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Cancer Immunology and Immunotherapy Center, Saint Savas Cancer Hospital, Athens, Greece
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, and GM-CSF. The process of the
activation of AWE-pulsed DCs by CD4+ T cells could be
inhibited with anti-CD40 ligand mAb. Moreover, the role of
CD4+ T cells in activating AWE-pulsed DCs was undertaken by
anti-CD40 mAb. Our data demonstrate for the first time in patients
with metastatic cancer the essential role of CD4+ Th
cell-activated DCs for optimal CD8+ T cell-mediated killing
of autologous tumors and provide the basis for the design of novel
protocols in cellular adoptive immunotherapy of cancer, utilizing
synthetic peptides capable of inducing T cell help in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although CD4+ Th cells possess a central role in regulating virtually all Ag-specific immune responses, their contribution in the initiation of antitumor immune responses has until recently been obscure. Strong evidence for the essential role of CD4+ T cells in antitumor immunity initially came from in vivo studies with murine tumor cell lines. After transfection with MHC class II genes, such tumor cells directly activated CD4+ T cells, resulting in tumor regression and induction of immunity against the wild-type MHC class II-negative tumors (1, 2, 3, 4). Tumor-specific CD4+ T cells restricted by MHC class II gene products have also been reported in patients with melanoma, lymphoma, sarcoma, colon cancer, and breast cancer (5, 6, 7). Responses of CD4+ T cells against tumor-specific peptide sequences expressed in an idiotypic IgM of a B cell lymphoma (8), bcr-abl fusion proteins (9), HPV type 16 E7 oncoprotein (10), and kras oncogene (11, 12, 13) have also been documented.
The essential role of CD4+ T cells providing help for maximal activation of tumor-specific CTL further emerged from cell-based vaccine models against MHC class II-negative murine tumors (14, 15, 16). Such studies indicated that tumor Ags released at the tumor site are taken up by macrophages, processed, and presented to CD4+ T cells, which in response produce and secrete lymphokines that activate tumor-specific CTL. In a recent report, Ossendorp et al. (17) demonstrated the induction of tumor-protective immunity by vaccination with a tumor-specific murine leukemia virus env-encoded Th peptide. In this way, strong protection was achieved against the highly aggressive MHC class II-negative RMA lymphoma line. These data provided indirect evidence for the cross-priming of tumor Ags by local APCs. In the same study as well as in others (18, 19), CD8+ T cells recognizing tumor Ags of viral origin were identified as the cytotoxic effector cells. Finally, in a murine model, Shoenberger et al. (20) demonstrated that in vivo generation of CTL specific for an adenovirus E18 protein-epitope requires cross-priming by host APC and is strictly helper dependent, because mice depleted of CD4+ Th cells were no longer capable of generating E18-specific CTL responses.
All these studies lead to the conclusion that induction of T cell help in vivo or cellular adoptive immunotherapy utilizing both tumor-specific helper and cytotoxic T cells are essential for improving clinical results in cancer immunotherapy. However, to apply such protocols in cancer treatment, the results obtained from the various experimental animal models must also be demonstrated in the human system. Herein, we report for the first time the absolute requirement of synergistic interactions accomplished by autologous tumor-specific CD4+ and CD8+ T cells and dendritic cells (DCs)2 for optimal killing of autologous tumor cells in patients with various types of metastatic cancer. CD4+ T cells were cross-primed against the MHC class II-negative tumors by autologous DCs. Our data also demonstrate that an initial interaction of CD4+ T cells with acid wash extract (AWE)-pulsed autologous DCs is required for providing all necessary conditions to the latter to induce optimal CTL priming. Blockade of the interaction between CD4+ T cells and DCs by anti-CD40 ligand (CD40L) mAb greatly inhibited CTL priming, which could be bypassed by signaling through CD40.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study included 19 patients (11 women, 8 men; medium age, 65
years; range, 45–79 years) with histologically proven malignant
tumors. All had measurable metastatic disease and fulfiled the
following criteria: Karnofsky performance status at least 80%;
bilirubin concentrations <1.7 ng/dl; creatinine <2.2 ng/dl; leukocyte
count >3000/µl; and platelet count >100,000/µl. They had not
received any antineoplastic therapy during the 3 wk preceding the onset
of the study. Patients’ HLA serotypes, in the same order as they
appear in Figs. 3
and 4 and in Tables II and III, are given in Table I. All patients were apprised of the
study, and consents were obtained consistent with the policies of St.
Savas Cancer Hospital. The clinical stage of disease of the
participating patients was as follows: stage III, n = 5
and stage IV, n = 14. Tumor grade III was scored in all
patients.
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Anti-CD83 conjugated with PE mAb was obtained from Caltag
Laboratories (Burlingame, CA). All other mAbs were purchased from
PharMingen (San Diego, CA). Anti-CD4, -CD8, -CD16, -CD20, -CD40, and
-CD80 mAbs as well as mAb to monomorphic determinants of HLA class I
and class II molecules were conjugated with FITC. Anti-CD3, -CD14
-CD54, -CD86, and HLA-DR mAbs were used conjugated with PE. For
blocking experiments, purified anti-CD40 (IgG1, clone 5C3),
anti-CD40L (CD154; IgG1, clone TRAP1), anti-HLA class I (IgG1,
clone G46-2.6), anti-HLA class II (IgG2b, clone TÜ36) and
isotype-matched mouse IgG1 (anti-trinitrophenol (TNP), clone
107.3), and mouse IgG2b (anti-TNP, clone 49.2) mAbs were used. The
latter two mAbs conjugated with PE as well as FITC-conjugated
anti-TNP mouse IgM (clone G155–228) mAb were also used as
isotype-matched negative controls in immunostaining experiments.
Anti-cytokine (IL-2, GM-CSF, TNF-, IFN-, and IL-4) mAbs were used
conjugated with PE. BB7.2 (anti-HLA-A2.1) mAb was kindly donated by
Professor H.-G. Rammensee (Department of Immunology, University of
Tübingen, Tübingen, Germany).
Preparation of effusion cells
Specimens of pleural effusions (300–500 ml) from 4 patients with lung adenocarcinoma and peritoneal effusions (1–2 l) from patients with melanoma (n = 1), breast cancer (n = 5), and ovarian cancer (n = 5) were centrifuged at 400 x g for 5 min to sediment cells. Malignant effusion-associated mononuclear cells (MEAMNC) and tumor cells were isolated from the cell pellet as previously described (21). In brief, 1 x 108 cells in 4–5 ml X-VIVO 15 medium (BioWhittaker, Walkersville, MD) were placed on top of a 75–100% discontinuous Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Sweden) and centrifuged at 200 x g for 25 min. Tumor cells (>80%) were collected from the top of the 75% Ficoll-Hypaque, and MEAMNC (>90%) were collected from the interface of 75% and 100% Ficoll-Hypaque. Both layers were washed once and resuspended in X-VIVO 15 medium supplemented with 1% heat-inactivated autologous serum. In agreement with our previous report (22), freshly isolated MEAMNC consisted mainly of T cells (>70% CD3+ cells) with almost equal numbers of CD3+CD8+ (30–35%) and CD3+CD4+ (34–39%) T cells. A substantial number of monocytes (CD14+ cells) was also detected (10–16%). Total MEAMNC or CD4+ and CD8+ T cells were used thereof as responders in the MLTC, whereas CD14+ cells were used to generate DCs. Total MEAMNC were also used as APC for restimulations during the MLTC. In all cases examined, the number of MEAMNC isolated from the effusions ranged from 175 to 900 x 106 and of tumor cells from 75 to 400 x 106. Aliquots of MEAMNC and autologous tumor cells were frozen in liquid N2. Phenotype analysis showed that all tumors expressed MHC class I molecules but were negative for MHC class II gene products.
Isolation of T cell subsets
CD4+ or CD8+ T cells were isolated from total MEAMNC using MACS CD4 or MACS CD8 Microbeads, respectively, according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). In brief, 1 x 107 MEAMNC were washed and resuspended in 80 µl PBS supplemented with 0.5% BSA and 2 mM EDTA (buffer A). To this, 20 µl MACS CD4 or MACS CD8 Microbeads were added, and the mixture was incubated for 15 min at 6–12°C. Cells were washed with buffer A, resuspended in 500 µl buffer A per 1 x 108 cells, and separated on positive selection columns placed in the magnetic field of a MACS separator (Miltenyi Biotec). Negative cells were allowed to flow through the column, centrifuged, and resuspended in X-VIVO 15 medium. The column was further removed from the MACS separator and placed on a suitable tube. The CD4+ or CD8+ T cell fraction was eluted with 1–2 ml buffer A and collected using a plunger provided with the column. For the isolation of highly purified CD4+ T cells, total MEAMNC were initially monocyte-depleted by plastic adherence (2 h at 37°C) before incubation with MACS CD4 Microbeads. The purity of isolated CD4+ or CD8+ T cells was in all cases >97%, whereas the negative fractions (i.e., CD4+ or CD8+ T cell-depleted MEAMNC) were totally devoid of CD4+ or CD8+ T cells, respectively. Purity of the isolated cell populations was tested by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA).
Monocyte isolation
CD14+ cells were isolated from total MEAMNC using the Monocyte Isolation Kit (Miltenyi Biotec), comprising a mixture of CD3, CD7, CD19, CD45RA, CD56, and anti-IgE Abs coupled to MACS Microbeads. The experimental procedure is identical with that described for CD4+ or CD8+ T cell isolation, the only difference being the addition of FcR blocking reagent before the addition of the Ab mixture. Monocytes (CD14+) thus isolated were highly pure (>98%), and no CD14+ cells could be detected in the MEAMNC-depleted fraction as analyzed by flow cytometry.
Preparation of AWE
This was performed as recently described (23) with modifications. Briefly, 2 x 107 tumor cells were washed with HBSS (Life Technologies, Gaithersburg, MD), followed by homogenization in 1 ml homogenization buffer (23). Eluates from cells were titrated with 10% trifluoroacetic acid and clarified by two successive centrifugations at 2,500 x g and 80,000 x g for 30 min and 5 h, respectively. The peptides were processed immediately on a SepPak C18 cartridge (Waters, Bedford, MA), equilibrated prior to use with 3 ml acetonitrile, followed by 3 ml deionized water. The eluate was allowed to flow through the cartridge by gravity, the column was washed with deionized water, and bound material was finally eluted with 2 ml 60% acetonitrile in deionized water and lyophilized in a Speed-Vac (Heto Lab Equipment, Allerod, Denmark). The dry product was reconstituted in HBSS and further processed on a Centricon centrifuge concentrator (Amicon, Beverly, MA) with a cutoff of 10 kDa by centrifugation at 2500 x g at 4°C for 2–3 h. The filtrate was aliquoted and stored at -20°C.
Generation of DCs
DCs were prepared as recently described (24).
Briefly, monocytes isolated from MEAMNC were grown for 7 days in X-VIVO
15 medium supplemented with 1% autologous serum, 800 U/ml rGM-CSF
(Schering-Plough, Brinny, Innishannon, Ireland) and 500 U/ml rIL-4 (R&D
Systems Europe, Abington, U.K.). rTNF- (R&D Systems Europe) was
added at 10 ng/ml for the last 24 h before culture termination.
The percentage of DCs recorded was >50%, as based on the expression
of a CD3-, CD14-,
CD16-, CD20-,
CD40+, CD80+,
CD83+, CD86+, and MHC class
II+ phenotype analyzed by flow cytometry.
Pulsing of DCs with AWE
AWE was added to 6-day cultured DCs along with rTNF-. The
amount of AWE (initially prepared from HLA-A2.1-positive patients) for
pulsing DCs was estimated based on its capacity to induce maximal
stabilization of HLA-A2.1 expression on T2 cells as detected with the
BB7.2 mAb (25). Thus, AWE extracted from 5 x
106 autologous tumor cells was added to 1 x
106 DCs. After a 24 h incubation in
CO2 incubators, cells were irradiated (4000 rad),
washed twice in medium, and used as AWE-pulsed DCs (AuTu-AWE-DC) in
experiments. In the experiments shown in Fig. 3
and presented in Table II, pulsing of a single donor’s DCs with
AWE from various allogeneic tumors was performed as follows. DCs
(1 x 104) were placed
in 100 µl X-VIVO 15 medium per well in V-bottom 96-well plates
(Costar, Cambridge, MA) along with 100 µl X-VIVO 15 medium containing
AWE extracted from 5 x 104 cells of each
allogeneic tumor. Every group consisted of triplicate cultures. Plates
were incubated for 24 h in CO2 incubators
and thereafter were irradiated (4000 rad). AWE-pulsed, irradiated DCs
were further gently washed twice (300 x g; 5 min),
resuspended in 100 µl fresh X-VIVO 15 medium, and coincubated with
autologous CD4+ T cells for the proliferation
assays (see below). A similar procedure was followed for the
cytotoxicity assays shown in Fig. 4 and presented in Table III except 1) the DCs were first labeled
with 51Cr and then incubated for 24 h with
AWE and 2) the isotope-labeled and pulsed DCs were not irradiated but
gently washed to remove excess of isotope before coincubation with the
effectors for the cytotoxicity assays (see below).
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Thawed autologous MEAMNC (10 x 106 cells/ml) were incubated for 24 h in CO2 incubators with AWE extracted from an equal number of autologous tumor cells. Cells were irradiated (4000 rad), washed, and used as stimulators.
Mixed lymphocyte tumor culture
Responder MEAMNC (2 x 106 cells/ml) were cocultured in 25-cm2 flasks (Costar) with 2 x 105 irradiated (10,000 rad) autologous tumor-stimulatory cells in a total volume of 5 ml X-VIVO 15 medium supplemented with 1% autologous serum, rIL-7 (20 ng/ml) (R&D Systems Europe), and 25 IU/ml rIL-2 (Cetus, Emeryville, CA) in CO2 incubators. Five days later, one-half of the medium was replenished with fresh medium containing 40 ng/ml rIL-7 and 50 IU/ml rIL-2. After 10 days of culture (=stimulation phase), recovered responders were washed and restimulated with thawed irradiated (4000 rad) autologous MEAMNC used as APC and autologous irradiated tumor cells (10,000 rad) at a cell ratio of 10:1:1, respectively. Preliminary experiments showed that the responder-stimulator ratio of 10:1 was optimal for sensitizing both CD4+ and CD8+ T cell responses in the MLTC (almost equally effective to 5:1 but superior to 20:1; data not shown) and therefore was used in all protocols. Fresh rIL-2 (25 IU/ml) was also added to the medium. Depending on the quality of each culture after the stimulation phase, viable responder cells were isolated over Ficoll-Hypaque. After two additional rounds of restimulation performed at 5-day intervals or as needed, cells were tested in proliferation and cytotoxicity assays.
To precisely analyze the role of CD4+ T cells as well as of DCs for the induction of cytotoxicity, MLTC were initiated with purified CD4+ and CD8+ T cells as responders (1 x 106 cells of each subset per ml) and irradiated (4000 rad) AWE-pulsed DCs (1 x 105 cells/ml) as stimulators. In this type of MLTC, control cultures consisted of (culture A) 1 x 106 CD8+ responder cells/ml and irradiated 1 x 105/ml AWE-pulsed DCs or (culture B) 1 x 106 CD8+ T cells/ml and 1 x 106 CD4+ T cells/ml pulsed with AWE. Restimulations were performed with AWE-pulsed irradiated (4000 rad) autologous MEAMNC. In all restimulations, AWE-pulsed MEAMNC in control cultures (A) were depleted of CD4+ T cells and in culture B of CD14+ cells to ensure the absolute exclusion of CD4+ T cells in culture A and CD14+ cells in culture B in any activation process of the CD8+ T cells during the restimulation phase of the MLTC. The ratio of responder cells to AWE-pulsed MEAMNC during restimulations was always kept at 10:1. Cytokines were added throughout the MLTC as already described.
In another series of experiments, MLTC were set up with responder MEAMNC depleted of either CD4+ T (culture A) or CD14+ (culture B) cells. Such MLTC were stimulated with AWE-pulsed DCs at a cell ratio of 10:1 (culture A) or with AWE-pulsed CD4+ T cells at the same ratio (culture B). Addition of cytokines and restimulations in both groups of MLTC were performed as described above.
Interaction of DC with autologous CD4+ T cells
DCs were prepared from thawed MEAMNC and pulsed with AWE from
the AuTu as previously described. One day before harvesting the
AuTu-AWE-DCs, a new aliquot of autologous MEAMNC was thawed; cells were
washed once and left overnight in a CO2 incubator
in X-VIVO 15 medium supplemented with 1% autologous serum.
CD4+ T cells were isolated from MEAMNC the day
after and incubated for 48 h with AuTu-AWE-DCs at a
CD4+:DC ratio of 5:1, in the presence or absence
of anti-CD40L mAb (10 µg/ml final concentration). In some groups,
AuTu-AWE-DCs were incubated for 48 h with anti-CD40 mAb (10
µg/ml) or in plain medium. The time period of 48 h to induce
activated DCs on incubation with CD4+ T cells or
anti-CD40 mAb was chosen based on findings from others (26, 27) who measured high levels of IL-12 release and potent T
cell-stimulatory capacity by similarly activated DCs. All incubations
were performed in 24-well plates (Costar) at a final volume of 2
ml/well, in CO2 incubators. Further, the
supernatant including the nonadherent CD4+ T
cells (with or without the mAbs) was decanted, and wells were washed
twice with X-VIVO 15 medium to remove residual
CD4+ T cells and/or excess mAbs. Treated DCs were
then detached by vigorous pipetting and, if necessary, with a rubber
policeman; thus, 
65–80% of the initially plated DCs could be
recovered. An aliquot of DCs was subjected to immunofluorescence
analysis (Table IV). MLTC were then
initiated by culturing the variously treated DCs (see also Fig. 6) with
autologous CD8+ T cells freshly isolated from
thawed MEAMNC at a cell ratio of 1:10 in the presence of IL-7 and IL-2
at the aforementioned concentrations. Restimulations were performed
with AWE-pulsed CD4+ T cell-depleted autologous
irradiated (4000 rad) MEAMNC.
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These were performed as described (22). Briefly, MLTC-activated CD8+ effector cells (1 x 106 cells/ml) were placed in 100-µl aliquots into wells of 96-well V-bottom plates (Costar). As targets, whole tumor cells or DCs pulsed with AWE were labeled with sodium [51Cr]chromate (Radiochemical Centre, Amersham, U.K.; 100–200 µCi isotope per 1–2 x 106 target cells) and added to the effectors at an E:T ratio of 20 or as otherwise indicated. Incubation was performed for 6 h in CO2 incubators. For blocking experiments, anti-MHC class I and isotype-matched control mAbs were added throughout the incubation period at a final concentration of 10 µg/ml. Supernatant (100 µl) was collected from each well, and the radioactivity was measured in a gamma counter (Packard, Downers Grove, IL).
Proliferation assays
CD4+ T cells isolated from 15–20-day MLTC
by immunomagnetic separation from total MEAMNC were cultured for 5 days
in X-VIVO 15 medium supplemented with 1% autologous serum in 96-well
V-bottom plates (Costar) at 4 x 104
cells/well with 1 x 104 irradiated (4000
rad) autologous DCs pulsed with AWE or with 1 x
104 autologous DCs (4000 rad) and 1 x
104 autologous tumor cells (10,000 rad) at a
final volume of 200 µl/well. The optimal numbers of cells used in the
proliferation assays (i.e., responder CD4+ T
cells and stimulatory AWE-pulsed DCs or tumor cells) were determined
from titration experiments (Fig. 1).
Anti-MHC class I or anti-MHC class II mAbs were added at a final
concentration of 10 µg/ml for the entire incubation period. Anti-TNP
mAb (isotype -matched to anti-MHC class II mAb) was also added at
the same concentration as for a control. Fifty microliters (=1 µCi)
[3H]TdR (Amersham) were added per well 24
h before culture termination. Incorporated
[3H]TdR in the DNA of proliferating cells was
measured in a beta counter (Packard). All cultures were performed in
triplicates, and results are expressed as cpm.
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CD8+ T cells cultured with the variously
treated autologous AWE-pulsed DC during the MLTC stimulation phase were
analyzed for intracellular expression of IL-2, IFN-, GM-CSF,
TNF-, and IL-4. To enhance intracellular fluorescence, protein
secretion was inhibited by the addition of 1 µM brefeldin A
(Sigma, St. Louis, MO) (28). Intracellular cytokine
staining was then analyzed as described (29). In brief,
cells were fixed with 4% paraformaldehyde for 10–15 min, and 0.1%
saponin in PBS was used to permeabilize the cell membrane. The
respective anti-cytokine mAbs were used at a concentration of 1
µg/ml. To detect cytokine-positive CD8+ T
cells, MLTC-recovered cells were stained for 30 min with a mixture of
FITC-conjugated anti-CD8 mAb and peridin-chlorophyl conjugate
(PerCP)-conjugated anti-CD3 mAb (Becton Dickinson, Mountain View,
CA), followed by a further 30 min incubation with purified
anti-cytokine mAbs conjugated with PE. All flow cytometry data were
calculated with LYSYS II (Becton Dickinson) software.
Phenotype analysis
Flow cytometry was conducted on a FACScan (Becton Dickinson) flow cytometer. Direct single-color immunofluorescence assays were performed with FITC- or PE-conjugated mAbs. Isotype-matched mAbs conjugated with FITC or PE were used as background.
Quantitation of cytokines in culture supernatants
ELISA kits specific for IL-2, TNF-, and IL-4 were obtained
from R&D Systems Europe. IFN- and GM-CSF were quantitated with ELISA
kits from Endogen (Boston, MA). Assays were performed according to the
manufacturers’ instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nineteen MLTC cultures were established from an equal number of
cancer patients using total MEAMNC as responder cells and autologous
tumor cells as stimulatory cells. On days 15–20 after culture
initiation, CD4+ T cells were isolated by
immunomagnetic separation and tested for proliferative responses
against the autologous tumor cells in the presence of autologous DCs as
APC (AuTu + DC) (Fig. 2A) or
against autologous DCs pulsed with AWE from the autologous tumor
(AuTu-AWE-DC) (Fig. 2B). CD4+ T cells
proliferated vigorously in both cases with comparable results. The
proliferative responses were blocked to a great extent by monomorphic
mAb to MHC class II molecules but remained unaffected in the presence
of an isotype-matched control mAb (anti-TNP) or anti-MHC class
I mAb (Fig. 2). To check the specificity for the autologous tumor
cells, MLTC-activated CD4+ T cells were tested
for proliferation in response to autologous DCs pulsed with AWE from
allogeneic metastatic tumors. The results from these experiments are
shown in Fig. 3 and summarized in Table II. A typical immune response profile is shown with donor 6 (melanoma
patient) whose CD4+ T cells proliferated only in
response to AuTu-AWE-DC. Similar results were also obtained with donors
2, 3, 4, 7 (lung adenocarcinoma), 8 (breast cancer), and 10 (ovarian
cancer) (Table II). An interesting situation appeared with donors 1
(breast cancer), 9, and 5 (both ovarian cancer), whose
CD4+ T cells, in addition to the AuTu-AWE-DC,
also recognized and proliferated in response to autologous DCs pulsed
with AWE from allogeneic tumor cells from the other two patients (Fig. 3 and Table II). Such proliferative responses, although weaker compared
with those stimulated by the AuTu-AWE-DC, still were significantly
higher over background values (not exceeding 800 cpm) and could be
inhibited with anti-MHC class II mAb (Fig. 3, B columns).
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Using the same MLTC cultures as above, we succeeded, after three
rounds of restimulation with autologous irradiated tumor cells and
MEAMNC as APC, to generate in vitro cytotoxicity specific for the
autologous tumor cells. As shown in Fig. 4, CD8+ T cells
isolated by immunomagnetic separation from MLTC-activated MEAMNC of
donor 6 lysed exclusively AuTu-AWE-DC (Fig. 4, column 6A). The
cytotoxic response was to a great extend inhibited by monomorphic mAb
to HLA class I molecules (Fig. 4, column 6B). Similar cytotoxicity
patterns were observed with the other donors (Table III), with the
exception of patients 1, 9, and 5 whose MLTC-activated
CD8+ T cells showed significant cross-reactivity
against autologous DCs pulsed with AWE from each other’s tumors (Fig. 4 and Table III). In all cases, cytotoxicity was blocked with MHC class
I mAb (Fig. 4, B columns). Similar cytotoxicity profiles were observed
when whole tumor cells were used as targets (Table III).
Requirement of both CD4+ T cells and APC in MLTC for optimal killing of autologous tumors by CD8+ CTL effectors
To directly analyze the role of CD4+ T cells
and APC in generating CD8+ T cell-mediated
autologous tumor-specific cytotoxicity during the MLTC, we set up in
vitro cultures with total MEAMNC or with MEAMNC depleted of either
CD4+ or CD14+ cells.
CD4+ T cell-depleted MEAMNC were stimulated with
AuTu-AWE-DC and restimulated with the irradiated autologous tumor and
autologous MEAMNC, which were also depleted of
CD4+ T cells to ensure the absence of
CD4+ T cells throughout the culture period. This
was necessary to avoid any CD4+ T cell-mediated
activation of the autologous CD8+ T cells later
in culture, after the initial stimulation phase (i.e., after the first
10 days of culture). Similarly, MLTC initiated with MEAMNC depleted of
CD14+ cells were stimulated with AWE-pulsed
CD4+ T cells and restimulated with
CD14+ cell-depleted MEAMNC to exclude any
involvement of monocytes or monocyte-derived DCs in the late activation
phase of autologous CD8+ T cells. As shown in
Fig. 5A,
CD8+ T cells isolated from MLTC set up with
unseparated MEAMNC lysed efficiently the autologous tumor targets
in an E:T ratio-dependent manner. In contrast, significantly less
killing was observed when CD4+ or
CD14+ cells were eliminated from cultures. These
results were confirmed in cultures from the same donors set up with
highly purified cell populations (Fig. 5B). Thus,
CD8+ T cells collected from MLTC cultures
initiated with CD4+ and
CD8+ T cells and AuTu-AWE-DC exhibited a high
killing rate of the autologous tumor targets. However, when MLTC were
initiated in the absence of CD4+ T cells, killing
was reduced by 53% at the highest E:T ratio. As already mentioned,
such CD4+ T cell-depleted MLTC were restimulated
with CD4+ T cell-depleted AWE-pulsed MEAMNC. An
even higher decrease in cytotoxicity (62% reduction) against the
autologous tumors was observed when the DCs were not included in the
MLTC (Fig. 5B). Also in this type of MLTC, restimulations
were performed with CD14+ cell-depleted
AWE-pulsed MEAMNC. CD8+ effectors collected from
both types of MLTC lysed their targets in an MHC class I-restricted
manner, as shown by the significant reduction of cytotoxicity at the
highest E:T ratio tested with an anti-MHC class I mAb (Fig. 5).
Cytotoxicity levels remained unaffected in the presence of a control
isotype-matched mAb, and only marginal killing was observed against the
control tumor cell line K562 (Fig. 5).
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The data thus far suggested that lysis of metastatic tumor cells
by autologous CD8+ CTL requires help provided by
CD4+ T cells and professional Ag-bearing APC
(DCs). To better understand the nature of cell-to-cell interactions
that take place after CD8+ T cell activation, we
performed the following series of experiments. AWE-pulsed DCs were
cultured for 48 h 1) with autologous CD4+ T
cells, 2) with autologous CD4+ T cells plus
anti-CD40L mAb, 3) with anti-CD40 mAb or 4) in plain medium.
After this preincubation period, nonadherent CD4+
T cells were removed, treated DCs were washed to remove residual
CD4+ T cells, and excess mAb (no mAb remained
bound on DCs after 48 h; data not shown) and then added to
autologous CD8+ T cells isolated from thawed
autologous MEAMNC. Restimulations were performed with thawed
CD4+ T cell-depleted MEAMNC pulsed with AWE as
described in Materials and Methods. As for a positive
control, CD4+ T cells from the preincubation
period were added back to the stimulation phase, and restimulations
were performed with total MEAMNC pulsed with AWE. As shown in Fig. 6, CD8+ T cells
derived from CD4+ cell-depleted MLTC cultures
that were stimulated with AuTu-AWE-DC preincubated with
CD4+ T cells or with anti-CD40 mAb exhibited
strong killing against the autologous tumor, comparable with that of
the positive control. AuTu-AWE-DC cultured with
CD4+ T cells plus anti-CD40L mAb or in plain
medium failed to efficiently stimulate autologous
CD8+ T cells during the initial MLTC phase,
resulting in markedly reduced cytotoxic responses against the
autologous tumor targets.
DCs can undergo phenotypic and functional changes after ligation of
their CD40 with T cell CD40L (26, 27). It was therefore of
interest to investigate whether the increased capacity of
AuTu-AWE-DCs to trigger cytotoxic responses against the autologous
tumor after preincubation with CD4+ T cells was
associated with modulation of surface Ags. We thus measured the levels
of adhesion, costimulatory, and HLA-DR Ags on DCs after 48 h
preincubation under the same culture conditions as described above.
Table IV shows that preincubation with CD4+ T
cells or anti-CD40 mAb highly increased the expression of CD54,
CD80, CD86, and HLA-DR molecules. In contrast, anti-CD40L mAb
blocked to almost baseline levels the enhancement of Ag expression
induced on preincubation with CD4+ T cells (Table IV). In both cases, DC preparations contained no
CD4+ T cells (data not shown).
Cytokine production by CD8+ T cells stimulated by variously treated autologous DC
CD8+ T cells cultured with AuTu-AWE-DC,
which were preincubated either with autologous
CD4+ T cells or with CD4+ T
cells and anti-CD40L mAb, were assessed by intracellular staining
and FACS analysis for cytokine production, namely IFN-, TNF-,
IL-2, GM-CSF, and IL-4. None of the CD8+ T cells
produced IL-4, whereas IL-2, IFN-, TNF-, and GM-CSF were detected
in a variable percentage of CD8+ T cells
stimulated with CD4+ T cell-preincubated
AuTu-AWE-DC (Fig. 7). Cytokine expression
in CD8+ T cells was drastically reduced when
preincubation of the AuTu-AWE-DC with CD4+ T
cells included also anti-CD40L mAb or when AuTu-AWE-DC were left in
plain medium (Fig. 7). Cytokine analysis in culture supernatants
revealed similar results (Table V). With
the use of commercially available ELISA kits, all four cytokines (i.e.,
IFN-, TNF-, IL-2, GM-CSF) were detected in varying
concentrations. In some instances, the concentrations were high (up to
100 ng/ml for IL-2). IL-4 levels were negligible (<1 ng/ml; data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our system, CD8+ T cells generated during MLTC
exhibited specific MHC class I-restricted recognition of the respective
autologous tumors. CD8+ T cells from three
patients (breast cancer, patient 1; ovarian cancer, patients 5 and 9)
responded to each other’s tumor cells and to unfractionated peptides
(AWE) extracted from the same tumors (Fig. 4 and Table III), confirming
data from other authors who demonstrated common tumor-associated Ags in
breast and ovarian cancer (30). Most of the tumor-derived
CTL peptide epitopes have been reported to be restricted by HLA-A2.1
(31). Because this particular allele is shared by all
three patients (Table I), we could speculate that their CTL recognize
common peptide(s) in its context. However, additional common CTL
epitopes could be restricted also by other alleles, such as HLA-A3
(expressed in patients 1 and 9) or HLA-Cw8 (expressed in patients 5 and
9), both of which have been recently reported to present several tumor
peptides to CTL (32, 33, 34). Our data provide also direct
proof that such a common antigenic system between breast and ovarian
tumors is recognized by tumor-specific CD4+ T
cells in an MHC class II-restricted manner (Fig. 3 and Table II). The
tumor-specific CD4+ T cells did not directly
recognize the autologous or the cross-reactive allogeneic tumors (all
of which are MHC class II negative) but required cross-priming of tumor
Ags by autologous APC (MEAMNC or DCs either pulsed with AWE or
cocultured with intact irradiated tumor cells). Recognition of tumor
peptides by CD4+ T cells has been shown to be
restricted by various MHC class II alleles, including HLA-DR4
(35, 36, 37), HLA-DR1 (38), HLA-DR11
(39), HLA-DR15 (40), and HLA-DQ7
(41). Shared expression of some of these alleles in
patients 1, 5, and 9 (e.g., all three patients share HLA-DQ7) may
adequately explain recognition of common peptide(s) by each other’s
CD4+ T cells. The successful establishment of
AWE-specific CD8+ CTL and
CD4+ clones followed by HPLC fractionation of the
tumor peptides included in AWE would be helpful to confirm the
cross-recognition of single antigenic peptide(s) by the T cells and to
identify the common antigenic system described herein.
The data presented also show that AWE-pulsed DCs on preincubation with CD4+ T cells or anti-CD40 mAb up-regulate adhesion and costimulatory molecules and are able to stimulate autologous CD8+ T cells directly, i.e., in the absence of CD4+ T cells. Such activated DCs are as stimulatory as those from MLTC cultures not devoid of CD4+ T cells. In other words, helper T cells need no longer communicate directly with the responding autologous CD8+ T cells. These results are consistent with recent reports demonstrating that ligation of CD40 on DCs with CD40L up-regulates the expression of ICAM-1, CD80, and CD86 molecules and also triggers the production of high levels of IL-12, resulting in the enhancement of their capacity to stimulate T cell-proliferative responses in vitro (26, 27) or to generate protective antitumor immunity in vivo (42). Binding of anti-MHC class II mAb on DCs also induces IL-12 release independently of CD40 ligation (26). Therefore, we did not attempt to block the CD4+ T cell-dependent activation of DC with anti-MHC class II mAb (as we did with anti-CD40L mAb), because this could possibly lead to a direct activation of DCs via ligation of MHC class II molecules.
Our data support the model of T cell help for CTLs proposed by others
in various experimental animal models (14, 15, 20), extend
this to the human system, and identify a series of cytokines (i.e.,
IFN-, IL-2, TNF-, and GM-CSF) produced by the CTL as a mechanism
via which activated AWE-pulsed DCs mediate CTL priming. The percentages
of CD8+ CTL that produced these cytokines
differed widely among the patients tested (almost 12–38% for IFN-,
IL-2, and TNF- and 6–17% for GM-CSF). A similar range of variation
was also observed when the same cytokines were measured in culture
supernatants. In both cases, cytokine production was largely inhibited
when the interaction of AWE-pulsed DCs with CD4+
T cells was blocked by anti-CD40L mAb. The above mentioned
cytokines have been shown to be produced by activated CTL
(43, 44, 45, 46) and also to be involved in cytolytic pathways as
immunoregulatory (46, 47) or effector molecules (46, 48, 49). In addition, we have recently shown (50)
that a synergism between all these cytokines (contained in culture
supernatants from activated mononuclear cells) results in efficient
lysis of both allogeneic and autologous tumor targets. Thus, such a
cytokine mixture produced by the CD8+ CTL would
provide the necessary conditions for optimal priming during the
stimulation phase and for efficient activation during restimulations,
leading ultimately to an effective antitumor immune response.
In this study, we have used unfractionated tumor-derived Ags present on intact tumor cells or extracted from them for T cell stimulation. The specificity of the immune responses for AWE preparations from the autologous tumor was demonstrated at the level of both CD4+ and CD8+ T cells in terms of proliferation and cytotoxicity, respectively. Thus, except for patients 1, 9, and 5, autologous tumor-specific CD4+ T cells proliferated in vitro in response to autologous DCs pulsed with AWE from the respective autologous, but not any allogeneic, metastatic tumors. Similarly, CD8+ CTL activated during the MLTC lysed at similar levels autologous tumor cells and DCs pulsed with autologous tumor-derived AWE. In a recent report (23), it was shown that highly tumorigenic virus-induced T cell lymphoma cells pulsed with AWE from immunogenic tumor cells became also immunogenic and induced potent specific CTL responses in vivo and in vitro. Thus, it appears that peptide mixtures isolated from tumor cells bypass the need for characterizing tumor-specific Ags and allow the use of vaccination protocols to several types of cancer where tumor-specific peptides have not yet been identified.
We (21, 22) and others (51) have used ascitic fluids as a source of T lymphocytes to generate CTL with specificity for the autologous tumor. As a novel approach, we have used ascitic fluids from patients with ovarian and breast cancer and melanoma and pleural effusions from patients with lung cancer as a source of both CD4+ and CD8+ T cells, as well as of DCs, to generate improved CTL responses against autologous tumor cells. Thus, ascites and pleural effusions-derived T cells and DCs may be successfully used for triggering tumor-specific CTL to be used in protocols aiming at cancer immunotherapy.
Taken together, our data provide direct evidence that CD4+ T cells are crucial participants in the development of cytotoxic responses against autologous metastatic tumors. Furthermore, because metastatic tumor cells mostly do not express HLA class II molecules, CD4+ T cells gain specificity for the autologous tumor through cross-priming of tumor Ags by autologous APC. In this context, the presence of tumor specific CD4+ T cells is essential at the site of Ag presentation to the immune system (e.g., at tumor-draining lymph nodes), where they can help amplify the activated CTL population. The further identification of tumor peptides with specificity for helper T cell activation will be important for engineering peptide constructs consisting of covalently linked "helper" and "cytotoxic" epitopes. Such peptide constructs have been successfully used for priming of anti-HIV CD8+ CTL in vivo (52). Moreover, recent evidence indicates that tumor-specific CD4+ T cells, in addition to simply providing help for CD8+ T cells, also recruit other antitumor effector cells such as eosinophils and macrophages that produce both superoxide and nitric oxide (53). Thus, vaccination strategies aiming at the specific activation of CD4+ T cells in vivo should be considered as essential for the induction of multiple effector mechanisms that can cooperate in the most effective killing of tumor deposits.
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Footnotes |
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2 Abbreviations used in this paper: DCs, dendritic cells; AWE, acid wash extracts; CD40L, CD40 ligand; MLTC, mixed lymphocyte tumor cultures; TNP, trinitrophenol; MEAMNC, malignant effusion-associated mononuclear cells; AuTu-AWE-DC, DCs pulsed with AWE from the autologous tumor.
Received for publication July 26, 1999. Accepted for publication January 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b32 peptide and antigen presenting cells exposed to b32 containing cell lysates. Blood 90:290.l) peptide vaccination of a patient, recognize 12Vl-dependent nested epitopes present within the vaccine peptide and kill autologous tumor cells carrying this mutation. Int. J. Cancer 72:784.[Medline]
Asp mutation is recognized by HLA-DQ7 restricted T cells in a patient with colorectal cancer: modifying effect of DQ7 on established cancers harbouring this mutation?. Int. J. Cancer 58:506.[Medline]
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), CD4+ T cells from the preincubation
system were added back to the culture during the stimulation phase.
After 10 days of incubation, 3 rounds of restimulation were
performed (restimulation phase) with MEAMNC depleted of
CD4+ T cells and pulsed with AWE (MEAMNC
(-CD4+) + AWE) except in group 
); CD4+ T cell-depleted MEAMNC
(
); CD14+ cell-depleted MEAMNC (•) (A);
or CD8+CD4+ T cells and AuTu-AWE-DC (
) was marginal in both
A and B. 
, Inhibition with
anti-MHC class I mAb. Inhibition with isotype-matched to
anti-MHC class I mAb at the same E:T ratio is also shown (
).
Results are given as mean percent cytotoxicity ± SD from four
experiments conducted with an equal number of patients.
). Treated AuTu-AWE-DC were
used to stimulate CD8+ T cells during the stimulation
phase. After 10 days, cells were fixed with paraformaldehyde and
permeabilized, followed by three-color staining with PE-labeled mAbs
against IFN-