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Immunology |
Units of Immunotherapy of Human Tumors [C. C., F. R., L. R., G. P.] and Immunohematology [A. M.], Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan, Italy, and Department of Oncology/Pathology, Unit of Experimental Oncology, Karolinska Institutet, S17176 Stockholm, Sweden [A-M. T. C., R. K.].
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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release by antimelanoma T cells by a
MHC-independent mechanism. In this case, however, higher doses of HSP70
were required. The capacity to activate class I-restricted, antitumor T
cells as well as antigen-presenting cells, together with the finding
that the HSP70 chaperoned peptide repertoire includes melanoma-shared
epitopes, holds promise for a HSP70-based cancer vaccine. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies aimed at evaluating the immunogenicity of murine tumors led to the discovery of the HSP.3 This family of chaperone proteins, the functions of which include sampling intracellular peptides derived from unfolded cytoplasmic proteins, displays a strong immunogenic potential, and by providing an immunogenic context for their complexed peptides, HSP can be considered adjuvant of mammalian origins (6) . In addition to their involvement in the degradation pathway of cellular proteins during stress condition, HSP70 assists the new synthesized proteins in their correct folding and is also directly involved in the translocation of proteins across membranes into different cellular compartments (7 , 8) . This large selection of physiological activities and the ability to traffic among different cell compartments make the repertoire of HSP70 chaperone peptides potentially wide and involving proteins having different subcellular localization.
HSPs obtained from tumors or virus-infected cells have been shown to induce CTL responses in vitro against a variety of antigens expressed in the cells from which HSPs have been purified. The specificity of the induced CTLs relies on the peptides chaperoned by these HSPs (9 , 10) . Murine studies have carefully assessed the usage of chaperone molecules as cancer vaccines, and their efficacy was demonstrated in tumors of different histologies in a prophylactic as well as in a therapeutic setting (11) . The immunogenicity of HSP70 and gp96 has been demonstrated for viral as well as tumor antigens in murine systems, but no data are as yet available for the immunogenic potential of human tumor-derived HSPs.
For human melanoma, a variety of tumor-associated peptides recognized by MHC-restricted T cells have been described recently (12) . Exploiting this knowledge and the availability of melanoma-specific T cells with defined peptide specificity, we decided to analyze the capacity of HSP70 melanoma-derived peptides to specifically activate T cells. We show here that HSP70 obtained from melanoma is able to reconstitute the epitope for HLA class I-restricted T-cell clones directed against melanoma differentiation antigens. The HSP70-mediated T-cell activation occurs via recognition of MHC molecules of the matched APCs pulsed with the melanoma-derived HSP70 and is strictly dependent on the presence of HSP chaperoned peptides. In addition, HSP peptide presentation to the antimelanoma-specific T-cell clones is likely to occur via cross-priming because MHC matching between melanoma cell lines used as a source of HSP70 and responding T-cell clones was not required. These findings provide a possible rationale for HSP-based vaccination in human cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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variable region 2 (data
not shown). The specificity of these effectors has been described
previously (13, 14, 15)
and is summarized in Table 1
. Melanoma cultures were maintained in RPMI 1640 with 10% FCS.
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SDS-PAGE and Immunoblot.
Samples of the purified HSP70 were run on SDS-PAGE 12.5% homogenous
gels. The gel was silver stained after partial transfer to polyvinyl
difluoride membrane (Bio-Rad Laboratories, Richmond, CA). The membrane
was blocked in 1% BSA at 4°C overnight, incubated for 1 h at
room temperature with the mouse anti-HSP/hsc70 antibody SPA-820
(StressGen Biotechnologies Corp., Victoria, British Columbia, Canada),
and washed three times for 5 min each with 0.5% Tween 20 in PBS. It
was then incubated with peroxidase-conjugated rabbit antimouse antibody
(Dakopatts, Denmark) for 1 h, washed as above, incubated with
peroxidase-conjugated swine antirabbit antibody (Dakopatts), and washed
again as described above. The blot was developed with 9 mg/ml
3,3'-diaminobenzidine (Sigma) and 0.018%
H2O2 in PBS for 10–50 s at
room temperature, washed in 0.5% Tween-PBS, and dried.
T-Cell Stimulation Assay.
Monocytes were prepared by adherence. Briefly, peripheral blood
monocyte cells of HLA-typed healthy donors were seeded in RPMI 1640
medium and let to adhere to plastic for 18 h. The adherent
cells were mechanically recovered and resuspended in RPMI 1640 and 10%
human serum. As control, the recovered cells were stained with
monoclonal antibody recognizing CD14 and HLA class II molecules. Flow
cytometric analysis of the stained cells showed that 23% of the cells
were CD14+ HLA class II+. Monocytes (4 x 105) were seeded in 96-well U-bottomed plates in
a volume of 30 µl. The indicated amount of HSP70 was added in 30
µl, and the incubation was performed for 2 h. Lymphocytes
(1 x 104
/wells) were then added
in 60 µl. Plates were incubated for 18 h at 37°C, supernatants
were collected, and their IFN- content was determined by
commercially available ELISA (Mabtech AB, Stockholm, Sweden).
Controls were performed by incubating T cells and stimulators with
medium alone or with HSP70 alone. The significance of IFN- release
in the presence of target cells in comparison with release in the
presence of medium alone was established by Student-Newman-Keuls
multiple range test at P = 0.01. For the
blocking experiments, before adding the effector cells, monocytes
loaded with HSP were further incubated for 1 h at 37°C with the
monoclonal antibody W6/32 (10 µg/ml) recognizing a monomorphic
determinant of class I HLA. The significance of inhibition of IFN-
release detected in the presence of antibodies was evaluated by
Student-Newmann-Keuls test (P = 0.01).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
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. The restriction
alleles for these CTL clones were HLA-A2 for MART-1, HLA-A3 and HLA-Cw8
for gp100, and HLA-Cw8 for TRP-2-specific clones, respectively (Table 1)
. HSP70 was purified from Me15392 autologous to the CTL DIL15392,
TB254, and TB327 and from Me501 autologous to the A42 CTLs. Taking
advantage of two alternative purification procedures, HSP70 complexed
with the endogenous associated peptides HSP70 (ADP) or with no
associated peptides HSP70 (ATP) was obtained from each melanoma cell
line. Monocytes purified from class I HLA-matched peripheral blood
monocyte cells of healthy donors were loaded with HSP70 (ADP) or HSP70
(ATP) and evaluated for the ability to activate
melanoma-specific CTLs. As shown in Fig. 2
, three of the four CTL clones, i.e., anti-gp100 CTL DIL15392
and TB254 as well as the anti-MART-1 A42, released IFN- in response
to stimulation with HSP70 (ADP)-loaded monocytes. Moreover, HSP70 (ADP)
activation was MHC class I dependent because the IFN- released by
the stimulated CTLs was significantly inhibited by incubation of APCs
with the anti-HLA-class I antibody W6/32. The level of cytokine
specifically induced by HSP70 stimulation was significantly different
among the CTLs tested, with the lowest release detected for the
anti-Mart-1-specific CTLs. This different level of CTL triggering could
reflect the different binding capacity of the tumor-derived epitopes
for the HSP70 binding groove.
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released was low. The HSP70 (ATP)-mediated activation was
diminished by the HLA class I-specific antibody, probably indicating
that in the ATP purification procedure, some HSP70 complexed with
endogenous peptides was still present as a contaminant. This
inhibition, however, failed to reach statistical significance. No
peptide-dependent, HSP70-mediated activation could be shown for the
clone TB327, because this clone was induced to release similar amounts
of IFN- by HSP70 (ADP) and HSP70 (ATP), and the
cytokine production was not MHC dependent.
To better define the role of chaperone-assisted peptides and the MHC
involvement in the HSP-mediated stimulation of the antimelanoma CTLs,
different doses of ADP- or ATP-purified HSP70 were compared for the
ability to induce cytokine production by the HLA-A3-restricted,
gp100-specific DIL15392 (Fig. 3)
and by the HLA-A2-restricted anti- MART-1 A42 CTLs (Fig. 4)
. A dose-dependent, MHC class I-restricted activation of both CTL
clones was achieved by the peptide containing HSP70 (ADP) (Figs. 3A
and 4A)
, whereas the amount of IFN- induced
by HSP70 (ATP) was not dependent on MHC class I molecules and was
clearly detectable only with the highest doses tested (Figs. 3B
and 4B)
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. No HLA class I-dependent IFN- release was induced by
clone A42 by MHC-unmatched APCs; the amount of cytokine in the medium
was comparable with the level achieved when the same clone was
stimulated by HLA-matched APCs loaded with HSP70 with no endogenous
peptide HSP70 (ATP) (Figs. 2
and 4B)
.
HSP70 Derived from Antigen-negative Cell Lines Does Not Activate
Antimelanoma CTLs.
To further support the notion that CTL activation occurs through
recognition of their nominal peptide chaperoned by HSP70 and presented
by the MHC molecules of the APCs, we analyzed the stimulatory activity
of HSP70 (ADP) obtained from antigen-negative sources. For this
purpose, HSP70 purified from LCL15392 not expressing any of the
melanoma differentiation antigens was used in a stimulation assay of
the anti-gp100, HLA-A3-restricted DIL15392 and HLA-A2 restricted
anti-MART-1 A42 CTLs. No specific activation occurred for any of the
antimelanoma CTLs tested (Fig. 5, A and B)
. For DIL15392, the low amount of
cytokine detectable in the medium was at least 3 logs lower than the
amount achieved by the specific peptide-dependent stimulation mediated
by the HSP70 (ADP) purified from the autologous tumor (see Fig. 2A
). In addition, the cytokine released in the medium by A42
was not MHC-restricted because similar background levels of IFN-
were produced when HLA-matched or HLA-unmatched monocytes (Fig. 5B)
were used as APCs.
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was released in a dose-dependent fashion, and cytokine production was
not inhibited by the incubation of the APCs with the anti-class I
antibody W6/32 (Fig. 5C)
. The activation of anti-Flu CTLs
was therefore mediated by a MHC-independent, antigen-unrelated
stimulation. In fact, HSP70 was purified from LCLs not infected by
influenza virus and therefore not expressing the epitope recognized by
the effector T cells used in the assay. These results reinforced the notion that HSP70 per se may exert a stimulatory activity for T cells and, in addition, indicate that the HSP70 (ADP) MHC-dependent stimulation of CTLs can occur only when the nominal epitope is included in the HSP70-chaperoned peptide repertoire.
HSP70-mediated T-Cell Activation Occurs via Cross-Priming.
Because HSP70-mediated T-cell activation was dependent on
peptides derived from shared tumor antigens, we evaluated the capacity
of HSP70 to stimulate across the MHC barriers. For this purpose,
DIL15392 was stimulated with HLA-A3+ monocytes loaded with HSP70
purified by ADP or ATP agarose from an allogeneic, HLA-A3-negative
melanoma expressing a high level of gp100 (data not shown). In the same
experiment, the stimulating ability of HSP70 purified from the
autologous melanoma was also evaluated as control. No difference in the
activation pattern of DIL15392 could be detected between the autologous
and allogeneic melanoma-derived HSP70, as evaluated by the amount of
IFN- released (Fig. 6A)
. Furthermore, the allogeneic HSP70 CTL stimulation was MHC
dependent (Fig. 6A)
and linked to the presence of chaperoned
peptides because peptide-free HSP70 (ATP) preparations failed to
significantly stimulate cytokine release by T cells (Fig. 6)
. The
cytokine release by DIL15392 was dependent on the dose of the
allogeneic purified HSP70 (ADP) used, and IFN- could be detected,
even at the lowest dose of 0.6 µg/ml (Fig. 6C)
. The
overall stimulating capacity of allogeneic HSP70 (ADP) was therefore
comparable with the stimulating capacity of autologous-derived HSP70
(ADP), strongly suggesting that HSP70-mediated T-cell activation
occurred via cross-priming.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we demonstrate that HSP70-complexes purified from human melanoma include peptides derived from normal differentiation proteins and that these epitopes, via APC presentation, may lead to the activation of the cognate T cells. In our experimental system, we were unable to check the presentation of "unique" peptides because no antimelanoma T cells recognizing unique antigens on the melanoma 15392 were available. Therefore, the relative abundance of shared versus unique epitopes in a human HSP70-chaperoned peptide repertoire could not be compared directly. Peptides derived from normal differentiation antigens should, however, be well represented in the HSP repertoire because four of five CTLs tested and recognizing differentiation antigens were stimulated by HSP70-derived peptides. Among the responding CTLs, the level of cytokine specifically induced by HSP70 stimulation was significantly different with the lower release detected for the anti-MART-1-specific CTLs. Unfortunately, we did not have the possibility to test the susceptibility of HSP70-mediated activation of CTLs expressing different T-cell receptors but recognizing the same epitope and therefore address the question of whether a different susceptibility to HSP70 stimulation could be an intrinsic characteristic of each CTL expressing a particular T-cell receptor.
T-cell clones recognizing peptides derived from MART-1, tyrosinase, and gp100 were activated in a MHC-restricted fashion by melanoma-derived HSP70 loaded on APCs, whereas the anti-TRP-2 clone was not. The melanoma cell lines used as a source for HSP70 were susceptible to lysis by TRP-2-specific CTLs, indicating that the level of expression of this protein was sufficient for the peptide to be presented by the MHC of tumor cells. We cannot, however, rule out the possibility that the abundance of HSP-TRP-2 peptide complexes is inadequate to sensitize monocytes for a T-cell-mediated recognition. Alternatively, the TRP-2 epitope cannot be allocated in the binding pocket of HSP70 because of structural constrains. Arguing against this possibility, however, both TRP-2 and gp100 HLA-Cw8 presented epitopes; the second, recognized by the T-cell clone TB254 susceptible to the HSP70-mediated activation, displays a similar primary structure (13) and is positioned inside an highly hydrophobic region of the respective molecules. Moreover, HSP70 is known to have a broad binding specificity that only requires the presence of a hydrophobic region composed of six or more amino acid residues (18) . Alternatively, the regions flanking the epitopes in the TRP-2 and in the gp100 molecules may directly contribute to or negatively influence the peptide/HSP70 binding, implying that the HSP70-chaperoned peptide is indeed a longer precursor of the nominal CTL epitope. Supporting this possibility, two histidines, not present in the corresponding gp100 sequence, are found in the NH2 or COOH terminus extended sequences of the TRP-2 epitope. These histidines may potentially interfere with the hydrophobic milieu required for binding.
This second hypothesis is in agreement with our data indicating the capacity of HSP70 to induce a CTL response across MHC barriers. MHC matching between melanoma cell lines used as source of HSP70 and responding T-cell clones is not required, suggesting that the HSP70-complexed peptide repertoire is not limited to the MHC class I ligands of the HSP70 donor cells. The HSP70-complexed peptides, therefore, should include precursor peptides that are then "trimmed" into the final CTL epitope. Experiments further dissecting the general mechanism involved in the HSP70-mediated activation of melanoma-specific T cells are in progress. The present data, however, show clearly that specific CTL stimulation of antimelanoma T cells takes place only in the presence of MHC-matched APC, whereas no specific activation can occur when unmatched monocytes are used. In addition, in the presence of the correct APC, HSP70 derived from autologous or allogeneic melanoma cells displayed the same efficacy in triggering the cognate CTLs. Such an efficient presentation also for peptides bound to HSP70 isolated from allogeneic melanomas cannot be the result of unspecific trimming of peptides by serum-derived proteases (19) and instead argues for a role of the APCs in the processing and presentation of the HSP70-chaperoned peptides to the MHC molecules. This is the first example indicating that HSP70 can elicit CTL responses via "cross-priming," as shown previously with gp96 (10 , 20) . The ability of the APCs to "trim" the HSP70-bound peptides to the size associated with MHC class I does not exclude that in some cases HSP70 could preferentially bind the final sized octameric or nonameric epitope, as shown recently for the Ld-restricted antigens of the RL1o mouse leukemia (21) .
Besides the MHC-restricted, peptide-dependent CTL induction,
tumor-derived HSP70 was also shown to activate antimelanoma T cells
in vitro by a mechanism that is not dependent on the
recognition of a specific peptide. In fact, IFN- production could be
achieved in all of the CTLs tested upon stimulation with HSP70 (ATP),
i.e., with HSP70 with no endogenously complexed peptides or
with HSP70 (ADP) derived from antigen-negative cells. This
peptide-independent activation was more evident with high doses of
HSPs, whereas the peptide and MHC-dependent activation generally
required lower amounts of HSP70. Furthermore, the peptide-independent,
HSP70-mediated activation required the presence of APCs excluding a
direct activation of T cells by HSP70. This unique feature of HSP70,
both being able to deliver antigenic peptides for uptake by APCs and to
act as an "intrinsic" adjuvant, is in line with data published
recently in a human setting (22)
and with our results in a
murine system. These latter results demonstrate that murine HSP70
efficiently induce bone marrow-derived dendritic cells to express high
levels of MHC and costimulatory molecules and to secrete
proinflammatory
cytokines.4
Similar to LPS, HSP70 treatment of bone marrow-derived dendritic cells
was dependent on the toll-like receptor-4 and resulted in the
activation of the nuclear factor-
B signaling pathway. Also, previous
findings obtained with gp96 have demonstrated in vivo and
in vitro activation of T cells after administration of
antigen-negative HSPs (23)
.
In conclusion, our data show that HSP70 derived from human melanoma can exert a pleiotropic triggering of antimelanoma T cells involving class I as well as MHC-independent pathways. We therefore suggest a model where cellular HSP70 is located at the interface between the different subsets of cells in the immune system including T cells and APC. When released by tumors undergoing necrosis, HSP70 may efficiently mediate the transfer of precursor peptides for HLA class I molecules to APCs and the induction of the final maturation of the APCs, ultimately supporting the generation and the maintenance of an efficient antitumor CTL response. Whether such a mechanism is active in vivo remains to be shown, but the ability of HSP70 to activate HLA class I-restricted antimelanoma T cells across the MHC barriers outlines an active role of HSP70 as a candidate vaccine in cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a Coordinated Grant
from Associazione Italiana per la Ricerca sul Cancro (Milan, Italy), by
grants from the Swedish Cancer Society, the Swedish Society for Medical
Research, the Cancer Society of Stockholm, the King Gustaf the Vth
Jubilee Fund, and by Grant QLK3-CT-1999-00064 from the European
Community. A-M. T. C. is a fellow of the Swedish Cancer Society and
has been supported by an ICRETT grant from Union International
Contre Cancer. 
2 To whom requests for reprints should be
addressed, at Istituto Nazionale Tumori, Via G. Venezian 1, 20133
Milan, Italy.
3 The abbreviations used are: HSP, heat shock
protein; APC, antigen-presenting cell; HLA, human leukocyte antigen;
LCL, lymphoblastoid cell line; ATP, adenosine triphosphate.
Received 4/25/00. Accepted 10/31/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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E. Linardakis, A. Bateman, V. Phan, A. Ahmed, M. Gough, K. Olivier, R. Kennedy, F. Errington, K. J. Harrington, A. Melcher, et al. Enhancing the Efficacy of a Weak Allogeneic Melanoma Vaccine by Viral Fusogenic Membrane Glycoprotein-mediated Tumor Cell-Tumor Cell Fusion Cancer Res., October 1, 2002; 62(19): 5495 - 5504. [Abstract] [Full Text] [PDF]
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B. Liu, A. M. DeFilippo, and Z. Li Overcoming Immune Tolerance to Cancer by Heat Shock Protein Vaccines Mol. Cancer Ther., October 1, 2002; 1(12): 1147 - 1151. [Abstract] [Full Text] [PDF]
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 A. Michils, D. Dutry, V. Z. de Beyl, M. Remmelink, V. de Maertelaer, and P. Rocmans Peripheral Blood Mononuclear Cell Proliferation to Heat Shock Protein-70 Derived from Autologous Lung Carcinoma Am. J. Respir. Crit. Care Med., September 1, 2002; 166(5): 749 - 753. [Abstract] [Full Text] [PDF]
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 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
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N. N. Panjwani, L. Popova, and P. K. Srivastava Heat Shock Proteins gp96 and hsp70 Activate the Release of Nitric Oxide by APCs J. Immunol., March 15, 2002; 168(6): 2997 - 3003. [Abstract] [Full Text] [PDF]
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) or in the presence (
) of the
anti-HLA-class I antibody W6/32. IFN-
). Monocytes
were then incubated overnight with 104 cells of the
HLA-A3-restricted DIL15392 CTL line recognizing gp100 antigen
(A), with the HLA-A2-restricted A42 CTLs recognizing
MART-1 (B), and with a polyclonal T-cell line
recognizing the HLA-A3-presented influenza nucleoprotein epitope
NP265–273 (C). The activation of anti-flu T-cell
line (C) was evaluated in the absence (
) of the anti-HLA-class I antibody W6/32. IFN-
) were loaded on HLA-A3+ monocytes
and then incubated overnight with the anti-gp100 HLA-A3-restricted CTL
DIL15392 (1 x 104). IFN-
). IFN-