| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* Unit of Immunotherapy of Human Tumor,
Gastrointestinal and Liver Surgery Unit, and
Colorectal Surgery Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy;
Medizinische Klinik III, University Hospital Benjamin Franklin, Berlin, Germany;
¶
Sigma-Tau ifr SpA, Rome, Italy;
||
Antigenics, New York, NY 10111; and
#
Center for Immunotherapy of Cancer and Infectious Disease, University of Connecticut, Farmington, CT 06030
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The evidence that the immunogenicity of tumor-derived hsp96 could stem from the ability of this protein to chaperone antigenic epitopes comes from both structural and immunological studies. Peptide-binding activity of hsp96 or other hsp family members (such as hsp70 or hsp90) has been directly demonstrated in vitro by using labeled peptide probes and by crystallographic studies, which allowed the identification of a possible hydrophobic binding pocket within the hsp96 molecule (6, 7, 8, 9). Additional direct evidence of this activity could come from the structural scrutiny of hsp96-associated peptides by mass spectroscopy. However, for technical difficulties related to hsp96 structure (2), efforts in identifying these peptides by biochemical means have been to date successful only in few cases (10, 11, 12, 13).
Nevertheless, the vast majority of data supporting the association of antigenic peptides with hsp96 derives from immunological studies in murine models, which demonstrated that preparations of these proteins purified either from virus-infected or from tumor cells could trigger Ag-specific T cell recognition when pulsed on appropriate MHC-matched APC, through a mechanism known as cross-priming (2, 14, 15). Recently, similar data have been obtained by our group in human melanoma-derived hsp70, which was found to be associated with antigenic peptides from the melanoma Ags gp100 and MART-1/Melan-A (hereafter MART-1) (16). However, no direct evidence on the chaperone properties of human tumor-derived hsp96, which belongs to a different hsp family and displays a diverse intracellular localization as compared with hsp70, is presently available. Because immunization with autologous tumor-derived hsp96 has recently started to be evaluated in clinical setting (2, 17, 18, 19), it is essential to prove that hsp96 purified from human cancer cells can actually transport immunogenic peptides for cross-presentation to tumor and Ag-specific T cells.
Thus, the purpose of the present study was to investigate whether hsp96 purified from melanoma or liver metastases of colon carcinoma cells did indeed contain immunogenic peptides derived from tumor Ags. To this aim, we used an immunological approach to evaluate the ability of tumor-derived hsp96 to reconstitute the epitopes recognized by CD8+ T cells specific for highly expressed melanoma or colon carcinoma Ags. Additionally, we investigated whether immunization with tumor-derived hsp96 could result in expansion of such specific T cells in the peripheral blood of patients with metastatic melanoma or liver metastases from colon carcinoma. In this study, we provide the first evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As Ag-specific T cells, we used: the HLA-A*0201-restricted MART-1-specific CD8+ T cell clone A42, autologous to 501mel (20) and recognizing the immunodominant epitope MART-127–35, and the CD8+ tumor-infiltrating lymphocyte (TIL) line (GDN-TIL), an oligoclonal T cell line recognizing MART-127–35 epitope in a HLA-A*0201-restricted fashion (20). PBMCs from HLA-A*0201+ melanoma or colon carcinoma patients were purified from peripheral blood by Ficoll gradient, aliquoted, and stored in liquid nitrogen. MART-1-, carcinoembryonic Ag (CEA)-, and epithelial cell adhesion molecule (EpCAM)-specific T cells were generated from PBMCs by in vitro peptide sensitization, as previously described (21). T cell cloning was performed by limiting dilution in the presence of OKT3 mAb. The anti-CEA T cell line 2708 was generated from PBMC of an HLA-A*0201+ healthy donor by in vitro peptide sensitization, and contained >90% of CD8+HLA-A*0201/CEA571–579 tetramer+ cells.
Melanoma, colon cancer cells, and other targets
The following targets of specific T cell recognition were used in the ELISPOT assay: 501mel, a melanoma line expressing both HLA-A*0201 and MART-1; the colon carcinoma line Colo 206, expressing HLA-A*0201, CEA, and EpCAM (22); and the TAP-deficient lymphoblastoid cell line T2, known to efficiently bind exogenously supplied peptides in the context of HLA-A*0201. The melanoma line Me 2658, expressing significant MART-1 levels, was used for hsp96 purification. Ag (i.e., MART-1, CEA, and Ep-CAM) expression was also evaluated in all tumor specimens (from melanoma or liver metastases from colon carcinoma) used for hsp96 purification. Immunohistochemistry on Formalin-fixed specimens was performed, as previously described (18), using the anti-MART-1 mAb (clone A103; Novocastra Labs, Newcastle upon Tyne, U.K.), the anti-CEA Ab (DAKO, Glostrup, Denmark), and the anti-EpCAM mAb (clone Ber-EP4; DAKO). MART-1 and EpCAM were highly expressed only in a fraction of melanoma (18) and colon carcinoma lesions (data not shown), respectively, including those from the patients developing anti-MART-1 and EpCAM T cell responses upon vaccination (i.e., melanoma patients 003, 007, and 020, and colon carcinoma patients 038 and 045). CEA was overexpressed in the totality of liver metastases from colon carcinoma (data not shown).
Hsp96 purification and vaccine treatment
Hsp96 was purified from tumor cells by affinity columns, as previously described (23). Purification was perfomed by Antigenics (Woburn, MA), starting from at least 3 g of nonnecrotic frozen tumor tissue or from a 3-g pellet of melanoma cells (501mel and Me 2658). Purification of hsp96 for clinical use was obtained under current Good Manufacturing Practice procedures; thus, all preparations contain no detectable level of endotoxin. For the immunization treatment, patients affected by stage IV melanoma (partially or radically resected for s.c. or visceral metastases) or patients with colon carcinoma (curatively resected for liver metastases) gave written informed consent to be vaccinated and fulfilled inclusion criteria required by two different clinical protocols, approved by the Internal Review Board and by the Independent Ethics Committee of the Istituto Nazionale Tumori of Milan (18, 19). Detailed vaccine schedule is reported elsewhere (18, 19). Briefly, patients received four weekly s.c. injections of autologous tumor-derived hsp96 (at 25 µg/injection), followed 1 or 2 mo later by a second cycle of four injections at 2-wk intervals.
Peptides
Peptides were synthesized using an Applied Biosystems 432 A peptide synthesizer (Foster City, CA), following a standard protocol, as previously described (22). The following HLA-A*0201-binding peptides were used: MART-1 p27–35 (AAGIGILTV), EpCAM p263–271 (GLKAGVIAV), and CEA p571–579 (YLSGANLNL, CAP-1). The peptide derived from Flu A matrix protein M1 (GILGFVFTL) was used as control Ag.
IFN- ELISPOT and 51Cr release assays
IFN- ELISPOT was performed, as previously described (18, 19), and evaluated by a computer-assisted ELISPOT reader (Bioline, AID, Torino, Italy). HLA-blocking experiments were conducted by preincubating target cells with the anti-class I HLA (-A,-B,-C) mAb W6.32 or the anti-HLA-DR mAb L243 (18). For detecting the presence of antigenic peptides in tumor-derived hsp96, monocytes were purified by adherence from PBMCs of HLA-A*0201+ donors and incubated in the presence of melanoma- or colon carcinoma-derived hsp96 (10 µg/ml), or with 5 µM peptide. After 2-h incubation, monocytes were transferred into ELISPOT plates (at 1 x 105 cells/well) containing specific T cell effectors at the appropriate concentration. For statistical evaluation, a t test for unpaired samples was used to compare pre- and postvaccine spots of the same patient, or to evaluate statistical significance of HLA-blocking experiments. Values of p < 0.05 were considered as statistically significant. Cytotoxicity was evaluated by a standard 51Cr release assay, as previously described (21). Monocytes, enriched by adherence from PBMC of HLA-A*0201+ or HLA-A*0201- healthy donors, were incubated for 2 h at 37°C with hsp96 from 501mel (at 10 µg/ml), labeled with 51Cr, washed, and used as targets (at 1000 cells/well) in a 6-h assay. For competition experiments, monocytes were pretreated with a 2-fold excess of hsp96 from colon carcinoma, or of BSA as negative control, washed several times, incubated with hsp96 from 501mel (at 10 µg/ml), and used as targets.
IFN- secretion assay
Monocytes, purified by adherence from PBMCs of HLA-A*0201+ donors, were incubated overnight at 37°C with hsp96 from colon carcinoma or melanoma cells (at 10 µg/ml), or with MART-127–35 or CEA571–579 peptides (10 µM). T cells specific for MART-1 (A42 T cell clone) or CEAs (oligoclonal T cell lines, composed by >90% CEA-specific CD8+ T cells, as assessed by HLA/tetramer staining) were then added. After 3 h at 37°C, nonadherent cells were collected and analyzed for IFN- secretion by the Miltenyi IFN- cytokine secretion assay (Miltenyi Biotec, Bergisch Gladbach, Germany), according to manufacturer’s procedures. Data were analyzed by FACSCalibur and CellQuest software (BD Biosciences, Milan, Italy).
Staining with HLA/peptide tetramers
HLA-A*0201/MART-126–35/2L tetramers, used for detecting anti-MART-1-specific T cells, were kindly provided by C. Giachino (Fondazione Maugeri, Pavia, Italy). HLA-A*0201/CEA571–579 tetramers and EpCAM263–271, used for staining colon carcinoma-reactive T cells, were produced by Beckman Coulter (San Diego, CA). Tetramer binding was evaluated after staining with the iMASC Gating Kit (Beckman Coulter), containing FITC anti-CD8 mAb, together with PC5 anti-CD4, CD13, and CD19 mAbs. Data are reported as percentage of tetramer+CD8+, CD4/CD13/CD19- cells. As negative control, we used iTAgTM HLA class I human negative tetramers streptavidin-PE (Beckman Coulter), developed for assessing the level of background PE fluorescence. They are loaded with a peptide that has been shown to tightly bind to HLA-A2 and has been proven not to be recognized by any T cells from HLA-A2+ individuals. Cells were analyzed using FACSCalibur and CellQuest software (BD Biosciences).
IFN- intracellular staining
IFN- intracellular staining was performed, as previously described (22). Briefly, PBMCs were stimulated with MART-1 peptide; then brefeldin A (Sigma-Aldrich, Deisenhofen, Germany) was added to block cytokine secretion. PBMCs were stained with PerCP anti-CD3, APC anti-CD8, PE anti-CD45RA, and PE anti-CCR7 mAbs (BD Biosciences). After lysing and permeabilizing the cells, IFN- was stained by incubation with FITC anti-IFN- mAb (BD Biosciences). Data acquisition was performed on FACSCalibur and analyzed using CellQuest software (BD Biosciences).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ag-specific T cells were used for detecting immunogenic epitopes in hsp96 extracted either from autologous or HLA-matched tumor cells. To this aim, we focused on three tumor Ags containing HLA-A*0201-restricted epitopes: 1) MART-1 (epitope 27–35), highly expressed by melanoma cells (20, 21); 2) CEA (epitope 571–579), overexpressed in several epithelial tumors, including colon carcinoma; and 3) EpCAM (epitope 263–271), an adhesion molecule overexpressed in colon carcinoma cells. The two latter peptides were recently reported to be target of spontaneous CD8+ T cell-mediated immune recognition in colon carcinoma patients (22). To show the presence in tumor-derived hsp96 of peptides originated from these Ags, epitope-specific T cells were incubated with monocytes from HLA-A*0201+ healthy donors, pulsed with hsp96 purified from melanoma or colon carcinoma cells, and their reactivity was measured by IFN- ELISPOT and 51Cr release assays. As shown in Fig. 1, both the anti-MART-1 T cell clone A42 (Fig. 1A) and the oligoclonal T cell line GDN-TIL (Fig. 1B) released considerable amounts of IFN- in response to monocytes pulsed with hsp96 from both the MART-1+ melanomas 501mel (autologous to A42) and Me2658. Such reaction was significantly blocked by the anti-HLA-A,-B,-C mAb W6.32, but not by the anti-HLA-DR mAb L243, indicating class I HLA-restricted T cell recognition and TCR involvement. A certain degree of cross-reactivity was observed in the presence of hsp96 purified from colon carcinoma cells, but such response was significantly weaker than that mediated by melanoma-derived hsp96 and could not be blocked by anti-HLA class I or II mAbs (Fig. 1). No cross-presentation was detected when HLA-A*0201-negative monocytes were used as APC (data not shown). To confirm that the oligoclonal anti-MART-1 T cell line GDN-TIL was recognizing HLA-A2/MART-1 complex on monocytes loaded with 501mel-derived hsp96, a cold target inhibition assay using T2 cells pulsed with MART-127–35 peptide or with CEA571–579, as negative control, was performed (Fig. 1C). A significant inhibition of the lysis of monocytes loaded with 501mel-derived hsp96 was achieved when T2 + MART-127–35, but not T2 + CEA571–579, was added to the assay at 20-fold excess. The killing by the GDN-TIL of HLA-A*0201+ monocytes pulsed with 501mel-derived hsp96 was reduced by the addition of irrelevant hsp96 (i.e., derived from MART-1-negative colon carcinoma cells), in a dose-dependent manner (Fig. 1D). In contrast, no competition was observed when a 2-fold excess of BSA was used as control (data not shown). These latter data suggest that colon carcinoma- and melanoma-derived hsp96 competed for the binding to monocytes through the same receptor, but only hsp96 from MART-1+ melanoma cells contain the relevant T cell epitope (i.e., MART-127–35).
|
in response to HLA-A*0201+ monocytes pulsed with hsp96 purified from autologous colon carcinoma cells, and not from allogeneic melanoma. Such recognition was significantly blocked by anti-HLA class I, but not by anti-HLA class II mAb. Overlapping results were obtained with EpCAM263–271-specific T cell line and CD8+ T cell clone (Fig. 2, C and D, respectively). Even in this case, HLA class I-restricted recognition of HLA-A*0201+ monocytes occurred only after pulsing with hsp96 from colon carcinoma cells. The amounts of IFN- released by anti-CEA and anti-EpCAM effectors were remarkably higher when APCs were pulsed with colon carcinoma-derived hsp96 as compared with the relevant peptide provided exogenously (Fig. 2).
|
secretion in 73% of CEA571–579-specific T cells, while no effect (2%) was seen in the presence of hsp96 from melanoma. The triggering of IFN- production mediated by colon carcinoma-derived hsp96 was significantly higher than that observed in the presence of the synthetic CEA571–579 peptide at the saturating dose of 10 µM (mediating cytokine secretion in only 12% T cells), and it was selectively detected in CEA-specific T cells, but not in the anti-MART-1 T cell clone. The more efficient pathway of presentation by hsp96 as compared with purified synthetic peptides was also evident with anti-MART-1 T cells (Fig. 3B), only 5% of which could be triggered to secrete IFN- by 10 µM MART-127–35 peptide, whereas hsp96 derived from the autologous melanoma cells mediated cytokine secretion in 14% cells. Hsp96-induced IFN- secretion by both anti-MART-1 and anti-CEA T cells was significantly blocked by anti-class I HLA mAb and could not be detected in the presence of HLA-A*0201-negative monocytes (data not shown).
|
Two phase I-II clinical trials based on vaccination with autologous tumor-derived hsp96 were recently performed in our Institute. In the first trial, 39 stage IV melanoma patients were treated according to a protocol described elsewhere in details (18) and summarized in Materials and Methods. The immunological monitoring, performed by IFN- ELISPOT, showed a significant increase of HLA class I-restricted T cell recognition of either autologous or allogeneic HLA-A-matched tumor cells in approximately half of the tested patients, as recently reported elsewhere (18).
Because our in vitro data suggested that tumor-derived hsp96 contained antigenic peptides, we evaluated whether T cells specific for MART-1 could have been expanded in vivo in HLA-A*0201+ patients vaccinated with autologous melanoma-derived hsp96. Indeed, among the five HLA-A*0201+ patients displaying increased CD8-mediated melanoma recognition after hsp96 vaccination (18), three showed a significant and specific increment of IFN- release in response to MART-127–35 peptide (Fig. 4), with unchanged T cell responses to the influenza-derived peptide M1Flu, used as irrelevant control. In contrast, in the remaining seven HLA-A*0201+ melanoma patients, which did not show any enhancement of CD8-mediated melanoma recognition after hsp96 vaccination (18), no sign of increased anti-MART-1 reactivity was detected (data not shown). The enhancement of IFN- release in response to MART-127–35 peptide occurred with different kinetics in these three cases, being detectable since the fourth week of treatment (i.e., at V5) in patients 007 and 020, and 1 mo after the end of the first vaccine cycle (V6) in patient 003 (Fig. 4). A significant boost of the IFN- release in response to the HLA-A*0201+ MART-1 melanoma cell line 501mel was additionally observed in all three patients (Fig. 4). Of note, patients 003 and 007 were the only 2 patients among 28 who responded clinically to the vaccine with a complete tumor regression (18).
|
release in response to both MART-1 peptide pulsed on T2 cells and MART-1+ HLA-A*0201+ melanoma cell line 501mel (Fig. 5C). Cytofluorimetric analysis for IFN- intracellular staining combined to phenotypic analysis was additionally performed to evaluate the phenotypic features of MART-1-specific T cells in patient 003. As reported in Fig. 5, D and F, T cells releasing cytokine in response to MART-1, and expanded in vivo by hsp96 vaccine (V6), showed a CD3+ CD45RA+ CCR7- phenotype, recently reported to be associated to lytic effector cells (24, 25). On the contrary, no IFN- production in response to MART-127–35 peptide could be detected in CD45RA-negative T cell subpopulations. A similar phenotypic pattern of MART-1-reactive CD8+ T cells was also observed in PBMC from patient 007 (data not shown).
|
|
A second clinical protocol of hsp96 vaccination was conducted in our Institute on 29 colon carcinoma patients surgically resected for liver metastases (19). On the basis of the data obtained in melanoma patients, we analyzed whether treatment with autologous tumor-derived hsp96 could lead to in vivo expansion of T cells recognizing the same CEA and EpCAM epitopes detected in vitro as bound to hsp96. Among the 5 HLA-A*0201+ patients displaying increased CD8-mediated colon carcinoma recognition after hsp96 vaccination (19), 2 developed a significant enhancement of CEA571–579 and EpCAM263–271 peptide recognition, paralleled by increase in the specific reactivity against HLA-A*0201+ CEA+ EpCAM+ colon carcinoma cells, as detected by IFN- ELISPOT (Fig. 6). On the contrary, no changes in the T cell reactivity against the influenza virus-derived epitope M1Flu were observed (data not shown). In the remaining four HLA-A*0201+ colon carcinoma patients, which did not show any enhancement of CD8-mediated tumor recognition after hsp96 vaccination (19), no sign of increased anti-CEA and anti-EpCAM reactivity was detected (data not shown).
|
release (Fig. 8, E and F). The relatively low tetramer-staining intensity observed in anti-CEA and anti-EpCAM T cells, as compared with that achieved with anti-MART-1 T cell cultures (Fig. 5A), could be a sign of limited TCR affinity and reduced peripheral frequency of these T cells, as already reported by others (26). In keeping with this hypothesis, we could not detect any reproducible increment of CEA571–579 and EpCAM263–271/tetramer staining in fresh PBMCs of these patients (data not shown). The observed lower frequency of T cells specific for CEA571–579 and EpCAM263–271, as compared with anti-MART-1 T cells, is to be ascribed to the strong variability in the peptide immunogenicity, MART-127–35 peptide being one of the most immunogenic T cell epitopes in cancer patients (21).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The antigenic transfer to APC mediated by hsp96 appeared to be superior to that achieved with exogenous peptide loading. This enhanced presentation was Ag dependent, i.e., it could be selectively detected when anti-MART-1 and anti-CEA or EpCAM T cells were activated by hsp96 derived from tumor cells expressing the relevant Ag (i.e., melanoma and colon carcinoma cells, respectively), and it could be specifically competed by excess of unrelated hsp96. This phenomenon could be ascribed to different and nonmutually exclusive mechanisms. An improved processing and HLA peptide loading mediated by hsp96, as compared with free peptide, can stem from the ability of hsp96 to access class I HLA presentation pathway through receptor-mediated uptake (2, 3), a mechanism that, at variance with nonspecific phagocytosis, operates with high efficiency even at low Ag concentrations (14). Furthermore, endogenous processing of exogenously provided epitopes is per se associated to improved CTL induction, as recently shown by Zwaveling et al. (27), who reported that vaccination with long peptides resulted in markedly enhanced T cell induction in comparison with the minimal CTL epitope vaccine, as a consequence of a preferable endocytosis, processing, and presentation by professional APC. Finally, the activatory functions that hsp96 plays on APCs (4) could eventually contribute in providing a more favorable environment for T cell activation.
Hence, hsp96 may represent an efficient and well-characterized tool for inducing class I HLA cross-presentation of tumor Ags from exogenous protein sources in human setting. Such presentation pathway, which is considered to play a key role in immune responses to tumors and viruses (28), seems to operate with low efficiency and through poorly understood mechanisms when exogenous Ags are given as tumor lysate or apoptotic bodies (29). The ability of hsp96 to simultaneously deliver antigenic peptides and appropriate activatory stimuli to APCs could provide an additional advantage upon the other cell-derived tumor Ag sources that, on the contrary, have been shown to potentially mediate suppressive effects on APC differentiation and maturation (30, 31).
We further demonstrated that hsp96 can activate and expand specific T lymphocytes in vivo. Among patients developing increased CD8 T cell-mediated recognition of autologous or HLA-matched allogeneic tumor cells upon vaccination with tumor-derived hsp96 (18, 19), expansion of T cells recognizing the same Ags detected in vitro in the hsp96 preparations was observed in a significant number of cases. Indeed, three of five melanoma patients displayed increased MART-1 activity, while enhanced T cell recognition of CEA/EpCAM Ags was seen in two of five colon carcinoma patients. Such reactivities, assessed by IFN- ELISPOT and HLA tetramer staining, were undetectable in PBMCs before vaccination, and involved T cells displaying a CD45RA+ CCR7- phenotype, recently proposed to characterize cytolytic effector T cells able to migrate into inflamed peripheral tissues (24, 25). Interestingly, hsp96-mediated T cell triggering appeared to be associated to in vivo tumor growth control, because four of the five patients showing expansion of Ag-specific T cells after hsp96 vaccination seemed to benefit from the treatment, with two complete responses in melanoma and two long-term disease-free survivals in colon carcinoma (18, 19). Ag-specific T cell effectors boosted by hsp96 immunization could thus be directly responsible for the in vivo tumor cell destruction, or may be alternatively just a marker of a potent activation of antitumor immune responses induced by hsp96 and possibly involving other unknown T cell reactivities. Indeed, given the physiological role of hsp96 as chaperone of the whole antigenic repertoire of the cell (2, 3, 4), it is conceivable that immunization with tumor-derived hsp96 may activate T cells directed to multiple antigenic determinants, including those derived from other shared as well as unique tumor Ags. Because the major rationale to the usage of autologous tumor-derived hsp96 consists in the possibility to immunize against a broad and patient-tailored Ag array, an extensive monitoring of different T cell reactivities directed toward immunogenic epitopes derived from shared tumor Ags and restricted by common HLA alleles would provide important information about the immunogenic potential of hsp96 vaccine. Preliminary experiments at this regard indeed show that increased CD8+ T cell responses directed against other common Ags (such as for instance the gp100-HLA-A*0301 epitope) (32) can be detected in hsp96-vaccinated melanoma patients (data not shown).
For its combined features of peptide vehicle and APC maturation promoter, hsp96 is deemed to be an appealing tool for immunization against tumor and infectious diseases. The results of the present study extend previous work in murine setting and provide strong evidence to the efficient chaperone activity of human tumor-derived hsp96, which could be exploited in the development of new and more effective immunotherapeutic strategies in cancer patients.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Licia Rivoltini, Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. E-mail address: licia.rivoltini{at}istitutotumori.mi.it
3 Abbreviations used in this paper: hsp, heat shock protein; CEA, carcinoembryonic Ag; EpCAM, epithelial cell adhesion molecule; TIL, tumor-infiltrating lymphocyte; MART-1, melanoma Ag recognized by T cell-1; Melan-A, melanoma Ag A.
Received for publication April 21, 2003. Accepted for publication July 22, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Related articles in The JI:
This article has been cited by other articles:
|
|
 |
|
  R. Shaykhiev and R. Bals Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis J. Leukoc. Biol., July 1, 2007; 82(1): 1 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Parmiani, A. De Filippo, L. Novellino, and C. Castelli Unique Human Tumor Antigens: Immunobiology and Use in Clinical Trials J. Immunol., February 15, 2007; 178(4): 1975 - 1979. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Di Pucchio, L. Pilla, I. Capone, M. Ferrantini, E. Montefiore, F. Urbani, R. Patuzzo, E. Pennacchioli, M. Santinami, A. Cova, et al. Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-{alpha} Results in the Activation of Specific CD8+ T Cells and Monocyte/Dendritic Cell Precursors. Cancer Res., May 1, 2006; 66(9): 4943 - 4951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. K. Sondak, M. S. Sabel, and J. J. Mule Allogeneic and autologous melanoma vaccines: where have we been and where are we going? Clin. Cancer Res., April 1, 2006; 12(7 Pt 2): 2337s - 2341s. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rosato, A. Zoso, S. D. Santa, G. Milan, P. Del Bianco, G. L. De Salvo, and P. Zanovello Predicting Tumor Outcome following Cancer Vaccination by Monitoring Quantitative and Qualitative CD8+ T Cell Parameters J. Immunol., February 1, 2006; 176(3): 1999 - 2006. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Cassatella, V. Huber, F. Calzetti, D. Margotto, N. Tamassia, G. Peri, A. Mantovani, L. Rivoltini, and C. Tecchio Interferon-activated neutrophils store a TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) intracellular pool that is readily mobilizable following exposure to proinflammatory mediators J. Leukoc. Biol., January 1, 2006; 79(1): 123 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Qian, S. Wang, J. Yang, J. Xie, P. Lin, M. E. Freeman III, and Q. Yi Targeting Heat Shock Proteins for Immunotherapy in Multiple Myeloma: Generation of Myeloma-Specific CTLs Using Dendritic Cells Pulsed with Tumor-Derived gp96 Clin. Cancer Res., December 15, 2005; 11(24): 8808 - 8815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. J. Quintana and I. R. Cohen Heat Shock Proteins as Endogenous Adjuvants in Sterile and Septic Inflammation J. Immunol., September 1, 2005; 175(5): 2777 - 2782. [Abstract] [Full Text] [PDF]
|
|
|
|
, 2-fold excess; 
, 1-fold excess), but not with the same excess of BSA (data not shown). GDN-TIL did not lyse HLA-A*0201- monocytes pulsed with 501mel-hsp96 (
). Results are representative of three independent experiments.
