CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells

Although they are considered as antigen-presenting cells, the role of antigen-unspecific B lymphocytes in antigen presentation and T-lymphocyte stimulation remains controversial. In this paper, we tested the capacity of normal human peripheral activated B cells to stimulate T cells using melanoma antigens or melanoma cell lysates. B lymphocytes activated through CD40 ligation and then pulsed with tumor antigens efficiently processed and presented MHC class II-restricted peptides to specific CD4(+) T-cell clones. This suggests that CD40-activated B cells have the functional and molecular competence to present MHC class II epitopes when pulsed with exogenous antigens, thereby making them a relevant source of antigen-presenting cells to generate T cells. To test this hypothesis, CD40-activated B cells were pulsed with a lysate prepared from melanoma cells and used to stimulate peripheral autologous T cells. Interestingly, T cells specific to melanoma antigens were generated. Additional analysis of these T-cell clones revealed that they recognized MHC class II-restricted epitopes from tyrosinase, a known melanoma tumor antigen. The efficient antigen presentation by antigen-unspecific activated B cells was correlated with a down-regulation in the expression of HLA-DO, a B cell-specific protein known to interfere with HLA-DM function. Because HLA-DM is important in MHC class II peptide loading, the observed decrease in HLA-DO may partially explain the enhanced antigen presentation after B-cell activation. Results globally suggest that when they are properly activated, antigen-unspecific B-lymphocytes can present exogenous antigens by MHC class II molecules and stimulate peripheral antigen-specific T cells. Antigen presentation by activated B cells could be exploited for immunotherapy by allowing the in vitro generation of T cells specific against antigens expressed by tumors or viruses.     

Generation of in vitro B-CLL specific HLA class I restricted CTL responses using autologous dendritic cells pulsed with necrotic tumor lysate

New approaches in the treatment of chronic B lymphocytic leukemia (B-CLL) have led to improved clinical response rates. In this setting there is a need to evaluate novel therapeutic approaches that aim to eradicate minimal residual B-CLL cells following an initial favorable response. The use of tumor lysate-pulsed dendritic cells (DC) represents a potentially important development in the field of cancer vaccination. B-CLL is ideally suited for DC-based vaccination since tumor cells are readily available (peripheral blood) and both known (tumor idiotype) and unknown antigens can be exploited to stimulate immune responses. In the current study we have evaluated the ability to stimulate in vitro autologous immune reactivity against target B-CLL cells using autologous DCs pulsed with B-CLL tumor lysate. Enhanced specific T cell IFN-gamma expression was detected in 9 of 14 patients evaluated. These responses were specific with increased levels of IFN-gamma mRNA measurable in T-cells stimulated with NC-DCs and not unpulsed DCs or DCs pulsed with normal B cell lysate. CTLs demonstrating increased levels of IFN-gamma mRNA also lysed autologous B-CLL targets cells in an MHC class 1-restricted manner by (51)chromium release assay. Priming target leukemic cells with CD40 ligand and IL-4 enhanced CTL killing. The effector CTL displayed negligible toxicity against NK susceptible target cells K-562 and spared CD19(+)CD5(-) normal B cells in cytotoxicity assays. The specificity of the CTL response was confirmed by blocking HLA class I molecules and cold target inhibition assays.     

Tumor-specific CD4+ T cells from a patient with renal cell carcinoma recognize diverse shared antigens

Murine models for immune-mediated tumor regression have defined an essential role for CD4+ T helper (Th) cells, but the contribution of these cells to antitumoral immune responses in humans remains poorly defined. Here, we investigated the Th cell response against the autologous tumor in a patient with metastasized renal cell carcinoma (RCC) exhibiting objective clinical response to immunotherapy. Peripheral blood T cells of the patient were repeatedly stimulated in vitro using either autologous IFNgamma-treated whole tumor cells or Epstein-Barr virus-immortalized B cells (EBV-B) pulsed with tumor cell lysate. CD4+ T-cell clones recognizing autologous tumor cells but not EBV-B cells were efficiently reactivated and expanded with both types of stimulator cells, establishing the latter as potentially useful for isolating CD4+ T cells reactive against MHC class II-negative tumors. Two T-cell clones from both stimulation protocol were further characterized. The restricting MHC class II molecules were defined by using allogeneic EBV-B cells pulsed with tumor lysate, and the expression pattern of the antigens was examined by analyzing lysates from normal kidney cells, allogeneic RCCs as well as tumors of different histologic origin. Furthermore, the subcellular localization of the antigens recognized by the T-cell clones was examined by fractionating the tumor lysate, and the Th phenotype was determined by assessing the cytokines released after T cell activation. These experiments show that a dual Th1/Th2, MHC class II-restricted T-helper-cell response against diverse shared tumor antigens has been elicited in this patient.     

Dead or alive: immunogenicity of human melanoma cells when presented by dendritic cells

Uveal melanoma is an aggressive malignancy with a poor prognosis despite current therapeutic intervention. These tumors have been shown to be antigenic because they express a number of melanoma-associated antigens and are therefore attractive targets for immunotherapy. Here, we investigated the immunogenicity of uveal melanoma cells that have undergone apoptosis and compared this with their necrotic or live counter-parts. The fate of the tumor antigens in these cells largely depends on their ability to be processed and phagocytosed by dendritic cells (DCs). Flow cytometric analysis shows that human DCs form conjugates more efficiently with dead uveal melanoma cells, and consequently these are effective stimuli of lymphocyte proliferation. However, only DCs pulsed with apoptotic cells were able to induce proliferation of CD8+ cytotoxic T cells and stimulate antigen-specific T cells. This study demonstrates for the first time that DCs derived from melanoma patients process and present antigens derived from both HLA-matched or HLA-mismatched human apoptotic tumor cells stimulating both CD4+ and CD8+ T cells. This approach may be important to the development of DC-based immunotherapies for melanoma.     

Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein

We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.     

Melanoma-specific CD4+ T lymphocytes recognize human melanoma antigens processed and presented by epstein-barr virus-transformed B cells

While much emphasis has been placed on the role of MHC class I-restricted CDS⁺ T cells in the recognition of tumor-specific antigens (Ag), evidence has accumulated that CD4⁺ T cells also play a critical role in the anti-tumor immune response. However, little information exists on the nature of MHC class II-restricted human tumor Ag. In an attempt to develop in vitro systems to characterize such Ag, we examined the ability of Epstein-Barr virus(EBV) transformed B cells to present melanoma-associated Ag to melanoma-specific CD4⁺ cells. CD4⁺ T cells cultured from lymphocytes infiltrating a s.c. melanoma metastasis secreted TNF-α and GM-CSF specifically in response to autologous cultured melanoma cells expressing MHC class II molecules. These CD4⁺ cells also recognized MHC class II-compatible EBV-B cells pulsed with extracts of autologous melanoma cells, but failed to recognize EBV-B cells pulsed with autologous non-transformed cells or a variety of allogeneic tumors or normal cells. B cells pre-fixed with paraformaldehyde were incapable of Ag presentation, suggesting that intracellular processing events were occurring. Antibody-blocking studies defined HLA-DR as the dominant if not exclusive restriction locus in this T-B interaction, and HLA-DR genocyping revealed DRBI 0404 to be the probable restriction element. In a second patient, a CD4⁺ T-cell clone cultured from a melanoma lesion recognized autologous tumor Ag presented by autologous EBV-B; no cross-reactivity was observed with the other tumor system investigated, nor with autologous CD4⁺ T cells specific for tetanus toxoid. These findings demonstrate that tumor Ag can be processed and presented by EBV-transformed B cells to MHC class II-restricted tumor-specific CD4⁺ T cells. They also provide a model system for direct identification of these tumor-derived antigens. © 1994 Wiley-Liss, Inc.     

In vitro generation of cytolytic T cells against human melanoma cells overexpressing HDM2

BACKGROUND: Previous experiments have shown that tumour-associated antigens can be exploited for a successful anti-tumour immunisation. Previous reports demonstrated that oncoprotein MDM2 (HDM2) contains two highly conserved MHC class I binding motifs, MDM2100 and MDM2441, and that dendritic cells (DC) presenting MDM2100 stimulate an effective CTL reaction against melanoma cells. MATERIALS AND METHODS: In this study, we investigated the CTL-inducing capacity of autologous human dendritic cells pulsed with fragment HDM2441. RESULTS: In vitro HDM2441-primed T lymphocytes revealed a strong proliferation activity, released Th-1-associated cytokines, and possessed an effective anti-tumour activity causing apoptosis in HDM2441-overexpressing melanoma cells. Cytotoxic assay demonstrated that in parallel to melanoma cells, up to 65% of primed Tcells also underwent apoptosis. CONCLUSION: These data suggest that HDM2441 may be exploited for broad-spectrum DC-based trials against metastatic melanomas overexpressing HDM2, and point out that the efficacy of such immunotherapeutical approaches may be limited via T cell apoptosis.     

Human dendritic cells pulsed with autologous Epstein-Barr virus transformed B-cell lymphoblastoid cell (BCL) lysate elicit a BCL specific MHC-class II restricted T-cell response

Epstein Barr Virus (EBV) associated lymphoproliferative disorders (LPD) express EBV latent antigens that are also expressed on normal B-cells transformed with EBV. This could potentially be exploited to develop immunotherapeutic strategies for LPD and other EBV associated malignancies. To this end we investigated the capacity of human monocyte derived dendritic cells (DC) pulsed with lysate from autologous EBV transformed B-cell lymphoblastoid cell (BCL) lysate to elicit an in vitro antitumor response. BCL lysate pulsed DC generate BCL specific cytotoxic lymphocytes, as lymphocytes primed with such DCs induce cytolysis of autologous (>60%) but not allogeneic BCL (<5%). In addition, lymphocytes primed with BCL lysate pulsed DC secrete gamma-IFN (3176 pg/ml). Whereas gamma-IFN production was markedly reduced (>99%) when BCL specific T-cells were stimulated by BCL lysate pulsed DC in the presence of blocking antibodies to HLA-DR, DP and DQ, use of antibodies to MHC class-I resulted in only a minimal reduction in gamma-IFN production (17%). These studies demonstrate that BCL lysate pulsed DC elicit a predominantly BCL specific, MHC class-Il restricted T cell response. This suggests that vaccination with autologous BCL lysate pulsed DC may represent a viable immunotherapeutic approach for the treatment of LPD.     

Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma

The primary goal of this Phase I study was to assess the safety and bioactivity of tumor lysate-pulsed dendritic cell (DC) vaccination to treat patients with glioblastoma multiforme and anaplastic astrocytoma. Adverse events, survival, and cytotoxicity against autologous tumor and tumor-associated antigens were measured. Fourteen patients were thrice vaccinated 2 weeks apart with autologous DCs pulsed with tumor lysate. Peripheral blood mononuclear cells were differentiated into phenotypically and functionally confirmed DCs. Vaccination with tumor lysate-pulsed DCs was safe, and no evidence of autoimmune disease was noted. Ten patients were tested for the development of cytotoxicity through a quantitative PCR-based assay. Six of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-gamma expression by peripheral blood mononuclear cells in response to tumor lysate after vaccination. Using HLA-restricted tetramer staining, we identified a significant expansion in CD8+ antigen-specific T-cell clones against one or more of tumor-associated antigens MAGE-1, gp100, and HER-2 after DC vaccination in four of nine patients. A significant CD8+ T-cell infiltrate was noted intratumorally in three of six patients who underwent reoperation. The median survival for patients with recurrent glioblastoma multiforme in this study (n = 8) was 133 weeks. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous tumor lysate-pulsed DC vaccine for patients with malignant glioma. We demonstrate for the first time the ability of an active immunotherapy strategy to generate antigen-specific cytotoxicity in brain tumor patients.     

Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen(s).

CTLs were generated in vitro from two healthy donors and one melanoma patient by stimulation of CD8+ T cells with autologous dendritic cells pulsed with natural melanoma peptides (NMPs), obtained by acid treatment of HLA-matched melanoma cells. CTLs showed MHC class I-restricted melanoma-specific cytolytic activity. Importantly, CTLs from the patient, induced with NMPs obtained from an allogeneic HLA-A-matched melanoma, killed the autologous tumor. COS-7 cells cotransfected with the cDNA of 13 melanoma antigens and the HLA-A1-restricting allele did not induce cytokines release from NMP-specific CTLs, suggesting that they recognize unidentified shared melanoma antigens and that they may be valuable for identification of new tumor antigens. These results strongly support the use of autologous and/or allogeneic NMP-pulsed dendritic cells as cancer vaccines in patients whose neoplasms do not express or have lost expression of known tumor antigens.     

The maturation of murine bone marrow-derived dendritic cells by tumor lysate uptake in vitro is not essential for cancer immunotherapy

Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) which are responsible for protection from tumor challenge and regression of established metastatic tumor. It has been hypothesized that tumor lysate contains factors that may modulate DC maturation. In this study, we examined whether the uptake of tumor lysate (MCA-102 fibrosarcoma) could modulate DC phenotypes in vitro and whether the administration in vivo of tumor lysate-pulsed DC (TP-DC) could elicit efficient tumor specific immune responses followed by a regression of established tumor burdens. It was investigated the uptake of tumor lysate by DC by means of flow cytometry and fluorescent microscope. Murine bone marrow-derived DC efficiently phagocytosed tumor lysate and after the uptake, the phenotype of TP-DC was surprisingly comparable to unpulsed-DC (UP-DC), exhibiting lower levels of CD80 (<51%), CD86 (<43%), and MHC class II (<59%). Also, TP-DC did not enhance secretion of IL-12p70 (UP- vs. TP; 54.5+/-6.4 vs. 50.5+/-4.8 pg/ml, respectively), contrary to those activated with LPS (113.6+/-16.8 pg/ml). However, TP-DC vaccination in vivo increased the IFN-gamma production from splenocytes higher than that of UP-DC (TP- vs. UP-DC; 41029+/-1523 vs. 4752+/-590 pg/ml, respectively). Furthermore, the administration of TP-DC enhanced specific T cell responses against MCA-102 fibrosarcoma. These results demonstrate that augmentation of DC phenotype and function in vitro is not necessarily a prerequisite for TP-DC vaccination to successfully promote anti-tumor immunity in vivo.     

Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients.

PURPOSE: We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. EXPERIMENTAL DESIGN: Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). RESULTS: All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells. CONCLUSIONS: We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical results.     

Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines

Dendritic cells (DCs) are potent antigen-presenting cells and play a pivotal role in T cell-mediated immunity. DCs have been shown to induce strong antitumor immune responses in vitro and in vivo, and their efficacy is being investigated in clinical trials. Compared with vaccination strategies directed against a single tumor antigen, tumor-cell lysate as the source of antigen offers the potential advantage of inducing a broad T-cell response against multiple known, as well as unknown, tumor-associated antigens expressed by the individual tumor. We used pancreatic carcinoma cell lines to develop an in vitro model for monitoring T-cell responses induced by lysate-pulsed DCs. Monocyte-derived DCs of HLA-A2(+) donors were pulsed with lysate generated from the HLA-A2(+) pancreatic carcinoma cell line Panc-1. In some experiments, the immunogenic protein keyhole limpet hemocyanin (KLH) was added to the lysate. Subsequently, the antigen-loaded DCs were activated with tumor necrosis factor-alpha and prostaglandin E(2). Autologous mononuclear cells were cocultured with DCs in the presence of low-dose interleukin (IL)-2 and IL-7 and were restimulated weekly with new DCs. High levels of IL-12 and IFN-gamma could be detected in the supernatants, indicating a T-helper type 1-type immune response. This cytokine profile was associated with the expression of the activation marker CD69 on both T helper and CTLs and with an antigen-induced proliferative T-cell response. After 4 weeks, CTL-mediated cytotoxicity was assessed. Tumor cell lysis was specific for Panc-1 tumor cells and was MHC class I-restricted. Cytokine secretion, CD69 expression of T cells, and antigen-induced T-cell proliferation correlated with the cytotoxic activity and were more pronounced when KLH was added to the lysate. This is the first study to show that T cells specific for pancreatic carcinoma cells can be generated in vitro by lysate-pulsed DCs and that the T-cell response can be enhanced by KLH. This in vitro model can be applied to compare different strategies in the development of DC-based tumor vaccines.     

Dendritic cells pulsed with viral oncolysates potently stimulate autologous T cells from cancer patients

We demonstrated before that primary operated breast cancer patients contain in their bone marrow (BM) cancer reactive memory T cells (MTC) which have to be re-activated to become tumor infiltrating effector cells. The aim of this study was to optimize an ex vivo stimulation protocol for MTC based on autologous dendritic cells (DC). As source of tumor antigens we used lysates from unmodified tumor cells or from tumor cells infected with Newcastle Disease Virus (NDV) which contain IFN-alpha inducing viral dsRNA as one danger signal. DC from breast cancer patients were pulsed with lysates from the MCF-7 breast cancer line (Tu-L) or from NDV infected MCF-7 cells (TuN-L, viral oncolysates) and compared for stimulatory capacity in an ELISPOT response of autologous BM derived MTC. To analyze potential further danger signals derived from NDV infection, we employed MALDI mass spectrometry, Western blots, FACS cytometry and ELISA tests. DC pulsed with viral oncolysates showed increased expression of co-stimulatory molecules in comparison to Tu-L pulsed DC and induced significantly higher ELISPOT MTC responses. Supernatants from co-cultures of MTC and TuN-L pulsed DC contained increased titers of IFN-alpha and IL-15. NDV infection of tumor cells resulted in a number of differences in protein expression including a heat-shock protein (HSP27) which became phosphorylated. The results suggest that a DC preparation pulsed with viral oncolysate includes danger signals (e.g. dsRNA, cytokines, HSP molecules) and is superior for MTC stimulation to a DC preparation pulsed with lysate from non-infected tumor cells.     

Murine dendritic cells transfected with human GP100 elicit both antigen-specific CD8(+) and CD4(+) T-cell responses and are more effective than DNA vaccines at generating anti-tumor immunity.

Dendritic cells (DCs) are potent inducers of cytotoxic T lymphocytes (CTLs) when pulsed with an antigenic peptide or tumor lysate. In this report, we have used liposome-mediated gene transfer to examine the ability of plasmid DNA encoding the human melanoma-associated antigen gp100 to elicit CD8(+) and CD4(+) T-cell responses. We also compared the efficacy between gp100 gene-modified DCs and naked DNA (pCDNA3/gp100)-based vaccines at inducing anti-tumor immunity. DCs were generated from murine bone marrow and transfected in vitro with plasmid DNA containing the gp100 gene. These gp100-modified DCs (DC/gps) were used to stimulate syngeneic naive spleen T cells in vitro or to immunize mice in vivo. Antigen-specific, MHC-restricted CTLs were generated when DC/gps were used to prime T cells both in vitro and in vivo. Thus, these CTLs were cytolytic for gp100-transfected syngeneic (H-2(b)) tumor MCA106 (MCA/gp) and vaccinia-pMel17/gp100-infected syngeneic B16 and MCA106, but not parental tumor MCA106 and B16, or gp100-transfected allogeneic tumor P815 (H-2(d)). Immunization with DC/gp protected mice from subsequent challenge with MCA/gp but not parental MCA106. Antibody-mediated T-cell subset depletion experiments demonstrate that induction of CTLs in vivo is dependent on both CD4(+) and CD8(+) T cells. Furthermore, DC/gp immunization elicits an antigen-specific CD4(+) T-cell response, suggesting that DC/gps present MHC class II epitopes to CD4(+) T cells. In addition, our data show that gene-modified, DC-based vaccines are more effective than the naked DNA-based vaccines at eliciting anti-tumor immunity in both prophylactic and therapeutic models. These results suggest that the use of DCs transfected with plasmid DNA containing a gene for TAA may be superior to peptide-pulsed DCs and naked DNA-based vaccines for immunotherapy and could provide an alternative strategy for tumor vaccine design.     

A CD4+, HLA-DR7-restricted T-helper lymphocyte clone recognizes an antigen shared by human malignant melanoma and glioma

CD4(+) Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4(+) Th cell clone (Th35-1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV-transformed B cells pulsed with melanoma tumor cell lysates. Th35-1A produces IFN-gamma (a Th1-type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II-restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II-matched and unmatched EBV-B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA-DR7 isolated from autologous EBV-B cells rendered the cell line stimulatory for Th35-1A cells. Specificity studies using autologous EBV-B cells (EBV-B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.     

Determinant spreading associated with clinical response in dendritic cell-based immunotherapy for malignant melanoma.

PURPOSE: The purpose of this study was to determine the toxicity and immunological effects of three different doses and two routes of administration of autologous dendritic cells (DCs) pulsed with the MART-1(27-35) immunodominant epitope. EXPERIMENTAL DESIGN: Eighteen HLA-A*0201-positive subjects with stage III-IV melanoma received three biweekly i.v. or intradermal injections of ex vivo generated myeloid DCs pulsed with MART-1(27-35) epitope. Repeated blood samples were processed to obtain peripheral blood mononuclear cells for immunological analysis using IFN-gamma ELISPOT, MHC class I tetramer, intracellular cytokine staining, and microcytotoxicity assays. RESULTS: The frequency of MART-1/Melan-A (MART-1) antigen-specific T cells in peripheral blood increased in all dose levels as assessed by ELISPOT and MHC class I tetramer assays, but without a clear dose-response effect. The intradermal route generated stronger MART-1 immunity compared with the i.v. route. MART-1-specific immunity did not correlate with clinical outcome in any of the four immunological assays used. However, analysis of determinant spreading to other melanoma antigens was noted in the only subject with complete response to this single-epitope immunization. CONCLUSIONS: Intradermal immunization with MART-1 peptide-pulsed DCs results in an increase in circulating IFN-gamma-producing, antigen-specific T cells. The frequency of these cells did not correlate with response. In contrast, spreading of immune reactivity to other melanoma antigens was only evident in a subject with a complete response, suggesting that determinant spreading may be an important factor of clinical response to this form of immunotherapy.     

MHC class II-transduced tumor cells originating in the immune-privileged eye prime and boost CD4(+) T lymphocytes that cross-react with primary and metastatic uveal melanoma cells

Uveal melanoma, the most common malignancy of the eye, has a 50% rate of liver metastases among patients with large primary tumors. Several therapies prolong survival of metastatic patients; however, none are curative and no patients survive. Therefore, we are exploring immunotherapy as an alternative or adjunctive treatment. Uveal melanoma may be particularly appropriate for immunotherapy because primary tumors arise in an immune-privileged site and may express antigens to which the host is not tolerized. We are developing MHC class II (MHC II)-matched allogeneic, cell-based uveal melanoma vaccines that activate CD4(+) T lymphocytes, which are key cells for optimizing CD8(+) T-cell immunity, facilitating immune memory, and preventing tolerance. Our previous studies showed that tumor cells genetically modified to express costimulatory and MHC II molecules syngeneic to the recipient are potent inducers of antitumor immunity. Because the MHC II-matched allogeneic vaccines do not express the accessory molecule, Invariant chain, they present MHC II-restricted peptides derived from endogenously encoded tumor antigens. We now report that MHC II-matched allogeneic vaccines, prepared from primary uveal melanomas that arise in the immune-privileged eye, prime and boost IFNgamma-secreting CD4(+) T cells from the peripheral blood of either healthy donors or uveal melanoma patients that cross-react with primary uveal melanomas from other patients and metastatic tumors. In contrast, vaccines prepared from metastatic cells in the liver are less effective at activating CD4(+) T cells, suggesting that tumor cells originating in immune-privileged sites may have enhanced capacity for inducing antitumor immunity and for serving as immunotherapeutic agents.     

Human autologous dendritic cell-glioma fusions: feasibility and capacity to stimulate T cells with proliferative and cytolytic activity

Summary Gliomas are the most common primary neoplasm of the central nervous system. The failure of conventional treatment modalities to improve outcome over the last two decades has led to interest in alternative treatment modalities. Dendritic cell (DC)-based immunotherapy has utilized DC pulsed with tumor lysate or peptide to induce an antitumor immune response mediated largely by CD8 T cells. While this has been effective in preclinical studies, clinical efficacy remains unproven. Recently, hybrid cells produced by fusions of tumor and autologous DC have demonstrated remarkable efficacy for stimulating an anti-tumor immune response in both preclinical and clinical studies of extra-cranial neoplasms. The advantage of generating such hybrid cells is that the entire cellular material of the tumor is processed and presented in both endogenous and exogenous pathways. This leads to activation of both MHC class I restricted CD8 cells as well as MHC class II restricted CD4 T cells. Here, we examinedin vitro T cell stimulatory capacity of autologous human DC-glioma fusion in comparison to DC loaded with apoptotic glioma. DC fused with autologous tumor or loaded with apoptotic tumor cells (DC/apo) were first used to stimulate autologous non-adherent peripheral blood mononuclear cells (PBMC),in vitro. The PBMC were then examined for phenotype (CD3, CD4, CD8) and intracellular IFN-γ using flow cytometry. Lymphocyte proliferation and cytolytic responses were also assessed. Lymphocytes stimulatedin vitro with fusion or DC/apo cells showed significantly enhanced cytotoxicity and proliferation against autologous tumor cells compared with PBMC stimulated with tumor cells or DC alone. Both strategies had similar efficacy. Tumor-cytolytic responses were enhanced by the addition of CD40 ligand (CD40L), and partially blocked by anti-MHC class I antibody. Flow cytometric analysis detected CD3⁺CD8⁺ T cells, which also stained positive for intracellular IFN-γ. The study suggests that DC/glioma fusion and DC/apo have comparable efficacy for stimulation of CTL with cytolytic and proliferative activity against human malignant gliomas. These findings may have implications for future studies of DC-based immunotherapy in malignant gliomas.