Antimelanoma activity of CTL generated from peripheral blood mononuclear cells after stimulation with autologous dendritic cells pulsed with melanoma gp100 peptide G209-2M is correlated to TCR avidity

Anchor residue-modified peptides derived from tumor-associated Ag have demonstrated success in engendering immune responses in clinical studies. However, tumor regression does not always correlate with immune responses. One hypothesis to explain this is that CTL resulting from such immunization approaches are variable in antitumor potency. In the present study, we evaluated this hypothesis by characterizing the activity of tumor-associated Ag-specific CTL. We chose an anchor residue-modified peptide from gp100, G209-2M, and used peptide-pulsed dendritic cells to generate CTL from PBMC of HLA-A2(+) normal donors. The specificities and avidities of the resulting CTL were evaluated. The results demonstrate that CTL generated by G209-2M can be classified into three categories: G209-2M-specific CTL which are cytotoxic only to G209-2M-pulsed targets; peptide-specific CTL which recognize both G209 and G209-2M peptides but not melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2(+)gp100(+) melanomas. CTL that kill only peptide-pulsed targets require a higher peptide concentration to mediate target lysis, whereas CTL that lyse melanomas need a lower peptide concentration. Increasing peptide density on melanomas by loading exogenous G209 peptide enhances their sensitivity to peptide-specific CTL. High avidity CTL clones also demonstrate potent antimelanoma activity in melanoma model in nude mice. Injection of G209 peptide around transplanted tumors significantly enhances the antitumor activity of low avidity CTL. These results suggest that peptide stimulation causes expansion of T cell populations with a range of avidities. Successful immunotherapy may require selective expansion of the higher-avidity CTL and intratumor injection of the peptide may enhance the effect of peptide vaccines.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

CD8+ T cells specific for the androgen receptor are common in patients with prostate cancer and are able to lyse prostate tumor cells

The androgen receptor (AR) is a hormone receptor that plays a critical role in prostate cancer, and depletion of its ligand has long been the cornerstone of treatment for metastatic disease. Here, we evaluate the AR ligand-binding domain (LBD) as an immunological target, seeking to identify HLA-A2-restricted epitopes recognized by T cells in prostate cancer patients. Ten AR LBD-derived, HLA-A2-binding peptides were identified and ranked with respect to HLA-A2 affinity and were used to culture peptide-specific T cells from HLA-A2+ prostate cancer patients. These T-cell cultures identified peptide-specific T cells specific for all ten peptides in at least one patient, and T cells specific for peptides AR805 and AR811 were detected in over half of patients. Peptide-specific CD8+ T-cell clones were then isolated and characterized for prostate cancer cytotoxicity and cytokine expression, identifying that AR805 and AR811 CD8+ T-cell clones could lyse prostate cancer cells in an HLA-A2-restricted fashion, but only AR811 CTL had polyfunctional cytokine expression. Epitopes were confirmed using immunization studies in HLA-A2 transgenic mice, in which the AR LBD is an autologous antigen with an identical protein sequence, which showed that mice immunized with AR811 developed peptide-specific CTL that lyse HLA-A2+ prostate cancer cells. These data show that AR805 and AR811 are HLA-A2-restricted epitopes for which CTL can be commonly detected in prostate cancer patients. Moreover, CTL responses specific for AR811 can be elicited by direct immunization of A2/DR1 mice. These findings suggest that it may be possible to elicit an anti-prostate tumor immune response by augmenting CTL populations using AR LBD-based vaccines.     

Generation of tumor-reactive CTL against the tumor-associated antigen HER2 using retrovirally transduced dendritic cells derived from CD34+ hemopoietic progenitor cells

Ag-specific CD8+ CTL are crucial for effective tumor rejection. Attempts to treat human malignancies by adoptive transfer of tumor-reactive CTL have been limited due to the difficulty of generating and expanding autologous CTL with defined Ag specificity. The current study examined whether human CTL can be generated against the tumor-associated Ag HER2 using autologous dendritic cells (DC) that had been genetically engineered to express HER2. DC progenitors were expanded by culturing CD34+ hemopoietic progenitor cells in the presence of the designer cytokine HyperIL-6. Proliferating precursor cells were infected by a retroviral vector encoding the HER2 Ag and further differentiated into CD83+ DC expressing high levels of MHC, adhesion, and costimulatory molecules. Retroviral transduction of DC resulted in the expression of the HER2 molecule with a transduction efficiency of 15%. HER2-transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2(369-377). HER2-transduced DC were used as professional APCs for stimulating autologous T lymphocytes. Following repetitive stimulation, a HER2-specific, HLA-A*0201-restricted CTL line was generated that was capable of lysing HLA-A*0201-matched tumor cells overexpressing HER2. A CD8+ T cell clone could be generated that displayed the same specificity pattern as the parenteral CTL line. The ability to generate and expand HER2-specific, MHC class I-restricted CTL clones using HER2-transduced autologous DC in vitro facilitates the development of adoptive T cell transfer for patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

Induction of primary human CD8+ T lymphocyte responses in vitro using dendritic cells

The ability of two different human professional APCs, specifically macrophages (Mphi) and dendritic cells (DC), to stimulate primary responses in human CD8+ T lymphocytes was examined using both allogeneic and Ag-pulsed autologous APCs. CTL responses in CD8+ T lymphocytes isolated from HIV-uninfected donors were evaluated against six different HIV epitopes that are restricted by four different HLA alleles using autologous human PBMC-derived Mphi and DCs for primary stimulation. In a side-by-side experiment, immature DCs, but not Mphi, were able to prime a CTL response against the B14-restricted p24gag 298-306 epitope; mature DCs were also able to prime a response against this epitope. In addition, DCs were capable of priming CD8+ CTL responses against the B8-restricted p24gag 259-267 epitope. In contrast, Mphi were unable to prime strong CTL responses against other epitopes. Since the Ag-specific cytotoxic responses required subsequent rounds of restimulation before they could be detected, the ability of the allogeneic Mphi and DCs to directly prime CD8+ T lymphocyte responses without subsequent restimulation was examined. Similar to the aforementioned peptide-specific results, DCs were more efficient than Mphi in priming both allogeneic proliferative and cytotoxic responses in human CD8+ T lymphocytes. Collectively, these results promote an enhanced status for DCs in the primary stimulation of human CD8+ T lymphocytes.     

Functional analysis of tumor-specific Th cell responses detected in melanoma patients after dendritic cell-based immunotherapy

Recently, we have demonstrated that tumor-specific CD4+ Th cell responses can be rapidly induced in advanced melanoma patients by vaccination with peptide-loaded monocyte-derived dendritic cells. Most patients showed a T cell reactivity against a melanoma Ag 3 (MAGE-3) peptide (MAGE-3(243-258)), which has been previously found to be presented by HLA-DP4 molecules. To analyze the functional and specificity profile of this in vivo T cell response in detail, peptide-specific CD4+ T cell clones were established from postvaccination blood samples of two HLA-DP4 patients. These T cell clones recognized not only peptide-loaded stimulator cells but also dendritic cells loaded with a recombinant MAGE-3 protein, demonstrating that these T cells were directed against a naturally processed MAGE-3 epitope. The isolated CD4+ Th cells showed a typical Th1 cytokine profile upon stimulation. From the first patient several CD4+ T cell clones recognizing the antigenic peptide used for vaccination in the context of HLA-DP4 were obtained, whereas we have isolated from the second patient CD4+ T cell clones which were restricted by HLA-DQB1*0604. Analyzing a panel of truncated peptides revealed that the CD4+ T cell clones recognized different core epitopes within the original peptide used for vaccination. Importantly, a DP4-restricted T cell clone was stimulated by dendritic cells loaded with apoptotic or necrotic tumor cells and even directly recognized HLA class II- and MAGE-3-expressing tumor cells. Moreover, these T cells exhibited cytolytic activity involving Fas-Fas ligand interactions. These findings support that vaccination-induced CD4+ Th cells might play an important functional role in antitumor immunity.     

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.     

Human tumor-derived heat shock protein 96 mediates in vitro activation and in vivo expansion of melanoma- and colon carcinoma-specific T cells

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8(+) T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8(+) T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-gamma 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-A(27-35) in three of five HLA-A*0201 melanoma patients, and of CEA(571-579) and EpCAM(263-271) 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.     

Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendritic cells

Radiation is generally considered to be an immunosuppressive agent that acts by killing radiosensitive lymphocytes. In this study, we demonstrate the noncytotoxic effects of ionizing radiation on MHC class I Ag presentation by bone marrow-derived dendritic cells (DCs) that have divergent consequences depending upon whether peptides are endogenously processed and loaded onto MHC class I molecules or are added exogenously. The endogenous pathway was examined using C57BL/6 murine DCs transduced with adenovirus to express the human melanoma/melanocyte Ag recognized by T cells (AdVMART1). Prior irradiation abrogated the ability of AdVMART1-transduced DCs to induce MART-1-specific T cell responses following their injection into mice. The ability of these same DCs to generate protective immunity against B16 melanoma, which expresses murine MART-1, was also abrogated by radiation. Failure of AdVMART1-transduced DCs to generate antitumor immunity following irradiation was not due to cytotoxicity or to radiation-induced block in DC maturation or loss in expression of MHC class I or costimulatory molecules. Expression of some of these molecules was affected, but because irradiation actually enhanced the ability of DCs to generate lymphocyte responses to the peptide MART-1(27-35) that is immunodominant in the context of HLA-A2.1, they were unlikely to be critical. The increase in lymphocyte reactivity generated by irradiated DCs pulsed with MART-1(27-35) also protected mice against growth of B16-A2/K(b) tumors in HLA-A2.1/K(b) transgenic mice. Taken together, these results suggest that radiation modulates MHC class I-mediated antitumor immunity by functionally affecting DC Ag presentation pathways.     

Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.     

Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34+ progenitor-derived dendritic cells

Between March 1999 and May 2000, 18 HLA-A*0201⁺ patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34⁺ hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-γ production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2–83), and the median event-free survival was 7 months (range: 2–83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.Joseph W. Fay and A. Karolina Palucka have equally contributed to this work     

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells

Our study demonstrates that tumor-derived heat shock protein (HSP)70 chaperones a tyrosinase peptide and mediates its transfer to human immature dendritic cells (DCs) by receptor-dependent uptake. Human tumor-derived HSP70 peptide complexes (HSP70-PC) thus have the immunogenic potential to instruct DCs to cross-present endogenously expressed, nonmutated, and tumor antigenic peptides that are shared among tumors of the melanocytic lineage for T cell recognition. T cell stimulation by HSP70-instructed DCs is dependent on the Ag bound to HSP70 in that only DCs incubated with HSP70-PC purified from tyrosinase-positive (HSP70-PC/tyr(+)) but not from tyrosinase-negative (HSP70-PC/tyr(-)) melanoma cells resulted in the specific activation of the HLA-A*0201-restricted tyrosinase peptide-specific cytotoxic T cell clone. HSP70-PC-mediated T cell stimulation is very efficient, delivering the tyrosinase peptide at concentrations as low as 30 ng/ml of HSP70-PC for T cell recognition. Receptor-dependent binding of HSP70-PC and active cell metabolism are prerequisites for MHC class I-restricted cross-presentation and T cell stimulation. T cell stimulation does not require external DC maturation signals (e.g., exogenously added TNF-alpha), suggesting that signaling DC maturation is an intrinsic property of the HSP70-PC itself and related to receptor-mediated binding. The cross-presentation of a shared human tumor Ag together with the exquisite efficacy are important new aspects for HSP70-based immunotherapy in clinical anti-cancer vaccination strategies, and suggest a potential extension of HSP70-based vaccination protocols from a patient-individual treatment modality to its use in an allogeneic setting.     

Allogeneic HLA-A*02-restricted WT1-specific T cells from mismatched donors are highly reactive but show off-target promiscuity

T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201-binding WT1(126-134) peptide were generated from both HLA-A*02-positive (self-HLA-restricted) and HLA-A*02-negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA-restricted WT1-specific clones only recognized WT1(126-134) with low avidities. In contrast, allo-HLA-restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02-positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201-binding peptides was investigated. The self-HLA-restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA-restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201-binding peptides. In conclusion, allogeneic HLA-A*02-restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.     

HER-2/neu-derived peptide 884–899 is expressed by human breast, colorectal and pancreatic adenocarcinomas and is recognized by in-vitro-induced specific CD4+ T cell clones

HER-2/neu peptides recognized in the context of HLA-DR molecules by CD4(+) Th lymphocytes on antigen-presenting cells have been identified. In this report, we demonstrate for the first time that HER-2/neu helper epitopes are also expressed on the surface of metastatic breast, colorectal and pancreatic carcinomas. Peripheral blood mononuclear cells from an HLA-DR4 healthy donor were used to induce HER-2/neu peptide-specific CD4(+) T cell clones by in vitro immunization with HER-2/neu peptide (884-899)-pulsed autologous dendritic cells (DCs). Strong proliferation and significant levels of IFN-gamma were induced by the CD4(+) T cell clones in response to specific stimulation with autologous DCs loaded with HER-2(884-899). Furthermore, these clones also recognized HER-2/neu(+) tumor cell lines, and tumor cells from breast, colorectal and pancreatic adenocarcinomas induced to express HLA-DR4, but also the HLA-DR4(+) melanoma cell line FM3 transfected to express HER-2/neu. The recognition of tumor cells was strongly inhibited by an anti-HLA-DR mAb. Taken altogether, we provide novel information for the role of HER-2(884-899) as a naturally processed epitope expressed by breast, colorectal and pancreatic carcinomas and the capacity of HER-2/neu protein to follow the endogenous class II processing pathway. Our results suggest that HER-2(884-899) might be attractive for broadly applicable vaccines and may prove useful for adoptive immunotherapy designed for breast, colorectal and pancreatic carcinomas.     

Forced expression of HLA-DM at the surface of dendritic cells increases loading of synthetic peptides on MHC class II molecules and modulates T cell responses

Adoptive transfer of autologous dendritic cells (DCs) loaded with tumor-associated CD4 and CD8 T cell epitopes represents a promising avenue for the immunotherapy of cancer. In an effort to increase the loading of therapeutic synthetic peptides on MHC II molecules, we used a mutant of HLA-DM (DMY) devoid of its lysosomal sorting motif and that accumulates at the cell surface. Transfection of DMY into HLA-DR(+) cells resulted in increased loading of the exogenously supplied HA(307-318) peptide, as well as increased stimulation of HA-specific T cells. Also, on transduction in mouse and human DCs, DMY increased loading of HEL(48-61) and of the tumor Ag-derived gp100(174-190) peptides, respectively. Interestingly, expression of DMY at the surface of APCs favored Th1 differentiation over Th2. Finally, we found that DMY(-) and DMY(+) mouse APCs differentially stimulated T cell hybridomas sensitive to the fine conformation of peptide-MHC II complexes. Taken together, our results suggest that the overexpression of HLA-DMY at the plasma membrane of DCs may improve quantitatively, but also qualitatively, the presentation of CD4 T cell epitopes in cellular vaccine therapies for cancer.     

Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

MHC-restricted recognition of autologous melanoma by tumor-specific cytotoxic T cells. Evidence for restriction by a dominant HLA-A allele.

Autologous melanoma-specific CTL recognize a common tumor-associated Ag (TAA) in the context of HLA class I antigens. We have demonstrated that HLA-A2 can be a restricting Ag and, in T cell lines homozygous for HLA-A2, that CTL can be generated by stimulation with HLA-A2 allogeneic melanomas. In the current study, we have investigated T cell lines from patients who are heterozygous at HLA-A region locus, to determine the relative importance of each A-region allele in this MHC-restricted recognition of tumor. We have shown that HLA-A1 can be a restricting Ag, and that allogeneic melanomas expressing HLA-A1 can substitute for the autologous tumor in the generation of HLA-A1-restricted CTL. However, when T cell lines express both HLA-A1 and HLA-A2, the HLA-A2 allele governed restriction of the melanoma TAA. Three autologous-stimulated HLA-A1, A2 CTL lines all demonstrated restriction by the HLA-A2 allele, when examined in cytotoxicity assays, cold-competition assays, and proliferation assays. There was no evidence of restriction by the second HLA-allele, HLA-A1. Although the autologous-stimulated CTL use a single A-region allele for tumor recognition, the autologous HLA-A1, A2 tumors are lysed by both HLA-A1-restricted and HLA-A2-restricted CTL. The dominance of restricting alleles was further demonstrated when HLA-matched allogeneic melanomas were used as the stimulating tumor to generate tumor-specific CTL. Stimulation of the heterozygous (HLA-A1, A2) lymphocytes with HLA-A2-matched allogeneic melanomas resulted in CTL specific for the autologous tumor, and restricted by the HLA-A2 Ag. However, stimulation with an HLA-A1-matched allogeneic melanoma failed to induce tumor-specific CTL restricted by the HLA-A1 Ag. The data suggest there is a dominance of HLA-A region Ag at the level of the T cell, such that only one is restricting in the recognition of the autologous melanoma. At the level of the tumor, however, the TAA is expressed in the context of both HLA-A region alleles. We can generate specific CTL from lymph node cells or PBL and HLA-A region matched allogeneic melanomas; however, because most patients are heterozygous at the HLA-A region locus, an understanding of the dominant restricting alleles must be obtained so that an appropriately matched allogeneic melanoma can be selected.