Noncovalent Association with Stress Protein
Facilitates Cross-Priming of CD8 + T Cells to
Tumor Cell Antigens by Dendritic Cells
This information is current as
of June 17, 2017.
Robert Kammerer, Detlef Stober, Petra Riedl, Claude
Oehninger, Reinhold Schirmbeck and Jörg Reimann
J Immunol 2002; 168:108-117; ;
doi: 10.4049/jimmunol.168.1.108
http://www.jimmunol.org/content/168/1/108
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References
Noncovalent Association with Stress Protein Facilitates
Cross-Priming of CD8ⴙ T Cells to Tumor Cell Antigens by
Dendritic Cells1
Robert Kammerer, Detlef Stober, Petra Riedl, Claude Oehninger, Reinhold Schirmbeck, and
Jörg Reimann2
A
ntigenic material taken up by APCs can be cross-presented to MHC class I-restricted T cells (1). This was
originally discovered in CTL responses against minor H
Ags (2). It has recently been recognized that cross-presentation of
Ags is involved in CD8⫹ T cell responses elicited by tumors (3–
6), bacterial (7, 8) and viral (9, 10) infections, or DNA vaccination
(11–13). Cross-presentation of Ag can either prime immunity or
induce tolerance (14). Most published data suggest that dendritic
cells (DC),3 but not macrophages, are the APC involved in crosspriming CTL. Cross-priming is involved in the uptake, processing,
and MHC class I-restricted presentation of apoptotic cell remnants
by DC, an approach interesting for the specific immunotherapy of
tumors (3, 4, 6, 15–18). The cell biology underlying the transfer of
Ag-containing material from apoptotic cells to DC is unresolved.
Understanding of this process may help to optimize immunotherapy protocols for the treatment of tumors.
Stress proteins (heat shock proteins (hsp)) of the hsp70 and
hsp90 family have been shown to facilitate CTL priming. Three
experimental approaches have demonstrated a role of hsp mole-
Department of Medical Microbiology, University of Ulm, Ulm, Germany
Received for publication May 23, 2001. Accepted for publication October 23, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by a grant from Wilhelm Sander Stiftung (to J.R. and R.S.)
and Deutsche Forschungsgemeinschaft (Schi505/2-1, to R.S.).
2
Address correspondence and reprint requests to Dr. Jörg Reimann, Department of
Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1,
D-89081 Ulm, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; BFA, brefeldin A; BM, bone
marrow; BMC, BM cell; cT, cytoplasmic T-Ag; cT272, N-terminal fragment of cTAg; CTLL, CTL line; eGFP, enhanced (mutant) GFP; FCM, flow cytometry; GFP,
green fluorescent protein; hsp, heat shock protein; NLS, nuclear location sequence;
T-Ag, large tumor Ag of SV40; wtT-Ag, wild-type T-Ag.
Copyright © 2002 by The American Association of Immunologists
cules in the loading of antigenic peptides to MHC class I molecules. When antigenic peptides are noncovalently loaded in vitro
or in vivo to purified hsp molecules and injected into mice, they
can prime CTL responses (19 –22). This procedure was pioneered
by Srivastava et al. (reviewed in Ref. 23). Alternatively, antigenic
peptides fused to hsp carriers and injected as recombinant fusion
proteins can elicit CTL responses (24 –28). The hsp70 fusion proteins efficiently elicit CD4⫹ Th cell-independent CTL responses,
suggesting that they are immunogenic vaccines useful for prophylaxis and therapy. A third approach developed by us involves the
expression of CTL epitopes in chimeric fusion proteins containing
an hsp-binding, N-terminal viral domain (29 –31). This allows high
level Ag expression in noncovalent molecules, but tight association with constitutively expressed, cytosolic hsp73 molecules. A
variety of large (40 –150 kDa) chimeric Ags have been expressed
in this way (R. Schirmbeck, unpublished observations). The intracellular, hsp-bound Ags are processed in a TAP-independent way
and can be efficiently recognized by CTL. DNA vaccines constructed on the basis of this expression system elicit CTL and
serum Ab responses (reviewed in Ref. 32).
In addition, DC secrete Ag-presenting vesicles, called exosomes, which contain functional MHC class I molecules, T cell
costimulatory molecules, hsc70 (hsp73), and antigenic peptides
(17, 33). Exosome-based cell-free vaccines induce CTL responses
in vivo and can eradicate or suppress the growth of established
murine tumors in a T cell-dependent manner (34). The hsp73 molecules selectively accumulate in exosomes and may play a role in
cross-priming. Exosomes can be taken up into the lumen of endocytic vesicles of DC, enter an endosome-to-cytosol transport, and
gain access to the cytosolic Ag-processing machinery and the conventional MHC class I Ag presentation pathway (35). These observations point to a role for hsp70 and hsp90 molecules in facilitating the processing and loading of antigenic peptides to MHC
0022-1767/02/$02.00
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A viral oncogene carrying well-defined Kb/Db-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp
association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Agexpressing tumor cells are taken up and processed by immature dendritic cells (DC), and the Kb/Db-binding epitopes T1, T2/3, and
T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor
cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than
when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-␥-producing CD8ⴙ T cells were elicited by a DNA
vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to
5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, Db/Kb-restricted IFN-␥-producing CD8ⴙ T cells were primed during
the growth of transfected H-2d Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F1(b ⴛ d) hosts.
Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo
of CTL by DC that process material from apoptotic cells. The Journal of Immunology, 2002, 168: 108 –117.
The Journal of Immunology
class I molecules and/or their immunogenic presentation, although
the mechanism that underlies these observations is not yet clear.
We describe in vitro and in vivo experiments to test whether antigenic material from tumor cells (containing MHC class I-binding
epitopes) taken up by DC can more efficiently cross-stimulate or
cross-prime CTL when expressed in association with hsp73.
Materials and Methods
Mice
C57BL/6JBom (B6) mice (H-2b), BALB/cJBom mice (H-2d), and
F1(BALB/c ⫻ C57BL/6) mice were bred and kept under standard
pathogen-free conditions in the animal colony of Ulm University (Ulm,
Germany). Breeding pairs of these inbred strains were obtained from
Bomholtgard (Ry, Denmark). C57BL/6J-Tap1tm1Arp (B6 TAP1⫺/⫺)
mice homozygous for the tap1 gene deletion were obtained from The
Jackson Laboratory (stock no. 002944, Bar Harbor, ME). Male and
female mice were used at 12–16 wk of age.
Cells and plasmids
Expression of T-Ag and its variants by tumor cells
Cells were transfected with plasmids using the Ca2PO4 method. Transfected cells were metabolically labeled for 2 h with [35S]methionine and
extracted with lysis buffer (120 mM NaCl, 1% aprotinin (Trasylol, Bayer,
Leverkusen, Germany), leupeptin, 0.5% Nonidet P-40, and 50 mM Trishydrochloride (pH 8.0)) for 30 min at 4°C. Extracts were cleared by centrifugation and immunoprecipitated for T-Ag using the mAb PAB108 (directed against the extreme N-terminus of the T-Ag) and protein
A-Sepharose. Immune complexes bound to protein A-Sepharose were purified with wash buffer (300 mM LiCl, 1% Nonidet P-40, and 100 mM
Tris-hydrochloride (pH 8.5)), followed by two washes with PBS and 0.1⫻
PBS. Immune complexes were recovered from protein A-Sepharose with
elution buffer (1.5% SDS, 5% ME, and 7 mM Tris-hydrochloride (pH 6.8))
and processed for SDS-PAGE. Levels of immunoprecipitated proteins
were analyzed either by Coomassie blue staining of the gels and/or by
Western blotting.
Generation of DC from bone marrow (BM)
The in vitro generation of DC from murine BM has been previously described (38). Briefly, BM cells (BMC) prepared from femurs were depleted
of CD4⫹ CD8⫹ B220⫹ and MHC class II⫹ cells by MACS sorting following the manufacturer’s instructions (bead-coupled Abs catalog no. 49201, 494-01, 495-01, and 524-01; Miltenyi Biotec, Bergisch-Gladbach, Germany). These BMC depleted of T cells, B cells, and maturing myeloid cells
were cultured at a density of 106 cells/ml in UltraCulture medium (catalog
no. 12–725F; BioWhittaker) supplemented with 5 ng/ml GM-CSF (catalog
no. 315-03, PeproTech, Rocky Hill, NJ), 2 mM glutamine, and antibiotics.
Cultures were incubated at 37°C in humidified air with 5% CO2. On days
3 and 5, cells were fed by medium exchange. On day 7 of culture, nonadherent cells were harvested, and CD11c⫹ cells were purified by MACS
(catalog no. 130-052-001, Miltenyi Biotec).
T-Ag-encoding DNA vaccines
SV40 T-Ag from the plasmid pTGIF (a gift from Dr. W. Deppert, Hamburg, Germany) was subcloned into the pCI vector. Similarly, the cT-Ag
was cloned into the pCI vector (31). For i.m. nucleic acid immunization,
we injected 50 ␮l PBS containing 1 ␮g/␮l plasmid DNA into each tibialis
anterior muscle. All mice received bilateral i.m. injections once as previously described (31, 37). Mice were inoculated with plasmid DNA using
the Helios Gene Gun system (catalog no. 165-2431 and –2432, Bio-Rad,
Munich, Germany). The manufacturer’s instructions were followed in coating DNA to gold particles and operating the gene gun. Mice were intradermally inoculated with DNA-coated 1-␮m gold particles into the shaved
abdominal skin using a helium pressure of 200 psi. Spleen cells obtained
14 days postvaccination were restimulated at weekly intervals with irradiated RBL5/T transfectants in RPMI 1640/10% FCS supplemented with 30
U/ml IL-2. To generate epitope-specific CTLL, spleen cells taken 10 days
postvaccination were restimulated in vitro with irradiated, peptide-pulsed
RBL5 cells. The cytolytic assay with peptide-pulsed targets demonstrated
that the CTLL displayed T1, T2/3, and/or T4 epitope specificity and the
respective (Kb or Db) restriction specificity.
Flow cytometric analyses
For surface staining cells were suspended in PBS/0.3% (w/v) BSA supplemented with 0.1% (w/v) sodium azide. Nonspecific binding of Abs to
FcR was blocked by preincubating cells with 1 ␮g/106 cells of the antiCD16/CD32 mAb 2.4G2 (catalog no. 01240D, BD PharMingen, San Diego, CA). Cells were incubated with 0.5 ␮g/106 cells of the relevant mAb
for 30 min at 4°C, washed twice, and subsequently incubated with a second-step reagent for 15 min at 4°C. Cells were washed twice and analyzed
on a FACScan (BD Biosciences, Mountain View, CA). Dead cells were
excluded by propidium iodide staining. The following reagents and mAbs
from BD PharMingen were used: PE-conjugated anti-I-Ab (catalog no.
06045A), biotinylated anti-H-2Db,d,k (catalog no. 06232D), PE-conjugated
anti-CD80 (B7-1; catalog no. 09605B), PE-conjugated anti-CD40 (catalog
no. 09665B), FITC-conjugated anti-CD86 (B7-2; catalog no. 09215B), and
FITC-conjugated and PE-conjugated anti-CD11c (catalog no. 553801 and
09705B). We furthermore used FITC-conjugated IgG1 mAb R3-34 (catalog no. 20614A), PE-conjugated IgG1 mAb R3-34 (catalog no. 20615A),
and streptavidin-Red670 (catalog no. 19543-024, Life Technologies,
Gaithersburg, MD).
Inducing apoptosis in tumor cells
Tumor cells were irradiated (1.5 mJ/cm2/s) for either 2 or 5 min using the
UV cross-linker 1800 (UV Stratalinker TM-1800, Stratagene). Cells were
suspended in PBS to exclude the UVB-absorbing effect of phenol red in
culture medium. Apoptosis was confirmed by annexin V staining. Cell
lysates were produced by exposing tumor cells to four rapid freeze-thaw
cycles until cell membrane integrity was lost. Cell debris was removed by
centrifugation (30 min at 5000 ⫻ g). Aliquots of the lysates were used to
pulse DCs.
Ag uptake by BMDC
Uptake of macromolecules was determined by incubating DC with 250
␮g/ml FITC-dextran (catalog no. D-1845; Sigma) for 2 h at either 37 or
4°C. Cells were washed and labeled with PE-conjugated anti-CD11c or
anti-MHC class II mAb. To study the uptake of apoptotic tumor cells by
DCs, tumor cells suspended in PBS were stained for 10 min at 37°C with
5 ␮M green CFSE dye (catalog no. C-1157, Molecular Probes). Cells were
washed three times in ice-cold PBS before induction of apoptosis. Apoptotic tumor cells incubated for 5 h postirradiation in medium were cocultured with DCs at different DC/tumor cell ratios at either 4 or 37°C. DCs
harvested from 18-h cocultures were stained by PE-labeled anti-CD11c or
anti-MHC II mAb. In two-color flow cytometric analyses we determined
the percentage of DC that had taken up green fluorescent material derived
from apoptotic tumor cells. DC incubated at 4°C showed no uptake of
material derived from tumor cells, indicating that we were dealing with
active, temperature-dependent uptake and not passive adsorption of material to the cell surface of DC.
Coculture of CTL with DC
DCs were harvested from cultures (in which they were pulsed with titrated
amounts of material from apoptotic tumor cells), extensively washed, and
cocultured at (2.5–5 ⫻ 104 DC/well) with CTL (1–2.5 ⫻ 104 CTL/well) in
96-well U-bottom plates for 24 h. Culture supernatants were harvested and
analyzed for IFN-␥ by ELISA. In some experiments DC were fixed with
1% paraformaldehyde in PBS for 10 min before the pulse with either remnants of apoptotic cells or peptides, washed, and cocultured with CTL.
Cytokines and cytokine detection by ELISA
For detection and capture of IFN-␥ in supernatants by conventional doublesandwich ELISA, we used the mAb R4-6A2 (catalog no. 18181D, BD
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The H-2d mastocytoma cell line P815 (TIB64) was obtained from the
American Tissue Culture Collection (Manassas, VA). The BALB/c-derived fibrosarcoma Meth-A was provided by Dr. W. Deppert (Hamburg,
Germany). The bovine papilloma virus-based vector BMGNeo, a gift from
Drs. Y. Karasuyama and F. Melchers (Basel, Switzerland), was used to
construct the BMG/large tumor Ag of SV40 (T-Ag).1 and BMG/cT-Ag.1
expression plasmids as previously described (29). The cT272-enhanced
(mutant) green fluorescent protein (eGFP)-encoding vector BMG/cT272eGFP was constructed by inserting the cT272-encoding region into the
commercially available pEGFP N1 vector (Clontech, Palo Alto, CA). The
entire cT272-eGFP-encoding cassette was subcloned into the BMG vector.
BMG/T, BMG/cT272-eGFP, or BMG/cT vector DNA or nonrecombinant
BMGNeo vector DNA was transfected into P815 or Meth-A cells as previously described (36, 37). Lines, clones, and subclones of H-2d P815/T
and P815/cT, and H-2d Meth-A/T and Meth-A/cT with stable expression of
the T-Ag protein were generated as previously described (29, 30, 36, 37).
Hybridomas producing the anti-CD4 mAb YTS 191.1 or the anti-CD8
mAb YTS 169.4 were used.
109
110
STRESS-PROTEIN-FACILITATED CROSS-PRESENTATION
PharMingen) and biotinylated mAb XMG1.2 (catalog no. 18112D, BD
PharMingen). Murine IFN-␥ was also obtained from BD PharMingen (catalog no. 19301T). Extinction was analyzed at 405/490 nm on a TECAN
microplate-ELISA reader (TECAN, Crailsheim, Germany) with the
EasyWin software (TECAN). The detection limit of the ELISA for
IFN-␥ was 20 pg/ml.
supernatant was collected for gamma radiation counting. The percent specific release was calculated as ((experimental release ⫺ spontaneous release)/(total release ⫺ spontaneous release)) ⫻ 100. Total counts were
measured by resuspending target cells. Spontaneously released counts were
always ⬍15% of the total counts. Data shown are the mean of triplicate
cultures. The SEM of triplicate data was always ⬍20% of the mean.
Tumor cell transplantation
Tumor cells were washed three times in PBS, and 50 ␮l of the cell suspension was injected s.c. into the shaved right flank. Experimental groups
consisted of four to six mice. Tumor development was followed by serial
measurements of tumor size at two perpendicular diameters.
Results
Determination of splenic CTL frequencies
We cloned different constructs encoding Ags with the N-terminal,
hsp-binding domain of the SV40 T-Ag into expression plasmids.
These included 1) the wild-type, nuclear 90-kDa T-Ag (wt-T); 2)
a mutant, cytoplasmic 85-kDa cT-Ag from which the NLS110 –152
was deleted (cT); and 3) the fusion protein cT272-eGFP in which
the N-terminus 1–272 aa of the cT-Ag fragment was fused inframe to eGFP. The maps of these three Ags with their Db- or
Kb-restricted T-Ag epitopes are shown in Fig. 1A.
P815 and Meth-A cells were transfected with plasmid DNA
encoding Ags with an N-terminal T-Ag domain. Stable transfectants were generated and cloned in vitro. From the lysate of
the stable transfectants, proteins of the expected sizes were
immunoprecipitated with an anti-T-Ag mAb that binds the
extreme N-terminus of this viral nucleoprotein (Fig. 1, B and
C). Anti-T-Ag mAb coprecipitated hsp73 from cells expressing
mutant cT-Ag or chimeric cT272-eGFP, but not from cells
expressing wtT-Ag (Fig. 1, B and C). Western bot analyses with
a panel of anti-hsp70 mAbs confirmed the association of mutant
T-Ag with hsp73, confirming our previously published data
(29 –31). Tumor cells stably transfected with the cT272-eGFPencoding plasmid DNA showed green cytoplasmic autofluorescence readily detectable by FCM (Fig. 1C). Hence, we generated a panel of stable transfectants from two different H-2d
tumor cell lines that expressed the wild-type, a mutant, or a
chimeric form of this viral oncogene.
6
CTL assays
Single-cell suspensions were prepared from spleens of mice in ␣-MEM
tissue culture medium supplemented with 10 mM HEPES buffer, 5 ⫻ 10⫺5
M 2-ME, antibiotics, and 10% (v/v) FCS (Life Technologies). A selected
batch of Con A-stimulated rat spleen cell supernatant (2%, v/v) was added
to the culture medium. Responder cells (3 ⫻ 107) were cocultured with 1 ⫻
106 irradiated, syngeneic RBL5/T transfectants or peptide-pulsed RBL5
cells. Coculture was performed in 10 ml medium in upright 25-cm2 tissue
culture flasks in a humidified atmosphere/5% CO2 at 37°C. After 5 days of
culture, CTL were harvested, washed, and assayed for specific cytolytic
reactivity. Serial dilutions of effector cells were cultured with 2 ⫻ 103
51Cr-labeled targets in 200-␮l round-bottom wells. Specific cytolytic activity of cells was tested in short term 51Cr release assays against transfected or peptide-pulsed targets. After a 4-h incubation at 37°C, 50 ␮l
FIGURE 1. Construction and expression of
Ags with the N-terminal, hsp-binding domain of
the SV40 T-Ag. A, Schematic representation of
wt nuclear T-Ag (wt-T), cT-Ag, and chimeric
cT272-GFP Ag (encoding the N-terminal 272 aa
of cT and the C-terminus of eGFP). The CTLdefined, Db- or Kb-restricted T-Ag epitopes,
hsp73 binding, and deletion of the nuclear location sequence (NLS) are indicated. B, Wt T-Ag
(not associated with hsp73) and (hsp73-associated) cT-Ag are stably expressed by transfected
P815 cells as detected after immunoprecipitation
with mAb PAB108 in Coomassie-stained gels.
Western analyses confirmed that the 70-kDa
band is hsp73 as described in detail previously
(29, 30). C, hsp-associated expression of cT272eGFP in transfected, radiolabeled Meth-A cells
was detected by immunoprecipitation with mAb
PAB108 in SDS-PAGE gels (a); eGFP expression by Meth-A cells was confirmed by FCM
analysis of transfectants (b).
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Spleen cells (10 /ml) were incubated for 60 min in RPMI medium with 5
␮g/ml of the indicated peptide in round-bottom 96-well plates. Thereafter,
5 ␮g/ml brefeldin A (BFA; catalog no. 15870, Sigma) was added, and the
cultures were incubated for an additional 4 h. Cells were harvested and
surface-stained with PE-conjugated anti-CD8 mAbs (catalog no. 01045B;
BD PharMingen). Surface-stained cells were fixed with 2% paraformaldehyde in PBS before intracellular staining for IFN-␥. Fixed cells were resuspended in permeabilization buffer (HBSS, 0.5% BSA, 0.5% saponin,
and 0.05% sodium azide) and incubated with FITC-conjugated anti-IFN-␥
mAb (catalog no. 55441, BD PharMingen) for 30 min at room temperature
and washed twice in permeabilization buffer. Stained cells were resuspended in PBS/0.3% (w/v) BSA supplemented with 0.1% (w/v) sodium
azide and analyzed by flow cytometry (FCM). The number of IFN-␥⫹ cells
in 105 CD8⫹ cells was determined.
Expression of wild-type, mutant, and chimeric SV40 T-Ag (T-Ag)
in tumor cell lines
The Journal of Immunology
Uptake of material from apoptotic tumor cells by immature DC
FIGURE 2. Uptake of tumor cell-derived material by
immature, bone marrow-derived DC in vitro. A, Immature
CD11c⫹ MHC-IIlow DC take up FITC-dextran. DC were
incubated with 0.25 mg/ml FITC-dextran for 2 h at either
37 or 4°C. Cells were washed and labeled with either PEconjugated anti-CD11c or PE-conjugated anti-MHC II
mAb. B, CFSE-labeled, apoptotic P815 cells were cocultured with DC (at a ratio of 1:1) for 16 h at 37 or 4°C,
harvested, washed twice, labeled with anti-MHC II mAb,
and analyzed by FCM by gating on the appropriate population of cells using forward and side scatter. DC cocultured with unlabeled, apoptotic P815 cells showed similar
fluorescence intensities than DC cocultured with CFSElabeled apoptotic P815 cells at 4°C. C and D, Immature
DC take up cT272-eGFP from apoptotic (C) or lysed (D)
Meth-A cells that express cT272-GFP. C, Apoptotic
Meth-A/cT272-eGFP cells were cocultured with DC (at a
ratio of 1:1) overnight and analyzed by FCM. The percentage of cells in each quadrant of the dot plots is indicated. A representative experiment of four experiments
with similar results is shown. D, Meth-A/cT272-eGFP
cells (107) were lysed by four rapid freeze-thaw cycles,
the lysates were cleared by centrifugation, and 100 ␮l of
serial dilutions were added to DC for 3 h at 37 or 4°C. DC
were harvested, washed twice, and analyzed by FCM. A
representative experiment of three experiments with similar results is shown.
Ags from apoptotic tumor cells taken up by DC are crosspresented to MHC class I-restricted CTL
Polyclonal and multispecific (short term) CTLL were generated
from vaccinated B6 mice as previously described (37). In an IFN-␥
release assay these CTL lines showed specific reactivity against
RBL5/T cells expressing T-Ag after transfection or against B6 DC
pulsed with T-Ag-derived, antigenic peptides (Fig. 3A). We tested
whether DC take up exogenous material from apoptotic tumor
cells and cross-present peptides generated by processing these exogenous tumor-associated Ags in the context of MHC class I molecules. CTLL released IFN-␥ in response to syngeneic DC that had
taken up material from allogeneic, T-Ag-expressing, apoptotic
P815 or Meth-A tumor cells (Fig. 3B). DC incubated with material
from nontransfected, apoptotic tumor cells did not stimulate cytokine release by CTLL (Fig. 3B). Apoptotic material from nontransfected or transfected tumor cells did not stimulate CTLL in the
absence of syngeneic DC (Fig. 3B). IFN-␥ release was blocked by
mAb to Db and Kb, demonstrating the MHC class I restriction of
the response (data not shown). When DC were incubated with
apoptotic material from transfected, allogeneic tumor cells at 4°C,
they did not acquire the capacity to cross-present epitopes to CTL
(data not shown). Similarly, paraformaldehyde-fixed DC incubated
with apoptotic material from transfected, allogeneic tumor cells at
37°C were not recognized by Kb/Db-restricted, T-Ag-specific
CTL, although peptide-pulsed, fixed DC stimulated CTL efficiently (Fig. 3C). The release of antigenic peptides by apoptotic
tumor cells that bind to surface MHC I molecules of DC is thus not
involved in this cross-presentation. Hence, uptake, processing, and
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Murine DC were generated from B6 bone marrow progenitors in
GM-CSF-supplemented, serum-free cultures using an established
protocol (38, 39). The large majority of the CD11c⫹ DC harvested
from day 7 cultures expressed low levels of MHC class II and
costimulator (CD40, CD80, CD86) molecules on the surface, indicating an immature phenotype. Only a minor fraction of 10 –20%
of the DC showed evidence of spontaneous maturation in culture
expressing high levels of MHC class II and costimulator molecules
on the surface. We measured FITC-dextran uptake by these marrow-derived DC. The majority of the immature (MHC-IIlow) DC,
but not the mature (MHC-IIhigh) DC, efficiently endocytosed
FITC-dextran at 37°C (Fig. 2A). Fluorescent material from CFSElabeled, apoptotic P815 or Meth-A tumor cells was efficiently
taken up by immature MHC-IIlow (but not mature MHC-IIhigh) DC
at 37°C (Fig. 2B). Uptake of FITC-dextran (Fig. 2A) or CFSElabeled, apoptotic tumor cells (Fig. 2B) was detected at 37°C, but
not at 4°C. Hence, immature, metabolically active DC take up
FITC-dextran or CFSE-labeled proteins from apoptotic tumor
cells. The autofluorescent protein cT272-eGFP was expressed by
transfected Meth-A tumor cells (Fig. 1C). This autofluorescent
protein was detected in FCM analyses of immature CD11c⫹
MHC-IIlow DC cocultured with apoptotic, cT272-eGFP-expressing Meth-A cells (Fig. 2C). Uptake of the autofluorescent protein
in tumor cell lysates was temperature and dose dependent (Fig.
2D). Hence, a substantial fraction of immature DC takes up hspbound, antigenic oncoprotein from apoptotic tumor cells or tumor
cell lysates.
111
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STRESS-PROTEIN-FACILITATED CROSS-PRESENTATION
T1, T2/3, and T4 epitopes are presented by transfected P815 and
Meth-A tumor cells expressing wt T-Ag or cT-Ag and by B6 DC
pulsed with the respective peptides. We tested whether the three
CTL-defined T1, T2/3, and T4 epitopes of T-Ag are presented by
DC that take up and process material from T-Ag-bearing tumor
cells. CTL lines specific for T1, T2/3, or T4 were established in
vitro. The Kb or Db restriction specificity of these lines was confirmed by mAb blocking studies (data not shown). B6-derived DC
cocultured with apoptotic P815 or Meth-A cells that expressed
T-Ag presented all three tested T-Ag epitopes to CTL (data not
shown). Hence, we did not find limitations in the repertoire of
epitopes that can be cross-presented by DC processing material
from apoptotic T-Ag-bearing tumor cells.
Cross-presentation of T-Ag from apoptotic tumor cells by DC is
TAP independent
presentation of exogenous material from apoptotic, allogeneic tumor cells by immature DC make them competent to specifically
activate CTL.
DC that have processed exogenous material from apoptotic
wtT-Ag- or cT-Ag-expressing tumor cells present the
Kb/Db-binding epitopes T1, T2/3, and T4
The SV40 T-Ag contains five CTL-defined epitopes (T1–T5) that
bind to either Kb or Db (40 – 42). The epitope T5 is subdominant
(and difficult to detect). The epitopes T2 and T3 are nested. The
hsp-associated (mutant) cT-Ag from apoptotic tumor cells is
more efficiently cross-presented by DC than non-hsp-associated
(native) T-Ag
We tested whether the relative efficiency of cross-presentation of
T-Ag epitopes differs when DC process apoptotic material and
lysates from tumor cells that express either native (non-hsp-associated) wt T-Ag, or mutant, hsp-associated cT-Ag. A constant
number of DC was pulsed either with titrated numbers of apoptotic
wtT-Ag-transfected or cT-Ag-transfected tumor cells or with titrated amounts of lysate from wt T-Ag-transfected or cT-Ag-transfected tumor cells. In the P815 and Meth-A tumor cell systems,
material from cT-Ag-expressing tumor cells was 2- to 8-fold more
efficient than material from wt T-Ag-expressing tumor cells in supporting cross-presentation of CTL epitopes by DC (Fig. 4). This
was reproducibly seen in four independent experiments. The expressions of wt T-Ag and cT-Ag by the transfected tumor cell lines
at the protein level were comparable (data not shown). Thus, different amounts of Ag expressed by the different tumor cell lines
cannot explain the differences in the efficiency of cross-presentation in this system. It is evident that hsp-associated Ag has a superior ability to cross-present epitopes to CTL compared with
non-hsp-associated Ag.
Further evidence for an involvement of cT/hsp complexes in
facilitating transfer of antigenic information from tumor cells to
DC was obtained when we treated lysates from tumor cells with 4
mM ATP before contact with DC (Fig. 5). As shown previously
(29), incubation with 4 mM ATP disrupts hsp/cT complexes that
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FIGURE 3. Ags from apoptotic tumor cells taken up by DC are crosspresented to CTL across an allo-barrier. C57BL/6 (H-2b)-derived DC were
cocultured with allogenic, apoptotic (H-2d) Meth-A or P815 tumor cells
expressing no viral Ag or expressing cT Ag after stable transfection. A,
Polyclonal, short-term T-Ag-specific H-2b CTLL released IFN-␥ when
cocultured with either transfected, T-Ag-expressing H-2b RBL5/T tumor
cells or BMDC pulsed with the T-Ag-derived peptides T1, T2/3, and T4.
Coculture of CTL with nonpulsed BMDC, BMDC pulsed with the irrelevant (Kb-binding) OVA peptide, or nontransfected RBL5 cells did not
stimulate IFN-␥ release by the CTL. B, The CTLL used released IFN-␥
when cocultured with H-2b DC pulsed with H-2d cT-Ag-expressing MethA/cT or P815/cT tumor cells. No IFN-␥ was released when CTL were
cocultured with allogeneic tumor cells without DC, or when CTL were
cocultured with syngeneic DC pulsed with material from nontransfected
Meth-A or P815 tumor cells. One experiment of five performed with similar results is shown. C, Paraformaldehyde-fixed BMDC pulsed with T-Agderived peptides stimulate IFN-␥ release by CTL. No IFN-␥ release was
observed when CTL were cocultured with nonpulsed, fixed BMDC or fixed
BMDC pulsed with either nontransfected or cT-Ag-transfected apoptotic
Meth-A tumor cells.
We tested whether processing of T-Ag-containing material from
apoptotic tumor cells by DC for MHC class I-restricted peptide
cross-presentation is TAP dependent. DC generated in vitro from
marrow of either TAP-competent or TAP-deficient (TAP1⫺/⫺
knockout) B6 mice were pulsed with transfected, apoptotic tumor
cells (that expressed either wt T-Ag or cT-Ag), washed, and cocultured with T-Ag-specific CTL. Comparable levels of IFN-␥ were
released by CTL that were cocultured with pulsed, TAP-competent
(3150 ⫾ 200 pg/ml) or TAP-deficient (2830 ⫾ 140 pg/ml) DC
(data not shown). Similar results were obtained in five independent
experiments in which apoptotic (transfected) P815 or Meth-A cells
expressing T-Ag were cocultured with TAP-competent or TAPdeficient DC, and the response was detected with different CTLL.
Hence, processing of apoptotic material from tumor cells for MHC
class I-restricted epitope presentation by B6 DC is at least in part
TAP independent in the T-Ag system. The data do not exclude the
possibility that a minor component of the processing of apoptotic
material for class I-restricted epitope presentation by DC is TAP
dependent.
The Journal of Immunology
can be immunoprecipitated from the lysate of transfected MethA/cT tumor cells (data not shown). Treatment of Meth-A/cT tumor
cell lysates with ATP before pulsing DC reduced cross-presentation to the level seen with cross-presentation of the same CTL
epitopes by DC pulsed with lysates from Meth-A/T tumor cells
(Fig. 5). Hence, disruption of hsp/cT complexes in the lysate of
tumor cells impairs the enhanced cross-presentation efficacy.
Specific priming of IFN␥-producing CD8⫹ T cells by DNA
vaccination is facilitated by hsp-associated T-Ag expression
B6 mice were vaccinated with pCI expression vector DNA encoding either wt T-Ag or (hsp-binding) cT-Ag. Mice were injected
once either i.m. with 100 ␮g nonpackaged DNA or intradermally
with 1 ␮g particle-coated DNA using the gene gun. The frequencies of class I-restricted, T-Ag-specific, IFN-␥-producing CD8⫹ T
cells were measured in the spleen 2 wk postvaccination; spleen
FIGURE 6. hsp-associated cT-Ag expressed by a DNA vaccine efficiently elicits specific CTL responses. Expression vectors pCI were used
that expressed either the non-hsp-binding wt T-Ag (pCI/T) or the mutant
hsp-binding cT-Ag (pCI/cT) or contained no insert (pCI). B6 mice were
vaccinated by a single i.m. injection of nonpackaged plasmid DNA (100
␮g/mouse) or a single i.d. injection of particle-coated DNA (1 ␮g/mouse)
with the gene gun. Spleen cells (4 ⫻ 106/ml) obtained 14 days postvaccination were restimulated with 5 ␮g T-Ag-derived peptides (T1, T2/3, and
T4) for 5 h (in the presence of BFA) before surface staining for CD8 and
intracellular staining for IFN-␥. We analyzed 105 CD8⫹ cells for IFN-␥
expression. Double-positive (CD8⫹IFN-␥⫹) cells were peptide-specific
CTL. The mean number of double-positive CD8⫹IFN-␥⫹ T cells/105
CD8⫹ spleen cells ⫾ SD of three individual mice are shown.
cells were restimulated in vitro for 5 h in the presence of BFA with
T-Ag-derived peptides, washed, surface stained for CD8, fixed,
and intracellularly stained for IFN-␥. The number of IFN-␥-producing CD8⫹ T cells per 105 splenic CD8⫹ T cells was measured
by FCM. The data in Fig. 6 show that 2- to 3-fold more T-Agspecific CTL were detectable in the spleen of mice that were immunized by cT-expressing plasmid DNA than in those expressing
wt T-Ag. This was most striking in vaccinations by the intradermal
route, in which Th2-biased immunity usually prevails. The stable
association of a viral Ag with hsp73 during its in situ expression
thus seems to facilitate priming of CTL.
A growing tumor cross-primes CTL
FIGURE 5. hsp-facilitated cross-presentation is inhibited by ATP treatment of tumor cell lysates. Tumor cell lysates (107 cells/ml) from T-Agtransfected or nontransfected Meth-A cells were not treated or were treated
with 4 mM ATP for 45– 60 min at 37°C to dissociate T-Ag/hsp70 complexes. The treated lysates were diluted in medium (1/4) and used to pulse
DC from H-2b mice for 3– 4 h. DC were washed and incubated for 16 h
before coculture with T-Ag-specific CTL (3 ⫻ 104) for 24 h. IFN-␥ release
was determined by ELISA. Results from one of three independent experiments with similar results are shown. The p value was calculated with a
paired t test using data from three independent experiments.
The nontransfected or transfected mastocytoma cell line P815 (derived from DBA/2 H-2d mice) was highly tumorigenic in syngeneic hosts; a single s.c. injection of 103 cells resulted in rapidly
progressing tumors in all transplanted mice (37, 43). Progressive,
lethal tumors developed when 107 nontransfected or transfected
Meth-A cells were injected s.c. into syngeneic BALB/c or
F1(BALB/c ⫻ B6) hosts. Transfer of 105 or 106 (nontransfected or
transfected) Meth-A cells into (semi)syngeneic hosts led to transient tumor growth, followed by subsequent rejection in most
adoptive hosts. Spontaneous regression of tumors derived from
(transfected or nontransfected) Meth-A tumor cells was dependent
on CD8⫹ and CD4⫹ T cells, as progressively growing tumors
developed in transplanted BALB/c or F1 hosts depleted of T cells
by in vivo Ab treatment (data not shown). Tumors developing in
F1 mice from transfected Meth-A/cT transplants were reproducibly
smaller and showed earlier regression (data not shown).
Tumors transiently outgrowing from Meth-A/T and Meth-A/cT
transplants cross-primed CTL (Fig. 7, A and B). Transfected H-2d
tumor cells growing in F1(d ⫻ b) hosts primed H-2b-restricted,
T-Ag-specific CTL that specifically lysed T-Ag-expressing H-2b
targets (Fig. 7A). This specific cytolytic reactivity of CTL was
lower when it was primed by a growing, T-Ag-expressing tumor
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FIGURE 4. Epitopes from hsp-associated (truncated) T-Ag derived
from apoptotic tumor cells are more efficiently cross-presented by DC than
native T-Ag (not associated with hsp). H-2b DC were pulsed with serial
dilutions of apoptotic Meth-A, Meth-A/T, or Meth-A/cT tumor cells (left
panel) or tumor cell lysate (right panel), washed, and cocultured with a
T-Ag-specific CTL. IFN-␥ was determined in supernatants after a 24-h
culture by ELISA. The results shown are representative for three independent experiments.
113
114
STRESS-PROTEIN-FACILITATED CROSS-PRESENTATION
than when it was primed by DNA-based vaccination. In addition,
cross-primed CTL from lymph node or spleen released IFN-␥
when specifically restimulated in vitro with T-Ag-expressing H-2b
stimulator cells (Fig. 7B). Similar data were obtained when 107
P815/T or P815/cT tumor cells were injected into allogeneic B6
mice (data not shown). These data indicate that Ags expressed
endogenously by a tumor growing in vivo can cross-prime a functional CTL response.
A growing tumor expressing hsp-associated cT-Ag more
efficiently cross-primes CTL than a growing tumor expressing
wild-type T-Ag
FIGURE 8. H-2d tumors expressing hsp-associated
T-Ag cross-prime T-Ag-specific, H-2b-restricted, IFN␥-producing CD8⫹ T cells efficiently. T-Ag-specific
CD8⫹ T cell frequencies were determined in spleens of
F1 mice transplanted with wtT-Ag- or cT-Ag-expressing
Meth-A cell transfectants. Mice transplanted with nontransfected Meth-A cells were the negative controls;
mice immunized with pCI/T were the positive controls.
Spleen cells (4 ⫻ 106) were incubated with 5 ␮g/ml T1,
T2/3, or T4 peptide (in the presence of BFA) for 5 h
before the surface staining for CD8 and intracellular
staining for IFN-␥. We analyzed 105 CD8⫹ T cells for
IFN-␥ expression. Double-positive CD8⫹IFN-␥⫹ T
cells were considered peptide-specific T cells. The mean
number of double-positive IFN-␥⫹ CD8⫹ T cells per
105 splenic CD8⫹ T cells ⫾ SD of three individual mice
are shown.
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FIGURE 7. Cross-presentation of T-Ag epitopes by tumor cells growing in vivo. Transfected or nontransfected Meth-A cells s.c. transferred into
F1 hosts (106 cells/mouse) induced transient tumor growth. Spleen cells or
lymph node (LN) cells from rejecting mice were restimulated in vitro with
RBL5/T cells. A, Splenic CTL derived from F1 mice transplanted with 106
transfected (but not nontransfected) Meth-A cells lysed RBL5/T targets;
nontransfected RBL5 targets were not lysed (data not shown). Splenic
CTLs from F1 mice immunized with pCI/T DNA vaccine were used as a
positive control. Data from a representative experiment from three independent experiments are shown. B, IFN-␥ release of spleen and LN cells
from mice transplanted with 106 Meth-A/cT cells and cocultured in vitro
with RBL5/T cells or RBL5 cells.
The data in Fig. 4 indicate that Meth-A/cT tumor cells cross-stimulate T-Ag-specific CTL responses in vitro more efficiently than
Meth-A/T tumor cells. Tumor regression was dependent on CD8⫹
T cells. F1 mice transplanted with Meth-A/cT tumor cells showed
less aggressive tumor growth and early regression than F1 mice
transplanted with Meth-A/T tumor cells. This indicates that tumor
growth-controlling T-Ag-specific CTL are primed in the system.
The frequencies of Kb- or Db-restricted CD8⫹ T cells specific for
the T1, T2/3, and T4 epitopes of the T-Ag cross-primed by a growing tumor were determined directly ex vivo. Spleen cells from F1
mice that rejected a nontransfected Meth-A, transfected Meth-A/T,
or transfected Meth-A/cT tumor or from mice that were vaccinated
with a pCI/T DNA vaccine were restimulated in vitro for 5 h with
the T1, T2/3, or T4 peptide, washed, surface stained for CD8,
fixed, and intracellularly stained for IFN-␥. The number of IFN␥-producing CD8⫹ T cells per 105 splenic CD8⫹ T cells was determined by FCM. In Meth-A/cT tumor-bearing F1 hosts we detected a larger number of T-Ag-specific CTL than in Meth-A/T
tumor-bearing animals (Fig. 8). The data shown in Fig. 8 demonstrate that 2- to 3-fold more CTL specific for all three T-Ag
epitopes tested were detected in the spleen of mice that rejected
Meth-A/cT tumors compared with mice that rejected Meth-A/T
tumors. These data confirm in a direct ex vivo readout that hspassociated cT-Ag more efficiently cross-primes CTL in vivo than
native T-Ag not associated with hsp. Taken together, these data
indicate that the association of Ag with hsp facilitates in vitro and
in vivo cross-priming of CTL to Ag endogenously expressed by
tumor cells.
The Journal of Immunology
Discussion
TGF-␤, PGs, and platelet-activating factor (53). C-reactive protein
and the classical complement components can promote noninflammatory clearance of apoptotic cells (59). In contrast, cytokines and
CD40-dependent signals can stimulate maturation of immunostimulatory DC that process engulfed material from apoptotic cells
(5, 59). We have demonstrated in vivo that CTL are cross-primed
against T-Ag epitopes in mice by growing Meth-A or P815 tumors.
It is unknown which signals drive the maturation of DC into an
immunostimulatory phenotype in this system. Stress proteins are
known to induce DC maturation and to be effective adjuvants for
vaccines (60 – 65). We propose that the associating of an oncogene
with hsp facilitates the generation of immunostimulatory DC required for cross-priming CTL and preventing induction of
tolerance.
Evidence has been reported that the stress protein gp96 participates in the cross-presentation of Ags of cellular origin (64). An
hsp70-like chaperone is involved in cross-priming T cell immunity
by DNA vaccination (63). hsp70 released from dying tumor cells
and taken up directly by DCs may be involved in direct chaperoning Ags into DCs (60). Exosomes, i.e., vesicles secreted by DC
upon fusion of late multivesicular endosomes with the plasma
membrane, induce potent anti-tumor immune responses in mice,
resulting in the regression of established tumors (34). A major
exosome component is hsp73 (hsc70) (33). hsp73 stably associated
with mutant or truncated T-Ag seems to play a role in Ag chaperoning. We have shown that stable, noncovalent binding of mutant, truncated, or chimeric T-Ag to hsp73 enhances expression,
facilitates access of the complexes to an alternative (TAP-independent) processing pathway for MHC class I-restricted peptide
presentation, and supports priming of Ab responses against endogenous Ags when used as a DNA vaccine (reviewed in Ref. 32).
Here we report a novel feature of the system: DC-dependent crosspriming of CTL was facilitated in vitro and in vivo by associating
a viral oncogene with hsp73. Although suggestive evidence for a
role of hsp molecules of the 70- and 90-kDa family is available
from different experimental systems, the molecular mechanism underlying these phenomena remains to be elucidated.
Is it the mutation, but not the hsp association, of T-Ag that
facilitates cross-presentation of its CTL epitopes in the tumor
model we studied? Mutant or truncated Ag (fragments) may give
rise to defective ribosomal products that may represent a major
source of antigenic peptides for MHC class I molecules (66). We
have shown that expression of C-terminal fragments of the T-Ag
(that contain some of the CTL epitopes studied here) is difficult to
achieve and does not lead to presentation of CTL epitopes (29, 30).
Although not formally excluded, a mutation or truncation per se
thus does not seem to convey to an Ag a greater efficacy to crosspresent its epitopes.
Our tumor transplantation experiments indicate that mice with a
transiently growing tumor expressing hsp-associated mutant viral
Ag contain 3-fold more oncogene-specific CTL than mice carrying
a tumor expressing the native (non-hsp-associated) variant of the
oncogene. Class I-restricted CTL against all three epitopes tested
were more frequent in mice carrying tumors expressing the hspbound cT-Ag variant. This was seen in transplantation experiments
using transfected Meth-A or P815 tumor cells. The Meth-A cell
inoculum of 106 cells/mouse allowed transient tumor growth followed by rejection of the tumor in ⬎90% of the transplanted mice.
All mice injected with 107 (transfected or nontransfected) Meth-A
cells developed progressively growing, lethal tumors. Mice that
had rejected a (transfected or nontransfected) Meth-A tumor transplant also rejected an inoculum of 107 Meth-A cells, suggesting
priming of tumor-associated Ag-specific immunity (data not
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The amino-phospholipid translocase-dependent appearance of
phosphatidylserine in the outer leaflet of the membrane of cells is
an early event signaling apoptosis (44). Apoptotic cells are rapidly
cleared by macrophages and DC that recognize them through
platelet-activating factor- and TGF-␤-dependent phosphatidylserine recognition (45), CD36 (46), ␣V␤5 (15) or ␣V␤3 integrins (46),
C-type lectin receptors, or Fc receptors (47). Uptake of apoptotic
cells or their remnants can operate through phagocytosis (15, 48),
endocytosis (17, 33), or macropinocytosis. Immature DC are most
efficient in engulfing remnants from apoptotic cells (15, 33), which
is confirmed by our data described in Fig. 2. Although Ag-containing material can be transferred between viable DC (18, 49),
and viable cell may transfer antigenic material to DC (18, 50 –52),
we detected no uptake of fluorescent protein by DC cocultured
with viable, transfected Meth-A/cT272-eGFP or P815/cT272eGFP tumor cells (data not shown).
Uptake of exogenous antigenic material by APC can lead to
processing and class I-restricted cross-presentation of epitopes to
CTL. Although macrophages efficiently take up apoptotic cells or
their remnants, they are deficient in cross-presenting Ags from
apoptotic cells to CTL (3, 15, 53). Exceptions to this rule have
been reported (48). Lymphoid (CD8␣⫹), but not myeloid
(CD8␣⫺), DC have been shown to cross-prime CTL (54). We used
myeloid DC generated in vitro from bone marrow for our crosspresentation experiments. Processing of exogenous material from
apoptotic cells for class I-restricted peptide presentation is in some
Ag systems apparently TAP dependent (17, 48, 55). Our data indicate that cross-presentation of Kb- and Db-binding T-Ag peptides
by DC is at least partially TAP-independent, confirming reports of
TAP-independent cross priming of CTL in a number of well-defined Ag systems (reviewed in Ref. 32). The nature of the Ag
and/or the functional state of the APC may play a role in regulating
TAP-dependent vs -independent epitope presentation. Uptake of
exogenous material from cells by DC stimulates CD4⫹ T cell responses (50, 56), and cross-priming CTL responses by apoptotic
tumor cells in vivo has been shown to be CD4⫹ Th cell dependent
(1, 5). Our data described here and published previously (37, 43)
demonstrate that the rejection of Meth-A and P815 tumors is
CD4⫹ and CD8⫹ T cell dependent, confirming the helper dependence of cross-primed CTL responses. We found that all three
epitopes of the T-Ag (T1, T2/3, T4) that bind to Db or Kb molecules are cross-presented to CTL in vitro and in vivo in the investigated tumor system. This is in contrast to the TAP-independent
presentation of endogenously expressed, hsp-associated T-Ag. We
have described that the two Db-restricted, but not the Kb-restricted,
T-Ag epitopes were efficiently expressed by TAP-deficient, transfected tumor cells (30). The described data indicate that all CTLdefined antigenic information of a viral oncogene expressed endogenously by tumor cells can be transferred by apoptotic tumor cells
to immature DC, processed, and cross-presented in the context of
MHC class I molecules to CTL.
Cross-presentation of CTL-defined epitopes does not usually
lead to cross-priming of T cells, but often induces tolerance (reviewed in Refs. 1 and 14). Maturation of immunostimulatory DC
could be induced by pulsing with material derived from necrotic
tumor cells, but not primary tissue cells or apoptotic cells, suggesting that necrosis (but not apoptosis) provides a control critical
for the initiation of immunity (6). Different anti-inflammatory effects of apoptotic cells have been described. CD95-mediated apoptosis of lymphoid cells leads to the rapid production of IL-10
(57, 58). Macrophages that have ingested apoptotic cells in vitro
inhibit proinflammatory cytokine production by the release of
115
116
shown). Tumor-draining DC have been shown to efficiently crossprime CTL in vivo to tumor-associated Ags, although the tumor
grows progressively and eventually kills its host. This suggests
failure to prevent tumor growth in the effector phase, but not in the
induction phase (67). In addition to facilitating Ag transfer between tumor cells and DCs and making DC immunostimulatory,
additional immunomodulatory protocols will be required to make
CTL-mediated tumor rejection more efficient.
Acknowledgments
We thankfully acknowledge the help of Drs. Mary Jo Wick and Alun Kirby
(Lund, Sweden) in introducing us to the determination of CTL frequencies
by FCM. We are grateful for the helpful comments and suggestions of
Dr. R. Kiessling (Karolinska Institute, Stockholm, Sweden).
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