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,
Departments of
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Human Genetics and
Medicine, and
Immunology Program, Memorial Sloan-Kettering Cancer Center and Weill Medical College of Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the requirement for interactions between immunizing DCs and T helper cells, we examined the function of MHC class II-peptide complexes on adoptively transferred DCs. These complexes govern interactions of the immunizing DCs with host MHC class II-restricted T cells. We used retroviral-mediated gene transfer to transduce a well-defined Ag encoding H-2Kb and I-Ab-restricted epitopes (13, 14) and thereby to coexpress MHC class I- and II-restricted epitopes in individual cells. To investigate further the importance of class II-restricted Ag presentation to recipient CD4+ helper T cells, we used DCs that lack MHC class II molecules on their surface. We demonstrate that autologous DCs lacking MHC class II molecules retain the ability to elicit strong CTL responses in vivo but, unlike MHC II+ DCs, fail to establish tumor immunity. Following the recent demonstration that CD40 activation of APCs can mimic the CD40/CD40 ligand (CD40L) interactions occurring between CD4+ T cells and DCs (15, 16, 17), we investigated whether CD40 activation of DCs lacking MHC II would alleviate the dependence of tumor rejection on DC-T helper cell interactions. To investigate what Ags could play a role in DC-T helper cell interactions, we compared DCs cultured in the presence of xenogeneic or syngeneic serum. We found that the latter are less potent in inducing tumor immunity. Taken together, our findings illustrate the value of enrolling CD4+ T cells to achieve antitumor immunity and underline the need to combine class I and class II epitopes appropriately to maximize the effectiveness of adoptively transferred DCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Female MHC class II-/- backcrossed to the C57BL/6 genetic background (N13) were purchased from Taconic (Germantown, NY) (18). All mice were used at the age of 5–8 wk and housed at the Central Animal Facility of Memorial Sloan-Kettering Cancer Center (MSKCC). EL4, a C57BL/6 thymoma, was obtained from the American Type Culture Collection (ATCC, Manassas, VA). MO4 (19), a chicken OVA-expressing subclone of B16, itself a C57BL/6-derived melanoma, was kindly provided by Dr. P. Srivastava, University of Connecticut Health Center, Farmington, CT.
Antigens
The H-2Kb-retricted peptide SIINFEKL, corresponding to amino acid residues 257–264 of chicken OVA (13) was synthesized in the Peptide Synthesis Facility at MSKCC and highly purified (>99%) as assessed by HPLC and amino acid analysis.
Retroviral vector construction
The retroviral vector SFG is derived from the Moloney murine leukemia virus (20). The vector SFG-OVA encoding the chicken OVA cDNA was constructed in several steps. First, the plasmid pAc-neo-OVA containing the OVA cDNA was cut partially with NcoI and then with PstI (419 bp). Another fragment was prepared by digesting pAc-neo-OVA with PstI and AlwnI (587 bp). A PCR reaction was performed to yield the 3' fragment from the AlwnI site (forward primer: 5'-CACATGCAGAAATCAATG-'3) to sequences extending the stop codon with addition of XhoI and BamHI sites (reverse primer: 5'-CCGCGGATCCTCGAGTTCTTTTAAGGGGAAACACATC-3'). The PCR product (190 bp) was digested with AlwnI and BamHI. The retroviral backbone was generated by cutting SFG-NTP (21) with NcoI and BamHI. The final product was sequenced to ascertain correct ligation of all the fragments. An ecotropic producer cell line was generated from the gp-E86 packaging cell line (22) as previously described (23).
Generation of bone marrow DCs and retroviral transduction
The procedure used for the generation of DCs in the presence of FBS (DCFBS) was that described by Inaba et al. (24) with some minor modifications. Briefly, cells expressing CD8, CD4, B220, and I-Ab were depleted using a mixture of mAbs and rabbit complement. The mAbs were 53.6 (anti-CD8), GK1.5 (anti-CD4), RA3-3A1/6.1 (anti-B220), and M5/114.15.2 (anti-I-A). 53.6 and GK1.5 were prepared in the Ab Core Facility at MSKCC; TIB146 and TIB120 were purchased from ATCC. Cells were plated in 60-mm petri dishes (3 x 106 cells/ml, 3.5 ml/dish) in RPMI 1640 medium supplemented with 10% FBS, and 500 U/ml recombinant murine GM-CSF and 50 U/ml recombinant murine IL-4 (Genzyme, Cambridge, MA). On day 2 of culture, nonadherent cells were removed, and 2/3 of fresh medium with cytokines was added. For gene transfer, nonadherent and adherent cells from day 2 cultures were collected and transferred onto a confluent plate of irradiated (30 Gy) producer cells. The medium was DC culture medium supplemented with Polybrene (4 µg/ml; Sigma, St. Louis, MO) and cytokines. After 48 h of cocultivation, nonadherent cells were removed and plated in fresh medium and cytokines. Gene transfer efficiency was measured by Southern blot analysis (21), using an OVA-specific fragment (+412 to +1168) as the probe. To generate DCs in syngeneic serum (DCB6S), FBS was substituted by 1% C57BL/6 serum. Serum was collected from adult 6- to 10-wk-old C57BL/6 mice and heat inactivated at 56°C for 30 min. In syngeneic serum, floating DCs appeared slightly earlier than in cultures containing FBS (our unpublished observations). DCB6S were therefore harvested on day 6 of culture and DCFBS on day 7. The DC yield from cultures in B6 serum was approximately 6- to 10-fold less than in cultures containing FBS.
Immunofluorescent staining and FACS analysis
Expression of cell-surface Ags on DCs was determined by cytofluorometric analysis with the following Abs: biotinylated mouse anti-MHC class II I-Ab (AFG-120) followed by PE-labeled streptavidin, FITC-labeled hamster anti-B7.1 (16-10A1), rat anti-B7.2 (GL-1), hamster anti-CD11c (HL3), hamster anti-ICAM-1 (3E2), and rat anti-B220 (RA3-6B2), all from PharMingen (San Diego, CA). Isotype-matched mAb were used as controls. All cells were stained in PBS containing 2% FCS for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/CD32 mAb (2.462, PharMingen) before staining. Acquisition was performed on a FACScan (Becton Dickinson, Mountain View, CA), using forward- and side-scatter characteristics to exclude dead cells. Data were analyzed using CellQuest (Becton Dickinson).
Mixed leukocyte reaction
Responder splenocytes from H-2Kd (CBA) mice were enriched for T cells by an initial plastic-adherence for 1 h at 37°C. The nonadherent cells were then opsonized with anti-B220 and I-A mAbs and removed using immunomagnetic beads (Dynall, Oslo, Norway). Responder T cells were cultured at 4 x 105 cells with 0.5 x 103-1.25 x 104 irradiated DCs for 4 days in flat-bottom microtiter plates. Proliferation of the responding T cells was based on the incorporation of [3H]thymidine (1 µCi/well) during the last 18 h of culture and measured in a Wallac (Gaithersburg, MD) scintillation counter.
CTL induction in mice
Naive adult C57BL/6 were injected i.v. with 105 SIINFEKL-pulsed or OVA-transduced DCs. DCs were washed extensively with HBSS before i.v. injection. Splenocytes were harvested 7 or 30 days later and restimulated in vitro for 5 days with irradiated SIINFEKL-pulsed C57BL/6 splenocytes in RPMI 1640, 10% heat-inactivated FCS, 50 µM 2-ME, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cytolytic activity was next measured in a standard 4-h 51Cr release assay.
Cell-mediated cytotoxicity assays
In vitro restimulated lymphocytes were tested for cytolytic activity in a standard 4-h 51Cr release assay against the C57BL/6 thymoma EL4, either alone or pulsed with SIINFEKL. Target cells were radiolabeled with 100 µCi Na251CrO4/2 x 106 cells for 1 h at 37°C. Peptide-pulsed EL4 cells were prepared by incubating EL4 cells with the radiolabel and SIINFEKL peptide at a concentration of 10 µg/ml. Maximum, spontaneous, and specific 51Cr release were determined in standard fashion.
Immunization of mice with DCs
Bone marrow-derived DC were generated as described above. Donors were either syngeneic wild-type C57BL/6 mice or congenic MHC II-/- mice. Recipient mice were treated with either PBS; DCs without peptide (DCFBS or DCB6S); DCs transduced with the OVA cDNA (DCFBS-OVA), or OVA-transduced MHC II-/- DCs (II-/-DCFBS-OVA); DCs pulsed with SIINFEKL (DCFBS/DCB6S-SIINFEKL), or MHC II-/- DCs pulsed with SIINFEKL (II-/-DCFBS-SIINFEKL). In some experiments, transduced II-/- DCs were activated with the agonistic activating anti-CD40 Ab IC10 or isotype-matched control Ab (25). Naive adult C57BL/6 mice were immunized once with 105 DCs and challenged 10 days later with a s.c. injection of 1 x 105 MO4 melanoma cells. Tumor growth and survival were monitored three times per week.
In vivo depletion of CD4+ T cells
Mice were injected i.v. with 250 µg of anti-CD4 mAb GK1.5 or control anti-DR-5 Ab (SFR3-DR-5). The first injection was given 6 days before inoculation of DCs, followed by three other injections at 3-day intervals. The specific depletion was >99% as determined by flow cytometry (data not shown).
CD40 activation of DCs
Day 6 DC cultures (1 x 106/ml) were treated with control Ig or the agonistic activation Ab IC10 (10 µg/ml; kindly provided by Dr. H.-C. Liou, Cornell University, New York, NY) for 24 h (25). DCs treated with IC10 formed densely packed clusters, whereas control-treated cultures exhibited relatively sparse aggregates. For surface phenotyping, cells were stained in PBS-2% FBS for 30 min at 4°C, using fluorochrome-conjugated mAbs specific for B7.1 (16-10A1), B7.2 (GL-1), and ICAM-1 (3E2) (PharMingen). Isotype-matched mAbs were used as controls. Fc receptors were blocked with anti-CD16/CD32 mAb (2.462, PharMingen) before staining. Acquisition was performed on a FACScan (Becton Dickinson) using forward- and side-scatter characteristics to exclude dead cells. Bcl-xL expression was detected by Western blot analysis. Cells were lysed, and 50 µg of protein from each sample was resolved on a 15% SDS-PAGE gel, probed for Bcl-xL (E20; Santa Cruz, Santa Cruz Biotechnology, CA), and detected with the appropriate HRP-conjugated secondary Ab.
Statistical analyses
Recipient mice were randomly assigned to the different treatment groups. To determine whether the different immunizations affected their survival rates, the nonparametric Wilcoxon rank sum test procedure was employed (26). Animals free of tumors by day 50 were then followed through days 80–100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of the cDNA encoding an Ag in DCs was compared with the
loading of DCs with a class I-restricted peptide for stimulation of
Ag-specific T cell responses and tumor immunity. We transduced bone
marrow-derived DCs with the cDNA of chicken OVA or pulsed them with the
OVA-encoded, Kb-restricted peptide SIINFEKL. The
former were transduced with a retroviral vector that placed OVA
expression under the transcriptional control of the retroviral long
terminal repeat. Mammalian cells transfected with the chicken OVA cDNA
secrete OVA (27, 28, 29), which is therefore available for
endocytic uptake and presentation by MHC class II molecules during and
after retroviral transduction. DCs sorted to high purity based on their
B7.2 marker expression (Fig. 1
A) bore on average one vector
copy per cell as shown by Southern blot analysis (Fig. 1B).
Peptide-loaded DCs were pulsed with peptide immediately before
injection and administered in an amount equal to the transduced DCs.
Transduced and peptide-loaded DCs showed the same phenotypic
characteristics in terms of cell surface expression of MHC class I,
class II, B7.1, B7.2, and ICAM-1 (data not shown). We consistently
found that syngeneic C57BL/6 mice immunized with a single dose of
105 OVA-transduced DCs were all protected against
a subsequent challenge with the OVA-transfected B16 melanoma MO4 (Fig. 1C). By comparison, about half of the animals immunized with
DCs pulsed with SIINFEKL were protected. Mock-transduced DCs pulsed
with the SIINFEKL peptide before injection achieved the same result
(Fig. 1C), establishing that retroviral transduction did not
in itself enhance DC function. Under these conditions, the transduced
DCs bearing the OVA cDNA encoding both MHC class I and class II
epitopes were therefore more potent in establishing tumor protection
than DCs presenting only the Kb-restricted
epitope.
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To address whether enhanced tumor protection was conferred by the
class II-restricted epitope encoded in the OVA cDNA, we transduced DCs
lacking MHC class II molecules on their surface
(II-/- DCs). As shown in Fig. 2A, the activity of DCs
lacking MHC class II was not only reduced but practically abolished
with respect to induction of tumor protection. The same was observed
when mice were immunized with SIINFEKL peptide-pulsed DCs lacking MHC
class II (Fig. 2B). MHC class II-/-
DCs either transduced with the OVA cDNA (Fig. 2A) or pulsed
with the SIINFEKL peptide (Fig. 2B) showed comparable
effectiveness, suggesting that presentation of the MHC class I epitope
was not functionally different. MHC class II molecules expressed on the
immunizing DCs were therefore essential to achieve tumor immunity,
suggesting that CD4+ lymphocytes engaged by these
MHC molecules play a key role in establishing tumor protection. The
involvement of CD4+ T cells was confirmed by in
vivo depletion of CD4+ T lymphocytes.
Administration of a CD4-specific mAb (GK1.5) during the induction phase
abolished tumor protection induced by peptide-pulsed DCs
(6) as well as by genetically-modified DCs (Fig. 2C). On the other hand, MHC II-/-
DCs either transduced with the OVA cDNA (Fig. 2A) or pulsed
with the SIINFEKL peptide (Fig. 2B) showed comparable
effectiveness, indicating that presentation of the MHC class I epitope
was not functionally different in these two cell populations.
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We next examined whether DCs lacking MHC class II could not induce
tumor immunity because of a failure to induce SIINFEKL-specific CTLs.
Mice were immunized with SIINFEKL-pulsed II+/+ or
II-/- DCs, and spleens were harvested 7 days
later to measure CTL activity. Mice immunized with DCs lacking or
expressing MHC class II displayed high levels of SIINFEKL-specific CTLs
(Fig. 3). Interestingly, we consistently
found that mice immunized with wild-type DCs had a high level of
background cytotoxic activity that was not observed in mice immunized
with DCs lacking MHC class II. These data indicate that DCs lacking MHC
class II molecules induce tumor-specific CTLs, but nonetheless fail to
establish protective immunity. When examining CTL activity on day 30
after immunization, it was apparent that mice immunized with
II+/+ DCs still had substantial levels of
SIINFEKL-specific CTLs, whereas mice immunized with
II-/- DCs did not (Fig. 3). Combined with the
observations shown in Fig. 2
, these findings suggested a correlation
between absence of CD4 activation, decreased long-term CTL activity,
and lack of tumor immunity.
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To investigate further the basis for suboptimal CTL activity in
mice immunized with II-/- DCs that could
account for the lack of tumor immunity, we activated the immunizing DCs
via their CD40 receptor. Such activation has indeed been shown to
support CTL induction in a CD4-independent fashion (9, 16, 17). Thus, CD40 activation would be expected to increase CTL
activity and perhaps overcome the lack of a concomitant CD4 response.
DCs treated with the agonistic anti-CD40 mAb IC10 were efficiently
activated as determined by up-regulation of cell-surface expression of
B7.1, B7.2, and ICAM-1 (from 64 to 120, 60 to 105, and 90 to 120,
respectively, with regard to mean log fluorescence) as well as
expression of Bcl-xL (Fig. 4A). The successful activation
of the II-/- DCs was also reflected in a
substantial increase in SIINFEKL-specific CTL activity (Fig. 4B). Mice given CD40-activated DCs showed SIINFEKL-specific
CTL activity in their spleen by day 30, whereas mice given DCs treated
with a control Ab did not (Fig. 4B). However, as shown in
Fig. 4C, CD40 activation of DCs lacking MHC class II did not
improve their ability to induce antitumor immunity. This indicated that
mimicry of CD40/CD40L interactions occurring between DCs and T helper
lymphocytes was not sufficient to restore tumor immunity.
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The observation that antitumor immunity conferred by DCs is
dependent on host CD4+ T cells and MHC class II
Ags expressed by DCs and host CD4+ cells raised a
paradox: How can DCs pulsed only with an MHC class I-restricted peptide
be effective? This prompted an investigation of the nature of Ags
presented by MHC class II molecules in that setting. The possible role
of Ags to which DCs are exposed during their culture before infusion
was examined. DCs are typically cultured in FBS, a rich source of
xenogeneic Ags. Conditions were established to generate bone
marrow-derived DCs in syngeneic C57BL/6 serum (B6S). DCs generated in
the presence of FBS or B6S, respectively DCFBS
and DCB6S, had identical profiles in terms of
expression of I-Ab, B7.1, B7.2, ICAM-1, and CD11c
(Fig. 5). Additionally
DCFBS and DCB6S showed
equal stimulatory capacity in allogeneic MLRs (Fig. 6). Furthermore, peptide-pulsed
DCFBS and DCB6S were
equally capable of inducing a specific CTL response against SIINFEKL,
but DCB6S did not generate the elevated
background cytotoxicity caused by DCFBS (data not
shown). By independent criteria, these DC populations appeared
equivalent in inducing proliferative and Ag-specific CTL responses.
However, as shown in Fig. 7, DCs grown in
syngeneic serum showed significantly decreased potency in the induction
of antitumor immunity (p < 0.01). This finding
indicates that FBS Ags presented by MHC class II molecules of cultured
DCs play an important role in establishing successful antitumor
immunity, likely via induction of CD4+ T cell
help and further activation of DCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further investigation of the SIINFEKL-specific CTL activity generated
by MHC class II-/- DCs shows that these CTLs
are short-lived compared with their counterparts induced by MHC class
II+/+ DCs. The presence of memory CTLs correlated
with tumor immunity. Indeed, mice immunized with MHC class
II+/+ DCs were protected against tumor challenges
(about one-half of them when treated with peptide-pulsed DCs and all of
them when given genetically modified DCs, Fig. 1) and displayed
substantial SIINFEKL-specific CTLs 30 days after primary immunization
(Fig. 3). Mice given MHC class II-/- DCs did
not develop tumor immunity (Fig. 2) and did not show SIINFEKL-specific
CTLs 30 days after primary immunization (Fig. 3). These data suggest
that conditions leading to the activation of CD4+
cells facilitated the generation of memory CTLs and tumor immunity.
This correlation, however, does not imply that the only function of the
activated CD4+ T cells is to provide some form of
help to CTL.
To begin to address the function of host CD4+ T
cells, we investigated whether their function is to activate DCs via
CD40/CD40L interactions. Indeed, it has been shown that T helper cells
can activate DCs via CD40 and increase their ability to stimulate CTLs
(15, 16, 17). It is therefore possible that CD40 activation of
immunizing DCs could reduce the CD4+ T cell
dependence of CTL responses against tumors. We asked whether CD40
activation could enhance the function of DCs that could not engage
CD4+ T cells via their TCR. After ex vivo CD40
activation, however, we found that II-/- DCs
still failed to establish tumor immunity (Fig. 4C), but the
CD40-activated II-/- DCs were now able to
induce SIINFEKL-specific CTLs that persisted to day 30 (Fig. 4B). The level of CTL activity was about 8- to 16-fold less
than that found in mice given an equal number of
II+/+ DCs (see Figs. 3 and 4B). This
observation does not dismiss the critical role played by CD40/CD40L
interactions, but rather suggests that the full effect of CD40
activation is dependent on active CD4+ T cell-DC
interactions and/or that CD4+ T cells exert other
important effects that may or may not be mediated via the DC. For
example, CD4+ T cells could sustain CTL function
by secreting IL-2 or by activating other effector cell types (12, 30). However, CD4+ T cells have been shown
not to be required in the effector phase of tumor immunity against
different B16 melanoma sublines (31, 32).
In the first interpretation, CD40 activation does not result in maximal DC activation. It is noteworthy that, in other studies investigating the role of CD40 in tumor immunity, CD40 activation was performed in the presence of CD4+ cells (33, 34, 35, 36, 37, 38). Thus, when CD40-/- mice immunized against a tumor failed to develop tumor immunity, CD40+/+ DCs restored immunity (38). However, this rescue took place in the presence of recipient CD4+ T cells, and thus suggests that CD40/CD40L interactions were necessary for tumor immunity, but not that they were sufficient. Likewise, when administration of anti-CD40 mAb in vivo was shown to reverse tolerance induction by a tumor-related class I peptide (37), this result was achieved in the presence of recipient CD4+ cells and tumor Ags, including tumor-derived MHC class II-restricted epitopes that could have been presented by cross-priming. In both instances, other molecular interactions between CD4+ T cells and DCs might have contributed to the immune responses in addition to the CD40/CD40L interactions. In one instance, CD40 treatment has been shown to delay tumor progression in mice immunized with II-/- DCs (38). However, a majority of animals rejected tumors without CD40 treatment in that particular model, suggesting that even a small increase in tumor-specific CTL activity may have been sufficient to prolong the delay in tumor progression. In our model, which uses an aggressive melanoma, the increase in CTL activity secondary to ex vivo CD40 treatment was not sufficient to overcome a lethal tumor challenge. Taken together, these results are consistent with an important role for molecular interactions in addition to those between CD40 and its ligand, e.g., those between MHC II and the TCR or between TRANCE and RANK (39, 40), in achieving maximal DC activation in vivo.
These findings have practical implications for the use of DCs to immunize against tumor cells. The data imply that the effectiveness of adoptively transferred DCs presenting a class I epitope is strongly enhanced by the MHC class II epitopes borne by the immunizing cells. In this interpretation, Ags present in FBS enable the infused DCs to interact with host CD4+ lymphocytes, which in turn increase the immunostimulatory function of the infused DCs and/or exert other antitumoral functions. The lesser tumor protection achieved with DCs cultured in C57BL/6 serum could be explained by the presentation of fewer and/or less antigenic class II-restricted helper epitopes (e.g., alternatively processed self Ags; Ref. 41). The exact nature of the bovine Ags active in our model and their relationship to the MO4 melanoma are difficult to specify. However, responses initiated against these Ags did not mediate tumor rejection, because immunized mice that rejected MO4 did not reject the parental B16 melanoma (data not shown). On the other hand, mice immunized with SIINFEKL-pulsed DCFBS remained fully capable of rejecting MO4 cells that were passaged in mice and briefly cultured in serum-free medium before inoculation (data not shown). These findings argue against a role for CTL responses against FBS Ags in tumor rejection and are consistent with a predominant role for the OVA-specific CTLs (4, 5). These results further caution against the clinical use of MHC class I peptide-pulsed DCs generated under serum-free or autologous serum-containing conditions in the absence of MHC class II-restricted helper epitopes.
These data demonstrate that immunization protocols using DCs are more powerful when not only MHC class I but also MHC class II molecules are Ag loaded. One effective approach is to transduce the entire cDNA of the Ag with a retroviral vector (42). An essential question awaiting resolution is to identify what MHC class II-restricted epitopes are best suited to sustain antitumoral cytotoxic responses, whether they need to be expressed by the tumor, or whether unrelated recall Ags or superantigens would be equally or more effective.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michel Sadelain, Box 182, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; EB, endogenous band; B6S, syngeneic C57BL/6 serum.
Received for publication August 30, 1999. Accepted for publication November 16, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. Loucka, G. Schlecht, J. Vodolanova, C. Leclerc, and P. Sebo Delivery of a MalE CD4+-T-Cell Epitope into the Major Histocompatibility Complex Class II Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase Infect. Immun., February 1, 2002; 70(2): 1002 - 1005. [Abstract] [Full Text] [PDF]
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