lack of
restorative effects of anti-CD40 treatment on CTL activity, we also
observed no beneficial effects of this treatment on cytotoxic
alloantibody production in CD154-deficient mice. Furthermore,
CD154+/+ mice treated with similar doses of FGK45
displayed partially suppressed titers of alloantibodies when compared
with the rat IgG controls. This negative effect of anti-CD40 mAb
treatment is likely due to excessive signaling of B cells through this
receptor. Although ligation of CD40 has been shown to be a critical
step in the generation of humoral immunity, hyperstimulation can lead
to a lack of Ab production due to an arrest of B cell terminal
differentiation (59). Therefore, caution should accompany
any attempt to boost cell-mediated immunity in human hyper-IgM syndrome
or AIDS patients with anti-CD40 Ab (or any other reagent capable of
ligating CD40) as it may be at the expense of the other arm of acquired
immunity, the Ab response.
We have previously reported that generation of CTL activity in the P815
tumor allograft model is CD4 dependent (34). However, this
dependency can be circumvented by immunizing CD4-depleted mice with
B7-transfected P815 cells as shown in Fig. 5B. We assume
that these tumor transfectants are capable of driving
CD8+ CTLp directly to become
CTLE because they possess both Ag
(H2-Dd) and costimulatory molecules (CD86) on
their cell surface. Therefore, it was totally unexpected to find that
CD86-P815-immunized CD154-deficient mice, in contrast to CD4-depleted
mice, did not generate comparable numbers of CTLE
to those observed in CD154+/+ mice. The only
difference in the CD8+ cells from each of these
mice was the inability of the CD154 knockout mice to express functional
CD154. However, this singular genetic defect negated the ability of the
P815-CD86 to generate competent CTL activity from these mice or
properly prime their T cells as reflected in the suppressed production
of Th1 cytokines. The data reflect that mice deficient for CD154 are
clearly not equivalent to CD4-depleted mice, possibly explaining the
differences in our inability to generate cell-mediated immunity in
CD154-/- mice treated with anti-CD40 when
compared with previous reports performed in CD4-depleted mice
(29, 30, 31).
The possible function of CD154 to transduce intracellular signals into the T cell remains to be proven. Several studies have provided some evidence that CD154 may serve as a receptor capable of transducing a costimulatory signal into the T cell. In these reports, it has been suggested that CD154 is a viable signal-transducing molecule in T cells, which, when ligated, is capable of activating a sphingomyelinase and releasing ceramide (60) and/or activating the kinases c-Jun N-terminal kinase/p38-K and protein kinase C (61, 62). A study by Blotta et al. demonstrated that cross-linking of CD3 and CD154 on CD4+ T cells enhances IL-4 production (63). Functional studies aimed at increasing tumor surveillance in mice suggested that CD40 transfection of poorly immunogenic tumors greatly enhances their clearance (R. J. Noelle, unpublished observations), possibly due to enhanced ligation of CD154 on CTLp. Similar results from Cayabyab et al. suggested that the CD40-CD154 costimulation pathway may have allowed for expansion of T cells after their interaction with CD40-bearing APCs (64). Recently, a study by Suzuki et al. demonstrated that reverse signaling through CD95 ligand, another member of the TNF superfamily to which CD154 belongs, is required for alloantigen-specific CTLs to achieve maximal proliferation (35). Taken together, these data suggest a possible role for CD154 in the generation of cell-mediated immunity by methods both indirect (i.e., induction of APC activity) and direct (i.e., transduction of essential priming signals into the T cell) following interaction with its receptor, CD40. Further studies directed at defining the potential signal-transducing capabilities of CD154 or associated intracellular proteins should further our understanding of the mechanisms involved in T cell activation while also providing additional therapeutic possibilities for immunocompromised individuals.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy I. Kerkvliet, Department of Environmental and Molecular Toxicology, ALS 1007, Oregon State University, Corvallis, OR 97331. E-mail address:
3 Abbreviations used in this paper: CTLE, effector cytotoxic T lymphocyte; CTLp, precursor cytotoxic T lymphocyte; MCF, mean channel fluorescence; GVHD, graft vs host disease; EAE, experimental allergic encephalomyelitis.
Received for publication January 11, 1999. Accepted for publication June 11, 1999.
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during the differentiation of cytotoxic T lymphocytes induced by allografts. J. Immunol. 148:2348.[Abstract]
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), CD154+/+ mice (
), and
C57BL/6 mice treated with 1.25 mg MR1 (
) or Hamster IgG (•) 10
days after injection with 1 x 107 allogeneic P815
tumor cells. B, Cytotoxic alloantibody titers were
determined on day 10 postimmunization from mice with functional
(CD154+/+ or hyper-IgG (HIgG) treatment) or disrupted
(CD154-/- or MR1 treatment) CD154:CD40. Data represent
mean ± SEM of six mice/treatment group; one representative
experiment of three is shown.
) during the
allogeneic response to P815 tumor cells. Spleen cells (1 x
107) were restimulated with 1 x 106 P815
tumor cells for 6 h, and the culture supernatants were assayed for
production of IL-2 (B) and IFN-
