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-Secreting, CD8+ CTL In Vivo1
Center for Gene Therapeutics, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting
CD8+ T cells developed in both wild-type and
IL-12-deficient backgrounds. Finally, CTL generated in the
IL-12-deficient environment were capable of protecting immunized mice
against tumor challenge, demonstrating that these CTL were fully
functional, despite the absence of IL-12 during the maturation process
in vivo. These results indicate that IL-12 is not critical for the
development of IFN- secreting, CD8+ T cells and that
another mechanism must be used by licensed DC to prime and activate
CTL. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(5). More recent studies have
demonstrated that, in fact, CD4+ T cells provide
help through direct interaction with the DC, not the CTL
(6). Ligation of CD40 on the DC by CD154 expressed on
activated CD4+ T cells promotes a functional
change in the DC, licensing the DC to prime CTL (7, 8).
Further evidence that CD4+ T cells provide help
through direct interaction with the DC comes from studies demonstrating
that the requirement for CD4+ T cell help during
CTL priming can be bypassed by the presence of a CD40 agonist
(9).
Understanding the signals used by a licensed DC to prime CTL will
have a tremendous impact on vaccine design. Although the molecular
changes that take place in the DC after CD40 ligation are still being
elucidated, one well-described effect is the induction of IL-12
(10, 11). IL-12 was originally identified as cytotoxic
lymphocyte maturation factor, and increased IL-12 production by DCs is
suggested to be a critical step in the process of CTL priming
(12). Furthermore, IL-12 is needed for maximal development
of IFN--secreting CTL (Tc1 cells) in vitro
(13, 14). Thus, incorporation of IL-12 or IL-12-inducing
agents into vaccines would be expected to enhance CTL generation, and
indeed, several reports have provided evidence of the adjuvant effects
of IL-12 (15, 16, 17, 18). We have recently demonstrated that in
the absence of IL-12, DCs loaded with the melanoma Ag gp100 can elicit
potent antitumor immunity, associated with a robust type 1 immune
response, suggesting that IL-12 expression by DC may not be absolutely
required for the development of cell-mediated immunity. However, the
major effector cell in the gp100 model is a CD4+
T cell, and because CD4+ T cell activation does
not appear to be CD40 dependent, it is quite possible that the
activation mechanism is IL-12 independent as well
(19).
IL-12 is used routinely as an indicator of DC activation and a
surrogate maker for type 1 T cell responses, yet the importance of
IL-12 in CTL priming in vivo is still unclear (20). To
investigate the requirement of IL-12 in CTL maturation and activation,
we have conducted a series of studies directed at evaluating
CD8+ CTL priming and activation in vivo.
Immunization of C57BL/6 mice with bone marrow-derived DCs (BMDCs)
loaded with an immunogenic Ag, chicken egg OVA, elicited a strong CTL
response that was CD4+ T cell and CD154
dependent, demonstrating that this model is representative of
CD4+ T cell-mediated DC licensing. In this
scenario, both wild-type and IL-12-deficient DCs can equally prime
CD8+ T cell effector function, as measured by
cytotoxic activity and Ag-specific IFN- production. Furthermore,
both classes of CTL (those generated in the presence of IL-12 and those
generated in the absence of IL-12) were able to protect immunized mice
from tumor challenge. These results indicate that although IL-12 may be
involved in the process of CTL maturation, its role is secondary to the
effect of DC licensing.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) mice aged 6–8 weeks were obtained from Charles River Laboratories (Wilmington, MA). IL-12 p40 gene knockout mice, IL-12 p40-/-, which do not produce bioactive IL-12, were kindly provided by Dr. J. Magram (Hoffmann-La Roche, Nutley, NJ; Ref. 21). B6,129S-H2dlAb1-Ea (MHC class II-/-), and C57BL/6J-Tnfsf5tm1Imx (CD40L-/-) mice (initially purchased from The Jackson Laboratory, Bar Harbor, ME) were bred in our pathogen-free animal facility. The tumor cell lines used were EL-4 (C57BL/6 thymoma) and EG7 (EL-4 transfected with the cDNA of chicken OVA). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. EG7 cells were maintained in medium containing 400 µg/ml G418 (Life Technologies, Grand Island, NY).
Peptides
The Kb-binding peptide of OVA (OVAp; SIINFEKL) and Db-binding peptide of the lymphocytic choriomeningitis virus glycoprotein (p33; KAVYNFATM) were purchased from American Peptide Company (Sunnyvale, CA) and William K. Warren Medical Research Institute (Oklahoma City, OK), respectively. Peptides were dissolved in distilled water and stored at -20°C.
Adenoviral vectors
A recombinant adenoviral vector expressing OVAp (SIINFEKL)
linked to the 
2-microglobulin signal sequence
(AdssOVA) was provided by Dr. M. J. Bevan (University of
Washington, Seattle, WA; Ref 22). AdLacZ
contains the gene for the Escherichia coli LacZ under
control of the CMV immediate early promoter and has been described
previously (23). Viruses were propagated on 293 cells and
purified by cesium chloride gradient centrifugation as described
previously (24).
Preparation of BMDC
In vitro expansion of BMDC has been described previously (19). Briefly, murine BM cells from C57BL/6 mice (IL-12+/+ DC) or IL-12-/- mice (IL-12-/- DC) were cultured in 24-well plates at 1 x 106 cells/well with RPMI 1640 medium containing 20 ng/ml recombinant murine GM-CSF and 10 ng/ml recombinant murine IL-4 (Schering-Plough Research Institute, Kenilworth, NJ). Two days after initial culture, cells were replated at 5 x 106 cells/well in 6-well plates with 50% of the fresh medium, and fresh cytokines were added. On day 5, DCs were harvested and purified on a 15% metrazimade column (Sigma) before immunization or phenotypic characterization.
FACS analysis
The phenotypic analysis of the DCs from C57BL/6 or IL-12-/- mice was performed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA). Cells were stained with the following Abs (obtained from BD PharMingen, San Diego, CA): FITC-conjugated anti-mouse CD40 (clone HM40–3); PE-conjugated anti-mouse CD86 (clone GL1); PE-conjugated anti-IAb (clone KH74); FITC-conjugated anti-mouse CD80 (clone 16–10A1); and FITC-conjugated anti-mouse CD11c (clone HL-3).
Immunization
Day 5 DCs were infected with adenovirus (Ad) vectors (multiplicity of infection of 50) or incubated with peptides (5 µg/ml) in culture medium for 2 h. Cells then were washed three times with PBS before immunization. To measure CTL priming, C57BL/6 and IL-12-/- mice were injected in each hind footpad with 5 x 105 Ag-loaded (Ad-infected or peptide-pulsed) IL-12+/+ or IL-12-/- DCs in 50 µl of PBS. Five days after immunization, lymphocytes were obtained from draining popliteal lymph nodes for cytotoxicity assay. In some experiments, MHC class II-deficient and CD154-deficient mice were immunized to determine the role of CD4+ T cells and CD40 ligation in the priming of CD8+ CTL. To measure antitumor immunity, C57BL/6 and IL-12-/- mice were immunized with 1 x 106 Ag-loaded wild-type or IL-12-/- DCs in 200 µl of PBS injected s.c. in the right hind flank. Fourteen days later, immunized animals were challenged by s.c. injection with 5 x 105 tumor cells (EL-4 or EG7). Tumor size in each group was monitored daily and measured twice a week.
Cytotoxicity assays
CTL lysis was assessed by a 4-h chromium release assay using 51Cr-labeled EL-4 cells in the presence or absence of 5 µg/ml OVAp or p33 peptide. To demonstrate effector phenotype, cytotoxic activity was blocked using hybridoma ascites fluid containing mAbs 53-6.72 (anti-CD8), GK1.5 (anti-CD4), or PK136 (anti-NK1.1). Percentage of specific 51Cr release was calculated as: (cpm experimental - cpm background/cpm maximum - cpm background) x 100%.
Intracellular cytokine staining
For staining of intracellular IFN-, popliteal lymphocytes
were harvested from mice 5 days after immunization with the DC/peptide
vaccines. Lymphocytes were incubated for 5 h in 24-well plate at a
concentration of 4 x 106 cells/well in 2 ml
of complete medium with 2.5 µg/ml indicated peptides and 0.5 mg/ml of
brefeldin A (Sigma). Cells were stained for intracellular IFN- using
the Cytofix/Cyto-perm kit (BD PharMingen) as specified by the
manufacturer. Briefly, stimulated cells were washed twice, Fc receptors
were blocked by incubation with rat anti-mouse CD16/CD32 (Fc Block;
PharMingen) for 20 min, and the cells were stained with FITC-labeled
rat anti-mouse CD8 (clone 53-6.7; BD PharMingen) for 30 min. After
fixation and permeabilization for 20 min, the cells were stained with
PE-labeled rat anti-mouse IFN- Ab (clone XMG1.2; BD PharMingen)
or an isotype control Ab (rat IgG1) for 30 min and then analyzed by
flow cytometry as described above. Numbers in FACS plots refer to
IFN-+ cells as percentage of the total
CD8+ population.
ELISA cytokine assay
To determine the cytokine profile in T cell cultures from primed
animals, splenocytes harvested from C57BL/6 or
IL-12-/- mice 14 days after s.c. immunization
and stimulated with irradiated EG7 cells at a 50:1 ratio in
complete medium for 72 h. Supernatants then were harvested
and analyzed for IFN- and IL-4 using ELISA kits from R&D Systems
(Minneapolis, MN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish an in vivo model of CTL priming, we have used
adoptive transfer of DC infected with an Ad expressing the SIINFEKL
peptide of OVA (AdssOVA) to provide a source of endogenous Ag for class
I presentation. As shown in Fig. 1
A, footpad injection of DC
infected with AdssOVA induced a strong CTL response in the popliteal
lymph nodes without the need for restimulation in vitro. The cytolytic
activity could be fully inhibited with anti-CD8, but not
anti-CD4 or anti-NK mAb, confirming that the effectors were
CD8+ T cells (Fig. 1B). Administration
of wild-type DC infected with AdssOVA failed to prime CTL in class II-
or CD154-deficient mice (Fig. 1C) demonstrating that: 1)
this model reflects a CD4+ T cell/CD40-dependent
mechanism of CTL priming; and 2) infection with Ad is not sufficient to
license DC for CTL activation.
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To determine the requirement of IL-12 in the priming of CTL after
DC licensing by CD4+ T cells, we have chosen to
compare wild-type DC to IL-12-deficient DC in the priming model
described in the previous paragraph. Because it has been reported that
DCs may respond to IL-12 in an autocrine/paracrine manner
(25), the phenotypes of wild-type and IL-12-deficient BMDC
were analyzed using flow cytometry to verify that any difference in CTL
priming is a consequence of the effects of IL-12 on T cells and not the
DC. Both IL-12+/+ and
IL-12-/- DCs appeared phenotypically similar,
displaying equivalent levels of MHC class II, CD40, CD80, and CD86
(Fig. 2). As expected, no IL-12
production was detected in the cultures of IL-12-deficient DCs, whereas
100–300 pg IL-12/106 cells/24 h was measured in
wild-type cultures (data not shown). These results demonstrate that
endogenous IL-12 is not critical for the differentiation of BMDCs.
Furthermore, both IL-12+/+ and
IL-12-/- DCs stimulate equivalent levels of
allogenic T cell proliferation in mixed lymphocyte reaction assays,
indicating that deficiency of IL-12 in DCs does not impair T cell
stimulatory capacity (data not shown).
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production
The production of IL-12 by activated DC has been suggested to
represent a critical early physiologic event in the development of
cellular immunity, especially with respect to optimal maturation of CTL
and the generation of Th1 cells. To determine the role of IL-12 in the
process of CTL priming, IL-12-deficient DC were transduced with AdssOVA
and adoptively transferred into IL-12-deficient mice. The results of
CTL priming in the absence of IL-12 was compared with identically
prepared DC from wild-type mice that were adoptively transferred into
wild-type hosts (Fig. 1
A). The absence of IL-12 had no
obvious effect on the cytotoxic activity of freshly primed CTL (Fig. 3A). Likewise, the CTL
response in wild-type mice was unaffected when
IL-12-/- DC were used for immunization,
indicating that IL-12-independent CTL activation is not a result of
compensatory mechanisms that may have developed in the
IL-12-/- mouse (data not shown). To determine
whether the absence of IL-12 resulted in reduced numbers of
Tc1 cells, we also measured Ag-specific IFN-
induction using intracellular stain (ICS). The ICS technique provided a
quantitative measure of CTL frequency in the freshly primed population.
Again, no difference was observed between wild-type and IL-12-deficient
animals in the frequency of IFN--producing
CD8+ T cells (Fig. 3B). Moreover, in a
restimulation assay, splenocytes from animals immunized with either
IL-12+/+ and IL-12-/- DCs
secreted large amounts of IFN- upon in vitro stimulation (Fig. 3C), whereas no IL-4 could be detected in both cases (data
not shown), indicating that the responsive T cells were primarily of
the T1 phenotype. Thus, IL-12 appears to be dispensable during the CTL
priming by virus-infected DC.
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Because Ad infection has been shown to impact on DC
function/maturation, it remains possible that Ad-infected DC may have
an altered phenotype, despite remaining dependent on
CD4+ T cells for licensing (26).
Therefore, IL-12+/+ or
IL-12-/- DCs were pulsed with OVAp and
adoptively transferred into wild-type and IL-12-deficient mice,
respectively. As with virally infected DC, the absence of IL-12 had no
impact on either the magnitude of cytolytic activity in freshly primed
CTL (Fig. 4A) or the frequency
of IFN--producing CD8+ T cells (Fig. 4B). These results indicate that the lack of IL-12
dependence during CTL priming by BMDC is not a result of the functional
changes of the DC due to Ad infection.
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A)
and IFN -producing CD8+ cells (Fig. 5B) was unaffected by the absence of IL-12. These results
suggest that although the DC are fully dependent on
CD4+ T cells and CD40 ligation for licensing,
even after virus infection, the CD4+ T cell/DC
interaction must invoke changes in the DC, other than IL-12 production,
that are critical for CTL priming.
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To evaluate the efficacy of CTL generated in the absence of IL-12
in a challenge model, mice immunized by our DC infected with AdssOVA
(DC/AdssOVA) were challenged with EG7 tumor cells, which express OVA as
a surrogate tumor Ag. Tumors grew in all unimmunized animals or animals
immunized with DC infected with an Ad expressing an irrelevant Ag, such
as E. coli -galactosidase (data not shown). Wild-type
mice vaccinated with wild-type DC/AdssOVA and
IL-12-/- mice immunized with
IL-12-/- DC/AdssOVA were both completely
protected from EG7 challenge. (Fig. 6A) Protection was Ag specific
because all animals challenged with EL-4 developed tumors. Similarly,
DC pulsed with OVAp were fully capable of protecting animals from tumor
challenge in the absence of IL-12 (Fig. 6B). In all cases,
tumor protection was CD8+ T cell dependent (data
not shown). These data demonstrate that CTL generated in the absence of
IL-12 are not only phenotypically equivalent to CTL from wild-type
animals, but they also are fully capable of protecting animals from
challenge.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have based our hypothesis for a central link between IL-12
production and CTL activation on the following data: 1) CD40 ligation
on DC is critical for the induction of CTL, and CD40 ligation induces
high-level IL-12 production in DC (11, 31, 32, 33); 2) IL-12
plays an important role in the development of type 1 T cell responses,
which are characterized by strong IFN- secretion and CTL activity
(12, 13, 14); 3) IL-12 treatment can enhance the development
of CTL in vitro/in vivo (34, 35, 36, 37); and 4) treatment of DC
with IL-12 improves their ability to activate CTL
(38).
By using adoptive transfer of BMDC, we have used an in vivo CTL priming
model to study the molecular requirements of CTL activation. In this
model, the DC require CD4+ T cells and CD154 to
successfully prime CTL, indicative of the CD4+ T
cell-dependent mechanism of DC licensing. Using p40-deficient mice, the
requirement of IL-12 could be evaluated in a background where IL-12 is
fully absent. DCs were loaded with Ag either by virus infection or
peptide loading, and we evaluated both Kb- and
Db-restricted CTL activation. Interestingly, the
DC pulsed with the class I binding peptides OVAp and p33 still required
CD4+ T cell help, even though these cells were
not pulsed with class II epitopes. Under these conditions, the FBS
additives used during the DC culture likely provide a source of MHC
class II peptides to engage CD4+ T cell help. In
all cases, the absence of IL-12 had no qualitative or quantitative
impact on the development of cytotoxic, IFN--secreting
CD8+ CTL. IL-12 independence does not seem to be
related to the maturation state of the DCs because we observed equal
enhancement of the CTL response in this model using LPS or CD154 trimer
to mature either IL-12+/+ or
IL-12-/- DCs (Y. Wan, unpublished
observations). Our results support the observation that the
CD4+ T cell and CD40 ligation pathways are
critically involved in the induction of CD8+ CTL,
but IL-12 production after CD40 ligation does not appear to be
necessary in the early phase of CD4 activation and subsequent CTL
priming. Thus, either IL-12 is not required for the differentiation and
maturation of Tc1 cells or secondary pathways
exist that can compensate for the absence of IL-12.
In support of a compensatory pathway, a recent report suggests that, in fact, IL-15, not IL-12, is responsible for enhanced CTL activation by CD154 trimer-treated DC using an in vitro system (39). By contrast, when the CD154 trimer was used to enhance DNA vaccination, the effect was IL-12 dependent (40). Likewise, Grufman and Karre found that p33-pulsed DC were unable to elicit CTL after adoptive transfer into IL-12-deficient animals (41). Thus, we had expected to observe a weakened CTL response in the IL-12-deficient mice. Because we observed no loss in CTL function, we interpret these results to indicate that the effects of IL-12 in the previous models may be secondary to the process of CTL priming. The study by Grufman and Karre (41) measured splenic CTL after restimulation in vitro, whereas our investigation focused on freshly primed CTL response within the draining lymph node. We currently are investigating the impact of IL-12 deficiency on CTL persistence and memory. One possible explanation for the discrepancy between the reports demonstrating a critical role for IL-12 and those indicating a secondary role may stem from the type of model used. In our model and the in vitro model, the early stages of the CTL response are being investigated, whereas the other models measured recall responses. Thus, perhaps IL-12 exerts its influence in the postpriming phase of the immune response.
The literature has mixed reports regarding the importance of IL-12 in
the development of CTL. Certainly, Tc1 cells can
be generated in vitro in the presence of IL-2 alone, suggesting
that IFN- secretion may be a default pathway during CTL priming.
Addition of IL-12 during in vitro differentiation greatly increases
IFN- production and cytolytic activity of Tc1
cells (13, 14). Likewise, the addition of IL-12 can
dramatically improve the development of antiviral CTL in vitro using
influenza-infected human DCs (37). In all of these cases,
IL-12 is added exogenously to the cultures, and therefore these
observations are not truly reflective of the impact of DC-derived IL-12
on CTL function. Interestingly, Bianchi et al. (25)
have recently reported that adoptive transfer of DCs pulsed with a
poorly immunogenic, class I-binding tumor peptide (P815AB) was unable
to elicit a T cell response unless the DC were treated with a CD40
agonist before adoptive transfer. Remarkably, they demonstrate that
neutralization of IL-12 during CD40 cross-linking in vitro abrogates
the adjuvant effect, and the addition of exogenous IL-12 can overcome
the requirement for a CD40 agonist, providing direct evidence that the
adjuvant effect of CD40 ligation can be due to increased autocrine
IL-12 (25). Similarly, Zitvogel et al. (42)
observed that neutralization of IL-12 at the time of immunization with
tumor peptide-pulsed DC could abrogate the protective response.
However, a fundamental difference between this model and other DC
immunization models outlined above is the nature of the Ag. To address
this possibility, we currently are investigating the influence of
endogenous or exogenous IL-12 on the immune outcome after adoptive
transfer of DCs loaded with a series of poorly immunogenic, MHC class
I-restricted peptides from melanoma compared with OVAp and
p33.
There is increasing interest in developing vaccines using DCs as a physiological adjuvant to enhance an Ag-specific immune response, especially a cell-mediated protective immunity. In this scenario, treatment of DC with IL-12 before inoculation, provision of exogenous IL-12 after immunization, and modification of the DC to express elevated levels of IL-12 have all been shown to enhance immune induction, particularly with weak Ags (16, 17, 18, 25). Although these results do not support a role for IL-12 in the process of CTL priming, they do demonstrate that the presence of IL-12 at the time of immune induction can strongly influence the outcome of the immune response and support the inclusion of IL-12, or IL-12-inducing agents, in vaccine formulations. However, from a biological point of view, the cellular target of the IL-12 remains to be determined, although it is tempting to speculate that IL-12 exerts its influence directly on the DC.
The results of these experiments provoke reconsideration of the
relationship between IL-12 production and DC phenotype. Accumulating
data have indicated that DCs are not only critical for the initiation
of T cell immunity, but these cells also determine the course of the
subsequent immune response (i.e., tolerance vs immunity; Ref.
30). However, the mechanisms by which DC can influence the
final outcome of a given immune response remain to be understood.
Distinct DC subsets have been identified, leading to the hypothesis
that the ability of DC to direct immunity is related to their
hemopoietic lineage. Although the correlation between DC subset and the
type of immune response remains controversial, the current belief is
that CD8
+ DC, but not
CD8- DC, are responsible for the induction of
cellular immunity in mice, based on the observations that IL-12
production is restricted to the CD8+ subset
(43, 44, 45, 46). Our BMDCs are largely
CD8-, yet they can stimulate strong cellular
immune responses upon adoptive transfer into recipient hosts. Moreover,
our DC promote a robust cellular response in an IL-12-independent
fashion, indicating that secretion of IL-12 by DCs may not necessarily
be a relevant measurement of the DC’s ability to elicit CTL-mediated
protective immunity.
Taken together, our data may offer new insight into the role of IL-12 in CD4-dependent mechanisms of DC licensing and facilitate the identification of other molecules in mediating CD4+ and CD8+ T cell interaction via DC activation. In addition, the selection of an appropriate DC phenotype to design cancer vaccines is a clinically relevant concern, and whether the ability to produce IL-12 should be used as a marker to distinguishing immunogenic and tolerogenic DCs requires further studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yonghong Wan, Department of Pathology and Molecular Medicine, McMaster University, HSC-4H21B, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. E-mail address: wanyong{at}mcmaster.ca
3 Abbreviations used in this paper: DC, dendritic cell; Tc1, IFN--secreting CTL; Ad, adenovirus; ICS, intracellular stain.
Received for publication July 25, 2001. Accepted for publication September 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production and type 1 cytokine responses. Immunity 4:471.[Medline]
+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587.This article has been cited by other articles:
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) and EL-4 cells (
).
B, Target cells were mixed with effectors at a ratio of
90:1. Abs against CD4, CD8, and NK1.1 were added to the wells 30 min
before the addition of the targets. C, Lymphocytes from
immunized wild-type (
), and
CD40L-deficient (
