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*
Clinical Immunology Section and
Medical Virology Section of Laboratory of Clinical Investigation,
Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, and
§
Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and
¶
Immunex Corporation, Seattle, WA 98101
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, cytolytic T cell activity, and IgG2a Ab. The
mechanism by which CD40LT DNA enhanced these responses was further
assessed by treating vaccinated mice with anti-IL-12 mAb or CTLA-4
Ig (CTLA4Ig). Production of IFN- and CTL activity was abrogated by
these treatments, suggesting that CD40LT DNA was mediating its effects
on IFN- and CTL activity through induction of IL-12 and enhancement
of B7 expression, respectively. Physiologic relevance for the ability
of CD40LT DNA to enhance immune responses by the aforementioned
pathways was shown in two in vivo models. First, with regard to CTL
activity, mice vaccinated with CD40LT DNA did not develop metastatic
tumor following challenge with lethal dose of tumor. Moreover, in a
mouse model requiring IL-12-dependent production of IFN-, mice
vaccinated with soluble Leishmania Ag and CD40LT DNA
were able to control infection with Leishmania major.
These data suggest that CD40LT DNA could be a useful vaccine adjuvant
for diseases requiring cellular and/or humoral
immunity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA immunization has recently been shown to induce potent humoral and cellular responses in vivo (4). More important, protective immunity for several infectious disease and tumor models has been demonstrated using this technology (4). Among the potential advantages of using plasmid DNA for a particular Ag rather than a purified protein plus adjuvant is that DNA can induce both MHC class I (5, 6, 7) and class II (8) responses. In addition, plasmid DNA itself contains specific immunostimulatory sequences (ISS)2 containing unmethylated CpG motifs that provide potent adjuvant effects in murine in vivo models (9, 10, 11, 12, 13, 14). Moreover, while in vitro data using PBMCs suggest that ISS are able to elicit inflammatory cytokines (14), it is not clear whether these ISS are as effective in inducing immune responses from human cells in vivo. Thus, because DNA vaccination still provides such an effective way to present Ag, several studies have utilized plasmid DNA encoding for cytokine or costimulatory molecules as an additional way to enhance the type and magnitude of the immune response.
The study reported in this work focuses on the ability of a novel
trimeric CD40 ligand DNA (CD40LT) to enhance Ag-specific Ab, cytokine,
and CTL responses following in vivo immunization. Previous work has
shown the CD40L/CD40 costimulatory pathway to be a central regulator of
both humoral and cellular immune responses (15, 16). CD40L is a type II
membrane glycoprotein with homology to TNF and lymphotoxin-
, which
are ligands known to exist as homotrimers (17, 18). The interaction of
CD40L and CD40 is both responsible for activation of B cells allowing
for isotype switching (19) and important in T cell activation (20) and
production of type 1 cytokines (IL-12, IFN-) in response to protein
Ags (21, 22, 23, 24, 25). Moreover, CD40L has been shown to play an important role
in CTL memory responses following viral infection (26). The mechanism
by which CD40L/CD40 regulates cellular responses is primarily through
induction of IL-12 and other inflammatory mediators from APCs (22, 23, 24, 25)
and/or through enhancement of the expression of various cell surface
molecules (e.g., B7-1, B7-2) (27, 28, 29, 30, 31). Previous work has revealed that
monomeric, dimeric, and trimeric CD40L constructs all bind to CD40
(18); however, there appears to be a greater capacity for trimeric
CD40L to trigger biologic responses (18). Thus, in this study, the
mechanism by which a trimeric CD40L DNA (CD40LT) induces enhancement of
both humoral and cellular immune responses in vivo was investigated.
The data show that the ability of CD40LT DNA to enhance cytolytic T
cell activity and cytokine responses is influenced by enhancement of B7
costimulation and production of IL-12, respectively. These mechanisms
were shown to be biologically important using a murine tumor and
infectious disease model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Division of Cancer Treatment, National Cancer Institute (Frederick, MD), and kept in National Institute of Allergy and Infectious Diseases Animal Care Facility under pathogen-free conditions. Mice used were between 6 and 8 wk of age.
Media and reagents
HBSS (Biofluids, Rockville, MD) was used as wash medium. RPMI 1640 (Biofluids) supplemented with 10% FCS (Biofluids), penicillin (100 U/ml), L-glutamine (2 mM), sodium pyruvate (1 mM), and 2-ME (0.005 mM) was used to culture splenocytes and lymph node cells.
Plasmid construction and purification
A plasmid encoding the Escherichia coli LacZ gene under the control of a CMV intermediate-early promoter, designated ß-gal DNA, was kindly provided by J. Haynes (PowderJect, Madison, WI). cDNA encoding murine CD40L fused to an IL-7 leader and a leucine zipper sequence (CD40LT) in plasmid vector (Bluescript11SK±) was kindly provided by M. Spriggs (Immunex, Seattle, WA). The CD40LT fusion protein insert was excised using NotI/KpnI and ligated into expression vector PcDNA-3, downstream of the CMV promoter (Invitrogen, San Diego, CA) and designated CD40 ligand/trimer DNA (CD40LT DNA). Immunoprecipitation of murine CD40LT from COS cells (transfected with the PcDNA-3 CD40LT DNA) using a mAb to the leucine zipper showed a 35-kDa band in lysates and supernatants of the transfected cells. A eukaryotic expression vector carrying IL-12 DNA was kindly provided by J. Haynes (PowderJect). IL-12 DNA was able to produce biologically active protein from supernatants of COS cells transfected with IL-12 DNA (data not shown). Plasmid DNA was purified by two cycles of cesium chloride gradient ultracentrifugation. The 260/280 ratios ranged from 1.8 to 2. The endotoxin content from purified plasmid DNA was found to be <5 endotoxin units/ml.
Immunization
Female BALB/c mice were injected s.c. in their hind footpads with 100 µg of ß-gal plasmid DNA in 50 µl of sterile PBS. Additionally, in some groups, ß-gal DNA was combined with 100 µg of plasmid DNA encoding for IL-12 or CD40LT DNA and injected as above. Mice were boosted 2 to 3 wk later with their initial regimen.
Treatment of mice with neutralizing Abs
Purified neutralizing mAb (1 mg/mouse i.p.) against murine IL-12 (hybridoma c17.8) was obtained from G. Trinchieri (Wistar Institute, Philadelphia, PA) and injected into mice at the time of initial vaccination and boost. CTLA4Ig (100 µg/mouse i.p.) and control L6Ig Ab (100 µg/mouse i.p.) were obtained from P. Linsley (Bristol-Meyers Squibb, Seattle, WA) and injected into mice at the time of vaccination and boost.
Cytokine production assay
Mice were euthanized, and spleens from individual mice were harvested 2 wk after the last vaccination. Single-cell preparations from spleens were plated in triplicate in a 96-well microtiter plate at 3 x 105 cells/200 µl. Recombinant ß-gal protein (20 µg/ml) (Sigma, St. Louis, MO) or human serum albumin (10 µg/ml) was added to cultures, and supernatants were collected 48 h later and stored at -20°C.
Measurement of cytokine production
Measurements of IFN- (32) and IL-4 were assessed by specific
ELISA. The lower limits of detection of IFN- and IL-4 were 30 and
1.5 pg/ml, respectively.
Measurement of ß-gal-specific Ab responses
ß-gal-specific Ab responses were measured using a sandwich ELISA. BALB/c mice were immunized as described above. Pooled serum samples (n = 5–10 mice per group) were obtained 2 wk after the last immunization and were analyzed for the presence of ß-gal-specific Abs. Briefly, 96-well microtiter Immulon-4 plates (Dynatech, Chantilly, VA) were coated with ß-gal protein (10 µg/ml) overnight at 4°C. Plates were blocked with 2% BSA/PBS at 37°C for 1 h to prevent nonspecific binding. Serum was added at serial fivefold dilutions (starting at 1/5) and incubated overnight at room temperature. Horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a (Southern Biotechnology Associates, Birmingham, AL) was added for 1 h at 37°C to detect the Ab complex immobilized to the wells. The resulting complex was detected by peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Absorbance was read on an ELISA plate reader (Dynatech) using a 410-nm filter referenced to 570 nm.
Cytolytic T cell 51Cr release assay
Primary lymphocyte effector cells were generated in vivo by immunization with ß-gal DNA with or without cytokine DNA. Secondary in vitro effector populations were generated by harvesting spleens 10 to 14 days after the second immunization and culturing single-cell suspension of splenocytes for 5 days in T-75 flasks (Nunc, Roskilde, Denmark) at a density of 3 x 106 cells/ml with 1 µg/ml of H-2 Ld-restricted synthetic peptide (33). A ß-gal-expressing tumor cell line (CT26.CL25) or peptide-pulsed CT26 wild-type cells were used as targets. Target cells were mixed with effector cells for 6 h at 37°C at the E:T ratios indicated. A 6-h 51Cr release assay was performed as previously described (33). Unpulsed CT26 wild-type target cells (CT26.WT) were used as negative controls for each experiment. In all experiments, lysis was <10% using unpulsed target wild-type cells.
Flow-cytometry analysis
Mice from each of the vaccination groups were euthanized 2 or 7 days after a DNA boost, and single-cell suspensions of spleens from two mice were pooled. Splenocytes (106) were stained with FITC-conjugated Abs to B7.1 or B7.2 and phycoerythrin-conjugated (I-Ad) anti-class II (PharMingen, San Diego, CA) and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Cells displaying typical lymphocyte and macrophage scatter were gated, and two-color dot plots were generated using CellQuest software.
Infectious challenge with L. major
Female BALB/c mice were injected in their hind footpads with 100 µg of plasmid DNA (IL-12, CD40LT, control PcDNA-3) in 50 µl of sterile PBS. Two days after the above injection, plasmid DNA was combined with 25 µg of protein Ag (SLA) and injected into the hind footpad as above. As a control, mice were injected as above with 25 µg of SLA with or without 2 µg of rIL-12 (Genetics Institute, Cambridge, MA). Mice were boosted 3 wk later with their initial regimen. Two weeks after the last vaccination, mice were challenged by injection in the hind footpad opposite that in which they received vaccination with 1 x 105 metacyclic promastigotes (34). Weekly footpad swelling measurements were recorded using a caliper.
Tumor challenge studies
CT26.WT is a clone of the N-nitroso-N-methylurethane-induced BALB/c (H-2d) undifferentiated colon carcinoma (33). Following transduction with a retrovirus encoding the LacZ gene, CT26.WT was subcloned to generate the ß-gal-expressing cell line CT26.CL25. CT26.CL25 was grown in complete media and 400 µg/ml of G418 (Life Technologies, Grand Island, NY). Mice were immunized with ß-gal DNA with or without CD40LT DNA, as described above, and challenged i.v. with 1.75 x 106 CT26.CL25 tumor cells. Twelve days later, lungs were harvested and pulmonary metastases were enumerated in a blind manner.
Statistics
Statistical evaluation of differences between means of experimental groups was done by analyses of variance and multiple Student’s t tests. A value of p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40L/CD40 interactions have been shown to be important regulators
in the induction of type 1 cytokine (IL-12, IFN-) responses (21, 22, 23, 24, 25, 35). Thus, it was of interest to evaluate whether CD40LT DNA, when
administered together with DNA for a specific Ag, could affect the
qualitative and/or quantitative immune response in vivo. In addition,
the ability of CD40LT DNA to effect production of Th1 cytokines was
directly compared with IL-12 DNA.
In the initial experiment, mice were injected s.c. with ß-gal DNA
with or without IL-12 DNA or CD40LT DNA and boosted 2 to 3 wk later
with the same regimen. Production of IFN- and IL-4 was assessed 2 wk
after the last vaccination by stimulating total splenocytes in vitro
with ß-gal or a control protein. As shown in Figure 1
A, mice injected with ß-gal
DNA plus IL-12 DNA or CD40LT DNA had increased production of
Ag-specific IFN- compared with mice injected with ß-gal DNA alone.
In contrast, mice vaccinated with ß-gal DNA and IL-12 DNA or CD40LT
DNA had a minimal increase in Ag-specific IL-4 production over mice
vaccinated with ß-gal DNA alone (Fig. 1B). These data
suggest that IL-12 and CD40LT DNA specifically enhance Th1 cytokine
production.
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As cytokines such as IL-4 and IFN- influence Ab class
switching, ß-gal-specific production of Ab subtypes was assessed to
provide an indirect but physiologic correlate of the pattern of
cytokine production in vivo. As shown in Figure 2A, mice vaccinated with
ß-gal DNA plus CD40LT DNA or IL-12 DNA had a substantially higher
(2–3 log) increase in the level of IgG2a compared with mice vaccinated
with ß-gal DNA alone. Furthermore, treatment of mice injected with
ß-gal plus CD40LT DNA or IL-12 DNA with anti-IL-12 mAb abrogated
much of the increase in IgG2a, providing evidence that induction of
IgG2a in vivo by these DNAs is due to induction of IL-12.
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B). Of interest, anti-IL-12 treatment reduced much of
the increase in IgG1 induced by IL-12 DNA and, to a lesser extent,
CD40LT DNA. CD40LT DNA and IL-12 DNA increase Ag-specific cytotoxic T cell responses
To study the effects that CD40LT DNA and IL-12 DNA had on
Ag-specific CTL responses, mice were immunized with ß-gal DNA with or
without IL-12 DNA or CD40LT DNA and boosted 2 to 3 wk later with the
same regimen. Two weeks after the boost, CTL responses were assessed.
As shown in Figure 3, cells from mice
injected with ß-gal DNA plus CD40LT or IL-12 DNA had enhanced CTL
responses compared with those from mice injected with ß-gal DNA
alone. Taken together with the previous figures, these data show that
CD40LT DNA enhances both humoral and cellular immune responses. The
mechanism by which CD40LT DNA mediates these responses is discussed
below.
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in an IL-12- and
B7-dependent manner
The ability of CD40L/CD40 stimulation to regulate type 1 (IFN-,
IL-12) cytokine production has been shown to occur through at least two
mechanisms (35), the first through the induction of IL-12 from APCs
(22, 23, 24, 25) and the second via the ability of CD40L/CD40 stimulation to
enhance expression of costimulatory cell surface molecules (e.g., B7-1,
B7-2), causing increased T cell stimulation and production of IFN-
(21, 35). To evaluate both of these mechanisms, mice were injected with
ß-gal DNA plus CD40LT DNA and treated with either anti-IL-12 or
CTLA4Ig at the time of each vaccination. Similar to the results seen
above, mice injected with ß-gal plus CD40LT DNA plus L6Ig (control
for CTLA4Ig) had an increase in IFN- compared with those injected
with ß-gal alone (Fig. 4). By contrast,
mice injected with ß-gal DNA plus CD40LT DNA and treated with
anti-IL-12 had >90% inhibition of IFN- production. These
results are consistent with the central role for CD40L/CD40 stimulation
in regulating IL-12 production in response to a protein Ag in vivo
(20).
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production
following vaccination with ß-gal with or without CD40LT DNA was
evaluated by treating mice with CTLA4Ig or a control Ab (L6Ig) at the
time of vaccination. All mice treated with CTLA4Ig had complete
inhibition of IFN- production. In the same experiment, CTLA4Ig also
inhibited ß-gal-specific in vitro proliferation (data not shown).
These data underscore the importance of B7/CD28 costimulation in
regulating primary T cell responses and show that the enhancement of
IFN- by CD40LT DNA is also dependent on B7 costimulation (see
below). CD40LT DNA enhances expression of costimulatory molecules B7-1 and B7-2
To further evaluate the mechanism by which CD40LT DNA enhances
immune responses in vivo, splenocytes from mice vaccinated with ß-gal
DNA with or without CD40LT DNA were analyzed by FACS for the expression
of B7-1 and B7-2. When examined 2 days after the second vaccination,
cells from mice vaccinated with ß-gal DNA plus CD40LT DNA had a two-
to threefold increase in the number of B7-1 and B7-2 MHC class
II-expressing cells compared with cells from mice vaccinated with
ß-gal DNA plus control DNA (Fig. 5A). There was also a
threefold increase in expression of B7-1 from splenocytes of mice
vaccinated with ß-gal plus CD40LT DNA when assessed 7 days after the
boost; however, there was a more modest increase in expression of B7-2
at this time (Fig. 5B). It should be noted that while the
fold increase in B7-1 was the same 2 and 7 days after the second
vaccination from mice injected with CD40LT DNA, the number of cells
expressing B7-1 was greater 7 days after the second vaccination.
Finally, the ability of CD40LT DNA to enhance expression of either B7-1
or B7-2 was completely abrogated by treatment of mice with CTLA4Ig,
but not with control L6Ig.
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The previous figures support the notion that CD40LT DNA enhances
CTL responses through a B7-dependent mechanism. Therefore, CTL
responses were assessed from mice vaccinated with ß-gal DNA plus
CD40LT DNA and treated with CTLA4Ig or control L6Ig. As shown in Figure 6, mice vaccinated with ß-gal plus
CD40LT DNA and treated with L6Ig had strong CTL responses, while mice
treated with CTLA4Ig had markedly diminished responses. Addition of
anti-CD8 Ab at the time of the CTL assay substantially diminished
CTL activity (p < 0.05), providing
evidence that the CTL activity was CD8 dependent. In contrast,
the addition of anti-CD4 mAb to cultures did not
significantly diminish CTL activity (p >
0.05). Moreover, using target cells pulsed with an MHC class
I-restricted peptide (ß-gal plus CD40LT plus L6Ig plus peptide)
provides additional evidence that the CTL activity is class I
restricted.
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Because CD40LT DNA induces strong CTL responses following in vitro
stimulation, it was of interest to see whether these responses were
functional in vivo. To address this, mice were vaccinated in a similar
manner as described above and then challenged i.v. with a
ß-gal-expressing undifferentiated adenocarcinoma 2 to 3 wk after the
boost. As shown in Table I, mice
vaccinated with ß-gal plus CD40LT DNA had little to no detectable
pulmonary metastases when assessed 10 days after challenge, while three
of five mice injected with ß-gal alone had detectable pulmonary
metastases.
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, it was demonstrated that CD40LT DNA induced an
IL-12-dependent enhancement in production of IFN-. To assess the
ability of CD40LT DNA to induce a protective immune response in a model
in which IL-12-dependent production of IFN- is required (36), the
murine model of L. major infection was used. Previous
work has shown that susceptible BALB/c mice vaccinated with either a
SLA or a cloned Leishmania protein with rIL-12 protein can
induce a protective immune response following infection (37, 38). In
the experiment shown in Figure 7, the
ability of either CD40LT DNA or IL-12 DNA to induce a protective immune
response when given in conjunction with SLA was compared with that of
SLA plus rIL-12 protein. Mice vaccinated either with SLA plus CD40LT
DNA or with IL-12 DNA effectively controlled infection with L.
major, as assessed by footpad swelling. This protection was
comparable with that achieved by SLA and rIL-12 protein. In addition,
the control of disease progression and reduction in parasite burden in
mice vaccinated with CD40LT DNA or IL-12 DNA were associated with an
increase in Ag-specific production of IFN- (data not shown). Thus,
these data are consistent with the premise that CD40LT DNA mediates a
protective immune response in vivo by enhancing IFN- production in
an IL-12-dependent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production; 3) CD40LT DNA enhances expression of B7 costimulatory
molecules and increases CTL activity in a B7-dependent manner; and 4)
CD40LT DNA induces protective immunity against tumor challenge or
L. major infection, providing functional evidence that
enhancement of CTL activity and IFN- production in vivo,
respectively, was physiologically relevant. The role of CD40LT and IL-12 DNA in regulating specific Ab isotypes in vivo
Mice vaccinated with ß-gal and CD40LT DNA had significant
increases in total Ig production compared with that of mice vaccinated
with ß-gal DNA only (data not shown). In evaluating specific Ab
isotypes, both CD40LT DNA and IL-12 DNA caused a 2- to 3-log increase
in IgG2a that was abrogated by treatment of mice with anti-IL-12.
These data are consistent with previous studies showing that
IL-12-dependent production of IFN- is a major regulator of IgG2a
production (40, 41, 42, 43, 44, 45). In contrast to the relatively straightforward role
of IL-12 and IFN- in regulating IgG2a isotype production, the
ability of cytokines to regulate IgG1 is less clear. Initial reports
showed that IL-4, but not IFN-, was a potent inducer of IgG1 (45, 46). In several of the studies alluded to above, mice injected with
IL-12 protein and various Ags had suppressed IgG1 production,
consistent with the cross-regulatory role of IFN- and IL-4 in
regulating IgG1 Ab production (40, 41, 42, 43, 44); however, in more recent
studies, immunization with IL-12 greatly enhanced IgG1 production (47, 48). These data, together with the observation that IgG1 Ab production
can occur in the absence of IL-4 (49), suggest that IgG1 production may
be a less specific marker for IL-4 in vivo and that IL-12 can enhance
its production depending on the type of adjuvant used (48) and the time
at which Ab is measured following immunization (42). In the studies
reported in this work, IL-12 and CD40LT DNA substantially increased
IgG1 compared with ß-gal DNA alone. Of interest, anti-IL-12
treatment completely abrogated the ability of IL-12 DNA to increase
IgG1 production, consistent with IL-12 having a role in augmenting IgG1
production. By contrast, anti-IL-12 treatment only partially
abrogated the CD40LT DNA-induced increase in IgG1. This latter
observation could be due to a direct effect of CD40L on B cells causing
an increase in all isotypes or to the fact that anti-IL-12
treatment of CD40LT DNA-vaccinated mice caused an increase in IL-4
production (data not shown). Overall, these data underscore the potent
effects that CD40L stimulation can have in eliciting potent Ab
responses.
CD40LT DNA enhances CTL activity in a B7/CD28-dependent manner
In this study, CD40LT DNA and IL-12 DNA increased Ag-specific CTL activity, consistent with previous reports (50, 51). The ability of CD40LT DNA to enhance CTL was inhibited by treatment of mice with CTLA4Ig, suggesting that B7 costimulation was critical for CTL induction. With regard to the effects of B7 stimulation on CTL induction, recent studies have shown that there may be differences in the ability of B7-1 and B7-2 DNA to enhance CTL responses (52, 53, 54). In this study, because there were increases in both B7-1 and B7-2 and the timing of peak expression varied with time, it is hard to distinguish which is more important in mediating CTL responses.
Functional role of CD40L DNA in vivo as a vaccine adjuvant
To assess whether the immune enhancement achieved by CD40LT DNA
was able to mediate protective immunity in vivo, we chose two
experimental models in which the mechanism of protection is well
defined. Previous work has shown that, following challenge with a
ß-gal-expressing adenocarcinoma, CD8-dependent CTL activity is
required to prevent metastatic lung disease (K.R.I., unpublished
observation). In these studies, mice vaccinated with ß-gal DNA
plus CD40LT DNA were protected from metastatic disease. CTL activity of
mice vaccinated with CD40LT DNA was inhibited by treatment with CTLA4Ig
in vivo or with anti-CD8 in vitro, suggesting that the mechanism of
tumor eradication is via a CD8-dependent pathway. Finally, mice
vaccinated with CD40LT DNA and treated with anti-IL-12 in vivo did
not have diminished CTL responses (data not shown), providing
additional evidence that the CD40LT DNA enhancement of CTL activity was
via the increase in B7 expression and not through an IL-12-dependent
mechanism. In regard to this latter point, recent work using IL-12 DNA
as an adjuvant showed that an in vitro CTL response directed against an
HIV envelope was inhibited by anti-IL-12 or anti-IFN-
treatment in vivo (51). This may reflect a need for IL-12 DNA-dependent
production of IFN-, which would induce optimal expression of
costimulatory molecules (i.e., B7) required for CTL activity.
To evaluate whether CD40LT DNA-induced production of IL-12 could
protect animals from infection, we used the experimental model of
L. major infection. Protective immunity achieved through
vaccination for this infection requires IL-12-dependent production of
IFN- (36). Vaccination of mice with SLA plus CD40LT DNA induced
protection similar to that achieved by SLA plus IL-12 DNA or IL-12
protein; it is therefore likely that CD40LT DNA mediates its effect
through the induction of Ag-specific IFN- (manuscript in
preparation).
Overall, the ability of CD40LT DNA to enhance a broad array of immune responses makes it a potent adjuvant for diseases requiring humoral and/or cellular immunity. Thus, for intracellular infections in which cellular immune responses are desirable (L. major, M. tuberculosis), CD40LT or IL-12 DNA could induce effective Th1 responses as well as CTL responses. Another potential infection in which CD40LT DNA would be a useful adjuvant is HIV. In this case, because it is not precisely known which immunologic correlates are required for protective immunity, it is likely that induction of both Ab and cellular responses would be desirable. One important caveat to using CD40LT DNA as an adjuvant is the potential for unregulated CD40L/CD40 stimulation. This could result in an excessive proinflammatory response, which may trigger or enhance an autoimmune process. In this regard, using a soluble CD40L agonist protein may provide a useful adjuvant without incurring any long-term effects.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ISS, immunostimulatory sequence; ß-gal, ß-galactosidase; CD40L, CD40 ligand; CD40LT, CD40 ligand/trimer; SLA, soluble Leishmania Ag.
Received for publication March 4, 1998. Accepted for publication June 25, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 P. W. Hellings, A. Kasran, D. Bullens, L. Overbergh, C. Mathieu, H. Heremans, P. Matthys, L. Boon, M. Jorissen, and J. L. Ceuppens IL-10- and IL-12-Independent Down-Regulation of Allergic Sensitization by Stimulation of CD40 Signaling J. Immunol., October 15, 2006; 177(8): 5138 - 5144. [Abstract] [Full Text] [PDF]
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J. T. Collins, J. Shi, B. E. Burrell, D. K. Bishop, and W. A. Dunnick Induced Expression of Murine {gamma}2a by CD40 Ligation Independently of IFN-{gamma} J. Immunol., October 15, 2006; 177(8): 5414 - 5419. [Abstract] [Full Text] [PDF]
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G. W. Stone, S. Barzee, V. Snarsky, K. Kee, C. A. Spina, X.-F. Yu, and R. S. Kornbluth Multimeric Soluble CD40 Ligand and GITR Ligand as Adjuvants for Human Immunodeficiency Virus DNA Vaccines J. Virol., February 15, 2006; 80(4): 1762 - 1772. [Abstract] [Full Text] [PDF]
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M. Chamekh, V. Vercruysse, M. Habib, M. Lorent, M. Goldman, A. Allaoui, and B. Vray Transfection of Trypanosoma cruzi with Host CD40 Ligand Results in Improved Control of Parasite Infection Infect. Immun., October 1, 2005; 73(10): 6552 - 6561. [Abstract] [Full Text] [PDF]
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M. G. Cusi, M. T. Del Vecchio, C. Terrosi, G. G. Savellini, G. Di Genova, M. La Placa, F. Fallarino, C. Moser, C. Cardone, G. Giorgi, et al. Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antigen Anticancer Vaccine J. Immunol., June 1, 2005; 174(11): 7210 - 7216. [Abstract] [Full Text] [PDF]
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I. N. Buhtoiarov, H. Lum, G. Berke, D. M. Paulnock, P. M. Sondel, and A. L. Rakhmilevich CD40 Ligation Activates Murine Macrophages via an IFN-{gamma}-Dependent Mechanism Resulting in Tumor Cell Destruction In Vitro J. Immunol., May 15, 2005; 174(10): 6013 - 6022. [Abstract] [Full Text] [PDF]
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R. Vankayalapati, P. Klucar, B. Wizel, S. E. Weis, B. Samten, H. Safi, H. Shams, and P. F. Barnes NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen J. Immunol., January 1, 2004; 172(1): 130 - 137. [Abstract] [Full Text] [PDF]
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Y. Miyahira, H. Akiba, M. Katae, K. Kubota, S. Kobayashi, T. Takeuchi, A. Garcia-Sastre, Y. Fukuchi, K. Okumura, H. Yagita, et al. Cutting Edge: A Potent Adjuvant Effect of Ligand to Receptor Activator of NF-{kappa}B Gene for Inducing Antigen-Specific CD8+ T Cell Response by DNA and Viral Vector Vaccination J. Immunol., December 15, 2003; 171(12): 6344 - 6348. [Abstract] [Full Text] [PDF]
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V. Bronte, S. Cingarlini, E. Apolloni, P. Serafini, I. Marigo, C. De Santo, B. Macino, O. Marin, and P. Zanovello Effective Genetic Vaccination with a Widely Shared Endogenous Retroviral Tumor Antigen Requires CD40 Stimulation during Tumor Rejection Phase J. Immunol., December 15, 2003; 171(12): 6396 - 6405. [Abstract] [Full Text] [PDF]
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P. Mendez-Samperio, H. Ayala, and A. Vazquez NF-{kappa}B Is Involved in Regulation of CD40 Ligand Expression on Mycobacterium bovis Bacillus Calmette-Guerin-Activated Human T Cells Clin. Vaccine Immunol., May 1, 2003; 10(3): 376 - 382. [Abstract] [Full Text] [PDF]
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B. Samten, B. Wizel, H. Shams, S. E. Weis, P. Klucar, S. Wu, R. Vankayalapati, E. K. Thomas, S. Okada, A. M. Krensky, et al. CD40 Ligand Trimer Enhances the Response of CD8+ T Cells to Mycobacterium tuberculosis J. Immunol., March 15, 2003; 170(6): 3180 - 3186. [Abstract] [Full Text] [PDF]
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S. Manoj, P. J. Griebel, L. A. Babiuk, and S. van Drunen Littel-van den Hurk Targeting with Bovine CD154 Enhances Humoral Immune Responses Induced by a DNA Vaccine in Sheep J. Immunol., January 15, 2003; 170(2): 989 - 996. [Abstract] [Full Text] [PDF]
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 J. D. Ahlers, I. M. Belyakov, M. Terabe, R. Koka, D. D. Donaldson, E. K. Thomas, and J. A. Berzofsky A push-pull approach to maximize vaccine efficacy: Abrogating suppression with an IL-13 inhibitor while augmenting help with granulocyte/macrophage colony-stimulating factor and CD40L PNAS, October 1, 2002; 99(20): 13020 - 13025. [Abstract] [Full Text] [PDF]
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M. A. McDowell, M. Marovich, R. Lira, M. Braun, and D. Sacks Leishmania Priming of Human Dendritic Cells for CD40 Ligand-Induced Interleukin-12p70 Secretion Is Strain and Species Dependent Infect. Immun., August 1, 2002; 70(8): 3994 - 4001. [Abstract] [Full Text] [PDF]
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C. Buning, K. Kruger, T. Sieber, D. Schoeler, and F. Schriever Increased Expression of CD40 Ligand on Activated T Cells of Patients with Colon Cancer Clin. Cancer Res., April 1, 2002; 8(4): 1147 - 1151. [Abstract] [Full Text] [PDF]
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 L. T. Nguyen, A. R. Elford, K. Murakami, K. M. Garza, S. P. Schoenberger, B. Odermatt, D. E. Speiser, and P. S. Ohashi Tumor Growth Enhances Cross-Presentation Leading to Limited T Cell Activation without Tolerance J. Exp. Med., February 11, 2002; 195(4): 423 - 435. [Abstract] [Full Text] [PDF]
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Y. Wan, L. Lu, J. L. Bramson, S. Baral, Q. Zhu, A. Pilon, and K. Dayball Dendritic Cell-Derived IL-12 Is Not Required for the Generation of Cytotoxic, IFN-{gamma}-Secreting, CD8+ CTL In Vivo J. Immunol., November 1, 2001; 167(9): 5027 - 5033. [Abstract] [Full Text] [PDF]
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R. Xiang, F. J. Primus, J. M. Ruehlmann, A. G. Niethammer, S. Silletti, H. N. Lode, C. S. Dolman, S. D. Gillies, and R. A. Reisfeld A Dual-Function DNA Vaccine Encoding Carcinoembryonic Antigen and CD40 Ligand Trimer Induces T Cell-Mediated Protective Immunity Against Colon Cancer in Carcinoembryonic Antigen-Transgenic Mice J. Immunol., October 15, 2001; 167(8): 4560 - 4565. [Abstract] [Full Text] [PDF]
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G. Chen, P. A. Darrah, and D. M. Mosser Vaccination against the Intracellular Pathogens Leishmania major and L. amazonensis by Directing CD40 Ligand to Macrophages Infect. Immun., May 1, 2001; 69(5): 3255 - 3263. [Abstract] [Full Text] [PDF]
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M. S. Rolph and S. H. E. Kaufmann CD40 Signaling Converts a Minimally Immunogenic Antigen into a Potent Vaccine Against the Intracellular Pathogen Listeria monocytogenes J. Immunol., April 15, 2001; 166(8): 5115 - 5121. [Abstract] [Full Text] [PDF]
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C. Demangel, U. Palendira, C. G. Feng, A. W. Heath, A. G. D. Bean, and W. J. Britton Stimulation of Dendritic Cells via CD40 Enhances Immune Responses to Mycobacterium tuberculosis Infection Infect. Immun., April 1, 2001; 69(4): 2456 - 2461. [Abstract] [Full Text] [PDF]
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C. I. Brodskyn, G. K. DeKrey, and R. G. Titus Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro Infect. Immun., February 1, 2001; 69(2): 665 - 672. [Abstract] [Full Text] [PDF]
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J. G. Turner, A. L. Rakhmilevich, L. Burdelya, Z. Neal, M. Imboden, P. M. Sondel, and H. Yu Anti-CD40 Antibody Induces Antitumor and Antimetastatic Effects: The Role of NK Cells J. Immunol., January 1, 2001; 166(1): 89 - 94. [Abstract] [Full Text] [PDF]
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 B. W. Tillman, T. L. Hayes, T. D. deGruijl, J. T. Douglas, and D. T. Curiel Adenoviral Vectors Targeted to CD40 Enhance the Efficacy of Dendritic Cell-based Vaccination against Human Papillomavirus 16-induced Tumor Cells in a Murine Model Cancer Res., October 1, 2000; 60(19): 5456 - 5463. [Abstract] [Full Text]
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 T. Kikuchi, M. A. S. Moore, and R. G. Crystal Dendritic cells modified to express CD40 ligand elicit therapeutic immunity against preexisting murine tumors Blood, July 1, 2000; 96(1): 91 - 99. [Abstract] [Full Text] [PDF]
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M. A. Marovich, M. A. McDowell, E. K. Thomas, and T. B. Nutman IL-12p70 Production by Leishmania major-Harboring Human Dendritic Cells Is a CD40/CD40 Ligand-Dependent Process J. Immunol., June 1, 2000; 164(11): 5858 - 5865. [Abstract] [Full Text] [PDF]
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R. A. Tripp, L. Jones, L. J. Anderson, and M. P. Brown CD40 Ligand (CD154) Enhances the Th1 and Antibody Responses to Respiratory Syncytial Virus in the BALB/c Mouse J. Immunol., June 1, 2000; 164(11): 5913 - 5921. [Abstract] [Full Text] [PDF]
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B. Samten, E. K. Thomas, J. Gong, and P. F. Barnes Depressed CD40 Ligand Expression Contributes to Reduced Gamma Interferon Production in Human Tuberculosis Infect. Immun., May 1, 2000; 68(5): 3002 - 3006. [Abstract] [Full Text] [PDF]
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J. T. Tan, J. K. Whitmire, K. Murali-Krishna, R. Ahmed, J. D. Altman, R. S. Mittler, A. Sette, T. C. Pearson, and C. P. Larsen 4-1BB Costimulation Is Required for Protective Anti-Viral Immunity After Peptide Vaccination J. Immunol., March 1, 2000; 164(5): 2320 - 2325. [Abstract] [Full Text] [PDF]
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R. M. Vabulas, H. Pircher, G. B. Lipford, H. Hacker, and H. Wagner CpG-DNA Activates In Vivo T Cell Epitope Presenting Dendritic Cells to Trigger Protective Antiviral Cytotoxic T Cell Responses J. Immunol., March 1, 2000; 164(5): 2372 - 2378. [Abstract] [Full Text] [PDF]
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P. J. Blair, J. L. Riley, D. M. Harlan, R. Abe, D. K. Tadaki, S. C. Hoffmann, L. White, T. Francomano, S. J. Perfetto, A. D. Kirk, et al. CD40 Ligand (CD154) Triggers a Short-Term CD4+ T Cell Activation Response That Results in Secretion of Immunomodulatory Cytokines and Apoptosis J. Exp. Med., February 21, 2000; 191(4): 651 - 660. [Abstract] [Full Text] [PDF]
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D. L. Martin, C. L. King, E. Pearlman, E. Strine, and F. P. Heinzel IFN-{gamma} Is Necessary But Not Sufficient for Anti-CD40 Antibody-Mediated Inhibition of the Th2 Response to Schistosoma mansoni Eggs J. Immunol., January 15, 2000; 164(2): 779 - 785. [Abstract] [Full Text] [PDF]
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W. W. Leitner, H. Ying, D. A. Driver, T. W. Dubensky, and N. P. Restifo Enhancement of Tumor-specific Immune Response with Plasmid DNA Replicon Vectors Cancer Res., January 1, 2000; 60(1): 51 - 55. [Abstract] [Full Text]
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K. Honey, S. P. Cobbold, and H. Waldmann CD40 Ligand Blockade Induces CD4+ T Cell Tolerance and Linked Suppression J. Immunol., November 1, 1999; 163(9): 4805 - 4810. [Abstract] [Full Text] [PDF]
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M. Perez-Melgosa, D. Hollenbaugh, and C. B. Wilson Cutting Edge: CD40 Ligand Is a Limiting Factor in the Humoral Response to T Cell-Dependent Antigens J. Immunol., August 1, 1999; 163(3): 1123 - 1127. [Abstract] [Full Text] [PDF]
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 W ROSENBERG Mechanisms of immune escape in viral hepatitis Gut, May 1, 1999; 44(5): 759 - 764. [Full Text] [PDF]
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