Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses

Immunotherapy trials targeting the induction of tumor-reactive T-cell responses in cancer patients appear to hold significant promise, Because nonmutated lineage-specific antigens and mutated idiotypic antigens may be coexpressed by tumor cells, the use of autologous tumor material to promote the broadest range of antitumor T-cell specificities has significant clinical potential in cancer vaccination trials. As a model for vaccination in the cancer setting, we chose to analyze the promotion of T-cell responses against Epstein-Barr virus (EBV)transformed B-lymphoblastoid cell line (B-LCL)-derived antigens in vitro. A series of bulk antigenic formats (freeze-thaw lysate, trifluoroacetic acid lysate, extracted membranes, affinity-purified MHC class I- and class Ii-presented peptides, acid-eluted peptides) prepared from EBV B-LCLs were tested for their ability to stimulate EBV B-LCL-reactive CD4(+) and CD8(+) T lymphocytes in vitro when pulsed onto autologous dendritic cells (DCs). DC presentation of freeze-thaw lysate material derived from (either autologous or allogeneic) EBV B-LCLs with an Mr of 10 kd or larger stimulated optimal anti-EBV B-LCL responsiveness from freshly isolated CD4(+) and CD8(+) peripheral blood T cells. These in vivo "memory" T-cell responses were observed only in EBV-seropositive donors. CD4(+) T-cell responses to lysate-pulsed DCs were Th1 type tie, strong interferon-gamma and weak interleukin-5 responses). While CD8(+) T-cell responses were also observed in interferon-gamma Elispot assays and in cytotoxicity assays, these responses were of low frequency unless the DC stimulators were induced to "mature" after being fed with tumor lysates, Optimal-length, naturally processed, and MHC class I- or class It-presented tumor peptides were comparatively poorly immunogenic in this model system. (C) 2000 by The American Society of Hematology

Identification of naturally processed and HLA-presented Epstein-Barr virus peptides recognized by CD4(+) or CD8(+) T lymphocytes from human blood

The broad clinical implementation of cancer vaccines targeting the induction of specific T cell-mediated immunity is hampered because T cell defined tumor-associated peptides are currently available for only a restricted range of tumor types. Current epitope identification strategies require a priori the generation of T "indicator" cell lines that specifically recognize the tumor antigenic epitope in in vitro assay systems. An alternative to this strategy is the use of "memory" T cells freshly isolated from the peripheral blood of patients with cancer in concert with sensitive effector cell readout assays (such as the cytokine enzyme-linked immunospot assay) and MS to identify relevant peptide epitopes. In a model system, we have evaluated the capacity of natural Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell line-extracted peptides to activate "memory" viral-specific CD4(+) or CD8(+) T cells freshly isolated from the blood of an EBV-seropositive individual using the IFN-gamma enzyme-linked immunospot assay. After HPLC fractionation and loading onto autologous dendritic cells, multiple naturally processed HLA class I and II-associated lymphoblastoid cell line-derived peptides were isolated that were capable of inducing IFN-gamma spot production by "memory" T lymphocytes. Using MS analysis on a HPLC fraction recognized by CD8(+) T cells, we were able to sequence natural 9-, 10-, and 11-mer peptides naturally processed from the latent EBV antigen LMP-2 (latent membrane protein-2) and presented in the context of HLA-A2. This approach provides a useful methodology for the future identification of MHC-presented viral and tumor epitopes using freshly isolated patient materials

Interferon-alpha (IFN-alpha) –conditioned DC Preferentially Stimulate Type-1 and Limit Treg-type In Vitro T-cell Responses From RCC Patients

Dendritic cells (DCs) are potent antigen presenting cells and represent attractive candidates for use in novel immunotherapies for patients with renal cell carcinoma (RCC), a disease that has proven refractory to conventional treatment modalities, such as chemotherapy and radiotherapy. Given the perceived need to augment antitumor type-l immunity (T(C)1 and T(h)1) as a therapeutic end point, and the known functional plasticity of DC populations that may display heterogeneous capacity to promote T-cell responses, we sought to identify a preferred DC preparation with this capacity. We compared 2 different preparations of monocyte-derived DC using interferon-alpha (IFN-alpha) (IFN-DC and alpha DCI) with classic DCs "matured" (mDCs) using interleukin-1 beta/interleukin-6/tumor necrosis factor-alpha/prostaglandin E-2, for their ability to promote autologous T(C)1 antitumor responses from RCC patients in vitro. IFN-alpha-conditioned DC promoted significantly higher numbers of RCC-specific CD8(+) T cells exhibiting a cytotoxic phenotype after in vitro stimulation (IVS) than cytokine cocktail-mDCs. Furthermore, IVS using IFN-DCs was able to diminish regulatory-type T cells among CD4(+) T-cell responder populations versus IVS using conventional mDC-based vaccines. These data emphasize an important role for IFN-a in modulating the immunologic functions of DCs toward a polarized DC1-type capable of coordinately promoting T(H)1- type and T(C)1-type T-cell mediated immunity and supports the translational development of patient-derived IFN-alpha-conditioned DC for use in novel immunotherapies for patients with RCC, and in whom, endogenous tumor-specific T(C)1 effector cells may be dysfunctional, anergic, or prone to undergo apoptosis

gp100/pmel17 and tyrosinase encode multiple epitopes recognized by Th1-type CD4+T cells.

CD4(+) T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as effector cells in the tumour microenvironment. In order to identify the turnout epitopes recognized by tumour-reactive human CD4+ T cells, we combined the use of an HLA-DR4/peptide binding algorithm with an IFN-gamma ELISPOT assay. Two known and three novel CD4+ T cell epitopes derived from the gp 100/pmel17 and tyrosinase mefanocyte-associated antigens were confirmed or identified. Of major interest, we determined that freshly-isolated PBMC frequencies of Th1-type CD4+ T recognizing these peptides are frequently elevated in HLA-DR4+ melanoma patients (but not normal donors) that are currently disease-free as a result of therapeutic intervention. Epitope-specific CD4+ T cells from normal DR4+ donors could be induced, however, after in vitro stimulation with autologous dendritic cell pulsed with antigens (peptides or antigen-positive melanoma lysates) or infected with recombinant vaccinia virus encoding the relevant antigen. Peptide-reactive CD4+ T cells also recognized HLA-DR4+ melanoma cell lines that constitutively express the relevant antigen. Based on these data, these epitopes may serve as potent vaccine components to promote clinically-relevant Th1-type CD4+ T cell effector function in situ. (C) 2001 Cancer Research Campaign

Dysfunctional DC subsets in RCC patients: Ex vivo correction to yield an effective anti-cancer vaccine

Dendritic cells (DCs) are potent antigen-presenting cells responsible for the activation and functional polarization of specific T cells. In patients with renal cell carcinoma (RCC) and other cancers, coordinate DC and T cell defects have been reported. In particular, DC and T cell functional subsets that are not conducive to tumor clearance are hypothesized to predominate in patients with advanced-stage disease. Two major peripheral blood DC subsets have been identified in humans: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) that are believed to mediate contrasting effects on cancer immunity. Given the lack of information regarding DC subsets in patients with RCC, in the present study we have investigated the comparative frequencies and activation states of mDC and pDC in peripheral blood, cancer tissues and lymph nodes of patients with RCC using flow cytometry and immunohistochemistry. Three monoclonal antibodies (mAbs) reactive against specific DC subsets (BDCA-2 or BDCA-4 for pDC and BDCA-1 and BDCA-3 which represent two distinct subsets of mDC, mDC1 and mDC2, respectively) were employed. We observed a significant reduction of both DC subsets in the peripheral blood of patients as compared to normal donors. Similarly, both mDC and pDC were recruited in large numbers into RCC tumor tissues, where they displayed an immature phenotype (DC-LAMP(-)) and appeared unable to differentiate into mature DC (CD83(+)) that were competent to migrate to draining lymph nodes. However, we were readily able to generate ex vivo mDC from RCC patients. These DC stimulated robust anti-tumor CTL in vitro and would be envisioned for use in DC-based vaccines applied in patients with RCC whose existing immune system is judged dysfunctional, anergic or prone to undergo apoptosis

MAGE-6 encodes HLA-DRbeta1*0401-presented epitopes recognized by CD4+ T cells from patients with melanoma or renal cell carcinoma

CD4+ T cells modulate the magnitude and durability of CTL responses in vivo and may serve as potent effector cells within the tumor microenvironment. The current study was undertaken to define novel epitopes from the broadly expressed tumor antigen MAGE-6 that are recognized by CD4+ T cells. We have combined the use of a HLA-DR4/peptide binding algorithm with the IFN-gamma enzyme-linked immunospot assay to identify four nonoverlapping sequences derived from the MAGE-6 protein that served as CD4+ T-cell epitopes in HLA-DR4+ donors. Strikingly, patients with active melanoma or renal cell carcinoma failed to secrete IFN-gamma in response to MAGE-6-derived epitopes, whereas both normal donors and cancer patients with no current evidence of disease were responsive, particularly after short-term in vitro stimulations with peptide-pulsed dendritic cells. Importantly, peptide-specific CD4+ T cells also recognized HLA-DRbeta1*0401+ tumor cells that constitutively expressed the MAGE-6 protein and autologous HLA-DRbeta1*0401+ dendritic cells transfected with MAGE-6 cDNA-elicited CD4+ T cells that reacted against individual peptide epitopes in vitro. These data suggest that MAGE-6-derived epitopes could serve as useful vaccine candidate components and may provide an immune-monitoring index of clinically important Th1-type immunity in patients with renal cell carcinoma or melanoma