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National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1*0701-restricted epitope of gp100. The minimal determinant of
this epitope was defined as gp100174–190
(TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally
transduced DCs have the capacity to present multiple MHC class I- and
class II-restricted peptides derived from a tumor Ag, thereby eliciting
a robust immune response against that Ag. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the cells of the immune system, dendritic cells (DCs)2 have a central role in primary activation of quiescent B and T lymphocytes (8) and may be useful for the generation of T cell-mediated immune responses against tumor Ags (9). Several approaches have been described that use tumor/DC fusions (10) or DCs pulsed with preparations derived from whole tumors such as total RNA (11) or apoptotic bodies (12, 13, 14). Although these strategies may induce immune responses against multiple relevant tumor Ags, the use of complex mixtures of proteins may result in competition and attenuation of the most potent immune interactions (15).
DCs have been manipulated to present tumor Ags by pulsing with MHC-restricted peptides (16, 17), but this approach limits the response to identified Ag epitopes and requires that patients express the appropriate HLA alleles. In addition, some epitopes presented by MHC molecules are modified post-transcriptionally (18, 19). Expressing whole tumor Ags in DCs may allow the presentation of multiple epitopes via MHC class I and possibly MHC class II molecules as well as undefined and post-transcriptionally modified epitopes.
Several strategies have been used to transfer genes into DCs, such as recombinant adenoviruses (20), recombinant poxviruses (21), or recombinant retroviruses (22). Because of the potential of viral Ags, often coexpressed in many vector systems, to be immunodominant over tumor Ags, we have developed methods to transduce DCs with recombinant retroviral vectors that solely express the gene of interest (22). Previously, we reported that retrovirally transduced DCs have the capacity to efficiently generate CD8+ T cells reactive against a single HLA-A2 epitope of MART-1 (22). In addition, retrovirally transduced DCs were effective in generating class I-restricted T cells and treating established lung metastases in an experimental murine model system (23). However, it is unclear whether retrovirally transduced DCs can present multiple MHC class I epitopes derived from a tumor Ag.
Besides the generation of class I-restricted T cells, retrovirally transduced DCs may be capable of presenting Ags in the context of class II. Recent observations suggest that the stimulation of both CD4+ and CD8+ T cells against tumor Ags can lead to an effective anti-tumor response (24, 25). Consequently, it is of particular importance to determine whether gene-modified DCs can generate T cells reactive against both MHC class I- and class II-restricted epitopes.
In the present study DCs were retrovirally transduced with the melanoma
differentiation Ag gp100 and used to stimulate T cells. The T cells
generated were analyzed for the capacity to recognize
multiple MHC class I and class II epitopes of gp100. The
gp100-transduced DCs were able to generate T cells recognizing three
distinct HLA-A2-restricted epitopes of the tumor Ag. Interestingly,
CD4+ T cells specific for a new HLA-DR1*0701
epitope of gp100 were generated. The ability of retrovirally transduced
DCs to generate T cells reactive against multiple MHC class I and class
II epitopes of a tumor Ag may be significant for in vitro analysis of
tumor Ags and, more importantly, for cancer patient immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Complete medium consisted of IMDM (Biofluids, Rockville,
MD and Life Technologies, Gaithersburg, MD) supplemented with 10%
human AB serum (male, heat inactivated; Gemini Bio-Products, Calabasas,
CA), 1 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin (all
from Biofluids), and 50 µg/ml gentamicin (Life Technologies). DC
medium consisted of complete medium supplemented with 100 ng/ml GM-CSF,
100 ng/ml TNF-
(both obtained from PeproTech, Rocky Hill, NJ), and
40 ng/ml stem cell factor (SCF; R&D Systems, Minneapolis, MN). T cell
medium consisted of complete medium supplemented with 60–300 IU/ml
rIL-2 (Chiron, Emeryville, CA). T cell clones and tumor-infiltrating
lymphocytes (TIL) 1520 were cultured in AIM-V medium (Life
Technologies) supplemented with 5% human serum, 1 mM glutamine, 50
U/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamicin.
rIL-2 at a concentration of 6000 IU/ml was added for TIL 1520 and at
300 IU/ml for the T cell clones.
Melanoma cell lines from patient A as well as cell lines 624.38 mel and 586 mel were developed at the Surgery Branch (National Cancer Institute, National Institutes of Health, Bethesda, MD) (26), and SK23 mel and MDA231 (breast cancer) were obtained from American Type Culture Collection (Manassas, VA). Because the melanoma line from patient A failed to express gp100, this line was transduced using a vesicular stomatitis virus (VSV)-pseudotyped retroviral vector expressing either gp100 or green fluorescent protein as described above. EBV-immortalized B cells were generated as previously described (26). Transduced cells were cultured in RPMI 1640-based medium containing 500 µg/ml geneticin sulfate (Life Technologies). All tumor and EBV-B cell lines were grown in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated FBS (Biofluids and Life Technologies) and antibiotics. T2 cells (American Type Culture Collection) are a TAP-deficient, HLA-A2-positive cell line and were grown in RPMI 1640 supplemented with 10% human AB serum (heat-inactivated) and antibiotics.
CD34+ hemopoietic progenitor cells were obtained from HLA-A2-positive patients undergoing treatment for melanoma as part of an institutional review board-approved protocol (Surgery Branch, National Cancer Institute). CD34+ cells were mobilized in peripheral blood by five daily s.c. injections of 10 µg/kg G-CSF (Neupogen; Amgen, Thousand Oaks, CA), followed by lymphocytopheresis to obtain PBMCs on day 6. CD34+ cells were selected by an immunoaffinity column (CellPro, Bothell, WA) and cryopreserved. A single leukapheresis typically yielded 2–5 x 108 CD34+ cells.
Retroviral vectors
The PG13-based gp100 retroviral packaging cell line was generated by inserting the complete gp100 cDNA into the retroviral vector SAMEN (27) as described previously (22). Producer cell medium consisted of DMEM (Life Technologies) supplemented with 10% heat-inactivated FBS and antibiotics.
The gp100-VSV-pseudotyped retroviral system was prepared by first inserting the complete gp100 sequence in the pCLNC retroviral plasmid (28). The pCLNC-gp100 and pMDG-VSV plasmids were cotransfected in 293-gag-pol packaging cells using Lipofectamine Plus (Life Technologies). The 293-gag-pol packaging cells, provided by I. Verma (The Salk Institute, La Jolla, CA), were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotics. Medium was changed 16 and 48 h after transfection. Culture supernatants were harvested on days 3 and 4 after transfection of the 293-gag-pol cells. Producer cells were removed from retroviral supernatant by filtration with a 0.2-µm filter (Nalgene, Rochester, NY). Supernatants were immediately frozen at -70°C for future use.
Transduction of CD34+-derived DCs
CD34+ cells were differentiated to DCs
according to the protocol previously described (22).
Briefly, CD34+ cells were thawed, washed in
complete medium, and plated at 5 x 106
cells/well in a six-well plate or at 5 x
105 cells/well in a 24-well plate in complete DC
medium containing TNF-, SCF, and GM-CSF. Cultured cells were
harvested on days 5 and 10, centrifuged, and resuspended in complete DC
medium with cytokines. Cells were used on day 14, and DC phenotype was
confirmed by morphological and FACS analyses (22).
For transduction with the PG13 system, CD34+ cells were cocultured with irradiated PG13-SAM-gp100-EN producer cells (31 Gy; 7 x 105 cells/well in a six-well plate) in complete DC medium with cytokines containing 8 µg/ml polybrene (Aldrich, Milwaukee, WI). After 30 h DCs were replated on fresh irradiated producer cells in DC medium including cytokines without polybrene for 24 h. On day 3, transduced DCs were resuspended in fresh complete DC medium in a six-well plate, and differentiation was completed as described above.
For transduction with the VSV-pseudotyped retroviruses, retroviral
supernatant was added to cultured CD34+ cells on
days 2 and 3 at a ratio of 1:1 with culture medium. GM-CSF, SCF,
TNF-, and polybrene were added, and cells were spun in the plate at
1000 x g for 1 h. On day 4 transduced DCs were
resuspended in fresh complete DC medium in a six-well plate, and
differentiation was completed as described above.
Mixed leukocyte reaction
MLR was performed as described previously (22) with minor modifications. Briefly, 1 x 105 allogeneic enriched T cells (human T cell immunoaffinity columns; R&D Systems) were cocultured with increasing numbers of irradiated (1500 rad) DCs in 96-well, flat-bottom tissue culture plates for 6 days. T cell proliferation was monitored by [3H]thymidine (DuPont-New England Nuclear, Boston, MA) incorporation for the last 16 h. Results were corrected for [3H]thymidine incorporation by irradiated DCs and T cells alone.
Stimulation of autologous T lymphocytes with transduced DCs
Autologous human PBMC were obtained from leukapheresis of patients at the Surgery Branch (National Cancer Institute) and were cryopreserved for use in experiments. T lymphocytes were isolated from PBMC using a human T cell immunoaffinity column (R&D Systems). Purified T cells (2 x 106) were cocultured with irradiated gp100-transduced DCs (2 x 105, 15 Gy) in a 24-well plate in 2 ml complete medium without rIL-2. rIL-2 (60–300 U/ml) was added on day 2, and the cells were diluted with fresh complete medium and rIL-2 to keep the cell concentration at 1–2 x 106 cell/ml. T cells from patient A were obtained before any nonsurgical treatment of melanoma and were stimulated once with gp100-transduced DCs. T lymphocytes from patient B were obtained 8 mo after immunization with gp100280–288 peptide in IFA (29). In some cultures a combination of soluble trimeric recombinant CD40 ligand (CD40L; Immunex, Seattle, WA) and LPS prepared from Salmonella typhimurium (Sigma, St. Louis, MO) was added to the T cell stimulation with the gp100-transduced DCs. CD40L, and LPS can increase the capacity of DCs to stimulate the generation of Ag-specific T cells (30).
Where indicated, the bulk T cell cultures were cloned by limiting dilution at 1, 2, or 10 cells/well in 96-well, round-bottom plates (31). Cloning was performed in complete medium using AIM-V medium supplemented with 5% human AB serum, 1 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 50 µg/ml gentamicin in the presence of 5 x 104 irradiated PBMC prepared from three different donors (from normal volunteers at the Clinical Center, National Institutes of Health), 30 ng/ml anti-CD3 (OKT3; Ortho-Biotech, Raritan, NJ), and 300 U/ml rIL-2. Relevant clones were expanded in T-25 tissue culture flasks at 1–5 x 105 cells/flask with 2.5 x 107 irradiated PBMC feeders prepared from three different donors (Blood Bank, Clinical Center, National Institutes of Health) and 30 ng/ml anti-CD3 in 25 ml of AIM-V complete medium. On day 2, 300 U/ml rIL-2 was added, and on day 5, 20 ml of medium was replaced with fresh AIM-V complete medium containing 300 U/ml rIL-2. On day 8, cells were counted, and the cell concentration was maintained at 1–2 x 106 cells/ml in AIM-V complete medium with 300 U/ml rIL-2.
T cell assays
T cells were analyzed for their capacity to
recognize tumors expressing gp100 and HLA-A2 or T2 cells pulsed with
gp100-derived HLA-A2 binding peptides
gp100154–162 (KTWGQYWQV),
gp100209–217 (ITDQVPFSV),
gp100280–288 (YLEPGPVTA) (32), or
an irrelevant HLA-A2-restricted peptide derived from the influenza
M-1 protein. T2 cells were pulsed with peptides at 1 µg/ml for 3
h at room temperature and washed twice with PBS. Peptide-pulsed T2 or
tumor cells (1 x 105) were incubated with
1 x 105 specific T cells in a 250-µl
volume in 96-well, flat-bottom plates. Supernatants were
harvested after 24 h, and human IFN-
was assayed by ELISA using
a commercially available kit (R&D Systems).
The gp100 peptide library consisted of 68 peptides of 17–21 residues
overlapping by 10–12 aa. The peptides were synthesized by solid phase
F-moc methodology as previously described (33). A control
peptide derived from the Ig 
-chain known to bind to HLA-DR1*0701
was also prepared (34). Recombinant gp100 protein was made
and purified as previously described (35). Recombinant
NY-ESO-1 protein is another tumor Ag (2) used as a
negative control and was made and purified as previously described
(36). EBV-B cells (1 x 105)
were pulsed with gp100-purified protein or peptides for 18 h in
96-well, flat-bottom plates, and T cells were added directly to the
pulsed B cells for a 24-h recognition assay. In some experiments EBV-B
cells (5 x 105) were pulsed in 48-well
plates for 18 h, and cells were washed twice with PBS. T cells
were then cocultured with the pulsed EBV-B cells in 96-well,
flat-bottom plates for 24 h. Supernatants were harvested, and
human IFN- was assayed by ELISA using a commercially available kit
(R&D Systems).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs derived from CD34+ hematopoietic stem
cells were generated according to the procedure previously described
(22). Because the CD34+ cells
actively proliferate upon differentiation into DCs, retroviral vectors
can be used to introduce genes in the genome. gp100 was used as a model
melanoma Ag and was cloned into retroviral vectors. T cells recognizing
an HLA-A2-restricted epitope of gp100 were used with transduced DCs to
monitor the expression and presentation of gp100 through MHC class I.
As shown in Fig. 1
A, two T
cell lines specific to an
HLA-A2-restricted epitope of
gp100209–217 recognized gp100-transduced DCs,
but not DCs transduced with the control retroviral vector. Similar
results were obtained using different CD34 donors.
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B, gp100
retrovirally transduced DCs equally stimulated the proliferation of
allogeneic T cells compared with DCs transduced with control vectors or
untransduced DCs. Because DCs were successfully transduced with gp100 without affecting their capacity to stimulate T cells, gp100-transduced DCs were next used in T cell sensitization studies.
Generation of T cells recognizing multiple HLA-A2-restricted epitopes of gp100
To determine whether gp100-transduced DCs could stimulate the
generation of specific CD8+ T cells recognizing
multiple epitopes within gp100, resting T cells from melanoma patients
were cultured with autologous gp100-transduced DCs. After one or two T
cell stimulations using transduced DCs, two of four patients tested had
T cells reactive against
HLA-A2+/gp100+ tumor cells
(Table I). T cell cultures derived from
patients A and B were reactive against two
HLA-A2+/gp100+ tumor cell
lines (624.38 mel and SK23 mel). These T cells failed to react with
HLA-A2-/gp100+ or
HLA-A2+/gp100- tumor cells
(586 mel and MDA231, respectively), confirming the HLA-A2 and gp100
specificity. Importantly, these cells recognized all three defined
HLA-A2-specific, gp100-restricted epitopes tested using peptides
gp100154–162,
gp100209–217, and
gp100280–288 when pulsed on T2 cells. Peptide
recognition using T cells from both patients were specific for gp100,
because they failed to react against an unrelated peptide from the
influenza M-1 protein. These results demonstrate that gp100-transduced
DCs can stimulate autologous T cells reactive against multiple
gp100-restricted HLA-A2 defined epitopes.
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1*0701 epitope of
gp100In addition to presentation of epitopes by MHC class I, transduced DCs may present peptides in the context of MHC class II molecules. T cell stimulations were next performed to determine whether gp100-transduced DCs could generate Ag-specific CD4+ T cells recognizing gp100 peptides presented by MHC class II. T cells from patient A were stimulated twice with DCs retrovirally transduced with gp100. Stimulated T cells were analyzed for their capacity to recognize a gp100-expressing autologous melanoma cell line in an MHC class II-restricted fashion. The cultured T cells were reactive against the autologous tumor line expressing HLA-DR and gp100, but not against gp100-negative melanoma cells or autologous cultured B cells (data not shown).
The reactive bulk T cell culture was cloned by limiting dilution, and
growing wells were screened using autologous melanoma cells expressing
gp100 or green fluorescent protein as a negative control. Forty-six of
167 wells were specifically reactive against the autologous melanoma
expressing gp100 and class II. The reactivity of nine representative
positive clones is presented in Fig. 2A along with four
representative negative wells (neg1 to neg4). Eight of the 46 positive
clones were expanded and further characterized. All eight clones were
CD4+ (data not shown), suggesting recognition of
gp100 peptides presented by MHC class II. To further characterize the
reactivity, some of the clones were cocultured with autologous B cells
pulsed with a bacterially produced gp100 protein. All the
CD4+ T cell clones tested secreted IFN- when
exposed to autologous B cells pulsed with gp100, but not those pulsed
with an irrelevant tumor Ag prepared using a similar procedure. The
results from two representative clones are presented in Fig. 2B. Clones 6 and 8 failed to react against NY-ESO-1, a
protein produced similarly to recombinant gp100. To identify the MHC
restriction element, Abs known to bind and block presentation by
defined MHC molecules were used. Recognition by clones 6 and 8 of
target cells pulsed with the gp100 protein was inhibited when using
blocking Abs against MHC class II and HLA-DR, but the recognition was
unchanged when using a blocking MHC class I Ab. Also, both clones
failed to recognize gp100 when pulsed on unmatched HLA-DR B cells. By
using HLA-DR-matched B cells, the restriction element was identified as
HLA-DR1*0701 (patient A is HLA-DR1*0701 and *1601; Fig. 3).
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A; data from two clones
shown). Using a gp100170–190-specific T cell
clone, a titration experiment using the
gp100170–190 peptide pulsed on EBV-B cells
revealed that recognition was undetectable below 4–10 µM (Fig. 4B). Interestingly, the
gp100170–190-specific T cell clone recognized B
cells pulsed with gp100 protein at a concentration as low as 22 nM. The
difference between the concentrations of protein and peptide required
for recognition may be due to the greater susceptibility of peptide to
degradation by serum proteases.
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1*0701 epitope. As shown in Table II, no peptides were better recognized at
10 or 1 µM compared with the gp100170–190
peptide. Finally, substitutions were made at position 5 of the
gp100174–194 peptide, presuming that this is
position 1 according to the defined motif for HLA-DR1*0701
(SYFPEITHI web site, http://syfpeithi.de/). However, none of the
peptides was better recognized when pulsed at 10 or 1 µM compared
with the wild-type gp100174–194 peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1*0701 epitope of gp100
was identified. Because 15–28% of the population express this HLA-DR
allele, this gp100 peptide represents a potential candidate for patient
immunization. To our knowledge this represents the first example of the
generation of CD4+ T cells recognizing a defined
HLA class II epitope using tumor Ag-transduced DCs.
The generation of CD4+ T cells using transduced
DCs suggests that peptides derived from the endogenously expressed
gp100 were presented by MHC class II molecules. Also, tumor cells
expressing MHC class II and gp100 were efficiently recognized when
using CD4+ T cells specific for an
HLA-DR1*0701 epitope of gp100. Classically, peptides presented by
MHC class I molecules are derived from endogenously expressed proteins,
while Ag presentation by MHC class II occurs mainly from exogenous Ags.
This study suggests that endogenously expressed gp100 can traffic to
the lysosomal/endocytic pathway to be presented by MHC class II.
Interestingly, a dileucine-based melanosomal transport signal motif
known to target proteins to the melanosome is present in gp100
(37). A similar motif derived from gp75/tyrosinase-related
protein-1 has been previously shown to be critical for targeting to the
lysosomal/endocytic compartment and for the presentation by MHC class
II (38). It is unclear whether all tumor Ags will
endogenously traffic to MHC class II, or whether this will require the
addition of specific signaling motifs.
It is not clear why gp100-specific CD8+ T cells were raised in only two of four patients tested. This may be due to patient-to-patient variability in the number of pre-existing T cells capable of recognizing tumors. In addition, in this study, only one or two in vitro T cell stimulations with DCs were used. Further rounds of stimulation may have resulted in the detection of specific T cells in additional patients.
The generation of both CD4+ and CD8+ T cells using transduced DCs could be important for an optimal anti-tumor immune response. For anti-viral responses and in autoimmunity, CD4+ T cells appear to help control and sustain CD8+ T cell activity (39, 40, 41). Recent observations suggest that CD4+ T cells help to enhance the anti-tumor activity of CD8+ T cells (24, 25). However, the precise mechanism of this help by CD4+ T cells is not well defined. Possible mechanisms may include the activation of APC through the expression of CD40L (42) or the secretion of cytokines such as IL-2. In addition, activated CD4+ T cells may have indirect effector functions, such as the recruitment of eosinophils and macrophages due to the secretion of both Th1- and Th2-type cytokines (25).
Therefore, the ability of transduced DCs to stimulate both CD4+ and CD8+ T cells could be exploited in tumor immunology in vitro for Ag characterization and in vivo for patient immunization. For example, in vitro strategies are needed to validate the immunologic relevance of candidate tumor Ags that are overexpressed in tumors compared with normal tissues. Retrovirally transduced DCs could be used to characterize the potential immunogenicity of candidate tumor Ags and may allow the identification of both MHC class I- and class II-restricted epitopes. Moreover, effective methods are needed to evaluate the efficiency of patient immunization following vaccination with defined tumor Ags. Retrovirally transduced DCs could be used for the in vitro analysis of peripheral blood precursor frequency of tumor-specific T cells before and after patient vaccination. Finally, because retrovirally transduced DCs are efficient at generating both CD4+ and CD8+ T cells in vitro, they may also have applications in direct patient immunization. Such an immunization strategy would require the mobilization of CD34+ hematopoietic stem cells from cancer patients, followed by retroviral transduction with the tumor Ag and in vitro differentiation to DCs. This approach would be feasible, because CD34+ cell mobilization, retroviral transduction, and treatment with cultured cells are all techniques that have been used clinically. Optimal DC maturational signals as well as the most effective schedule and route of immunization remain to be defined.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; SCF, stem cell factor; TIL, tumor-infiltrating lymphocytes; VSV, vesicular stomatitis virus.
Received for publication June 14, 2001. Accepted for publication August 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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