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*
Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, United Kingdom;
The Chiron Vaccines Immunological Research Institute, Via Fiorentina, Siena, Italy; and
Department of Medical Microbiology and Immunology, University of Goteborg, Goteborg, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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enzyme-linked immunospot assays indicated that
IFN--/- and IL-12 p40-/- gene knockout
mice developed CTL responses equivalent to those detected in normal
C57BL/6 mice. The results highlight the versatility of toxin-based
adjuvants and suggest that LT potentiates CTL responses independently
of IL-12 and IFN- and probably by a mechanism unrelated to
cross-priming. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LTR72 and LTK63 can adjuvant systemic and local humoral immune responses to coadministered Ags after vaginal, i.n., or oral immunization (4, 5). However, individual LT derivatives are associated with different levels of adjuvant activity. For example, LTR72 is as effective as LT in inducing Ab production to bystander Ags (4). LTK63 has reduced adjuvant activity, compared with LTR72 but is significantly more active than LT-B, which is a poor mucosal adjuvant (9). The usefulness of attenuated toxins based on LT and CT is highlighted by their capacity to elicit protective immune responses to Ags from Helicobacter pylori (10), Salmonella dublin (11), Streptococcus pneumoniae (12), and Candida albicans (13).
CTLs play a critical role in controlling many infections and may
enhance the success of a vaccine against mucosally transmitted
pathogens (14). Relatively little work has been performed
on the ability of LT and CT derivatives to prime CTLs. Bowen et al.
(15) reported that oral or i.v. coadministration of CT
with OVA-primed mice for OVA-specific CTL. CT has also been used to
generate CTLs against a HIV peptide and a peptide from OVA
(16). LT and LTK63 were similarly shown to adjuvant CTL
responses to a coadministered class I binding peptide after i.n.
immunization (17). In this paper we compare the potential
of several different derivatives of ADP-ribosylating toxins as mucosal
adjuvants for CD8+ CTL induction. OVA, and the
immunodominant H-2b-restricted class I binding
epitope from OVA (OVA257–264), were used as
model bystander Ags. The contribution of CD4+ T
cells and the type I cytokines IL-12 and IFN- to OVA-specific CTL
induction was investigated in Ab depleted and gene knockout mice,
respectively. The results, using a variety of immunization routes,
indicated that nontoxic mutants of LT differ in their ability to
adjuvant CTL responses. Furthermore the adjuvant affect of LT for CTL
responses operates via a mechanism largely independent of
CD4+ T cell help, IL-12, and IFN-.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mycoplasma-free EL4 cells, which only express MHC class I (18), were maintained in RPMI 1640 containing 10% FCS (Sigma, St. Louis, MO), 5 x 10-5 M 2-ME, 2 mM L-glutamine (Sigma), 100 U penicillin/ml (Sigma), and 100 µg/ml streptomycin (cRPMI)(Sigma). E.G7-OVA, a subclone of EL4, stably transfected with the gene encoding OVA (18) (kind gift from A. Mowat, University of Glasgow, Glasgow, U.K.) were maintained in cRPMI containing 200 µg/ml G418 (Life Technologies, Paisley, Scotland). All cells were cultured in humidified air with 5% CO2 at 37°C.
Ags and Abs
OVA (grade V) and wild-type CT were purchased from Sigma. Wild-type porcine LT, LTK63, LTR72, and rLT-B were purified as described (3). CTA1-DD, which consists of the ADP-ribosylating A1 subunit of CT linked to the two D repeats of S. aureus protein A, was supplied by Nils Lycke and prepared as described (8). The immunodominant OVA octapeptide (OVA257–264) with the sequence SIINFEKL was purchased from Zinsser Analytic (Maidenhead, Berkshire, U.K.) and was >95% pure. OVA257–264 is known to be a naturally processed peptide derived from OVA expressed in mouse cells in the context of Kb (19). The sequence of OVA257–264 was verified by electron spray mass spectroscopy. The GK1.5 hybridoma producing an anti-mouse CD4 mAb was a kind gift from C. Engwerda (London School of Tropical Medicine and Hygiene, London, U.K.). GK1.5 mAbs were produced as ascites in Rag-1-/- mice. Magnetic bead conjugated anti-CD4 and anti-CD8 Abs used for in vitro cell depletions were purchased from Dynal (Wirral, Merseyside, U.K.). FITC conjugated anti-CD4 (clone RM4-4), CD8 (clone 53-6.7), and anti-CD45R (B220) were purchased from PharMingen (Oxford, Oxfordshire, U.K.) and used according to the manufacturers instructions.
ELISA
OVA-specific Abs were measured in sera of immunized mice. A solution of OVA (60 µg/ml) in carbonate buffer (pH 9.6) was used to coat 96-well ELISA plates (Life Technologies) (100 µl/well) which were then incubated overnight at 4°C. After washing with PBS and Tween 20, wells were blocked by addition of 1.5% (w/v) BSA in PBS for 1 h. Plates were washed twice with PBS and Tween 20 before sera from individual mice was added and serially diluted in PBS and Tween 20 containing 0.2% (w/v) BSA and incubated for 2 h at 37°C. The wells were washed with PBS and Tween 20 before addition of 100 µl of rabbit anti-mouse Ig-HRP conjugate (Dako, Buckinghampshire, U.K.) diluted 1/1000 in PBS and Tween 20 containing 0.2% (w/v) BSA for 2 h at 37°C. Finally, after washing with PBS and Tween 20, Ag-bound Ab was detected by addition of o-phenylenediamine substrate (Sigma) and the A490 read. Titers were determined arbitrarily as the reciprocal of the serum dilution corresponding to an optical density of 0.3. The minimum detectable titer was 100.
Mice and immunizations
C57BL/6 mice were purchased from Harlan Olac (Bichester, U.K.)
and were used between 6 and 8 wk of age. IL-12
p40-/- (20) and IFN-
(21) gene knockout mice were purchased from The Jackson
Laboratory (Bar Harbor, ME) and bred at B
K Universal (Hull, U.K.)
under containment conditions. Gene knockout mice were used between
6–12 wk of age. For i.n. immunizations, groups of mice
(n = 3) were lightly anesthetized with gaseous
halothane, and 30 µl of Ag in PBS applied to the nasal nares. Mice
were i.n. immunized on day 0 and 14 and killed on day 21–23. For s.c.
immunization, groups of mice (n = 3) were injected s.c.
on the left side of the abdomen with 150 µl of Ag mixture in PBS. As
per i.n. immunization, mice were s.c. immunized on day 0 and 14 and
killed on day 21–23. For oral immunizations, Ags were diluted in 0.1 M
sodium bicarbonate and administered in a 200 µl volume directly into
the stomachs of unanesthetized groups of mice (n = 4)
via a gavage needle. Mice were orally immunized on day 0, 9, and 18 and
killed on day 25. A group of mice (n = 3) s.c.
immunized with 200 µl of an emulsion of CFA containing 20 µg of
OVA257–264 7–14 days before killing served as a
positive control group for the induction of OVA-specific CTL in all
experiments. All mice were bled from the tail vein before being
killed.
Restimulation and 51Cr release assays
Spleens and superficial cervical lymph nodes (sCLN) were aseptically removed from mice that had been killed by cervical dislocation or exsanguinated under terminal anesthesia. Single cell suspensions from pooled sCLNs (n = 3), or pooled spleens (n = 3), were prepared by passing organs through 100-µm nylon sieves (Marathon Laboratories, London, U.K.). After lysis of splenic erythrocytes with Tris-ammonium chloride, a total of 3.5 x 107 leukocytes from the spleen or sCLN were cultured for 6 days in upright T25 tissue culture flasks in 10 ml of cRPMI in the presence of 3 x 106 gamma-irradiated (20,000 rads) E.G7-OVA cells. Graded numbers of effector cells, consisting of cells which remained viable after the culture period, were harvested and cocultured in triplicate with 5,000 or 10,000 Na51CrO4-labeled EL4 or E.G7-OVA target cells for 4–5 h at the indicated E:T ratios in 200 µl cRPMI. A total of 100 µl of culture supernatant was then removed into 100 µl of OptiPhase "Hisafe" scintillation mixture (Fisher Chemicals, Leicestershire, U.K.), and the amount of Na51CrO4 was measured using a 1450 Microbeta liquid scintillation counter (Wallac, Milton Keynes, U.K.). The percentage killing was calculated using the following formula: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100, where spontaneous release represents the counts obtained when the target cells were incubated in culture medium without effectors and maximum release represents the counts obtained when the target cells were lysed with 1% Triton X-100. Each experiment was repeated at least twice.
Enzyme-linked immunospot (ELISPOT) assays
The ELISPOT assays were performed as previously described
(22) and all Abs were purchased from PharMingen. Briefly,
nitrocellulose-based 96-well microtiter plates (Multiscreen-HA,
Millipore, Hertfordshire, U.K.) were coated overnight at 4°C with 50
µl/well of either 4 µg/ml anti-IFN- (R46A2) or 4 µg/ml
anti-IL-4 (BVD4-1D11) mAb diluted in carbonate buffer (pH 9.6).
After washing three times with filtered PBS, all wells were blocked
with 200 µl of cRPMI for 2–3 h at 37°C. Following removal of the
blocking medium, spleen cells from individual mice were added to the
wells in duplicate (maximum 5 x 105
cells/well in 200 µl), serially diluted, and incubated for 20 h
at 37°C in 5% CO2 in the presence or absence
of 1 µM OVA257–264 peptide. Cells were removed
by washing three times with PBS, followed by a further three times with
PBS and Tween 20 (0.05% v/v); then 50 µl of the biotinylated
anti-IFN- (XMG1.2) or anti-IL-4 (BVD6-24G2) Abs (1 µg/ml
in filtered PBS and Tween 20) was added to each well for 2 h.
After washing plates five times with filtered PBS and Tween 20, a
1/1000 dilution of Extravadin-alkaline phosphatase (Sigma) was added to
all wells for 1–2 h at room temperature. Finally, after washing three
times with PBS and Tween 20 and once with PBS alone,
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Fast
BCIP/NBT; Sigma) dissolved in 10 ml of distilled water was added as
substrate. Spots, representing single IFN- or IL-4-producing cells,
were counted using a dissecting microscope. The number of
peptide-specific spot-forming cells (SFCs) was determined by
subtracting the number of spots obtained with cells stimulated with
medium from those stimulated with
OVA257–264.
Depletion of CD4+ T cells
C57BL/6 mice, 6–8 wk old, were depleted of CD4+ T cells by i.p. administration of 0.5 mg GK1.5 mAb on day 2 and day 4 before primary immunization; depletion was maintained by administering 0.5 mg mAb on day 5, 10, and 15 after primary immunization. Control mice were administered 0.5 mg rat IgG (Sigma) on the same days. The efficiency of depletion was assessed by staining spleen and sCLN cells, with the noncompeting FITC labeled anti-CD4 mAb (clone RM4-4) (PharMingen). Appropriate FITC-labeled isotype-control Abs were used in all experiments. Stained cells were analyzed on a FACsort flow cytometer (Becton Dickinson, San Diego, CA).
Statistical analysis
Mean IFN- ELISPOT numbers were compared using the
nonparametric Mann-Whitney t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies showed that LT and CT can adjuvant CTL responses to i.n. coadministered peptides (16, 23). To establish whether this observation extended to i.n. coadministered whole proteins and to facilitate a direct comparison of LT and CT as adjuvants for CTL, whole OVA, or OVA257–264 were used as model bystander Ags in immunization experiments. Standard 51Cr release assays were used to detect CTL activity from splenocytes and cells from the sCLN. In preliminary single dose i.n. immunization experiments, inconsistent OVA-specific CTL responses were elicited using LT and CT as adjuvants. This was overcome by boosting mice 14 days after the initial immunization.
Mice i.n. immunized twice, 14 days apart, with 1 µg of LT or CT mixed
with 100 µg of OVA or 40 µg of OVA257–264,
mounted OVA-specific CTL responses which were consistently detected in
the spleens and sCLNs of immunized animals (Fig. 1
). Mice immunized with 1 µg of LT
mixed with 2 µg of OVA257–264 (an equimolar
amount of OVA257–264 epitope to that present in
100 µg of whole OVA) also mounted OVA-specific CTL responses, which
were detected in spleens (data not shown).
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). Intranasal immunization of mice
with 100 µg of OVA alone or 40 µg of
OVA257–264 alone consistently failed to elicit a
detectable CTL response in either the spleen (Fig. 1) or the sCLN (data
not shown). LT adjuvants MHC class I restricted CD8+ CTL responses
To identify the T cell population with cytotoxic activity, either CD8+ or CD4+ T cells were depleted from restimulated spleen cell populations prepared from mice i.n. immunized with 1 µg of LT mixed with OVA or OVA257–264. Depletion of CD8+ T cells, but not CD4+ T cells, immediately before coculturing them with chromium-labeled targets abrogated all lytic activity (data not shown).
LTR72 and LTK63 but not rLT-B adjuvant OVA-specific CTL responses after i.n. immunization
The adjuvant properties attributed to LT are reportedly enhanced
by the ADP-ribosyltransferase activity associated with the A subunit
(4). The role of this enzymatic activity and LT-A itself
in promoting CD8+ CTL responses to OVA or
OVA257–264 was assessed in immunization studies
using attenuated LT derivatives with reduced (LTR72) or absent (LTK63
and rLT-B) enzymatic activity. Initial experiments using 1 µg of
mutant toxin as adjuvant in i.n. immunization experiments failed to
consistently elicit OVA-specific CTL responses. However, i.n.
immunization of mice with 10 µg of LTK63 or 10 µg of LTR72 mixed
with 100 µg of OVA or 40 µg of OVA257–264
elicited OVA-specific CTL responses which were reproducibly detected by
51Cr release assay (Fig. 2). In contrast, i.n. coimmunization of
mice with 10 µg of rLT-B mixed with 100 µg of OVA or 40 µg of
OVA257–264 failed to elicit significant CTL
responses (Fig. 2).
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ELISPOTs
were performed in parallel with 51Cr-release
assays using splenocytes from selected groups of i.n. immunized mice.
The results (Fig. 2D) demonstrated that i.n.
coadministration of OVA or OVA257–264 with LT or
LTK63, but not rLT-B, elicited
OVA257–264-specific T cells which produced
IFN- upon secondary restimulation in vitro. LT, CT, and LTR72, but not LTK63 or LT-B, adjuvant OVA-specific CTL responses after oral immunization
The capacity of LT derivatives to adjuvant OVA-specific CTL
responses by the oral route of immunization was explored. LT, and to a
lesser extent a CT control, adjuvanted splenic CTL responses after oral
coimmunization of 10 µg of toxin with 5 mg of OVA (Fig. 3). The OVA-specific CTL responses
elicited using 10 µg of LTR72 as adjuvant was comparable to that
elicited by 10 µg of wild-type LT (Fig. 3). In contrast, oral
administration of LTK63 or rLT-B with 5 mg of OVA failed to elicit
detectable CTL responses (Fig. 3). Oral immunization of mice with 5 mg
of OVA alone failed to elicit a CTL response (Fig. 3).
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Mice immunized s.c. with 100 µg of OVA mixed with 1 µg of the
ADP-ribosylating molecules LT or CT, or 20 µg of CTA1-DD, all mounted
OVA-specific CTL responses that were detected in the spleen (Fig. 4). Similarly, mice immunized s.c. with
100 µg of OVA mixed with 1 µg of the attenuated toxins LTR72 or
LTK63 also mounted OVA-specific CTL responses. Conversely, immunization
of mice with 1 µg of rLT-B mixed with 100 µg of OVA, or 100 µg of
OVA alone, failed to elicit a CTL response (Fig. 4). Immunization of
mice with LT, CT, or CTA1-DD alone (data not shown) did not elicit
OVA-specific CTL responses.
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CD4+ T cell help is a common but not universal requirement for CTL responses (24). The role of CD4+ T cells in the induction of OVA-specific CD8+ CTL responses using LT as an adjuvant was assessed by depleting mice of CD4+ T cells with the mAb GK1.5. Mice were injected twice with 0.5 mg of GK1.5 or rat IgG (control group) before being i.n. immunized twice, 14 days apart, with 1 µg of LT mixed with 100 µg of OVA or 40 µg of OVA257–264. Mice were injected again with 0.5 mg of the GK1.5 mAb or rat IgG on day 5, 10, and 15 postprimary immunization. The efficiency of CD4+ T cell depletion was assessed on day 0, 10, and 21 by flow cytometric analysis (using a noncompeting anti-CD4 mAb) of lymphocytes recovered from the spleens and sCLNs of randomly selected mice. In two separate experiments, between 93 and 96% of CD4+ T cells were depleted from both the spleen and sCLN at the time points studied.
Depletion of CD4+ T cells did not prevent the
induction of an OVA-specific CD8+ CTL response in
mice i.n. immunized with LT mixed with OVA or
OVA257–264 (Fig. 5). In contrast, the serum Ab response to
OVA, which in rat IgG-treated mice exceeded a titer of
105, was completely abrogated in CD4-depleted
mice (Fig. 5K). Mice administered GK1.5 or rat IgG and i.n.
immunized with 100 µg of OVA alone or 40 µg of
OVA257–264 alone failed to mount significant
OVA-specific CTL responses (Fig. 5). As previously described
(25), OVA-specific CTL responses resulting from s.c.
immunization of mice with OVA257–264 emulsified
in CFA were independent of CD4+ T cell help (Fig. 5).
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IL-12 and IFN- play central roles in the development of
Th1-type immune responses and, potentially, in Th cell-independent CTL
generation (26). The contribution of IL-12 and IFN- to
the induction of OVA-specific CD8+ CTL
responses was assessed in mice deficient in the IL-12 p40 subunit
and IFN- gene respectively. C57BL/6 mice
(H-2b), IL-12 p40-/- mice
(H-2b) and IFN--/-
mice (H-2b) were immunized i.n. with 1 µg of LT
mixed with 100 µg of OVA or 40 µg of
OVA257–264. Additional groups of normal and gene
knockout mice were immunized i.n. with 100 µg of OVA alone or 40 µg
of OVA257–264 alone.
In multiple experiments, i.n. immunization of IL-12
p40-/- and IFN--/-
mice elicited OVA-specific CTL responses that were similar in
magnitude, as shown by 51Cr release assay, to
that detected in C57BL/6 control mice (Fig. 6). The induction of OVA-specific T cell
responses in IL-12 p40-/- mice was also
assessed by IFN- and IL-4 ELISPOT. The IFN- ELISPOT results
indicated that IL-12 p40-/- mice, i.n.
immunized with LT mixed with OVA or OVA257–264,
had fewer numbers of OVA257–264 peptide-specific
IFN- SFCs when compared with normal control mice. However, these
differences were not statistically different (Fig. 7). In contrast, IL-12
p40-/- mice immunized with
OVA257–264 emulsified in CFA had significantly
fewer peptide-specific splenic T cells producing IFN- when compared
with similarly immunized normal control mice (p
< 0.05) (Fig. 7).
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-/- mice (58 ± 46
SFC/106 splenocytes), was not significantly
different to that found in i.n. immunized C57BL/6 mice (86 ± 34
SFC/106 splenocytes). The frequency of IL-4 SFCs
did not increase when spleen cells from immunized IL-12
p40-/- mice, IFN--/-
mice, or normal C57BL/6 mice were restimulated with
OVA257–264 (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This represents the first comparative study of LT, rLT-B and
nontoxic LT mutant derivatives as adjuvants for CTL responses and the
first attempt at delineating the immunological mechanisms through which
wild-type LT adjuvants whole Ag and peptide-specific CTL responses. The availability of recombinant derivatives of LT which are either devoid or reduced in enzymatic activity (LTK63 and LTR72), or which lack the enzymatic A domain (rLT-B) has facilitated a dissection of the properties of this toxin which endow it with adjuvant activity. The inability of rLT-B to adjuvant CTL responses to i.n., orally, or s.c. codelivered OVA suggests that the A subunit of LT endows the toxin with characteristics essential for promoting CTL responses. The ADP-ribosyltransferase activity associated with the A subunit in wild-type LT appears to enhance the adjuvant potential of the toxin, but is nevertheless nonessential for adjuvanticity because LTK63, which is devoid of ADP-ribosyltransferase activity, adjuvants mucosal and systemic Ab (5), and CTL responses to i.n. or s.c. coadministered OVA. The function of the mutated A subunit in LTK63 may be to enhance the half-life of the holotoxin in vivo and thereby increase the hosts exposure to the affects mediated by LTK63 binding its ubiquitously found glycosphingolipid receptors. These effects may include up-regulated expression of MHC class II and co-stimulatory molecules on B cells (27) or stimulating cellular synthesis of arachidonic acid metabolites (e.g., PGE2) (28). Alternatively, the A subunit in LTK63 may possess other immunomodulating properties which, in addition to affects mediated by toxin ligation of receptor, are critical for adjuvanting CTL responses to bystander proteins or peptides. These immunomodulatory properties could potentially be mediated through binding of the A subunit to ADP-ribosylation factors (ARFs), an interaction that has been shown to occur outside the NAD binding cleft (29). While speculative, ARF binding to the A subunit may lead to alterations in vesicular membrane trafficking in both endocytic and exocytic pathways of host cells, because ARFs are recognized as being essential mediators of these events (30, 31).
The results of this study indicated that LTK63 was unable to adjuvant splenic CTL responses after oral coimmunization with OVA. It is unclear whether this represents a definitive inability to adjuvant CTL responses perorally, or whether a CTL response may be elicited by increasing the amount of LTK63 coadministered with the bystander Ag. In any case, the result suggests that toxins with either partial (e.g., LTR72) or wild-type levels (e.g., LT, CT) of ADP-ribosyltransferase activity are better oral adjuvants for CTL responses.
The novel finding that CTA1-DD and CT can adjuvant CTL responses after i.n. and s.c. coimmunization with whole OVA highlights the immunological potency of molecules with ADP-ribosyltransferase activity. CTA1-DD is a potent activator of B cells and strongly up-regulates costimulatory molecules, in particular B.7, on their surface (8). That the CTA1-DD adjuvant fails to prime CD4+ T cells in B cell-deficient mice, whereas it is highly effective in wild-type mice (N. Lycke, unpublished observation), indicates that this targeted adjuvant appears to act via B cells to induce naive CD4+ T cells. This mechanism of T cell induction may also apply to CTA1-DD in its effect as an adjuvant for OVA-specific MHC class I-restricted CTL responses.
The capacity of CT to augment CTL responses to OVA257–264 after i.n. immunization, or whole OVA after oral coimmunization, concurs with previous studies of this toxin (15, 16). Interestingly, although numerous studies have described CT as a potent inducer of Th2-type immune responses (6, 32), this study, like others (15, 16, 33), showed CT to be a useful adjuvant for CTL responses; a response not normally associated with strong Th2-type immune responses.
CD4+ T cell lymphocytes play a central role in the regulation of the immune response. The role of CD4+ T cells in the induction and maintenance of CD8+ CTL responses appears to depend on the system in which they are studied. CD8+ CTL responses which result from cross priming, as occurs after DNA vaccination (34), or injection of cell associated Ags (25, 35), is clearly dependent on CD4+ T cell help. Elegant studies indicate that this CD4+ T cell help is mediated through CD154 signaling of CD40 on the host APC (35, 36, 37). In studies of CTL responses to certain viruses however, CD4+ T cell help is not critical for the induction of CD8+ CTL, but they may contribute to maintenance of memory CTL responses (38, 39). Similarly, induction of CD8+ CTL responses by injection of class I binding peptides in strong adjuvants is not dependent on CD4+ T cell help (25), but their long-term maintenance can require CD4+ T cells (40). The results of this study showed that LT, like CFA, can elicit CTL responses to OVA or OVA257–264 independently of CD4+ T cell help. This result suggests that LT directly or indirectly activates host APCs (thereby obviating the need for this activation to occur via CD4+ T cells), such that they are now competent to prime an OVA-specific CD8+ T cell response. The identity of the APC which primes the CD8+ T cell response after i.n immunization with CT and OVA257–264 has been suggested in a previous study. Porgador et al. (41) showed that following intranasal coimmunization of OVA257–264 with CT, only dendritic cells (DCs) from the nasal associated lymphoid tissue could present OVA257–264 to OVA-specific T cells in vitro, suggesting that DCs may also be the APC responsible for the presentation of OVA257–264 in vivo. Whether DCs can also present antigenic peptides from soluble OVA has not been determined, although previous studies have established that DCs can take up OVA via macropinocytosis in vitro and subsequently prime an Ag-specific CTL response following adoptive transfer in vivo (42).
The rationale for examining the role of IL-12 in the induction of
OVA-specific CTL responses stems from previous reports indicating that
IL-12 is essential for CTL responses to mucosally delivered peptide
immunogens (33) and contributes to OVA-specific CTL
responses induced by immune stimulating complexes (43).
Further, a role for IL-12 in CTL induction is implied by the
observation that supply of exogenous IL-12 at the time of immunization,
either in the form of recombinant protein (44) or encoded
in a DNA vaccine (45, 46), enhances Ag-specific CTL
responses. Conversely, however, IL-12 p40 gene knockout mice have no
defect in allogeneic CTL responses (47) or in the CTL
response that develops following a viral infection (48).
Interestingly, the adjuvanticity of CT has also been reported to be
unaffected in IL-12-deficient mice (49), although CTL
induction was not examined. Our data suggest that IL-12 is unimportant
or redundant for the induction of OVA-specific CTL responses when LT is
used as an i.n. adjuvant or when CFA is used as a parenteral adjuvant
(Fig. 6). Furthermore, our IFN- ELISPOT results suggests the
presence of an IL-12-independent mechanism through which
CD8+ T cells expressing IFN- (but not IL-4)
can be elicited (Fig. 7). IL-12-independent mechanisms of inducing Th1
CD4+ T cell responses have also been described
(48, 50), and together these results imply that the
requirement for IL-12 in priming T cells which express IFN- is
dependent on the nature and composition of the Ag and the cytokine
environment in which Ag is presented to naive
CD8+ T cells.
Like IL-12, IFN- has also variously been described as essential
(51) and alternatively, dispensable for the induction of
CTL in vivo (52). Our data, using
IFN--/- mice, suggests that LT (and CFA)
potentiate OVA-specific CTL responses independently of IFN-, and
also imply that the cytotoxic function of these T cells is not impaired
by their inability to make IFN-. IFN--/-
mice with acute viral infections (53, 54) also mount
normal CTL responses, suggesting IFN- may be unimportant or
redundant for CD8+ CTL responses when Ag is
presented in an environment rich in inflammatory cytokines and
mediators. Interestingly, following immunization of
IFN--/- mice, the number of
OVA257–264-specific CD8+ T
cells making IL-4 did not increase, suggesting that despite the absence
of IFN-, the cytokine profile of the responding
OVA257–264-specific CD8+ T
cells was not biased toward expression of a typical type II
cytokine.
There exist many experimental strategies that can be used to elicit Ag-specific CTL responses in model systems. The attractiveness of using molecularly defined, nontoxic adjuvants based on LT lies in their capacity to elicit CTL responses by mucosal immunization. That mucosally elicited CTL mediates more effective resistance to a mucosal pathogen than systemically elicited CTL has already been demonstrated in one system (14). The authors are currently extending this area of research using nontoxic derivatives of LT.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cameron Simmons, Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London SW7 2AZ, U.K. E-mail address:
3 Abbreviations used in this paper: CT, cholera toxin; LT, E. coli labile-toxin; i.n., intranasal; sCLN, superficial cervical lymph nodes; ELISPOT, enzyme-linked immunospot; SCF, spot-forming cell.
Received for publication August 5, 1999. Accepted for publication October 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  J. Dietrich, C. Andersen, R. Rappuoli, T. M. Doherty, C. G. Jensen, and P. Andersen Mucosal Administration of Ag85B-ESAT-6 Protects against Infection with Mycobacterium tuberculosis and Boosts Prior Bacillus Calmette-Guerin Immunity J. Immunol., November 1, 2006; 177(9): 6353 - 6360. [Abstract] [Full Text] [PDF]
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C. Luci, C. Hervouet, D. Rousseau, J. Holmgren, C. Czerkinsky, and F. Anjuere Dendritic Cell-Mediated Induction of Mucosal Cytotoxic Responses following Intravaginal Immunization with the Nontoxic B Subunit of Cholera Toxin. J. Immunol., March 1, 2006; 176(5): 2749 - 2757. [Abstract] [Full Text] [PDF]
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S. E. Applequist, E. Rollman, M. D. Wareing, M. Liden, B. Rozell, J. Hinkula, and H.-G. Ljunggren Activation of Innate Immunity, Inflammation, and Potentiation of DNA Vaccination through Mammalian Expression of the TLR5 Agonist Flagellin J. Immunol., September 15, 2005; 175(6): 3882 - 3891. [Abstract] [Full Text] [PDF]
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M. M. Gherardi, E. Perez-Jimenez, J. L. Najera, and M. Esteban Induction of HIV Immunity in the Genital Tract After Intranasal Delivery of a MVA Vector: Enhanced Immunogenicity After DNA Prime-Modified Vaccinia Virus Ankara Boost Immunization Schedule J. Immunol., May 15, 2004; 172(10): 6209 - 6220. [Abstract] [Full Text] [PDF]
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) and E.G7-OVA (•) cells.
Coimmunization of 1 µg of LT with 100 µg of OVA or 40 µg of
OVA257–264 elicited OVA-specific CTLs in spleens
(A) and sCLNs (B). Splenic CTLs were also
detected when 1 µg of CT (C) or 20 µg of CTA1-DD
(D) was used to adjuvant whole OVA; however, only CT
adjuvanted OVA257–264. Immunization of mice with OVA alone
or OVA257–264 alone failed to elicit detectable
OVA-specific CTLs (E). The results are representative of
two to four separate experiments.
) or
OVA257–264 (
